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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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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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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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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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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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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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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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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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Na DH, Cui S, Fang X, Lee H, Eum SH, Shin YJ, Lim SW, Yang CW, Chung BH. Advancements in Research on Genetic Kidney Diseases Using Human-Induced Pluripotent Stem Cell-Derived Kidney Organoids. Cells 2024; 13:1190. [PMID: 39056771 PMCID: PMC11274677 DOI: 10.3390/cells13141190] [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: 05/14/2024] [Revised: 07/09/2024] [Accepted: 07/11/2024] [Indexed: 07/28/2024] Open
Abstract
Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area.
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Affiliation(s)
- Do Hyun Na
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
- Division of Nephrology, Department of Internal Medicine, Seoul St. Mary’s Hospital, The College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
| | - Sheng Cui
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
| | - Xianying Fang
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
| | - Hanbi Lee
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
- Division of Nephrology, Department of Internal Medicine, Seoul St. Mary’s Hospital, The College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
| | - Sang Hun Eum
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
- Division of Nephrology, Department of Internal Medicine, Incheon St. Mary’s Hospital, The College of Medicine, The Catholic University of Korea, Incheon 21431, Republic of Korea
| | - Yoo Jin Shin
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
| | - Sun Woo Lim
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
| | - Chul Woo Yang
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
- Division of Nephrology, Department of Internal Medicine, Seoul St. Mary’s Hospital, The College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
| | - Byung Ha Chung
- Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea; (D.H.N.); (S.C.); (X.F.); (H.L.); (S.H.E.); (Y.J.S.); (S.W.L.); (C.W.Y.)
- Division of Nephrology, Department of Internal Medicine, Seoul St. Mary’s Hospital, The College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
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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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11
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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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12
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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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13
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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: 1] [Impact Index Per Article: 1.0] [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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14
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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: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [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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15
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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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16
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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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17
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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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18
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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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19
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Matsui K, Yamanaka S, Chen S, Matsumoto N, Morimoto K, Kinoshita Y, Inage Y, Saito Y, Takamura T, Fujimoto T, Tajiri S, Matsumoto K, Kobayashi E, Yokoo T. Long-term viable chimeric nephrons generated from progenitor cells are a reliable model in cisplatin-induced toxicity. Commun Biol 2023; 6:1097. [PMID: 37898693 PMCID: PMC10613230 DOI: 10.1038/s42003-023-05484-9] [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: 06/27/2023] [Accepted: 10/18/2023] [Indexed: 10/30/2023] Open
Abstract
Kidney organoids have shown promise as evaluation tools, but their in vitro maturity remains limited. Transplantation into adult mice has aided in maturation; however, their lack of urinary tract connection limits long-term viability. Thus, long-term viable generated nephrons have not been demonstrated. In this study, we present an approachable method in which mouse and rat renal progenitor cells are injected into the developing kidneys of neonatal mice, resulting in the generation of chimeric nephrons integrated with the host urinary tracts. These chimeric nephrons exhibit similar maturation to the host nephrons, long-term viability with excretion and reabsorption functions, and cisplatin-induced renal injury in both acute and chronic phases, as confirmed by single-cell RNA-sequencing. Additionally, induced human nephron progenitor cells differentiate into nephrons within the neonatal kidneys. Collectively, neonatal injection represents a promising approach for in vivo nephron generation, with potential applications in kidney regeneration, drug screening, and pathological analysis.
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Affiliation(s)
- Kenji Matsui
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Shuichiro Yamanaka
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan.
| | - Sandy Chen
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Naoto Matsumoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Keita Morimoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Yoshitaka Kinoshita
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
- Department of Urology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8654, Japan
| | - Yuka Inage
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
- Department of Pediatrics, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Yatsumu Saito
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Tsuyoshi Takamura
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Toshinari Fujimoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Susumu Tajiri
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Kei Matsumoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Eiji Kobayashi
- Department of Kidney Regenerative Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan
| | - Takashi Yokoo
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan.
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20
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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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21
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Matsumoto N, Yamanaka S, Morimoto K, Matsui K, Nishimura S, Kinoshita Y, Inage Y, Fujimori K, Kuroda T, Saito Y, Takamura T, Fujimoto T, Tajiri S, Matsumoto K, Inoue M, Kobayashi E, Yokoo T. Evaluation of the ability of human induced nephron progenitor cells to form chimeric renal organoids using mouse embryonic renal progenitor cells. Biochem Biophys Res Commun 2023; 662:18-25. [PMID: 37094429 DOI: 10.1016/j.bbrc.2023.04.052] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2023] [Accepted: 04/17/2023] [Indexed: 04/26/2023]
Abstract
The number of patients with end-stage renal failure is increasing annually worldwide and the problem is compounded by a shortage of renal transplantation donors. In our previous research, we have shown that transplantation of renal progenitor cells into the nephrogenic region of heterologous fetuses can induce the development of nephrons. We have also developed transgenic mice in which specific renal progenitor cells can be removed by drugs. By combining these two technologies, we have succeeded in generating human-mouse chimeric kidneys in fetal mice. We hope to apply these technologies to regenerative medicine. The quality of nephron progenitor cells (NPCs) derived from human pluripotent stem cells is important for the generation of chimeric kidneys, but there is currently no simple evaluation system for the chimerogenic potential of human NPCs. In this study, we focused on the fact that the re-aggregation of mouse renal progenitor cells can be used for nephron formation, even when merged into single cells. First, we examined the conditions under which nephron formation is likely to occur in mice during re-aggregation. Next, to improve the differentiation potential of human NPCs derived from pluripotent stem cells, NPCs were sorted using Integrin subunit alpha 8 (ITGA8). Finally, we demonstrated chimera formation between different species by mixing mouse cells with purified, selectively-induced human NPCs under optimum conditions. We observed these chimeric organoids at different time points to learn about these human-mouse chimeric structures at various stages of renal development. We found that the rate of chimera formation was affected by the purity of the human NPCs and the cell ratios used. We demonstrated that chimeric nephrons can be generated using a simple model, even between distant species. We believe that this admixture of human and mouse renal progenitor cells is a promising technology with potential application for the evaluation of the chimera formation abilities of NPCs.
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Affiliation(s)
- Naoto Matsumoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Shuichiro Yamanaka
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan.
| | - Keita Morimoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Kenji Matsui
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Sandy Nishimura
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Yoshitaka Kinoshita
- Department of Kidney Regenerative Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan; Department of Urology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-8654, Japan
| | - Yuka Inage
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan; Department of Pediatrics, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Koki Fujimori
- Sumitomo Pharma, Co., Ltd., 2-6-8, Doshomachi, Chuo-ku, Osaka, 541-0045, Japan
| | - Takao Kuroda
- Sumitomo Pharma, Co., Ltd., 2-6-8, Doshomachi, Chuo-ku, Osaka, 541-0045, Japan
| | - Yatsumu Saito
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Tsuyoshi Takamura
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Toshinari Fujimoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Susumu Tajiri
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Kei Matsumoto
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Makoto Inoue
- Sumitomo Pharma, Co., Ltd., 2-6-8, Doshomachi, Chuo-ku, Osaka, 541-0045, Japan
| | - Eiji Kobayashi
- Department of Kidney Regenerative Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan
| | - Takashi Yokoo
- Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo, 105-8461, Japan.
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22
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Xi J, Zheng W, Chen M, Zou Q, Tang C, Zhou X. Genetically engineered pigs for xenotransplantation: Hopes and challenges. Front Cell Dev Biol 2023; 10:1093534. [PMID: 36712969 PMCID: PMC9878146 DOI: 10.3389/fcell.2022.1093534] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Accepted: 12/31/2022] [Indexed: 01/14/2023] Open
Abstract
The shortage of donor resources has greatly limited the application of clinical xenotransplantation. As such, genetically engineered pigs are expected to be an ideal organ source for xenotransplantation. Most current studies mainly focus on genetically modifying organs or tissues from donor pigs to reduce or prevent attack by the human immune system. Another potential organ source is interspecies chimeras. In this paper, we reviewed the progress of the genetically engineered pigs from the view of immunologic barriers and strategies, and discussed the possibility and challenges of the interspecies chimeras.
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23
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Development of a Method for the In Vivo Generation of Allogeneic Hearts in Chimeric Mouse Embryos. Int J Mol Sci 2023; 24:ijms24021163. [PMID: 36674675 PMCID: PMC9865658 DOI: 10.3390/ijms24021163] [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: 11/03/2022] [Revised: 12/05/2022] [Accepted: 01/05/2023] [Indexed: 01/11/2023] Open
Abstract
Worldwide, there is a great gap between the demand and supply of organs for transplantations. Organs generated from the patients' cells would not only solve the problem of transplant availability but also overcome the complication of incompatibility and tissue rejection by the host immune system. One of the most promising methods tested for the production of organs in vivo is blastocyst complementation (BC). Regrettably, BC is not suitable for the creation of hearts. We have developed a novel method, induced blastocyst complementation (iBC), to surpass this shortcoming. By applying iBC, we generated chimeric mouse embryos, made up of "host" and "donor" cells. We used a specific cardiac enhancer to drive the expression of the diphtheria toxin gene (dtA) in the "host" cells, so that these cells are depleted from the developing hearts, which now consist of "donor" cells. This is a proof-of-concept study, showing that it is possible to produce allogeneic and ultimately, xenogeneic hearts in chimeric organisms. The ultimate goal is to generate, in the future, human hearts in big animals such as pigs, from the patients' cells, for transplantations. Such a system would generate transplants in a relatively short amount of time, improving the quality of life for countless patients around the world.
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24
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Var SR, Strell P, Johnson ST, Roman A, Vasilakos Z, Low WC. Transplanting Microglia for Treating CNS Injuries and Neurological Diseases and Disorders, and Prospects for Generating Exogenic Microglia. Cell Transplant 2023; 32:9636897231171001. [PMID: 37254858 PMCID: PMC10236244 DOI: 10.1177/09636897231171001] [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: 01/26/2023] [Revised: 03/18/2023] [Accepted: 04/05/2023] [Indexed: 06/01/2023] Open
Abstract
Microglia are associated with a wide range of both neuroprotective and neuroinflammatory functions in the central nervous system (CNS) during development and throughout lifespan. Chronically activated and dysfunctional microglia are found in many diseases and disorders, such as Alzheimer's disease, Parkinson's disease, and CNS-related injuries, and can accelerate or worsen the condition. Transplantation studies designed to replace and supplement dysfunctional microglia with healthy microglia offer a promising strategy for addressing microglia-mediated neuroinflammation and pathologies. This review will cover microglial involvement in neurological diseases and disorders and CNS-related injuries, current microglial transplantation strategies, and different approaches and considerations for generating exogenic microglia.
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Affiliation(s)
- Susanna R. Var
- Department of Neurosurgery, Medical
School, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, Medical School,
University of Minnesota, Minneapolis, MN, USA
| | - Phoebe Strell
- Stem Cell Institute, Medical School,
University of Minnesota, Minneapolis, MN, USA
- Department of Veterinary and Biomedical
Sciences, University of Minnesota, Minneapolis, MN, USA
| | - Sether T. Johnson
- Department of Neurosurgery, Medical
School, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, Medical School,
University of Minnesota, Minneapolis, MN, USA
| | - Alex Roman
- Department of Neuroscience, University
of Minnesota, Minneapolis, MN, USA
| | - Zoey Vasilakos
- Stem Cell Institute, Medical School,
University of Minnesota, Minneapolis, MN, USA
- Department of Neuroscience, University
of Minnesota, Minneapolis, MN, USA
| | - Walter C. Low
- Department of Neurosurgery, Medical
School, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, Medical School,
University of Minnesota, Minneapolis, MN, USA
- Department of Veterinary and Biomedical
Sciences, University of Minnesota, Minneapolis, MN, USA
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25
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Muacevic A, Adler JR, Ajmal M, Nawaz G. Organ Regeneration Through Stem Cells and Tissue Engineering. Cureus 2023; 15:e34336. [PMID: 36865965 PMCID: PMC9973391 DOI: 10.7759/cureus.34336] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/29/2023] [Indexed: 01/30/2023] Open
Abstract
Loss of organ and tissue due to injuries or diseases led to the development of regenerative therapies to decrease reliance on organ transplantations. It deals with employing the self-renewal ability of stem cells to differentiate into numerous lineages to assist in providing effective treatment for a range of various injuries and diseases. Regenerative engineering of organs or tissues represents an ever-expanding field that is aimed at developing biological replacements for dysfunctional organs or injured tissues. The critical issue, however, with the engineering of organs outside the human body is the insufficient availability of human cells, the absence of a suitable matrix with the same architecture and composition as the target tissue, and the maintenance of organ viability in the absence of the blood supply. The issue regarding the maintenance of the engineered organ viability can be solved using bioreactors consisting of mediums with defined chemical composition, i.e., nutrients, cofactors, and growth factors that can successively sustain the target cell's viability. Engineered extracellular matrices and stem cells to regenerate organs outside the human body are also being used. Clinically, various adult stem cell therapies are readily under practice. This review will focus on the regeneration of organs through various types of stem cells and tissue engineering techniques.
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26
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Shetty A, Lim S, Strell P, Steer CJ, Rivera-Mulia JC, Low WC. In Silico Stage-Matching of Human, Marmoset, Mouse, and Pig Embryos to Enhance Organ Development Through Interspecies Chimerism. Cell Transplant 2023; 32:9636897231158728. [PMID: 36929807 PMCID: PMC10026093 DOI: 10.1177/09636897231158728] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 01/31/2023] [Accepted: 02/04/2023] [Indexed: 03/18/2023] Open
Abstract
Currently, there is a significant shortage of transplantable organs for patients in need. Interspecies chimerism and blastocyst complementation are alternatives for generating transplantable human organs in host animals such as pigs to meet this shortage. While successful interspecies chimerism and organ generation have been observed between evolutionarily close species such as rat and mouse, barriers still exist for more distant species pairs such as human-mouse, marmoset-mouse, human-pig, and others. One of the proposed barriers to chimerism is the difference in developmental stages between the donor cells and the host embryo at the time the cells are introduced into the host embryo. Hence, there is a logical effort to stage-match the donor cells with the host embryos for enhancing interspecies chimerism. In this study, we used an in silico approach to simultaneously stage-match the early developing embryos of four species, including human, marmoset, mouse, and pig based on transcriptome similarities. We used an unsupervised clustering algorithm to simultaneously stage-match all four species as well as Spearman's correlation analyses to stage-match pairs of donor-host species. From our stage-matching analyses, we found that the four stages that best matched with each other are the human blastocyst (E6/E7), the gastrulating mouse embryo (E6-E6.75), the marmoset late inner cell mass, and the pig late blastocyst. We further demonstrated that human pluripotent stem cells best matched with the mouse post-implantation stages. We also performed ontology analysis of the genes upregulated and commonly expressed between donor-host species pairs at their best matched stages. The stage-matching results predicted by this study will inform in vivo and in vitro interspecies chimerism and blastocyst complementation studies and can be used to match donor cells with host embryos between multiple species pairs to enhance chimerism for organogenesis.
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Affiliation(s)
- Anala Shetty
- Molecular, Cellular, Developmental
Biology, and Genetics Graduate Program, University of Minnesota, Minneapolis, MN,
USA
| | - Seunghyun Lim
- Bioinformatics and Computational
Biology Graduate Program, University of Minnesota, Minneapolis, MN, USA
| | - Phoebe Strell
- Comparative and Molecular Biosciences
Graduate Program, University of Minnesota, Minneapolis, MN, USA
| | - Clifford J. Steer
- Molecular, Cellular, Developmental
Biology, and Genetics Graduate Program, University of Minnesota, Minneapolis, MN,
USA
- Department of Medicine, University of
Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of
Minnesota, Minneapolis, MN, USA
| | - Juan Carlos Rivera-Mulia
- Molecular, Cellular, Developmental
Biology, and Genetics Graduate Program, University of Minnesota, Minneapolis, MN,
USA
- Stem Cell Institute, University of
Minnesota, Minneapolis, MN, USA
- Department of Biochemistry, Molecular
Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - Walter C. Low
- Molecular, Cellular, Developmental
Biology, and Genetics Graduate Program, University of Minnesota, Minneapolis, MN,
USA
- Bioinformatics and Computational
Biology Graduate Program, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of
Minnesota, Minneapolis, MN, USA
- Department of Neurosurgery, University
of Minnesota, Minneapolis, MN, USA
- Graduate Program in Neuroscience,
University of Minnesota, Minneapolis, MN, USA
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27
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Brown JL, Voth JP, Person K, Low WC. A Technological and Regulatory Review on Human-Animal Chimera Research: The Current Landscape of Biology, Law, and Public Opinion. Cell Transplant 2023; 32:9636897231183112. [PMID: 37599386 PMCID: PMC10467371 DOI: 10.1177/09636897231183112] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2022] [Revised: 05/20/2023] [Accepted: 06/04/2023] [Indexed: 08/22/2023] Open
Abstract
Organ transplantation is a highly utilized treatment for many medical conditions, yet the number of patients waiting for organs far exceeds the number available. The challenges and limitations currently associated with organ transplantation and technological advances in gene editing techniques have led scientists to pursue alternate solutions to the donor organ shortage. Growing human organs in animals and harvesting those organs for transplantation into humans is one such solution. These chimeric animals usually have certain genes necessary for a specific organ's development inhibited at an early developmental stage, followed by the addition of cultured pluripotent human cells to fill that developmental niche. The result is a chimeric animal that contains human organs which are available for transplant into a patient, circumventing some of the limitations currently involved in donor organ transplantation. In this review, we will discuss both the current scientific and legal landscape of human-animal chimera (HAC) research. We present an overview of the technological advances that allow for the creation of HACs, the patents that currently exist on these methods, as well as current public attitude and understanding that can influence HAC research policy. We complement our scientific and public attitude discussion with a regulatory overview of chimera research at both the national and state level, while also contrasting current U.S. legislation with regulations in other countries. Overall, we provide a comprehensive analysis of the legal and scientific barriers to conducting research on HACs for the generation of transplantable human organs, as well as provide recommendations for the future.
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Affiliation(s)
- Jennifer L. Brown
- Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Institute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Law School, University of Minnesota, Minneapolis, MN, USA
| | - Joseph P. Voth
- Department of Neuroscience, University of Washington, Seattle, WA, USA
- Medical Scientist Training Program, University of Washington, Seattle, WA, USA
| | - Kennedy Person
- Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
- 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, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
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28
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Zhao L, Li M, Yin Z, Lv L, Zhou M, Wang Y, Zhang M, Guo T, Guo X, Liu H, Cheng L, Liang X, Duo S, Li R. Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2. Cells 2022; 11:cells11233874. [PMID: 36497134 PMCID: PMC9740088 DOI: 10.3390/cells11233874] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2022] [Revised: 11/28/2022] [Accepted: 11/29/2022] [Indexed: 12/04/2022] Open
Abstract
A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.
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Affiliation(s)
- Lihua Zhao
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China
| | - Meishuang Li
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Zhibao Yin
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Limin Lv
- Laboratory Animal Center, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Meng Zhou
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Yixi Wang
- Laboratory Animal Center, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Manling Zhang
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Tianxu Guo
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Xiyun Guo
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Han Liu
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Linxin Cheng
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
| | - Xiubin Liang
- Department of Pathophysiology, Nanjing Medical University, Nanjing 211166, China
| | - Shuguang Duo
- Laboratory Animal Center, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Correspondence: (S.D.); (R.L.)
| | - Rongfeng Li
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
- Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China
- Correspondence: (S.D.); (R.L.)
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29
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Abstract
Embryonic development and cell specification have been viewed as an epigenetically rigid process. Through accumulation of irreversible epigenetic marks, the differentiation process has been considered unidirectional, and once completed cell specification would be permanent and stable. However, somatic cell nuclear transfer that involved the implantation of a somatic nucleus into a previously enucleated oocyte accomplished in amphibians in the 1950s and in mammals in the late 1990s-resulting in the birth of "Dolly the sheep"-clearly showed that "terminal" differentiation is reversible. In parallel, work on lineage-determining factors like MyoD revealed surprising potential to modulate lineage identity in somatic cells. This work culminated in the discovery that a set of four defined factors can reprogram fibroblasts into induced pluripotent stem (iPS) cells, which were shown to be molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells, thus essentially showing that defined factors can induce authentic reprogramming without the need of oocytes. This concept was further extended when it was shown that fibroblasts can be directly converted into neurons, showing induced lineage conversion is possible even between cells representing two different germ layers. These findings suggest that "everything is possible" (i.e., once key lineage reprogramming factors are identified, cells should be able to convert into any desired lineage).
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Affiliation(s)
- Hannah Shelby
- Departments of Pathology and Chemical and Systems Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Tara Shelby
- Departments of Pathology and Chemical and Systems Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Marius Wernig
- Departments of Pathology and Chemical and Systems Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA
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30
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Zvick J, Tarnowska-Sengül M, Ghosh A, Bundschuh N, Gjonlleshaj P, Hinte LC, Trautmann CL, Noé F, Qabrati X, Domenig SA, Kim I, Hennek T, von Meyenn F, Bar-Nur O. Exclusive generation of rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells. Stem Cell Reports 2022; 17:1942-1958. [PMID: 35931077 PMCID: PMC9481912 DOI: 10.1016/j.stemcr.2022.07.005] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 07/06/2022] [Accepted: 07/07/2022] [Indexed: 11/17/2022] Open
Abstract
Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.
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Affiliation(s)
- Joel Zvick
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Monika Tarnowska-Sengül
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Adhideb Ghosh
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland; Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich 8057, Switzerland
| | - Nicola Bundschuh
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Pjeter Gjonlleshaj
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Laura C Hinte
- Laboratory of Nutrition and Metabolic Epigenetics, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Christine L Trautmann
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Falko Noé
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland; Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich 8057, Switzerland
| | - Xhem Qabrati
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Seraina A Domenig
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Inseon Kim
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Thomas Hennek
- ETH Phenomics Center, ETH Zurich, Zurich 8049, Switzerland
| | - Ferdinand von Meyenn
- Laboratory of Nutrition and Metabolic Epigenetics, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Ori Bar-Nur
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland.
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Huang B, Zeng Z, Zhang CC, Schreiber ME, Li Z. Approaches to kidney replacement therapies—opportunities and challenges. Front Cell Dev Biol 2022; 10:953408. [PMID: 35982852 PMCID: PMC9380013 DOI: 10.3389/fcell.2022.953408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2022] [Accepted: 07/01/2022] [Indexed: 11/29/2022] Open
Abstract
One out of seven people develop chronic kidney disease (CKD). When kidney function continues to decline, CKD patients may develop end-stage renal disease (ESRD, or kidney failure). More than 2 out of 1,000 adults develop ESRD and these patients must live on dialysis or get a kidney transplant to survive. Each year, more than $51 billion is spent to treat patients with ESRD in the United States. In addition, ESRD greatly reduces longevity and quality of life for patients. Compared to dialysis, kidney transplant offers the best chance of survival, but few donor organs are available. Thus, there is an urgent need for innovative solutions that address the shortage of kidneys available for transplantation. Here we summarize the status of current approaches that are being developed to solve the shortage of donor kidneys. These include the bioartificial kidney approach which aims to make a portable dialysis device, the recellularization approach which utilizes native kidney scaffold to make an engineered kidney, the stem cell-based approach which aims to generate a kidney de novo by recapitulating normal kidney organogenesis, the xenotransplantation approach which has the goal to make immunocompatible pig kidneys for transplantation, and the interspecies chimera approach which has potential to generate a human kidney in a host animal. We also discuss the interconnections among the different approaches, and the remaining challenges of translating these approaches into novel therapies.
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Affiliation(s)
- Biao Huang
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Zipeng Zeng
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Chennan C. Zhang
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Megan E. Schreiber
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Zhongwei Li
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- *Correspondence: Zhongwei Li,
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Moya-Jódar M, Coppiello G, Rodríguez-Madoz JR, Abizanda G, Barlabé P, Vilas-Zornoza A, Ullate-Agote A, Luongo C, Rodríguez-Tobón E, Navarro-Serna S, París-Oller E, Oficialdegui M, Carvajal-Vergara X, Ordovás L, Prósper F, García-Vázquez FA, Aranguren XL. One-Step In Vitro Generation of ETV2-Null Pig Embryos. Animals (Basel) 2022; 12:ani12141829. [PMID: 35883376 PMCID: PMC9311767 DOI: 10.3390/ani12141829] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2022] [Revised: 07/05/2022] [Accepted: 07/14/2022] [Indexed: 11/16/2022] Open
Abstract
Simple Summary One of the latest goals in regenerative medicine is to use pluripotent stem cells to generate whole organs in vivo through the blastocyst complementation technique. This method consists of the microinjection of pluripotent stem cells into preimplantation embryos that have been genetically modified to ablate the development of a target organ. By taking advantage of the spatiotemporal clues present in the developing embryo, pluripotent stem cells are able to colonize the empty developmental niche and create the missing organ. Combining human pluripotent stem cells with genetically engineered pig embryos, it would be possible to obtain humanized organs that could be used for transplantation, and, therefore, solve the worldwide issue of insufficient availability of transplantable organs. As endothelial cells play a critical role in xenotransplantation rejection in all organs, in this study, we optimized a protocol to generate a vascular-disabled preimplantation pig embryo using the CRISPR/Cas9 system. This protocol could be used to generate avascular embryos for blastocyst complementation experiments and work towards the generation of rejection-free humanized organs in pigs. Abstract Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.
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Affiliation(s)
- Marta Moya-Jódar
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
| | - Giulia Coppiello
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
| | - Juan Roberto Rodríguez-Madoz
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
| | - Gloria Abizanda
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
| | - Paula Barlabé
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
| | - Amaia Vilas-Zornoza
- Advanced Genomics Laboratory, Program of Hemato-Oncology, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain;
| | - Asier Ullate-Agote
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
- Advanced Genomics Laboratory, Program of Hemato-Oncology, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain;
| | - Chiara Luongo
- Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain; (C.L.); (E.R.-T.); (S.N.-S.); (E.P.-O.)
- Institute for Biomedical Research of Murcia, IMIB-Arrixaca, 30100 Murcia, Spain
| | - Ernesto Rodríguez-Tobón
- Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain; (C.L.); (E.R.-T.); (S.N.-S.); (E.P.-O.)
- Institute for Biomedical Research of Murcia, IMIB-Arrixaca, 30100 Murcia, Spain
| | - Sergio Navarro-Serna
- Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain; (C.L.); (E.R.-T.); (S.N.-S.); (E.P.-O.)
- Institute for Biomedical Research of Murcia, IMIB-Arrixaca, 30100 Murcia, Spain
| | - Evelyne París-Oller
- Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain; (C.L.); (E.R.-T.); (S.N.-S.); (E.P.-O.)
- Institute for Biomedical Research of Murcia, IMIB-Arrixaca, 30100 Murcia, Spain
| | | | - Xonia Carvajal-Vergara
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
| | - Laura Ordovás
- Aragon Agency for Research and Development (ARAID), 50018 Zaragoza, Spain;
- Biomedical Signal Interpretation and Computational Simulation (BSICoS), Institute of Engineering Research (I3A), University of Zaragoza & Instituto de Investigación Sanitaria (IIS), 50018 Zaragoza, Spain
| | - Felipe Prósper
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), 28029 Madrid, Spain
- Instituto de Investigación Sanitaria de Navarra (IdiSNA), 31008 Pamplona, Spain
- Department of Hematology and Cell Therapy, Clínica Universidad de Navarra, 31008 Pamplona, Spain
| | - Francisco Alberto García-Vázquez
- Department of Physiology, Veterinary School, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, 30100 Murcia, Spain; (C.L.); (E.R.-T.); (S.N.-S.); (E.P.-O.)
- Institute for Biomedical Research of Murcia, IMIB-Arrixaca, 30100 Murcia, Spain
- Correspondence: (F.A.G.-V.); (X.L.A.)
| | - Xabier L. Aranguren
- Program of Regenerative Medicine, Centre for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain; (M.M.-J.); (G.C.); (J.R.R.-M.); (G.A.); (P.B.); (A.U.-A.); (X.C.-V.); (F.P.)
- Correspondence: (F.A.G.-V.); (X.L.A.)
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Tsuji K, Kitamura S, Wada J. Potential Strategies for Kidney Regeneration With Stem Cells: An Overview. Front Cell Dev Biol 2022; 10:892356. [PMID: 35586342 PMCID: PMC9108336 DOI: 10.3389/fcell.2022.892356] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Accepted: 04/19/2022] [Indexed: 11/28/2022] Open
Abstract
Kidney diseases are a major health problem worldwide. Despite advances in drug therapies, they are only capable of slowing the progression of kidney diseases. Accordingly, potential kidney regeneration strategies with stem cells have begun to be explored. There are two different directions for regenerative strategies, de novo whole kidney fabrication with stem cells, and stem cell therapy. De novo whole kidney strategies include: 1) decellularized scaffold technology, 2) 3D bioprinting based on engineering technology, 3) kidney organoid fabrication, 4) blastocyst complementation with chimeric technology, and 5) the organogenic niche method. Meanwhile, stem cell therapy strategies include 1) injection of stem cells, including mesenchymal stem cells, nephron progenitor cells, adult kidney stem cells and multi-lineage differentiating stress enduring cells, and 2) injection of protective factors secreted from these stem cells, including growth factors, chemokines, and extracellular vesicles containing microRNAs, mRNAs and proteins. Over the past few decades, there have been remarkable step-by-step developments in these strategies. Here, we review the current advances in the potential strategies for kidney regeneration using stem cells, along with their challenges for possible clinical use in the future.
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Fukunaga S, Ogawa N, Matsumoto A, Ito T, Tanabe K, Otani H. Administration of retinoic acid to pregnant mice increases the number of fetal mouse glomeruli. Biochem Biophys Rep 2022; 30:101245. [PMID: 35280524 PMCID: PMC8907684 DOI: 10.1016/j.bbrep.2022.101245] [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: 01/15/2022] [Revised: 02/25/2022] [Accepted: 03/04/2022] [Indexed: 11/17/2022] Open
Abstract
The prevalence of chronic kidney disease (CKD) is increasing worldwide, and CKD is a serious global health problem. Low glomerular number is one of the risk factors for CKD; therefore, the glomerular number is associated with the risk of CKD. Increasing the glomerular number above normal levels may reduce the risk of CKD. It has been reported that, in vitro, the addition of retinoic acid (RA) to the culture medium increases the glomerular number. However, there is no report of an increase in glomerular number above normal levels with the addition of RA in vivo. In this study, RA (20 mg/kg) was administered intraperitoneally to pregnant mice once at embryonic day (E) 10.5, E12.5, E14.5, or E16.5. The fetuses were harvested at E18.5 and fetal mouse kidneys were evaluated. Fetal kidney volume and weight were significantly increased in the E16.5 group compared to the control group. The total glomerular number in the E16.5 group was also approximately 1.46 times higher than that in the control group. In summary, we established a method to increase the glomerular number in the fetal kidney by administration of RA to pregnant mice at E16.5. These results will facilitate the investigation of whether CKD risk is reduced when the glomerular number increases above normal.
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Affiliation(s)
- Shohei Fukunaga
- Department of Internal Medicine IV, Shimane University Faculty of Medicine, Izumo, Shimane, 693-8501, Japan
| | - Noriko Ogawa
- Department of Developmental Biology, Shimane University Faculty of Medicine, Izumo, Shimane, 693-8501, Japan
| | - Akihiro Matsumoto
- Department of Developmental Biology, Shimane University Faculty of Medicine, Izumo, Shimane, 693-8501, Japan
| | - Takafumi Ito
- Department of Internal Medicine IV, Shimane University Faculty of Medicine, Izumo, Shimane, 693-8501, Japan
| | - Kazuaki Tanabe
- Department of Internal Medicine IV, Shimane University Faculty of Medicine, Izumo, Shimane, 693-8501, Japan
| | - Hiroki Otani
- Department of Developmental Biology, Shimane University Faculty of Medicine, Izumo, Shimane, 693-8501, Japan
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In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation. Sci Rep 2022; 12:3242. [PMID: 35217706 PMCID: PMC8881621 DOI: 10.1038/s41598-022-07159-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Accepted: 02/07/2022] [Indexed: 11/19/2022] Open
Abstract
Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.
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Generation of chimeric kidneys using progenitor cell replacement: Oshima Award Address 2021. Clin Exp Nephrol 2022; 26:491-500. [PMID: 35138500 PMCID: PMC9114015 DOI: 10.1007/s10157-022-02191-3] [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: 12/23/2021] [Accepted: 01/28/2022] [Indexed: 11/03/2022]
Abstract
It is believed that the development of new renal replacement therapy (RRT) will increase treatment options for end-stage kidney disease and help reduce the mismatch between supply and demand. Technological advancement in the development of kidney organoids derived from pluripotent stem cells and xenotransplantation using porcine kidneys has been accelerated by a convergence of technological innovations, including the discovery of induced pluripotent stem cells and genome editing, and improvement of analysis techniques such as single-cell ribonucleic acid sequencing. Given the difficulty associated with kidney regeneration, hybrid kidneys are studied as an innovative approach that involves the use of stem cells to generate kidneys, with animal fetal kidneys used as a scaffold. Hybrid kidney technology entails the application of local chimerism for the generation of chimeric kidneys from exogenous renal progenitor cells by borrowing complex nephrogenesis programs from the developmental environment of heterologous animals. Hybrid kidneys can also utilize the urinary tract and bladder tissue of animal fetuses for urine excretion. Generating nephrons from syngeneic stem cells to increase self-cell ratio in xeno-tissues can reduce the risk of xeno-rejection. We showed that nephrons can be generated by ablation of host nephron progenitor cells (NPCs) in the nephron development region of animals and replacing them with exogenous NPCs. This progenitor cell replacement is the basis of hybrid kidney regeneration from progenitor cells using chimera technology. The goal of xeno-regenerative medicine using hybrid kidneys is to overcome serious organ shortage.
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Little MH, Humphreys BD. Regrow or Repair: An Update on Potential Regenerative Therapies for the Kidney. J Am Soc Nephrol 2022; 33:15-32. [PMID: 34789545 PMCID: PMC8763179 DOI: 10.1681/asn.2021081073] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Fifteen years ago, this journal published a review outlining future options for regenerating the kidney. At that time, stem cell populations were being identified in multiple tissues, the concept of stem cell recruitment to a site of injury was of great interest, and the possibility of postnatal renal stem cells was growing in momentum. Since that time, we have seen the advent of human induced pluripotent stem cells, substantial advances in our capacity to both sequence and edit the genome, global and spatial transcriptional analysis down to the single-cell level, and a pandemic that has challenged our delivery of health care to all. This article will look back over this period of time to see how our view of kidney development, disease, repair, and regeneration has changed and envision a future for kidney regeneration and repair over the next 15 years.
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Affiliation(s)
- Melissa H. Little
- Murdoch Children’s Research Institute, Parkville, Melbourne, Victoria, Australia,Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, Melbourne, Victoria, Australia,Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Melbourne, Victoria, Australia
| | - Benjamin D. Humphreys
- Division of Nephrology, Department of Medicine, Washington University in St. Louis School of Medicine, Missouri,Department of Developmental Biology, Washington University in St. Louis School of Medicine, Missouri
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Kano M, Mizutani E, Homma S, Masaki H, Nakauchi H. Xenotransplantation and interspecies organogenesis: current status and issues. Front Endocrinol (Lausanne) 2022; 13:963282. [PMID: 35992127 PMCID: PMC9388829 DOI: 10.3389/fendo.2022.963282] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Accepted: 07/06/2022] [Indexed: 12/04/2022] Open
Abstract
Pancreas (and islet) transplantation is the only curative treatment for type 1 diabetes patients whose β-cell functions have been abolished. However, the lack of donor organs has been the major hurdle to save a large number of patients. Therefore, transplantation of animal organs is expected to be an alternative method to solve the serious shortage of donor organs. More recently, a method to generate organs from pluripotent stem cells inside the body of other species has been developed. This interspecies organ generation using blastocyst complementation (BC) is expected to be the next-generation regenerative medicine. Here, we describe the recent advances and future prospects for these two approaches.
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Affiliation(s)
- Mayuko Kano
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
| | - Eiji Mizutani
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
- Laboratory of Stem Cell Therapy, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Shota Homma
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States
| | - Hideki Masaki
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
- *Correspondence: Hiromitsu Nakauchi, ; Hideki Masaki,
| | - Hiromitsu Nakauchi
- Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States
- *Correspondence: Hiromitsu Nakauchi, ; Hideki Masaki,
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Xie Y, Yao J, Jin W, Ren L, Li X. Induction and Maturation of Hepatocyte-Like Cells In Vitro: Focus on Technological Advances and Challenges. Front Cell Dev Biol 2021; 9:765980. [PMID: 34901010 PMCID: PMC8662991 DOI: 10.3389/fcell.2021.765980] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2021] [Accepted: 11/08/2021] [Indexed: 12/17/2022] Open
Abstract
Limited by the poor proliferation and restricted sources of adult hepatocytes, there is an urgent need to find substitutes for proliferation and cultivation of mature hepatocytes in vitro for use in disease treatment, drug approval, and toxicity testing. Hepatocyte-like cells (HLCs), which originate from undifferentiated stem cells or modified adult cells, are considered good candidates because of their advantages in terms of cell source and in vitro expansion ability. However, the majority of induced HLCs are in an immature state, and their degree of differentiation is heterogeneous, diminishing their usability in basic research and limiting their clinical application. Therefore, various methods have been developed to promote the maturation of HLCs, including chemical approaches, alteration of cell culture systems, and genetic manipulation, to meet the needs of in vivo transplantation and in vitro model establishment. This review proposes different cell types for the induction of HLCs, and provide a comprehensive overview of various techniques to promote the generation and maturation of HLCs in vitro.
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Affiliation(s)
- Ye Xie
- The First Clinical Medical College, Lanzhou University, Lanzhou, China
| | - Jia Yao
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,Key Laboratory of Biotherapy and Regenerative Medicine of Gansu Province, Lanzhou, China
| | - Weilin Jin
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,Institute of Cancer Neuroscience, The First Hospital of Lanzhou University, Lanzhou, China.,The Medical Frontier Innovation Research Center, The First Hospital of Lanzhou University, Lanzhou, China
| | - Longfei Ren
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,The Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, China
| | - Xun Li
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,Key Laboratory of Biotherapy and Regenerative Medicine of Gansu Province, Lanzhou, China.,The Medical Frontier Innovation Research Center, The First Hospital of Lanzhou University, Lanzhou, China.,The Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, China.,Hepatopancreatobiliary Surgery Institute of Gansu Province, Lanzhou, China
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40
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Founta KM, Papanayotou C. In Vivo Generation of Organs by Blastocyst Complementation: Advances and Challenges. Int J Stem Cells 2021; 15:113-121. [PMID: 34711704 PMCID: PMC9148837 DOI: 10.15283/ijsc21122] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Revised: 08/05/2021] [Accepted: 08/08/2021] [Indexed: 11/09/2022] Open
Abstract
The ultimate goal of regenerative medicine is to replace damaged cells, tissues or whole organs, in order to restore their proper function. Stem cell related technologies promise to generate transplants from the patients' own cells. Novel approaches such as blastocyst complementation combined with genome editing open up new perspectives for organ replacement therapies. This review summarizes recent advances in the field and highlights the challenges that still remain to be addressed.
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Affiliation(s)
- Konstantina-Maria Founta
- Department of Basic Science, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
| | - Costis Papanayotou
- Department of Basic Science, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
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41
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Garry DJ, Garry MG. Interspecies chimeras as a platform for exogenic organ production and transplantation. Exp Biol Med (Maywood) 2021; 246:1838-1844. [PMID: 34165344 DOI: 10.1177/15353702211024948] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Chronic diseases are associated with considerable morbidity and mortality. Therefore, new therapeutic strategies are warranted. Here, we provide a brief review outlining the rationale and feasibility for the generation of intraspecies and interspecies chimeras, which one day may serve as a platform for organ transplantation. These strategies are further associated with consideration of scientific and ethical issues.
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Affiliation(s)
- Daniel J Garry
- Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.,Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA.,Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN 55455, USA.,NorthStar Genomics, LLC, Eagan, MN 55122, USA
| | - Mary G Garry
- Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.,Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA.,Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN 55455, USA.,NorthStar Genomics, LLC, Eagan, MN 55122, USA
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42
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Zheng C, Ballard EB, Wu J. The road to generating transplantable organs: from blastocyst complementation to interspecies chimeras. Development 2021; 148:dev195792. [PMID: 34132325 PMCID: PMC10656466 DOI: 10.1242/dev.195792] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Growing human organs in animals sounds like something from the realm of science fiction, but it may one day become a reality through a technique known as interspecies blastocyst complementation. This technique, which was originally developed to study gene function in development, involves injecting donor pluripotent stem cells into an organogenesis-disabled host embryo, allowing the donor cells to compensate for missing organs or tissues. Although interspecies blastocyst complementation has been achieved between closely related species, such as mice and rats, the situation becomes much more difficult for species that are far apart on the evolutionary tree. This is presumably because of layers of xenogeneic barriers that are a result of divergent evolution. In this Review, we discuss the current status of blastocyst complementation approaches and, in light of recent progress, elaborate on the keys to success for interspecies blastocyst complementation and organ generation.
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Affiliation(s)
- Canbin Zheng
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Department of Microsurgery, Orthopaedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
| | - Emily B. Ballard
- 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
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43
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Kitahara A, Ran Q, Oda K, Yasue A, Abe M, Ye X, Sasaoka T, Tsuchida M, Sakimura K, Ajioka Y, Saijo Y, Zhou Q. Generation of Lungs by Blastocyst Complementation in Apneumic Fgf10-Deficient Mice. Cell Rep 2021; 31:107626. [PMID: 32402288 DOI: 10.1016/j.celrep.2020.107626] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 04/09/2020] [Accepted: 04/18/2020] [Indexed: 01/13/2023] Open
Abstract
The shortage of donor lungs hinders lung transplantation, the only definitive option for patients with end-stage lung disease. Blastocyst complementation enables the generation of transplantable organs from pluripotent stem cells (PSCs) in animal models. Pancreases and kidneys have been generated from PSCs by blastocyst complementation in rodent models. Here, we report the generation of lungs using mouse embryonic stem cells (ESCs) in apneumic Fgf10 Ex1mut/Ex3mutmice by blastocyst complementation. Complementation with ESCs enables Fgf10-deficient mice to survive to adulthood without abnormalities. Both the generated lung alveolar parenchyma and the interstitial portions, including vascular endothelial cells, vascular and parabronchial smooth muscle cells, and connective tissue, largely originate from the injected ESCs. These data suggest that Fgf10 Ex1mut/Ex3mutblastocysts provide an organ niche for lung generation and that blastocyst complementation could be a viable approach for generating whole lungs.
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Affiliation(s)
- Akihiko Kitahara
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan; Division of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Qingsong Ran
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Kanako Oda
- Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Akihiro Yasue
- Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
| | - Manabu Abe
- Department of Cellular Neurobiology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Xulu Ye
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Toshikuni Sasaoka
- Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Masanori Tsuchida
- Division of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Kenji Sakimura
- Department of Animal Model Development, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Yoichi Ajioka
- Division of Molecular and Diagnostic Pathology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Yasuo Saijo
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan.
| | - Qiliang Zhou
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan.
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Ruiz-Estevez M, Crane AT, Rodriguez-Villamil P, Ongaratto FL, You Y, Steevens AR, Hill C, Goldsmith T, Webster DA, Sherry L, Lim S, Denman N, Low WC, Carlson DF, Dutton JR, Steer CJ, Gafni O. Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs. Stem Cell Res Ther 2021; 12:292. [PMID: 34011403 PMCID: PMC8132445 DOI: 10.1186/s13287-021-02348-z] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2021] [Accepted: 04/19/2021] [Indexed: 11/10/2022] Open
Abstract
Background There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. Methods The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. Results Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. Conclusions This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02348-z.
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Affiliation(s)
- M Ruiz-Estevez
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - A T Crane
- Department of Neurosurgery, University of Minnesota, Minneapolis, USA.,Stem Cell Institute, University of Minnesota, Minneapolis, USA
| | - P Rodriguez-Villamil
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - F L Ongaratto
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - Yun You
- Mouse Genetics Laboratory, University of Minnesota, Minneapolis, USA
| | - A R Steevens
- Department of Neurosurgery, University of Minnesota, Minneapolis, USA.,Stem Cell Institute, University of Minnesota, Minneapolis, USA
| | - C Hill
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - T Goldsmith
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - D A Webster
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - L Sherry
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - S Lim
- Bioinformatics and Computational Biology Program, University of Minnesota, Minneapolis, USA
| | - N Denman
- Stem Cell Institute, University of Minnesota, Minneapolis, USA.,Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, USA
| | - W C Low
- Department of Neurosurgery, University of Minnesota, Minneapolis, USA.,Stem Cell Institute, University of Minnesota, Minneapolis, USA
| | - D F Carlson
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA
| | - J R Dutton
- Stem Cell Institute, University of Minnesota, Minneapolis, USA.,Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, USA
| | - C J Steer
- Stem Cell Institute, University of Minnesota, Minneapolis, USA. .,Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, USA. .,Department of Medicine, University of Minnesota, 420 Delaware Street SE, MMC 36, Minneapolis, MN, 55455, USA.
| | - O Gafni
- Recombinetics Inc., Stem Cell Technologies, 3388 Mike Collins Drive, Eagan, MN, 55121, USA.
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45
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Ballard EB, Wu J. Growth Competition in Interspecies Chimeras: A New Paradigm for Blastocyst Complementation. Cell Stem Cell 2021; 28:3-5. [PMID: 33417870 DOI: 10.1016/j.stem.2020.12.011] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Blastocyst complementation represents a powerful technique for interspecies organogenesis but is limited by low chimerism due to developmental incompatibilities. In this issue of Cell Stem Cell,Nishimura et al. (2021) circumvent early developmental barriers by disabling Igf1r in host embryos, conferring donor cells with a growth advantage from mid-gestation onward.
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Affiliation(s)
- Emily B Ballard
- 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.
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46
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Fráguas-Eggenschwiler M, Eggenschwiler R, Söllner JH, Cortnumme L, Vondran FWR, Cantz T, Ott M, Niemann H. Direct conversion of porcine primary fibroblasts into hepatocyte-like cells. Sci Rep 2021; 11:9334. [PMID: 33927320 PMCID: PMC8085017 DOI: 10.1038/s41598-021-88727-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Accepted: 04/12/2021] [Indexed: 01/01/2023] Open
Abstract
The pig is an important model organism for biomedical research, mainly due to its extensive genetic, physiological and anatomical similarities with humans. Until date, direct conversion of somatic cells into hepatocyte-like cells (iHeps) has only been achieved in rodents and human cells. Here, we employed lentiviral vectors to screen a panel of 12 hepatic transcription factors (TF) for their potential to convert porcine fibroblasts into hepatocyte-like cells. We demonstrate for the first time, hepatic conversion of porcine somatic cells by over-expression of CEBPα, FOXA1 and HNF4α2 (3TF-piHeps). Reprogrammed 3TF-piHeps display a hepatocyte-like morphology and show functional characteristics of hepatic cells, including albumin secretion, Dil-AcLDL uptake, storage of lipids and glycogen and activity of cytochrome P450 enzymes CYP1A2 and CYP2C33 (CYP2C9 in humans). Moreover, we show that markers of mature hepatocytes are highly expressed in 3TF-piHeps, while fibroblastic markers are reduced. We envision piHeps as useful cell sources for future studies on drug metabolism and toxicity as well as in vitro models for investigation of pig-to-human infectious diseases.
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Affiliation(s)
- Mariane Fráguas-Eggenschwiler
- Gastroenterology, Hepatology and Endocrinology Department, Hannover Medical School, Hannover, Germany. .,Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany.
| | - Reto Eggenschwiler
- Gastroenterology, Hepatology and Endocrinology Department, Hannover Medical School, Hannover, Germany.,Translational Hepatology and Stem Cell Biology, REBIRTH - Research Center for Translational Regenerative Medicine and Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Jenny-Helena Söllner
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Mariensee, Neustadt, Germany
| | - Leon Cortnumme
- Translational Hepatology and Stem Cell Biology, REBIRTH - Research Center for Translational Regenerative Medicine and Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Florian W R Vondran
- Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany.,German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Tübingen, Germany
| | - Tobias Cantz
- Gastroenterology, Hepatology and Endocrinology Department, Hannover Medical School, Hannover, Germany.,Translational Hepatology and Stem Cell Biology, REBIRTH - Research Center for Translational Regenerative Medicine and Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Michael Ott
- Gastroenterology, Hepatology and Endocrinology Department, Hannover Medical School, Hannover, Germany.,Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Heiner Niemann
- Gastroenterology, Hepatology and Endocrinology Department, Hannover Medical School, Hannover, Germany. .,Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany.
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47
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Alle Q, Le Borgne E, Milhavet O, Lemaitre JM. Reprogramming: Emerging Strategies to Rejuvenate Aging Cells and Tissues. Int J Mol Sci 2021; 22:3990. [PMID: 33924362 PMCID: PMC8070588 DOI: 10.3390/ijms22083990] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 04/06/2021] [Accepted: 04/06/2021] [Indexed: 12/12/2022] Open
Abstract
Aging is associated with a progressive and functional decline of all tissues and a striking increase in many "age-related diseases". Although aging has long been considered an inevitable process, strategies to delay and potentially even reverse the aging process have recently been developed. Here, we review emerging rejuvenation strategies that are based on reprogramming toward pluripotency. Some of these approaches may eventually lead to medical applications to improve healthspan and longevity.
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Affiliation(s)
- Quentin Alle
- IRMB, University of Montpellier, INSERM, 34295 Montpellier, France; (Q.A.); (E.L.B.)
| | - Enora Le Borgne
- IRMB, University of Montpellier, INSERM, 34295 Montpellier, France; (Q.A.); (E.L.B.)
| | - Ollivier Milhavet
- IRMB, University of Montpellier, INSERM, CNRS, 34295 Montpellier, France
| | - Jean-Marc Lemaitre
- IRMB, University of Montpellier, INSERM, 34295 Montpellier, France; (Q.A.); (E.L.B.)
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48
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Osafune K. Regenerative treatments for kidney diseases: The closest and fastest strategies to solving related medical and economic problems. Artif Organs 2021; 45:447-453. [PMID: 33590913 DOI: 10.1111/aor.13943] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 01/19/2021] [Accepted: 02/11/2021] [Indexed: 01/15/2023]
Abstract
Recent advances in developmental biology and stem cell biology have led to the increased availability of extrarenal stem cells, including mesenchymal/stromal stem cells (MSCs), renal stem or progenitor cells isolated from embryonic and adult kidneys, and kidney lineage cells or tissues generated from human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human-induced pluripotent stem cells. Regenerative medicine strategies for kidney diseases are largely categorized into the transplantation of reconstructed kidney organs and cell therapies. Reconstruction is being attempted by hPSC-derived kidney lineage cells with various strategies, such as self-organization, interspecies blastocyst complementation, utilization of a xenogeneic organ niche, decellularization and repopulation, and 3D bioprinting. However, cell therapies using extrarenal stem cells, such as MSCs, and renal stem or progenitor cells derived from embryonic and adult kidneys or differentiated from hPSCs have been investigated in animal models of both acute kidney injury and chronic kidney disease. Indeed, multiple clinical trials using MSCs, bone marrow stem cells, and kidney-derived cells have already been carried out. This review summarizes the current status and future perspective of kidney regenerative medicine strategies and discusses the closest and fastest strategies to solving the medical and economic problems associated with kidney diseases.
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Affiliation(s)
- Kenji Osafune
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
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49
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Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats. Nat Commun 2021; 12:1328. [PMID: 33637711 PMCID: PMC7910474 DOI: 10.1038/s41467-021-21557-x] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Accepted: 01/29/2021] [Indexed: 02/06/2023] Open
Abstract
Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.
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50
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Steevens AR, Griesbach MW, You Y, Dutton JR, Low WC, Santi PA. Generation of inner ear sensory neurons using blastocyst complementation in a Neurog1 +/--deficient mouse. Stem Cell Res Ther 2021; 12:122. [PMID: 33579352 PMCID: PMC7881691 DOI: 10.1186/s13287-021-02184-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Accepted: 01/24/2021] [Indexed: 11/10/2022] Open
Abstract
This research is the first to produce induced pluripotent stem cell-derived inner ear sensory neurons in the Neurog1+/− heterozygote mouse using blastocyst complementation. Additionally, this approach corrected non-sensory deficits associated with Neurog1 heterozygosity, indicating that complementation is specific to endogenous Neurog1 function. This work validates the use of blastocyst complementation as a tool to create novel insight into the function of developmental genes and highlights blastocyst complementation as a potential platform for generating chimeric inner ear cell types that can be transplanted into damaged inner ears to improve hearing.
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Affiliation(s)
- Aleta R Steevens
- Department of Ophthalmology, University of Minnesota, Minneapolis, MN, USA. .,Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA. .,Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA.
| | | | - Yun You
- Mouse Genetics Laboratory, University of Minnesota, Minneapolis, MN, USA
| | - James R Dutton
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Walter C Low
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA.,Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Peter A Santi
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA.,Department of Otolaryngology, University of Minnesota, Minneapolis, MN, USA
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