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Barlabé P, Aranguren XL, Coppiello G. Blastocyst complementation: current progress and future directions in xenogeneic organogenesis. Stem Cell Res Ther 2025; 16:321. [PMID: 40551151 PMCID: PMC12186422 DOI: 10.1186/s13287-025-04426-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2025] [Accepted: 05/27/2025] [Indexed: 06/28/2025] Open
Abstract
The generation of organs derived from pluripotent stem cells can be achieved in vivo through the blastocyst complementation technique. This method is based on the introduction of pluripotent stem cells into organogenesis-disabled pre-implantation embryos, where environmental signals instruct donor cells to colonize the vacant niche and to develop into the missing organ. When applied interspecies, this approach has the potential to produce human organs in genetically engineered livestock, offering a promising solution to the global transplants' shortage crisis. In this review, we summarize the current progress in blastocyst complementation research and highlight the key challenges that must be addressed to advance this field.
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Affiliation(s)
- Paula Barlabé
- Biomedical Engineering Program, Enabling Technologies Division, CIMA Universidad de Navarra, 31008, Pamplona, Spain
| | - Xabier L Aranguren
- Biomedical Engineering Program, Enabling Technologies Division, CIMA Universidad de Navarra, 31008, Pamplona, Spain.
| | - Giulia Coppiello
- Biomedical Engineering Program, Enabling Technologies Division, CIMA Universidad de Navarra, 31008, Pamplona, Spain.
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2
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Cao J, Guo Z, Xu X, Li P, Fang Y, Deng S. Advances in CRISPR-Cas9 in lineage tracing of model animals. Animal Model Exp Med 2025. [PMID: 40491322 DOI: 10.1002/ame2.70033] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2024] [Accepted: 04/28/2025] [Indexed: 06/11/2025] Open
Abstract
Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree. However, traditional methods have limitations of poor stability and insufficient resolution. As an efficient and flexible gene editing tool, CRISPR-Cas9 system has been widely used in biological research. Furthermore, CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins, reporter genes, or DNA barcodes for high-throughput sequencing, enabling precise lineage analysis, significantly improving precision and resolution, and expanding its application range. In this review, we summarize applications of CRISPR-Cas9 system in cell lineage tracing, with special emphasis on its successful applications in traditional model animals (e.g., zebrafish and mice), large animal models (pigs), and human cells or organoids. We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.
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Affiliation(s)
- Jingchao Cao
- College of Animal Science and Technology, China Agricultural University, Beijing, China
| | - Zihang Guo
- National Center of Technology Innovation for Animal Model, National Human Diseases Animal Model Resource Center, National Health Commission of China (NHC) Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China
| | - Xueling Xu
- College of Bee Science and Biomedicine, Fujian Agriculture and Forestry University, Fuzhou, China
| | - Pan Li
- Xianghu Laboratory, Hangzhou, China
| | - Yi Fang
- Key Lab of Animal Production, Product Quality and Security, Ministry of Education, Jilin Agricultural University, Changchun, China
| | - Shoulong Deng
- National Center of Technology Innovation for Animal Model, National Human Diseases Animal Model Resource Center, National Health Commission of China (NHC) Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China
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3
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Garry DJ, Garry MG, Nakauchi H, Masaki H, Sachs DH, Weiner JI, Reichart D, Wolf E. Allogeneic, Xenogeneic, and Exogenic Hearts for Transplantation. Methodist Debakey Cardiovasc J 2025; 21:92-99. [PMID: 40384731 PMCID: PMC12082467 DOI: 10.14797/mdcvj.1590] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2025] [Accepted: 04/07/2025] [Indexed: 05/20/2025] Open
Abstract
The only curative therapy for end-stage heart failure is orthotopic allogeneic heart transplantation. This therapy has extended the survival of patients worldwide but is limited due to the scarcity of donor organs. Potential alternative donor sources of organs for transplantation include genetically-modified (GM) large animal donors (ie, xenografts) and human organs developed in large animal hosts. These strategies utilize gene editing and somatic cell nuclear transfer technologies to engineer partially or completely humanized organs. Preclinical xenotransplantation studies of GM pig hearts into baboons have already provided an important clinical foundation, as two patients have received cardiac xenografts from GM pigs and have survived for up to 2 months. Additional issues need to be addressed in order for patients to survive more than 1 year, which would make these strategies clinically applicable. Thus, in combination with immunosuppression agents, xenogeneic and exogenic organ sources hold tremendous promise for an unlimited and transformative supply of organs for transplantation.
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Affiliation(s)
- Daniel J. Garry
- Stem Cell Institute, IN
- Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota, US
| | - Mary G. Garry
- Stem Cell Institute, IN
- Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota, US
| | - Hiromitsu Nakauchi
- University of Tokyo, Tokyo, JP
- Institute of Science Tokyo (formerly Tokyo Medical and Dental University), Tokyo, JP
- Stanford University School of Medicine, Stanford, California, US
| | - Hideki Masaki
- University of Tokyo, Tokyo, JP
- Institute of Science Tokyo (formerly Tokyo Medical and Dental University) Tokyo, JP
| | - David H. Sachs
- Vagelos College of Physicians and Surgeons, Columbia University, New York, New York, US
- Massachusetts General Hospital, Boston, Massachusetts, US
| | - Joshua I. Weiner
- Vagelos College of Physicians and Surgeons, Columbia University, New York, New York, US
| | - Daniel Reichart
- University Hospital, LMU Munich, Munich, DE
- Gene Center and Center for Innovative Medical Models (CiMM), DE
- Interfaculty Center for Endocrine and Cardiovascular Disease Network Modelling and Clinical Transfer (ICONLMU), LMU Munich, Munich, DE
| | - Eckhard Wolf
- Gene Center and Center for Innovative Medical Models (CiMM), DE
- Interfaculty Center for Endocrine and Cardiovascular Disease Network Modelling and Clinical Transfer (ICONLMU), LMU Munich, Munich, DE
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4
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Zimmerlin L, Angarita A, Park TS, Evans-Moses R, Thomas J, Yan S, Uribe I, Vegas I, Kochendoerfer C, Buys W, Leung AKL, Zambidis ET. Proteogenomic reprogramming to a functional human blastomere-like stem cell state via a PARP-DUX4 regulatory axis. Cell Rep 2025; 44:115671. [PMID: 40338744 DOI: 10.1016/j.celrep.2025.115671] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2024] [Revised: 02/17/2025] [Accepted: 04/16/2025] [Indexed: 05/10/2025] Open
Abstract
Here, we show that conventional human pluripotent stem cells cultured with non-specific tankyrase-PARP1-inhibited conditions underwent proteogenomic reprogramming to functional blastomere-like tankyrase/PARP inhibitor-regulated naive stem cells (TIRN-SC). TIRN-SCs concurrently expressed hundreds of pioneer factors in hybrid 2C-8C-morula-ICM programs that were augmented by induced expression of DUX4. Injection of TIRN-SCs into 8C-staged murine embryos equipotently differentiated human cells to the extra-embryonic and embryonic compartments of chimeric blastocysts and fetuses. Ectopic expression of murine-E-Cadherin in TIRN-SCs further enhanced interspecific chimeric tissue targeting. TIRN-SC-derived trophoblast stem cells efficiently generated placental chimeras. Proteome-ubiquitinome analyses revealed increased TNKS and reduced PARP1 levels and an ADP-ribosylation-deficient, hyper-ubiquitinated proteome that impacted expression of both tankyrase and PARP1 substrates. ChIP-seq of NANOG-SOX2-OCT4 and PARP1 (NSOP) revealed genome-wide NSOP co-binding at DUX4-accessible enhancers of embryonic lineage factors; suggesting a DUX4-NSOP axis regulated TIRN-SC lineage plasticity. TIRN-SCs may serve as valuable models for studying the proteogenomic regulation of pre-lineage human embryogenesis. VIDEO ABSTRACT.
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Affiliation(s)
- Ludovic Zimmerlin
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Ariana Angarita
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Tea Soon Park
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Rebecca Evans-Moses
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Justin Thomas
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Sirui Yan
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Isabel Uribe
- Departments of Biochemistry and Molecular Biology, The Johns Hopkins School of Public Health, Baltimore, MD, USA
| | - Isabella Vegas
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Clara Kochendoerfer
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Willem Buys
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Anthony K L Leung
- Departments of Biochemistry and Molecular Biology, The Johns Hopkins School of Public Health, Baltimore, MD, USA
| | - Elias T Zambidis
- Institute for Cell Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD, USA.
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Kardeh S, Mazloomrezaei M, Hosseini A. Scaling Autologous Epidermal Cell Therapies: iPSC-Derived Keratinocytes and In Vivo Chimerism for Skin Regeneration. Exp Dermatol 2025; 34:e70107. [PMID: 40289411 DOI: 10.1111/exd.70107] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2024] [Revised: 04/13/2025] [Accepted: 04/17/2025] [Indexed: 04/30/2025]
Abstract
Severe skin injuries and genetic disorders such as epidermolysis bullosa present significant clinical challenges due to limitations in current epidermal replacement therapies. While promising, cultured epithelial autografts (CEAs) suffer from prolonged culture times, cellular senescence, and low-quality clinical outcomes, limiting their widespread application. Recent advancements in iPSC-derived keratinocytes (iKeratinocytes) and in vivo chimerism offer transformative potential for scalable and personalised skin regeneration. Advances in understanding transcriptional networks, mRNA delivery, CRISPR-based genome editing, and automated biomanufacturing processes can enable improved and efficient protocols for generating iKeratinocytes. Despite these advances, there are still challenges for scaling iKeratinocytes, including optimising xeno-free culture systems and developing reproducible methods for generating multilayered skin with appendages. Interspecies chimerism utilising lineage-specific ablation systems and targeted in utero delivery of organ progenitor cells can enable human epidermal tissue development within animal hosts, offering an alternative novel platform for scaling epidermal cell and skin generation. This method, however, requires further refinements for complete ablation and detachment of target cells in the animal hosts and improved human cell integration in chimeric models. Together, iKeratinocytes and in vivo chimerism hold great promise for advancing autologous epidermal cell therapies and enabling broader clinical adoption and improved outcomes for patients with severe skin injuries and genetic disorders.
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Affiliation(s)
- Sina Kardeh
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
- Harvard Medical School, Boston, Massachusetts, USA
- Shriners Hospital for Children, Boston, Massachusetts, USA
| | - Mohsen Mazloomrezaei
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
- Harvard Medical School, Boston, Massachusetts, USA
- Shriners Hospital for Children, Boston, Massachusetts, USA
| | - Ahmad Hosseini
- Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA
- Harvard Medical School, Boston, Massachusetts, USA
- Shriners Hospital for Children, Boston, Massachusetts, USA
- Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, Boston, Massachusetts, USA
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Natsume R, Murata K, Taketsuru H, Hirayama R, Iwasaki T, Yamashiro H, Takao K, Nakatsukasa E, Abe M, Sakimura K. Fertilizable Rat Sperm Is Generated in Mice Using Blastocyst Complementation: An Efficient Method for Producing Rats With ES Cell Traits. Genes Cells 2025; 30:e70024. [PMID: 40401572 DOI: 10.1111/gtc.70024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2025] [Revised: 04/22/2025] [Accepted: 04/30/2025] [Indexed: 05/23/2025]
Abstract
We developed a novel approach for generating rat offspring using rat embryonic stem (ES) cell-derived sperm produced in mice with the blastocyst complementation method. By optimizing culture conditions, we established naïve male rat ES cells from two transgenic rat strains expressing EGFP and Venus fluorescence, respectively. The pluripotency of these cells was confirmed by the formation of germline chimeras. These ES cells were then injected into blastocysts of germ cell-deficient mice, which resulted in chimeric mice with the ability to produce rat-derived sperm. Histological analysis confirmed the presence of seminiferous tubules and spermatozoa, which are morphologically characteristic of rats, in the chimeric testes. To evaluate the fertilization potential of the chimeric mouse sperm, we performed intracytoplasmic sperm injection (ICSI) to rat oocytes and successfully produced viable offspring carrying ES cell-derived traits. This method eliminates concerns regarding host cell contribution, as all sperm in the chimeras originate from rats, enabling the use of nonfluorescent cells. Furthermore, the absence of competition with host cells is expected to enhance sperm production efficiency. By utilizing germ cell-deficient mice as recipients, this approach offers a cost-effective and efficient strategy for generating genetically modified rats, addressing key limitations in rat ES cell-based genetic engineering.
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Affiliation(s)
- Rie Natsume
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
| | - Kosuke Murata
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
- Graduate School of Science and Technology, Niigata University, Niigata, Japan
| | - Hiroaki Taketsuru
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
- Department of Comparative & Experimental Medicine, Brain Research Institute, Niigata University, Niigata, Japan
| | - Runa Hirayama
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
- Department of Behavioral Physiology Graduate School of Innovative Life Science, University of Toyama, Toyama, Japan
| | - Tsugumi Iwasaki
- Graduate School of Science and Technology, Niigata University, Niigata, Japan
| | - Hideaki Yamashiro
- Graduate School of Science and Technology, Niigata University, Niigata, Japan
| | - Keizo Takao
- Department of Behavioral Physiology Graduate School of Innovative Life Science, University of Toyama, Toyama, Japan
- Department of Behavioral Physiology, Faculty of Medicine, University of Toyama, Toyama, Japan
- Research Center for Idling Brain Science, University of Toyama, Toyama, Japan
| | - Ena Nakatsukasa
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
| | - Manabu Abe
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
| | - Kenji Sakimura
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
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7
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Guo X, Wang X, Wang J, Ma M, Ren Q. Current Development of iPSC-Based Modeling in Neurodegenerative Diseases. Int J Mol Sci 2025; 26:3774. [PMID: 40332425 PMCID: PMC12027653 DOI: 10.3390/ijms26083774] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2025] [Revised: 04/08/2025] [Accepted: 04/09/2025] [Indexed: 05/08/2025] Open
Abstract
Over the past two decades, significant advancements have been made in the induced pluripotent stem cell (iPSC) technology. These developments have enabled the broader application of iPSCs in neuroscience, improved our understanding of disease pathogenesis, and advanced the investigation of therapeutic targets and methods. Specifically, optimizations in reprogramming protocols, coupled with improved neuronal differentiation and maturation techniques, have greatly facilitated the generation of iPSC-derived neural cells. The integration of the cerebral organoid technology and CRISPR/Cas9 genome editing has further propelled the application of iPSCs in neurodegenerative diseases to a new stage. Patient-derived or CRISPR-edited cerebral neurons and organoids now serve as ideal disease models, contributing to our understanding of disease pathophysiology and identifying novel therapeutic targets and candidates. In this review, we examine the development of iPSC-based models in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease.
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Affiliation(s)
- Xiangge Guo
- Department of Human Anatomy, Hebei Medical University, Shijiazhuang 050017, China; (X.G.); (X.W.); (J.W.)
| | - Xumeng Wang
- Department of Human Anatomy, Hebei Medical University, Shijiazhuang 050017, China; (X.G.); (X.W.); (J.W.)
| | - Jiaxuan Wang
- Department of Human Anatomy, Hebei Medical University, Shijiazhuang 050017, China; (X.G.); (X.W.); (J.W.)
| | - Min Ma
- Department of Human Anatomy, Hebei Medical University, Shijiazhuang 050017, China; (X.G.); (X.W.); (J.W.)
- Human Brain Bank, Hebei Medical University, Shijiazhuang 050017, China
| | - Qian Ren
- Department of Human Anatomy, Hebei Medical University, Shijiazhuang 050017, China; (X.G.); (X.W.); (J.W.)
- The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang 050017, China
- Hebei Key Laboratory of Neurodegenerative Disease Mechanism, Hebei Medical University, Shijiazhuang 050017, China
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Strell P, Waldron MA, Johnson S, Shetty A, Crane AT, Steer CJ, Low WC. Characterization of the intraspecies chimeric mouse brain at embryonic day 12.5. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.31.646380. [PMID: 40236149 PMCID: PMC11996362 DOI: 10.1101/2025.03.31.646380] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
Incidence of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis have increased dramatically as life expectancy at birth has risen year-over-year and the population ages. Neurological changes within the central nervous system, specifically the brain, include cell loss and deterioration that impact motor function, memory, executive function, and mood. Available treatments are limited and often only address symptomatic manifestations of the disease rather than disease progression. Cell transplantation therapy has shown promise for treating neurodegenerative diseases, but a source of autologous cells is required. Blastocyst complementation provides an innovative method for generating those autologous neural cells. By injecting mouse induced pluripotent stem cells (iPSCs) into a wild type (WT) mouse blastocyst, we generated a chimeric mouse brain derived of both donor and host neuronal and non-neuronal cells. An embryonic day 12.5 (E12.5), automated image analysis of mouse-mouse chimeric brains showed the presence of GFP-labeled donor-derived dopaminergic and serotonergic neuronal precursors. GFP-labeled donor-derived cholinergic precursor neurons and non-neuronal microglia-like and macrophage-like cells were also observed using more conventional imaging analysis software. This work demonstrates that the generation of mouse-mouse chimeric neural cells is possible; and that characterization of early neuronal and non-neuronal precursors provides a first step towards utilizing these cells for cell transplantation therapies for neurodegenerative diseases.
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Henden C, Fjerdingstad HB, Bjørnsen EG, Thiruchelvam-Kyle L, Daws MR, Inngjerdingen M, Glover JC, Dissen E. NK-cell cytotoxicity toward pluripotent stem cells and their neural progeny: impacts of activating and inhibitory receptors and KIR/HLA mismatch. Stem Cells 2025; 43:sxae083. [PMID: 39708357 PMCID: PMC11929945 DOI: 10.1093/stmcls/sxae083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Accepted: 11/14/2024] [Indexed: 12/23/2024]
Abstract
Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK-cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2, and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2, or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS-cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS-cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2-stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS-cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK-cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.
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Affiliation(s)
- Camilla Henden
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Hege B Fjerdingstad
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
- Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, N-0317 Oslo, Norway
| | - Elisabeth G Bjørnsen
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Lavanya Thiruchelvam-Kyle
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Michael R Daws
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Marit Inngjerdingen
- Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, N-0317 Oslo, Norway
| | - Joel C Glover
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
- Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, N-0317 Oslo, Norway
| | - Erik Dissen
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
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10
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Bigliardi E, Shetty AV, Low WC, Steer CJ. Interspecies Blastocyst Complementation and the Genesis of Chimeric Solid Human Organs. Genes (Basel) 2025; 16:215. [PMID: 40004544 PMCID: PMC11854981 DOI: 10.3390/genes16020215] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2025] [Revised: 02/06/2025] [Accepted: 02/09/2025] [Indexed: 02/27/2025] Open
Abstract
Solid organ transplantation remains a life-saving treatment for patients worldwide. Unfortunately, the supply of donor organs cannot meet the current need, making the search for alternative sources even more essential. Xenotransplantation using sophisticated genetic engineering techniques to delete and overexpress specific genes in the donor animal has been investigated as a possible option. However, the use of exogenous tissue presents another host of obstacles, particularly regarding organ rejection. Given these limitations, interspecies blastocyst complementation in combination with precise gene knockouts presents a unique, promising pathway for the transplant organ shortage. In recent years, great advancements have been made in the field, with encouraging results in producing a donor-derived organ in a chimeric host. That said, one of the major barriers to successful interspecies chimerism is the mismatch in the developmental stages of the donor and the host cells in the chimeric embryo. Another major barrier to successful chimerism is the mismatch in the developmental speeds between the donor and host cells in the chimeric embryos. This review outlines 19 studies in which blastocyst complementation was used to generate solid organs. In particular, the genesis of the liver, lung, kidney, pancreas, heart, thyroid, thymus and parathyroids was investigated. Of the 19 studies, 7 included an interspecies model. Of the 7, one was completed using human donor cells in a pig host, and all others were rat-mouse chimeras. While very promising results have been demonstrated, with great advancements in the field, several challenges continue to persist. In particular, successful chimerism, organ generation and donor contribution, synchronized donor-host development, as well as ethical concerns regarding human-animal chimeras remain important aspects that will need to be addressed in future research.
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Affiliation(s)
- Elena Bigliardi
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN 55455, USA;
| | - Anala V. Shetty
- Molecular, Cellular, Developmental Biology, and Genetics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA;
- Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - Walter C. Low
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN 55455, USA;
- Molecular, Cellular, Developmental Biology, and Genetics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA;
- Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - Clifford J. Steer
- Molecular, Cellular, Developmental Biology, and Genetics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA;
- Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
- Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
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11
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Wang LN, Jia JS, Yang XL, Wen YT, Liu JX, Li DK, Chen XR, Wang JH, Li JK, Huang ZX, Yao KT. Foxa1 disruption enhances human cell integration in human-mouse interspecies chimeras. Cell Tissue Res 2025; 399:231-245. [PMID: 39708115 DOI: 10.1007/s00441-024-03941-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2024] [Accepted: 11/25/2024] [Indexed: 12/23/2024]
Abstract
Blastocyst complementation can potentially generate a rodent model with humanized nasopharyngeal epithelium (NE) that supports sustained Epstein-Barr virus (EBV) infection, enabling comprehensive studies of EBV biology in nasopharyngeal carcinoma. However, during this process, the specific gene knockouts required to establish a developmental niche for NE remain unclear. We performed bioinformatics analyses and generated Foxa1 mutant mice to confirm that Foxa1 disruption could potentially create a developmental niche for NE. Subsequently, MYD88-inactivated human pluripotent stem cells (hPSCs) were constructed and complemented with Foxa1-deficient mouse blastocysts, with Nosip-deficient mouse blastocysts as a control. The chimerism of human cells in mouse embryos was evaluated from E8.5 to E12.5 using genomic DNA PCR and immunohistochemistry. Our bioinformatics analysis indicated that the expression patterns of Foxa1 in E8.5 to E16.5 mouse embryos underscore its critical role in NE development. The generated mice with Foxa1 disordered region mutations displayed morphological abnormality in NE, suggesting Foxa1-knockouts could potentially establish a developmental niche for NE. In chimeric assays, human cells integrated into 80.00% of Foxa1-deficient embryos, compared with the 4.17% in controls. Immunohistochemistry results revealed robust proliferation of human cells in Foxa1-deficient mouse embryos. However, chimeras from Foxa1-deficient mouse embryos did not survive beyond E10.5, hindering the evaluation of human cell integration in mouse NE. Foxa1 disruption in mouse embryos significantly enhances the integration of human cells in human-mouse interspecies chimeras, thereby facilitating the generation of endoderm-derived organs through blastocyst complementation. Overcoming chimeras' embryonic lethality is crucial for successfully generating humanized NE in Foxa1-deficient mouse embryos.
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Affiliation(s)
- Li-Na Wang
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
- Department of Oncology, School of Medicine, Guangzhou First People's Hospital, Southern China University of Technology, Guangzhou, 510180, China
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China
| | - Jun-Shuang Jia
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China
| | - Xing-Long Yang
- Department of Radiation Oncology, Affiliated Cancer Hospital and Institute of Guangzhou Medical University, Guangzhou, 510095, China
| | - Yue-Ting Wen
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China
| | - Jing-Xian Liu
- Department of Oncology, Shenzen Hospital of Southern Medical University, Shenzhen, 518110, China
| | - Deng-Ke Li
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China
| | - Xing-Rui Chen
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China
| | - Jia-Hong Wang
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China
| | - Ji-Ke Li
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China
| | - Zhong-Xi Huang
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China.
| | - Kai-Tai Yao
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.
- Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Guangzhou, 510515, China.
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12
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Yoneyama Y, Zhang RR, Maezawa M, Masaki H, Kimura M, Cai Y, Adam M, Parameswaran S, Mizuno N, Bhadury J, Maezawa S, Ochiai H, Nakauchi H, Potter SS, Weirauch MT, Takebe T. Intercellular mRNA transfer alters the human pluripotent stem cell state. Proc Natl Acad Sci U S A 2025; 122:e2413351122. [PMID: 39841146 PMCID: PMC11789055 DOI: 10.1073/pnas.2413351122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2024] [Accepted: 12/07/2024] [Indexed: 01/23/2025] Open
Abstract
Intercellular transmission of messenger RNA (mRNA) is being explored in mammalian species using immortal cell lines. Here, we uncover an intercellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells under the condition impermissible for primed hPSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis shows the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 d of coculture with mouse embryonic stem cells, surface marker analysis and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naïve-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naïve-like conversion. Thus, interspecies mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes, which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and interspecies cellular communications.
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Affiliation(s)
- Yosuke Yoneyama
- Human Biology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, Bunkyo-ku, Tokyo113-8510, Japan
- Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Osaka565-0871, Japan
| | - Ran-Ran Zhang
- Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
| | - Mari Maezawa
- Human Biology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, Bunkyo-ku, Tokyo113-8510, Japan
| | - Hideki Masaki
- Stem Cell Therapy Division, Institute of Integrated Research, Institute of Science Tokyo, Bunkyo-ku, Tokyo113-8510, Japan
| | - Masaki Kimura
- Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
| | - Yuqi Cai
- Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
| | - Mike Adam
- Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
| | - Sreeja Parameswaran
- Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
| | - Naoaki Mizuno
- Stem Cell Therapy Division, Institute of Integrated Research, Institute of Science Tokyo, Bunkyo-ku, Tokyo113-8510, Japan
| | - Joydeep Bhadury
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA94305
| | - So Maezawa
- Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba278-8510, Japan
| | - Hiroshi Ochiai
- Division of Gene Expression Dynamics, Medical Institute of Bioregulation, Kyushu University, Fukuoka812-0054, Japan
| | - Hiromitsu Nakauchi
- Stem Cell Therapy Division, Institute of Integrated Research, Institute of Science Tokyo, Bunkyo-ku, Tokyo113-8510, Japan
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA94305
| | - S. Steven Potter
- Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
| | - Matthew T. Weirauch
- Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH45229-3039
| | - Takanori Takebe
- Human Biology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, Bunkyo-ku, Tokyo113-8510, Japan
- Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Osaka565-0871, Japan
- Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH45229-3039
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229-3039
- World Premier International Research Center Initiative Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka565-0871, Japan
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13
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Var SR, Strell P, Shetty A, Roman A, Clark IH, Crane AT, Dunbar GL, Fink K, Grande AW, Parr AM, Rossignol J, Sanberg PR, Zhao LR, Zholudeva LV, Low WC, American Society for Neural Therapy and Repair Task Force. Research Guideline Recommendations for Research on Stem Cells, Human Embryos, and Gene Editing. Cell Transplant 2025; 34:9636897241312793. [PMID: 40007211 PMCID: PMC11863228 DOI: 10.1177/09636897241312793] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2024] [Revised: 12/17/2024] [Accepted: 12/20/2024] [Indexed: 02/27/2025] Open
Abstract
Recent advances in biomedical technologies have extended the boundaries of previously established regulatory guidelines pertaining to stem cell research. These guidelines constrained the study of human pluripotent stem cells (hPSCs) and their derivatives from use under various conditions, including the introduction of hPSCs into the brains of host animals because of concerns of humanizing the brains of animal species. Other guidelines constrained the use of hPSCs in creating human-animal chimeras because of the potential contribution of human stem cells not only to the brain but also to the germline. Some regulatory guidelines forbid the growing of human embryos ex vivo beyond the stage of primitive streak development because of concerns regarding the creation of human forms of life ex vivo. At the subcellular level, there are guidelines regulating the transfer of mitochondria within human embryos. At the molecular level, there are guidelines regulating genome editing to prevent permanent genetic alterations in germline cells. These and other issues related to stem cells have been reviewed, and new research guidelines established by the International Society for Stem Cell Research (ISSCR) for its membership. Because many of the recommended changes by the ISSCR impact research being conducted by members of the American Society for Neural Therapy and Repair (ASNTR), the ASNTR established a task force to review relevant recommendations by the ISSCR to determine which new guidelines to adopt for research conducted by the ASNTR society membership. The final ASNTR recommendations are presented in this document.
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Affiliation(s)
- Susanna R. Var
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Phoebe Strell
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
- Department of Comparative and Molecular Biosciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA
| | - Anala Shetty
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
- Department of Molecular, Cellular, Developmental Biology, and Genetics, University of Minnesota, Minneapolis, MN, USA
| | - Alex Roman
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
| | - Isaac H. Clark
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
| | - Andrew T. Crane
- Department of Medicine, University of Minnesota, Minneapolis, MN, USA
| | - Gary L. Dunbar
- Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
| | - Kyle Fink
- Department of Neurology, University of California, Davis, Davis, CA, USA
| | - Andrew W. Grande
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
| | - Ann M. Parr
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
| | - Julien Rossignol
- College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
| | - Paul R. Sanberg
- Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA
| | - Li-Ru Zhao
- Department of Neurosurgery, The State University of New York Upstate Medical University, Syracuse, NY, USA
| | | | - Walter C. Low
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
- Department of Comparative and Molecular Biosciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA
- Department of Molecular, Cellular, Developmental Biology, and Genetics, University of Minnesota, Minneapolis, MN, USA
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
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14
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Yuri S, Arisawa N, Kitamuro K, Isotani A. Blastocyst complementation-based rat-derived heart generation reveals cardiac anomaly barriers to interspecies chimera development. iScience 2024; 27:111414. [PMID: 39687030 PMCID: PMC11647242 DOI: 10.1016/j.isci.2024.111414] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2024] [Revised: 08/27/2024] [Accepted: 11/14/2024] [Indexed: 12/18/2024] Open
Abstract
The use of pluripotent stem cells (PSCs) to generate functional organs via blastocyst complementation is a cutting-edge strategy in regenerative medicine. However, existing models that use this method for heart generation do not meet expectations owing to the complexity of heart development. Here, we investigated a Mesp1/2 deficient mouse model, which is characterized by abnormalities in the cardiac mesodermal cells. The injection of either mouse or rat PSCs into Mesp1/2 deficient mouse blastocysts led to successful heart generation. In chimeras, the resulting hearts were predominantly composed of rat cells; however, their functionality was limited to the embryonic developmental stage on day 12.5. These results present the functional limitation of the xenogeneic heart, which poses a significant challenge to the development in mouse-rat chimeras.
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Affiliation(s)
- Shunsuke Yuri
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
- Laboratory of Experimental Animals, Research Institution, National Center for Geriatrics and Gerontology, 7-430 Morioka-cho, Obu, Aichi 474-8511, Japan
| | - Norie Arisawa
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
| | - Kohei Kitamuro
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
| | - Ayako Isotani
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
- Life Science Collaboration Center (LiSCo), Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
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15
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Zhang H, Wei Y, Wang Y, Liang J, Hou Y, Nie X, Hou J. Emerging Diabetes Therapies: Regenerating Pancreatic β Cells. TISSUE ENGINEERING. PART B, REVIEWS 2024; 30:644-656. [PMID: 39276101 DOI: 10.1089/ten.teb.2024.0041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/16/2024]
Abstract
The incidence of diabetes mellitus (DM) is steadily increasing annually, with 537 million diabetic patients as of 2021. Restoring diminished β cell mass or impaired islet function is crucial in treating DM, particularly type 1 DM. However, the regenerative capacity of islet β cells, which primarily produce insulin, is severely limited, and natural regeneration is only observed in young rodents or children. Hence, there is an urgent need to develop advanced therapeutic approaches that can regenerate endogenous β cells or replace them with stem cell (SC)-derived or engineered β-like cells. Current strategies for treating insulin-dependent DM mainly include promoting the self-replication of endogenous β cells, inducing SC differentiation, reprogramming non-β cells into β-like cells, and generating pancreatic-like organoids through cell-based intervention. In this Review, we discuss the current state of the art in these approaches, describe associated challenges, propose potential solutions, and highlight ongoing efforts to optimize β cell or islet transplantation and related clinical trials. These effective cell-based therapies will generate a sustainable source of functional β cells for transplantation and lay strong foundations for future curative treatments for DM.
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Affiliation(s)
- Haojie Zhang
- Key Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, School of Basic Medical Sciences, Henan University, Kaifeng, China
| | - Yaxin Wei
- Key Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, School of Basic Medical Sciences, Henan University, Kaifeng, China
| | - Yubo Wang
- Key Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, School of Basic Medical Sciences, Henan University, Kaifeng, China
| | - Jialin Liang
- Key Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, School of Basic Medical Sciences, Henan University, Kaifeng, China
| | - Yifan Hou
- Kaifeng 155 Hospital, China RongTong Medical Healthcare Group Co. Ltd., Kaifeng, China
- Department of Urinary Surgery, Henan Provincial Research Center for the Prevention and Diagnosis of Prostate Diseases, Huaihe Hospital, Henan University, Kaifeng, China
| | - Xiaobo Nie
- Key Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, School of Basic Medical Sciences, Henan University, Kaifeng, China
- Department of Urinary Surgery, Henan Provincial Research Center for the Prevention and Diagnosis of Prostate Diseases, Huaihe Hospital, Henan University, Kaifeng, China
| | - Junqing Hou
- Kaifeng 155 Hospital, China RongTong Medical Healthcare Group Co. Ltd., Kaifeng, China
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16
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Simpson SG, Park KE, Yeddula SGR, Waters J, Scimeca E, Poonooru RR, Etches R, Telugu BP. Blastocyst complementation generates exogenous donor-derived liver in ahepatic pigs. FASEB J 2024; 38:e70161. [PMID: 39530535 DOI: 10.1096/fj.202401244r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2024] [Revised: 09/04/2024] [Accepted: 10/24/2024] [Indexed: 11/16/2024]
Abstract
Liver diseases are one of the leading causes of morbidity and mortality worldwide. Globally, liver diseases are responsible for approximately 2 million deaths annually (1 of every 25 deaths). Many of the patients with chronic liver diseases can benefit from organ transplantation. However, stringent criteria for placement on organ transplantation waitlist and chronic shortage of organs preclude access to patients. To bridge the shortfall, generation of chimeric human organs in pigs has long been considered as an alternative. Here, we report feasibility of the approach by generating chimeric livers in pigs using a conditional blastocyst complementation approach that creates a vacant niche in chimeric hosts, enabling the initiation of organogenesis through donor-derived pluripotent cells. Porcine fetal fibroblasts were sequentially targeted for knockin of CRE into the endogenous FOXA3 locus (FOXA3CRE) followed by floxing of exon 1 of HHEX (FOXA3CREHHEXloxP/loxP) locus. The conditional HHEX knockout and constitutive GFP donor (COL1ACAG:LACZ 2A EGFP) were used as nuclear donors to generate host embryos by somatic cell nuclear transfer, and complemented and transferred into estrus synchronized surrogates. In the resulting fetuses, donor EGFP blastomeres reconstituted hepatocytes as confirmed by immunohistochemistry. These results potentially pave the way for exogenous donor-derived hepatogenesis in large animal models.
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Affiliation(s)
- Sean G Simpson
- RenOVAte Biosciences Inc, Reisterstown, Maryland, USA
- Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| | - Ki-Eun Park
- RenOVAte Biosciences Inc, Reisterstown, Maryland, USA
- Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| | | | - Jerel Waters
- RenOVAte Biosciences Inc, Reisterstown, Maryland, USA
- Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| | - Erin Scimeca
- RenOVAte Biosciences Inc, Reisterstown, Maryland, USA
| | | | - Rob Etches
- RenOVAte Biosciences Inc, Reisterstown, Maryland, USA
| | - Bhanu P Telugu
- RenOVAte Biosciences Inc, Reisterstown, Maryland, USA
- Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
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17
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Baker JC. Placental Evolution: Innovating How to Feed Babies. Annu Rev Genet 2024; 58:391-408. [PMID: 39227137 DOI: 10.1146/annurev-genet-111523-102135] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/05/2024]
Abstract
The evolution of the placenta was transformative. It changed how offspring are fed during gestation from depositing all the resources into an egg to continually supplying resources throughout gestation. Placental evolution is infinitely complex, with many moving parts, but at the core it is driven by a conflict over resources between the mother and the baby, which sets up a Red Queen race, fueling rapid diversification of morphological, cellular, and genetic forms. Placentas from even closely related species are highly divergent in form and function, and many cellular processes are distinct. If we could extract the entirety of genomic information for placentas across all species, including the many hundreds that have evolved in fish and reptiles, we could find their shared commonality, and that would tell us which of the many pieces really matter. We do not have this information, but we do have clues. Convergent evolution mechanisms were repeatedly used in the placenta, including the intense selective pressure to co-opt an envelope protein to build a multinucleated syncytium, the use of the same hormones and structural proteins in placentas derived from separate embryonic origins that arose hundreds of millions of years apart, and the co-option of endogenous retroviruses to form capsids as a way of transport and as mutagens to form new enhancers. As a result, the placental genome is the Wild West of biology, set up to rapidly change, adapt, and innovate. This ability to adapt facilitated the evolution of big babies with big brains and will continue to support offspring and their mothers in our ever-changing global environment.
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Affiliation(s)
- Julie C Baker
- Department of Genetics, Stanford University, Stanford, California, USA;
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18
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Fujimori K, Yamanaka S, Shimada K, Matsui K, Kawagoe S, Kuroda T, Ikeda A, Inoue M, Kobayashi E, Yokoo T. Generation of human-pig chimeric renal organoids using iPSC technology. Commun Biol 2024; 7:1278. [PMID: 39375428 PMCID: PMC11458617 DOI: 10.1038/s42003-024-06986-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2024] [Accepted: 09/28/2024] [Indexed: 10/09/2024] Open
Abstract
Porcine organs and human induced pluripotent stem cell (iPSC)-derived organoids as alternative organs for human transplantation have garnered attention, but both face technical challenges. Interspecies chimeric organ production using human iPSCs shows promise in overcoming these challenges. Our group successfully generated chimeric renal organoids using human iPSC-derived nephron progenitor cells (NPCs) and fetal mouse kidneys. However, the current technology is limited to rodents. Therefore, this study focused on producing human-pig chimeric renal organoids, as pigs are the most promising species for xenotransplantation. Modification of existing culture systems enables continuous renal development in both species, resulting in the successful creation of human-pig chimeric renal organoids. Moreover, this method can be applied to generate humanized xenogeneic kidneys for future clinical applications. This study provides evidence that optimizing culture conditions enables the early-stage kidney development beyond species barriers, thus laying the foundation for accelerating research on humanized xenogeneic kidney fabrication for clinical purposes.
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Affiliation(s)
| | - Shuichiro Yamanaka
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan.
| | | | - Kenji Matsui
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan
| | - Shiho Kawagoe
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan
| | | | | | | | - Eiji Kobayashi
- Department of Kidney Regenerative Medicine, The Jikei University School of Medicine, Tokyo, Japan
| | - Takashi Yokoo
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan.
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19
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Ballard E, Sakurai M, Yu L, Liu L, Oura S, Huang J, Wu J. Incompatibility in cell adhesion constitutes a barrier to interspecies chimerism. Cell Stem Cell 2024; 31:1419-1426.e7. [PMID: 39181131 DOI: 10.1016/j.stem.2024.07.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 06/05/2024] [Accepted: 07/30/2024] [Indexed: 08/27/2024]
Abstract
Interspecies blastocyst complementation holds great potential to address the global shortage of transplantable organs by growing human organs in animals. However, a major challenge in this approach is the limited chimerism of human cells in evolutionarily distant animal hosts due to various xenogeneic barriers. Here, we reveal that human pluripotent stem cells (PSCs) struggle to adhere to animal PSCs. To overcome this barrier, we developed a synthetic biology strategy that leverages nanobody-antigen interactions to enhance interspecies cell adhesion. We engineered cells to express nanobodies and their corresponding antigens on their outer membranes, significantly improving adhesion between different species' PSCs during in vitro assays and increasing the chimerism of human PSCs in mouse embryos. Studying and manipulating interspecies pluripotent cell adhesion will provide valuable insights into cell interaction dynamics during chimera formation and early embryogenesis.
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Affiliation(s)
- Emily Ballard
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Masahiro Sakurai
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Leqian Yu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Lizhong Liu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Seiya Oura
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jia Huang
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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20
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Shimizu D, Miura A, Mori M. The perspective for next-generation lung replacement therapies: functional whole lung generation by blastocyst complementation. Curr Opin Organ Transplant 2024; 29:340-348. [PMID: 39150364 DOI: 10.1097/mot.0000000000001169] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/17/2024]
Abstract
PURPOSE OF REVIEW Blastocyst complementation represents a promising frontier in next-generation lung replacement therapies. This review aims to elucidate the future prospects of lung blastocyst complementation within clinical settings, summarizing the latest studies on generating functional lungs through this technique. It also explores and discusses host animal selection relevant to interspecific chimera formation, a challenge integral to creating functional human lungs via blastocyst complementation. RECENT FINDINGS Various gene mutations have been utilized to create vacant lung niches, enhancing the efficacy of donor cell contribution to the complemented lungs in rodent models. By controlling the lineage to induce gene mutations, chimerism in both the lung epithelium and mesenchyme has been improved. Interspecific blastocyst complementation underscores the complexity of developmental programs across species, with several genes identified that enhance chimera formation between humans and other mammals. SUMMARY While functional lungs have been generated via intraspecies blastocyst complementation, the generation of functional interspecific lungs remains unrealized. Addressing the challenges of controlling the host lung niche and selecting host animals relevant to interspecific barriers between donor human and host cells is critical to enabling the generation of functional humanized or entire human lungs in large animals.
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Affiliation(s)
- Dai Shimizu
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, New York, USA
- Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
| | - Akihiro Miura
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, New York, USA
- Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
| | - Munemasa Mori
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, New York, USA
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21
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Ahmadzada B, Felgendreff P, Minshew AM, Amiot BP, Nyberg SL. Producing Human Livers From Human Stem Cells Via Blastocyst Complementation. CURRENT OPINION IN BIOMEDICAL ENGINEERING 2024; 31:100537. [PMID: 38854436 PMCID: PMC11160964 DOI: 10.1016/j.cobme.2024.100537] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/11/2024]
Abstract
The need for organ transplants exceeds donor organ availability. In the quest to solve this shortage, the most remarkable area of advancement is organ production through the use of chimeric embryos, commonly known as blastocyst complementation. This technique involves the combination of different species to generate chimeras, where the extent of donor cell contribution to the desired tissue or organ can be regulated. However, ethical concerns arise with the use of brain tissue in such chimeras. Furthermore, the ratio of contributed cells to host animal cells in the chimeric system is low in the production of chimeras associated with cell apoptosis. This review discusses the latest innovations in blastocyst complementation and highlights the progress made in creating organs for transplant.
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Affiliation(s)
- Boyukkhanim Ahmadzada
- Research Trainee in the Division of Surgery Research (Ahmadzada; limited tenure), Artificial Liver and Liver Transplantation Laboratory (Minshew, Amiot, and Nyberg), and Division of Surgery Research (Nyberg), Mayo Clinic, Rochester, Minnesota, USA; Research Fellow in the Division of Surgery Research (Felgendreff), Mayo Clinic School of Graduate Medical Education, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, USA. Dr Felgendreff is also affiliated with the Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
| | - Philipp Felgendreff
- Research Trainee in the Division of Surgery Research (Ahmadzada; limited tenure), Artificial Liver and Liver Transplantation Laboratory (Minshew, Amiot, and Nyberg), and Division of Surgery Research (Nyberg), Mayo Clinic, Rochester, Minnesota, USA; Research Fellow in the Division of Surgery Research (Felgendreff), Mayo Clinic School of Graduate Medical Education, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, USA. Dr Felgendreff is also affiliated with the Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
| | - Anna M Minshew
- Research Trainee in the Division of Surgery Research (Ahmadzada; limited tenure), Artificial Liver and Liver Transplantation Laboratory (Minshew, Amiot, and Nyberg), and Division of Surgery Research (Nyberg), Mayo Clinic, Rochester, Minnesota, USA; Research Fellow in the Division of Surgery Research (Felgendreff), Mayo Clinic School of Graduate Medical Education, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, USA. Dr Felgendreff is also affiliated with the Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
| | - Bruce P Amiot
- Research Trainee in the Division of Surgery Research (Ahmadzada; limited tenure), Artificial Liver and Liver Transplantation Laboratory (Minshew, Amiot, and Nyberg), and Division of Surgery Research (Nyberg), Mayo Clinic, Rochester, Minnesota, USA; Research Fellow in the Division of Surgery Research (Felgendreff), Mayo Clinic School of Graduate Medical Education, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, USA. Dr Felgendreff is also affiliated with the Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
| | - Scott L Nyberg
- Research Trainee in the Division of Surgery Research (Ahmadzada; limited tenure), Artificial Liver and Liver Transplantation Laboratory (Minshew, Amiot, and Nyberg), and Division of Surgery Research (Nyberg), Mayo Clinic, Rochester, Minnesota, USA; Research Fellow in the Division of Surgery Research (Felgendreff), Mayo Clinic School of Graduate Medical Education, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, USA. Dr Felgendreff is also affiliated with the Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany
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22
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Navarro M, Laiz-Quiroga L, Blüguermann C, Mutto A. Livestock embryonic stem cells for reproductive biotechniques and genetic improvement. Anim Reprod 2024; 21:e20240029. [PMID: 39175999 PMCID: PMC11340801 DOI: 10.1590/1984-3143-ar2024-0029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2024] [Accepted: 05/27/2024] [Indexed: 08/24/2024] Open
Abstract
Embryonic stem cells (ESCs) have proven to be a great in vitro model that faithfully recapitulates the events that occur during in vivo embryogenesis, making them a unique tool to study the cellular and molecular mechanisms that define tissue specification during embryonic development. Livestock ESCs are particularly attractive and have broad prospects including drug selection and human disease modeling, improvement of reproductive biotechniques and agriculture-related applications such as production of genetically modified animals. While mice and human ESCs have been established many years ago, no significant advances were made in livestock species until recently. Nowadays, livestock ESCs are available from cattle, pigs, sheep, horses and rabbits with different states of pluripotency. In this review, we summarize the current advances on livestock ESCs establishment and maintenance along with their present and future applications.
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Affiliation(s)
- Micaela Navarro
- Laboratorio de Biotecnologías aplicadas a la Reproducción Animal, Instituto de Investigaciones Biotecnológicas “Dr. Rodolfo Ugalde”, Universidad Nacional de General San Martín, Buenos Aires, Argentina
| | - Lucia Laiz-Quiroga
- Laboratorio de Biotecnologías aplicadas a la Reproducción Animal, Instituto de Investigaciones Biotecnológicas “Dr. Rodolfo Ugalde”, Universidad Nacional de General San Martín, Buenos Aires, Argentina
| | - Carolina Blüguermann
- Laboratorio de Biotecnologías aplicadas a la Reproducción Animal, Instituto de Investigaciones Biotecnológicas “Dr. Rodolfo Ugalde”, Universidad Nacional de General San Martín, Buenos Aires, Argentina
| | - Adrián Mutto
- Laboratorio de Biotecnologías aplicadas a la Reproducción Animal, Instituto de Investigaciones Biotecnológicas “Dr. Rodolfo Ugalde”, Universidad Nacional de General San Martín, Buenos Aires, Argentina
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23
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Kano M. Parathyroid Gland Generation from Pluripotent Stem Cells. Endocrinol Metab (Seoul) 2024; 39:552-558. [PMID: 38853617 PMCID: PMC11375298 DOI: 10.3803/enm.2024.1989] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Accepted: 05/07/2024] [Indexed: 06/11/2024] Open
Abstract
Patients with permanent hypoparathyroidism require lifelong treatment. Current replacement therapies sometimes have adverse effects (e.g., hypercalciuria and chronic kidney disease). Generating parathyroid glands (PTGs) from the patient's own induced pluripotent stem cells (PSCs), with transplantation of these PTGs, would be an effective treatment option. Multiple methods for generating PTGs from PSCs have been reported. One major trend is in vitro differentiation of PSCs into PTGs. Another is in vivo generation of PSC-derived PTGs by injecting PSCs into PTG-deficient embryos. This review discusses current achievements and challenges in present and future PTG regenerative medicine.
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Affiliation(s)
- Mayuko Kano
- Department of Metabolism and Endocrinology, St. Marianna University School of Medicine, Kawasaki, Japan
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24
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Wen B, Li E, Wang G, Kalin TR, Gao D, Lu P, Kalin TV, Kalinichenko VV. CRISPR-Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat. Am J Respir Crit Care Med 2024; 210:167-177. [PMID: 38507610 PMCID: PMC11273307 DOI: 10.1164/rccm.202306-0964oc] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Accepted: 03/20/2024] [Indexed: 03/22/2024] Open
Abstract
Rationale: Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat. Objective: We sought to generate the mouse lung in a rat. Methods: Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat one-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESCs into Nkx2-1-deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing. Measurements and Main Results: Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9-mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1-/- blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse-rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat. Conclusions: A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors."
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Affiliation(s)
- Bingqiang Wen
- Phoenix Children’s Research Institute, Department of Child Health, College of Medicine Phoenix, University of Arizona, Phoenix, Arizona
| | - Enhong Li
- Phoenix Children’s Research Institute, Department of Child Health, College of Medicine Phoenix, University of Arizona, Phoenix, Arizona
| | | | - Timothy R. Kalin
- College of Arts and Sciences, University of Cincinnati, Cincinnati, Ohio
| | - Dengfeng Gao
- State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing, China; and
| | - Peixin Lu
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
| | - Tanya V. Kalin
- Phoenix Children’s Research Institute, Department of Child Health, College of Medicine Phoenix, University of Arizona, Phoenix, Arizona
- Division of Pulmonary Biology and
| | - Vladimir V. Kalinichenko
- Phoenix Children’s Research Institute, Department of Child Health, College of Medicine Phoenix, University of Arizona, Phoenix, Arizona
- Division of Neonatology, Phoenix Children’s Hospital, Phoenix, Arizona
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25
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Mori M, Cardoso WV. Can a Rat Breathe through a Mouse's Lung? Am J Respir Crit Care Med 2024; 210:133-134. [PMID: 38701370 PMCID: PMC11273309 DOI: 10.1164/rccm.202404-0706ed] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Accepted: 05/02/2024] [Indexed: 05/05/2024] Open
Affiliation(s)
- Munemasa Mori
- Department of Medicine Columbia University Irving Medical Center New York, New York
| | - Wellington V Cardoso
- Department of Medicine
- Department of Genetics and Development Columbia University Irving Medical Center New York, New York
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26
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Pluchino S, Lombardi I. Crossing species boundaries in regenerative neuroscience with rat-mouse brain chimeras. Lab Anim (NY) 2024; 53:179-180. [PMID: 38886566 PMCID: PMC11216989 DOI: 10.1038/s41684-024-01394-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/20/2024]
Affiliation(s)
- Stefano Pluchino
- Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, Cambridge, UK.
| | - Ivan Lombardi
- Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, Cambridge, UK
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, Milan, Italy
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27
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Yoneyama Y, Zhang RR, Kimura M, Cai Y, Adam M, Parameswaran S, Masaki H, Mizuno N, Bhadury J, Maezawa S, Ochiai H, Nakauchi H, Potter SS, Weirauch MT, Takebe T. Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.27.600209. [PMID: 38979277 PMCID: PMC11230441 DOI: 10.1101/2024.06.27.600209] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/10/2024]
Abstract
Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 days of mESC culture, surface marker analysis, and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naïve-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naïve-like conversion. Thus, inter-species mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes(4), which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and inter-species cellular communications.
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Affiliation(s)
- Yosuke Yoneyama
- Institute of Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
- Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
| | - Ran-Ran Zhang
- Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
| | - Masaki Kimura
- Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
| | - Yuqi Cai
- Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
| | - Mike Adam
- Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
| | - Sreeja Parameswaran
- Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
| | - Hideki Masaki
- Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan
| | - Naoaki Mizuno
- Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan
| | - Joydeep Bhadury
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA
| | - So Maezawa
- Faculty of Science and Technology, Department of Applied Biological Science, Tokyo University of Science, Chiba, 278-8510, Japan
| | - Hiroshi Ochiai
- Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-0054, Japan
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo, 108-8639, Japan
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA
| | - S. Steven Potter
- Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
| | - Matthew T. Weirauch
- Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
- Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
- Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
| | - Takanori Takebe
- Institute of Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
- Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
- Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
- Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
- Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka 565-0871, Japan
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28
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Tanaka J, Miura A, Shimamura Y, Hwang Y, Shimizu D, Kondo Y, Sawada A, Sarmah H, Ninish Z, Mishima K, Mori M. Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation. Cell Rep 2024; 43:114340. [PMID: 38865239 PMCID: PMC11580835 DOI: 10.1016/j.celrep.2024.114340] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Revised: 04/25/2024] [Accepted: 05/23/2024] [Indexed: 06/14/2024] Open
Abstract
Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.
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Affiliation(s)
- Junichi Tanaka
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA; Division of Pathology, Department of Oral Diagnostic Sciences, Showa University School of Dentistry, Tokyo 142-8555, Japan.
| | - Akihiro Miura
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
| | - Yuko Shimamura
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
| | - Youngmin Hwang
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
| | - Dai Shimizu
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
| | - Yuri Kondo
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
| | - Anri Sawada
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
| | - Hemanta Sarmah
- Columbia Stem Cell Initiative, Stem Cell Core, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Zurab Ninish
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
| | - Kenji Mishima
- Division of Pathology, Department of Oral Diagnostic Sciences, Showa University School of Dentistry, Tokyo 142-8555, Japan
| | - Munemasa Mori
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA.
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29
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Wu J, Fu J. Toward developing human organs via embryo models and chimeras. Cell 2024; 187:3194-3219. [PMID: 38906095 PMCID: PMC11239105 DOI: 10.1016/j.cell.2024.05.027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Revised: 02/02/2024] [Accepted: 05/14/2024] [Indexed: 06/23/2024]
Abstract
Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting, and organoids, only offer partial solutions. This perspective focuses on two promising approaches emerging for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outlining future prospects for advancing both fields toward the generation of human tissues and organs for basic research and translational applications.
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Affiliation(s)
- Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
| | - Jianping Fu
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
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30
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Furlanetto F, Frank S, Karow M. Unlocking the potential of SY-stem cells. Development 2024; 151:dev203086. [PMID: 38895963 DOI: 10.1242/dev.203086] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/21/2024]
Abstract
The sixth SY-Stem Symposium, jointly organized by the Research Institute of Molecular Pathology and the Institute of Molecular Biotechnology took place in Vienna in March 2024. Again, aspiring new group leaders were given a stage to present their work and vision of their labs. To round up the excellent program, the scientific organizers included renowned keynote speakers. Here, we provide a summary of the talks covering topics such as early embryogenesis, nervous system development and disease, regeneration and the latest technologies.
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Affiliation(s)
- Federica Furlanetto
- Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg,Fahrstrasse 17, 91054 Erlangen, Germany
| | - Sarah Frank
- Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg,Fahrstrasse 17, 91054 Erlangen, Germany
| | - Marisa Karow
- Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg,Fahrstrasse 17, 91054 Erlangen, Germany
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31
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Huang C, Jiang H, Dong J, Jiang L, Li J, Xu J, Cui T, Wang L, Li X, Feng G, Zhang Y, Li T, Li W, Zhou Q. Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis. Stem Cell Reports 2024; 19:877-889. [PMID: 38729156 PMCID: PMC11390683 DOI: 10.1016/j.stemcr.2024.04.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2024] [Revised: 04/14/2024] [Accepted: 04/15/2024] [Indexed: 05/12/2024] Open
Abstract
Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.
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Affiliation(s)
- Cheng Huang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Haiping Jiang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jingxi Dong
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Liyuan Jiang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jie Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jing Xu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Tongtong Cui
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Leyun Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Xin Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Guihai Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Ying Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Tianda Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
| | - Qi Zhou
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
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Nagaya M, Uchikura A, Nakano K, Watanabe M, Matsunari H, Umeyama K, Mizuno N, Nishimura T, Nakauchi H, Nagashima H. Generation of insulin-like growth factor 1 receptor-knockout pigs as a potential system for interspecies organogenesis. Regen Ther 2024; 26:783-791. [PMID: 39309395 PMCID: PMC11416208 DOI: 10.1016/j.reth.2024.08.025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2024] [Revised: 08/27/2024] [Accepted: 08/29/2024] [Indexed: 09/25/2024] Open
Abstract
BACKGROUND To overcome organ shortage during transplantation, interspecies organ generation via blastocyst complementation has been proposed, although not yet in evolutionarily distant species. To establish high levels of chimerism, low chimerism is required early in development, followed by high chimerism, to effectively complement the organ niche. Very few human cells are expected to contribute to chimerism in heterologous animals. Previous studies had demonstrated increased donor chimerism in both intra- and interspecies chimeras in rodents, using insulin-like growth factor 1 receptor (Igf1r) knockout (KO) mice; deletion of the Igf1r gene in the mouse host embryo created a cell-competitive niche. The current study aimed to generate IGF1R-KO pigs and evaluate whether they have the same phenotype as Igf1r-KO mice. METHODS To generate IGF1R-KO pigs, genome-editing molecules were injected into the cytoplasm of pig zygotes. The fetuses were evaluated at 104 days of gestation. RESULTS IGF1R-KO pigs were generated successfully. Their phenotypes were almost identical to those of Igf1r-KO mice, including small lungs and enlarged endodermal organs in fetuses, and they were highly reproducible. CONCLUSIONS Pigs may allow the generation of organs using blastocyst complementation with developmentally-compatible xenogeneic pluripotent stem cells over a large evolutionary distance.
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Affiliation(s)
- Masaki Nagaya
- Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
| | - Ayuko Uchikura
- Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
| | - Kazuaki Nakano
- Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
- PorMedTec Co. Ltd., 2-3227 Mita, Tama-ku, Kawasaki, Kanagawa, 214-0034, Japan
| | - Masahito Watanabe
- Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
- PorMedTec Co. Ltd., 2-3227 Mita, Tama-ku, Kawasaki, Kanagawa, 214-0034, Japan
| | - Hitomi Matsunari
- Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
- PorMedTec Co. Ltd., 2-3227 Mita, Tama-ku, Kawasaki, Kanagawa, 214-0034, Japan
| | - Kazuhiro Umeyama
- Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
- PorMedTec Co. Ltd., 2-3227 Mita, Tama-ku, Kawasaki, Kanagawa, 214-0034, Japan
| | - Naoaki Mizuno
- Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
- Stem Cell Therapy Laboratory, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8510 Tokyo, Japan
| | - Toshiya Nishimura
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA94305, USA
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
- Stem Cell Therapy Laboratory, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8510 Tokyo, Japan
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA94305, USA
| | - Hiroshi Nagashima
- Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
- Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan
- PorMedTec Co. Ltd., 2-3227 Mita, Tama-ku, Kawasaki, Kanagawa, 214-0034, Japan
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Li B, Kwon C. Mesendodermal cells fail to contribute to heart formation following blastocyst injection. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.22.595392. [PMID: 38826381 PMCID: PMC11142170 DOI: 10.1101/2024.05.22.595392] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/04/2024]
Abstract
Blastocyst complementation offers an opportunity for generating transplantable whole organs from donor sources. Pluripotent stem cells (PSCs) have traditionally served as the primary donor cells due to their ability to differentiate into any type of body cell. However, the use of PSCs raises ethical concerns, particularly regarding their uncontrollable differentiation potential to undesired cell lineages such as brain and germline cells. To address this issue, various strategies have been explored, including the use of genetically modified PSCs with restricted lineage potential or lineage-specified progenitor cells as donors. In this study, we tested whether nascent mesendodermal cells (MECs), which appear during early gastrulation, can be used as donor cells. To do this, we induced Bry-GFP+ MECs from mouse embryonic stem cells (ESCs) and introduced them into the blastocyst. While donor ESCs gave rise to various regions of embryos, including the heart, Bry-GFP+ MECs failed to contribute to the host embryos. This finding suggests that MECs, despite being specified from PSCs within a few days, lack the capacity to assimilate into the developing embryo.
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Affiliation(s)
- Biyi Li
- Division of Cardiology, Department of Medicine, Department of Biomedical Engineering, Department of Cell Biology, Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Chulan Kwon
- Division of Cardiology, Department of Medicine, Department of Biomedical Engineering, Department of Cell Biology, Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD, USA
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Haideri T, Lin J, Bao X, Lian XL. MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells. Stem Cell Reports 2024; 19:744-757. [PMID: 38579711 PMCID: PMC11103783 DOI: 10.1016/j.stemcr.2024.03.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2024] [Revised: 03/08/2024] [Accepted: 03/08/2024] [Indexed: 04/07/2024] Open
Abstract
Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.
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Affiliation(s)
- Tahir Haideri
- Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802, USA
| | - Jirong Lin
- Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802, USA
| | - Xiaoping Bao
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN 47907, USA.
| | - Xiaojun Lance Lian
- Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802, USA; Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802, USA; Department of Biology, Pennsylvania State University, University Park, PA 16802, USA.
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35
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Lim JJY, Murata Y, Yuri S, Kitamuro K, Kawai T, Isotani A. Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system. Sci Rep 2024; 14:10636. [PMID: 38724644 PMCID: PMC11082136 DOI: 10.1038/s41598-024-61167-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2023] [Accepted: 05/02/2024] [Indexed: 05/12/2024] Open
Abstract
Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNAms system that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNAms at the Rosa26 locus (Foxn1Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNAms system because Rosa26_ms could be maintained as homozygous.
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Affiliation(s)
- Jonathan Jun-Yong Lim
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore
| | - Yamato Murata
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
| | - Shunsuke Yuri
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
| | - Kohei Kitamuro
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
| | - Taro Kawai
- Laboratory of Molecular Immunobiology, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
- Life Science Collaboration Center (LiSCo), Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan
| | - Ayako Isotani
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan.
- Life Science Collaboration Center (LiSCo), Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan.
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Lenardič A, Domenig SA, Zvick J, Bundschuh N, Tarnowska-Sengül M, Furrer R, Noé F, Trautmann CL, Ghosh A, Bacchin G, Gjonlleshaj P, Qabrati X, Masschelein E, De Bock K, Handschin C, Bar-Nur O. Generation of allogeneic and xenogeneic functional muscle stem cells for intramuscular transplantation. J Clin Invest 2024; 134:e166998. [PMID: 38713532 PMCID: PMC11178549 DOI: 10.1172/jci166998] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2022] [Accepted: 04/23/2024] [Indexed: 05/09/2024] Open
Abstract
Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected Dmdmdx mouse induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Notably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.
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MESH Headings
- Animals
- Mice
- Muscular Dystrophy, Duchenne/therapy
- Muscular Dystrophy, Duchenne/genetics
- Induced Pluripotent Stem Cells/transplantation
- Induced Pluripotent Stem Cells/cytology
- Induced Pluripotent Stem Cells/metabolism
- Rats
- Satellite Cells, Skeletal Muscle/transplantation
- Satellite Cells, Skeletal Muscle/metabolism
- Satellite Cells, Skeletal Muscle/cytology
- Stem Cell Transplantation
- Humans
- Dystrophin/genetics
- Dystrophin/metabolism
- Muscle, Skeletal/metabolism
- Muscle, Skeletal/cytology
- Mice, Inbred mdx
- Heterografts
- Transplantation, Heterologous
- Injections, Intramuscular
- Transplantation, Homologous
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Affiliation(s)
- Ajda Lenardič
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Seraina A. Domenig
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Joel Zvick
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Nicola Bundschuh
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Monika Tarnowska-Sengül
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | | | - Falko Noé
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
- Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich, Switzerland
| | - Christine L. Trautmann
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Adhideb Ghosh
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
- Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich, Switzerland
| | - Giada Bacchin
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Pjeter Gjonlleshaj
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Xhem Qabrati
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Evi Masschelein
- Laboratory of Exercise and Health, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | - Katrien De Bock
- Laboratory of Exercise and Health, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
| | | | - Ori Bar-Nur
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach, Switzerland
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Nagano H, Mizuno N, Sato H, Mizutani E, Yanagida A, Kano M, Kasai M, Yamamoto H, Watanabe M, Suchy F, Masaki H, Nakauchi H. Skin graft with dermis and appendages generated in vivo by cell competition. Nat Commun 2024; 15:3366. [PMID: 38684678 PMCID: PMC11058811 DOI: 10.1038/s41467-024-47527-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2023] [Accepted: 04/03/2024] [Indexed: 05/02/2024] Open
Abstract
Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 knockout embryos' dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos' dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.
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Affiliation(s)
- Hisato Nagano
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
- Department of Plastic and Reconstructive Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan
| | - Naoaki Mizuno
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
- Department of Experimental Animal Model for Human Disease, Center for Experimental Animals, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
| | - Hideyuki Sato
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Eiji Mizutani
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
- Laboratory of Stem Cell Therapy, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8577, Japan
| | - Ayaka Yanagida
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
- Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan
| | - Mayuko Kano
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
- Metabolism and Endocrinology, Department of Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa, 216-8511, Japan
| | - Mariko Kasai
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Hiromi Yamamoto
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Motoo Watanabe
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Fabian Suchy
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Hideki Masaki
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA.
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Reardon S. Rat neurons repair mouse brains - and restore sense of smell. Nature 2024:10.1038/d41586-024-01222-1. [PMID: 38664559 DOI: 10.1038/d41586-024-01222-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/03/2024]
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Throesch BT, Bin Imtiaz MK, Muñoz-Castañeda R, Sakurai M, Hartzell AL, James KN, Rodriguez AR, Martin G, Lippi G, Kupriyanov S, Wu Z, Osten P, Izpisua Belmonte JC, Wu J, Baldwin KK. Functional sensory circuits built from neurons of two species. Cell 2024; 187:2143-2157.e15. [PMID: 38670072 PMCID: PMC11293795 DOI: 10.1016/j.cell.2024.03.042] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2023] [Revised: 01/18/2024] [Accepted: 03/26/2024] [Indexed: 04/28/2024]
Abstract
A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.
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Affiliation(s)
- Benjamin T Throesch
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA; Neuroscience Graduate Program, University of California, San Diego, La Jolla, San Diego, CA, USA
| | - Muhammad Khadeesh Bin Imtiaz
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | | | - Masahiro Sakurai
- Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Andrea L Hartzell
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Kiely N James
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA; Neuroscience Graduate Program, University of California, San Diego, La Jolla, San Diego, CA, USA
| | - Alberto R Rodriguez
- Mouse Genetics Core, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Greg Martin
- Mouse Genetics Core, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Giordano Lippi
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Sergey Kupriyanov
- Mouse Genetics Core, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Zhuhao Wu
- Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Pavel Osten
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
| | - Juan Carlos Izpisua Belmonte
- Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Altos Labs, San Diego, CA, USA
| | - Jun Wu
- Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
| | - Kristin K Baldwin
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA; Neuroscience Graduate Program, University of California, San Diego, La Jolla, San Diego, CA, USA; Department of Genetics and Development, Columbia Stem Cell Initiative, Columbia University Medical Center, New York, NY, USA.
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40
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Huang J, He B, Yang X, Long X, Wei Y, Li L, Tang M, Gao Y, Fang Y, Ying W, Wang Z, Li C, Zhou Y, Li S, Shi L, Choi S, Zhou H, Guo F, Yang H, Wu J. Generation of rat forebrain tissues in mice. Cell 2024; 187:2129-2142.e17. [PMID: 38670071 PMCID: PMC11646705 DOI: 10.1016/j.cell.2024.03.017] [Citation(s) in RCA: 11] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Revised: 11/14/2023] [Accepted: 03/13/2024] [Indexed: 04/28/2024]
Abstract
Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.
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Affiliation(s)
- Jia Huang
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Bingbing He
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiali Yang
- Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of MARA, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China
| | - Xin Long
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yinghui Wei
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Leijie Li
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Min Tang
- Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Yanxia Gao
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yuan Fang
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Wenqin Ying
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Zikang Wang
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Chao Li
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yingsi Zhou
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Shuaishuai Li
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Linyu Shi
- Huidagene Therapeutics Co., Ltd, Shanghai 200131, China
| | - Seungwon Choi
- Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Haibo Zhou
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
| | - Fan Guo
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China.
| | - Hui Yang
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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41
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Dardano M, Lebek T, H. C. Tsang I. Exploring stem cell frontiers: definitions, challenges, and perspectives for regenerative medicine. Biol Open 2024; 13:bio060245. [PMID: 38592154 PMCID: PMC11033525 DOI: 10.1242/bio.060245] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/10/2024] Open
Abstract
Each year, the European Summer School on Stem Cell Biology and Regenerative Medicine (SCSS) attracts early-career researchers and actively practicing clinicians who specialise in stem cell and regenerative biology. The 16th edition of this influential course took place from 12th to 19th September 2023 on the charming Greek island of Spetses. Focusing on important concepts and recent advances in stem cells, the distinguished faculty included experts spanning the spectrum from fundamental research to clinical trials to market-approved therapies. Alongside an academically intensive programme that bridges the various contexts of stem cell research, delegates were encouraged to critically address relevant questions in stem cell biology and medicine, including broader societal implications. Here, we present a comprehensive overview and key highlights from the SCSS 2023.
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Affiliation(s)
- Miriana Dardano
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover 30625, Germany
| | - Tamina Lebek
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, School of Biological Sciences, The University of Edinburgh, Edinburgh EH16 4UU, UK
| | - Ingrid H. C. Tsang
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen N DK-2200, Denmark
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42
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Blake MJ, Steer CJ. Chimeric Livers: Interspecies Blastocyst Complementation and Xenotransplantation for End-Stage Liver Disease. Hepat Med 2024; 16:11-29. [PMID: 38379783 PMCID: PMC10878318 DOI: 10.2147/hmer.s440697] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2023] [Accepted: 02/10/2024] [Indexed: 02/22/2024] Open
Abstract
Orthotopic liver transplantation (OLT) currently serves as the sole definitive treatment for thousands of patients suffering from end-stage liver disease; and the existing supply of donor livers for OLT is drastically outpaced by the increasing demand. To alleviate this significant gap in treatment, several experimental approaches have been devised with the aim of either offering interim support to patients waiting on the transplant list or bioengineering complete livers for OLT by infusing them with fresh hepatic cells. Recently, interspecies blastocyst complementation has emerged as a promising method for generating complete organs in utero over a short timeframe. When coupled with gene editing technology, it has brought about a potentially revolutionary transformation in regenerative medicine. Blastocyst complementation harbors notable potential for generating complete human livers in large animals, which could be used for xenotransplantation in humans, addressing the scarcity of livers for OLT. Nevertheless, substantial experimental and ethical challenges still need to be overcome to produce human livers in larger domestic animals like pigs. This review compiles the current understanding of interspecies blastocyst complementation and outlines future possibilities for liver xenotransplantation in humans.
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Affiliation(s)
- Madelyn J Blake
- Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA
| | - Clifford J Steer
- Departments of Medicine, and Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN, USA
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43
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Wang H, Yin X, Xu J, Chen L, Karuppagounder SS, Xu E, Mao X, Dawson VL, Dawson TM. Interspecies chimerism with human embryonic stem cells generates functional human dopamine neurons at low efficiency. Stem Cell Reports 2024; 19:54-67. [PMID: 38134925 PMCID: PMC10828682 DOI: 10.1016/j.stemcr.2023.11.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Revised: 11/24/2023] [Accepted: 11/27/2023] [Indexed: 12/24/2023] Open
Abstract
Interspecies chimeras offer great potential for regenerative medicine and the creation of human disease models. Whether human pluripotent stem cell-derived neurons in an interspecies chimera can differentiate into functional neurons and integrate into host neural circuity is not known. Here, we show, using Engrailed 1 (En1) as a development niche, that human naive-like embryonic stem cells (ESCs) can incorporate into embryonic and adult mouse brains. Human-derived neurons including tyrosine hydroxylase (TH)+ neurons integrate into the mouse brain at low efficiency. These TH+ neurons have electrophysiologic properties consistent with their human origin. In addition, these human-derived neurons in the mouse brain accumulate pathologic phosphorylated α-synuclein in response to α-synuclein preformed fibrils. Optimization of human/mouse chimeras could be used to study human neuronal differentiation and human brain disorders.
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Affiliation(s)
- Hu Wang
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Xiling Yin
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Jinchong Xu
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Li Chen
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Senthilkumar S Karuppagounder
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Enquan Xu
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Xiaobo Mao
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Valina L Dawson
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
| | - Ted M Dawson
- Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA 70130-2685, USA; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
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Roman A, Huntemer-Silveira A, Waldron MA, Khalid Z, Blake J, Parr AM, Low WC. Cell Transplantation for Repair of the Spinal Cord and Prospects for Generating Region-Specific Exogenic Neuronal Cells. Cell Transplant 2024; 33:9636897241241998. [PMID: 38590295 PMCID: PMC11005494 DOI: 10.1177/09636897241241998] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Revised: 03/05/2024] [Accepted: 03/11/2024] [Indexed: 04/10/2024] Open
Abstract
Spinal cord injury (SCI) is associated with currently irreversible consequences in several functional components of the central nervous system. Despite the severity of injury, there remains no approved treatment to restore function. However, with a growing number of preclinical studies and clinical trials, cell transplantation has gained significant potential as a treatment for SCI. Researchers have identified several cell types as potential candidates for transplantation. To optimize successful functional outcomes after transplantation, one key factor concerns generating neuronal cells with regional and subtype specificity, thus calling on the developmental transcriptome patterning of spinal cord cells. A potential source of spinal cord cells for transplantation is the generation of exogenic neuronal progenitor cells via the emerging technologies of gene editing and blastocyst complementation. This review highlights the use of cell transplantation to treat SCI in the context of relevant developmental gene expression patterns useful for producing regionally specific exogenic spinal cells via in vitro differentiation and blastocyst complementation.
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Affiliation(s)
- Alex Roman
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Department of Neurosurgery, Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Anne Huntemer-Silveira
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
| | - Madison A. Waldron
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
| | - Zainab Khalid
- Department of Neurosurgery, Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Jeffrey Blake
- Department of Neurosurgery, Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Ann M. Parr
- Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Department of Neurosurgery, Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Walter C. Low
- Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Department of Neurosurgery, Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
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45
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Abe T, Sarentonglaga B, Nagao Y. Advancements in medical research using fetal sheep: Implications for human health and treatment methods. Anim Sci J 2024; 95:e13945. [PMID: 38651196 DOI: 10.1111/asj.13945] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2024] [Revised: 03/13/2024] [Accepted: 03/27/2024] [Indexed: 04/25/2024]
Abstract
Sheep are typically considered as industrial animals that provide wool and meals. However, they play a significant role in medical research in addition to their conventional use. Notably, sheep fetuses are resistant to surgical invasions and can endure numerous manipulations, such as needle puncture and cell transplantation, and surgical operations requiring exposure beyond the uterus. Based on these distinguishing characteristics, we established a chimeric sheep model capable of producing human/monkey pluripotent cell-derived blood cells via the fetal liver. Furthermore, sheep have become crucial as human fetal models, acting as platforms for developing and improving techniques for intrauterine surgery to address congenital disorders and clarifying the complex pharmacokinetic interactions between mothers and their fetuses. This study emphasizes the significant contributions of fetal sheep to advancing human disease understanding and treatment strategies, highlighting their unique characteristics that are not present in other animals.
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Affiliation(s)
- Tomoyuki Abe
- Open Science Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan
| | | | - Yoshikazu Nagao
- Department of Agriculture, Utsunomiya University, Tochigi, Japan
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46
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Yuri S, Murase Y, Isotani A. Generation of rat-derived lung epithelial cells in Fgfr2b-deficient mice retains species-specific development. Development 2024; 151:dev202081. [PMID: 38179792 DOI: 10.1242/dev.202081] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2023] [Accepted: 11/29/2023] [Indexed: 01/06/2024]
Abstract
Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, although successful lung formation using interspecies chimeric animals has not yet been achieved. Here, we applied a reverse-blastocyst complementation method to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. These findings provide useful insights to overcome the barrier of species-specific developmental timing to generate functional lungs in interspecies chimeras.
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Affiliation(s)
- Shunsuke Yuri
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
| | - Yuki Murase
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
| | - Ayako Isotani
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
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47
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Coppiello G, Barlabé P, Moya-Jódar M, Abizanda G, Pogontke C, Barreda C, Iglesias E, Linares J, Arellano-Viera E, Larequi E, San Martín-Úriz P, Carvajal-Vergara X, Pelacho B, Mazo MM, Pérez-Pomares JM, Ruiz-Villalba A, Ullate-Agote A, Prósper F, Aranguren XL. Generation of heart and vascular system in rodents by blastocyst complementation. Dev Cell 2023; 58:2881-2895.e7. [PMID: 37967560 DOI: 10.1016/j.devcel.2023.10.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Revised: 07/10/2023] [Accepted: 10/23/2023] [Indexed: 11/17/2023]
Abstract
Generating organs from stem cells through blastocyst complementation is a promising approach to meet the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here, we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation, we rescued heart and vascular system development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs.
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Affiliation(s)
- Giulia Coppiello
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain.
| | - Paula Barlabé
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Marta Moya-Jódar
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Gloria Abizanda
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain; Cell Therapy Area, Clínica Universidad de Navarra, Pamplona 31008, Spain
| | - Cristina Pogontke
- Department of Animal Biology, University of Málaga, Málaga 29010, Spain; Biomedical Research Institute of Málaga (IBIMA-Plataforma BIONAND), Málaga 29590, Spain
| | - Carolina Barreda
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Elena Iglesias
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Javier Linares
- Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, 01307 Dresden, Germany
| | | | - Eduardo Larequi
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Patxi San Martín-Úriz
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Xonia Carvajal-Vergara
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Beatriz Pelacho
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Manuel Maria Mazo
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain; Cell Therapy Area, Clínica Universidad de Navarra, Pamplona 31008, Spain
| | - José Maria Pérez-Pomares
- Department of Animal Biology, University of Málaga, Málaga 29010, Spain; Biomedical Research Institute of Málaga (IBIMA-Plataforma BIONAND), Málaga 29590, Spain
| | - Adrián Ruiz-Villalba
- Department of Animal Biology, University of Málaga, Málaga 29010, Spain; Biomedical Research Institute of Málaga (IBIMA-Plataforma BIONAND), Málaga 29590, Spain
| | - Asier Ullate-Agote
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain
| | - Felipe Prósper
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain; Hematology and Cell Therapy Service, Cancer Center Clínica Universidad de Navarra (CCUN), IdISNA, Pamplona 31008, Spain; Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, Madrid 28029, Spain; Red Española de Terapias Avanzadas (RICORS-TERAV), Madrid 28029, Spain
| | - Xabier L Aranguren
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain.
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Tanaka J, Miura A, Shimamura Y, Hwang Y, Shimizu D, Kondo Y, Sawada A, Sarmah H, Ninish Z, Mishima K, Mori M. Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.13.566845. [PMID: 38014349 PMCID: PMC10680620 DOI: 10.1101/2023.11.13.566845] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2023]
Abstract
Various patients suffer from dry mouth due to salivary gland dysfunction. Whole salivary gland generation and transplantation is a potential therapy to resolve this issue. However, the lineage permissible to design the entire salivary gland generation has been enigmatic. Here, we discovered Foxa2 as a lineage critical for generating a salivary gland via conditional blastocyst complementation (CBC). Foxa2 linage, but not Shh nor Pitx2, initiated to label between the boundary region of the endodermal and the ectodermal oral mucosa before primordial salivary gland formation, resulting in marking the entire salivary gland. The salivary gland was agenesis by depleting Fgfr2 under the Foxa2 lineage in the mice. We rescued this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts. Those mice survived until adulthood with normal salivary glands compatible in size compared with littermate controls. These results indicated that CBC-based salivary gland generation is promising for next-generation cell-based therapy.
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49
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Miura A, Sarmah H, Tanaka J, Hwang Y, Sawada A, Shimamura Y, Otoshi T, Kondo Y, Fang Y, Shimizu D, Ninish Z, Suer JL, Dubois NC, Davis J, Toyooka S, Wu J, Que J, Hawkins FJ, Lin CS, Mori M. Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung. eLife 2023; 12:e86105. [PMID: 37861292 PMCID: PMC10642968 DOI: 10.7554/elife.86105] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Accepted: 10/19/2023] [Indexed: 10/21/2023] Open
Abstract
Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.
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Affiliation(s)
- Akihiro Miura
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
- Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
| | - Hemanta Sarmah
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Junichi Tanaka
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Youngmin Hwang
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Anri Sawada
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Yuko Shimamura
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Takehiro Otoshi
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Yuri Kondo
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Yinshan Fang
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Dai Shimizu
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Zurab Ninish
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Jake Le Suer
- The Pulmonary Center and Department of Medicine, Boston University School of MedicineBostonUnited States
- Center for Regenerative Medicine, Boston University and Boston Medical CenterBostonUnited States
| | - Nicole C Dubois
- Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount SinaiNew YorkUnited States
| | - Jennifer Davis
- Department of Pathology, University of WashingtonSeattleUnited States
| | - Shinichi Toyooka
- Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical CenterDallasUnited States
| | - Jianwen Que
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
| | - Finn J Hawkins
- The Pulmonary Center and Department of Medicine, Boston University School of MedicineBostonUnited States
- Center for Regenerative Medicine, Boston University and Boston Medical CenterBostonUnited States
| | - Chyuan-Sheng Lin
- Bernard and Shirlee Brown Glaucoma Laboratory, Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University Irving Medical CenterNew YorkUnited States
| | - Munemasa Mori
- Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical CenterNew YorkUnited States
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50
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Wang J, Xie W, Li N, Li W, Zhang Z, Fan N, Ouyang Z, Zhao Y, Lai C, Li H, Chen M, Quan L, Li Y, Jiang Y, Jia W, Fu L, Mazid MA, Zhu Y, Maxwell PH, Pan G, Esteban MA, Dai Z, Lai L. Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation. Cell Stem Cell 2023; 30:1235-1245.e6. [PMID: 37683604 DOI: 10.1016/j.stem.2023.08.003] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2022] [Revised: 06/12/2023] [Accepted: 08/07/2023] [Indexed: 09/10/2023]
Abstract
Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.
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Affiliation(s)
- Jiaowei Wang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100039, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China
| | - Wenguang Xie
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China
| | - Nan Li
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China; Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Wuyi University, Jiangmen 529020, China
| | - Wenjuan Li
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Zhishuai Zhang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Nana Fan
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China
| | - Zhen Ouyang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China; Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Wuyi University, Jiangmen 529020, China
| | - Yu Zhao
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China; Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Wuyi University, Jiangmen 529020, China
| | - Chengdan Lai
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China; Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Wuyi University, Jiangmen 529020, China
| | - Hao Li
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Mengqi Chen
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100039, China
| | - Longquan Quan
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China
| | - Yunpan Li
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yu Jiang
- Jilin Provincial Key Laboratory of Animal Embryo Engineering, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130012, China
| | - Wenqi Jia
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100039, China; Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Lixin Fu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Md Abdul Mazid
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yanling Zhu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Patrick H Maxwell
- School of Clinical Medicine, University of Cambridge, Cambridge CB2 0ST, UK
| | - Guangjin Pan
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Institute of Stem Cells and Regeneration, Chinese Academy of Sciences, Beijing 100039, China.
| | - Miguel A Esteban
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Jilin Provincial Key Laboratory of Animal Embryo Engineering, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130012, China; Institute of Stem Cells and Regeneration, Chinese Academy of Sciences, Beijing 100039, China.
| | - Zhen Dai
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China.
| | - Liangxue Lai
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya 572000, China; Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Wuyi University, Jiangmen 529020, China; Jilin Provincial Key Laboratory of Animal Embryo Engineering, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130012, China; Institute of Stem Cells and Regeneration, Chinese Academy of Sciences, Beijing 100039, China; Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences, Guangzhou 510530, China.
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