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Prakash N, Cha Y, Koh WG, Park H, Bello AB, Lee SH. Derivation of Mesenchymal Stem Cells through Sequential Presentation of Growth Factors via Gelatin Microparticles in Pluripotent Stem Cell Spheroids. Biomater Res 2025; 29:0184. [PMID: 40303481 PMCID: PMC12038162 DOI: 10.34133/bmr.0184] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2024] [Revised: 03/10/2025] [Accepted: 03/15/2025] [Indexed: 05/02/2025] Open
Abstract
The use of mesenchymal stem cells (MSCs) in regenerative medicine has gained considerable attention in recent years with the development of clinically relevant MSCs from induced pluripotent stem cells (iPSCs) and embryonic stem cells. Through sequential presentations of appropriate growth factors (GFs), iPSCs can be differentiated into mesodermal cells and then into MSCs. Furthermore, the formation of 3-dimensional cell spheroids, known as embryoid bodies, can be used to mimic in vivo conditions. However, the compact nature of embryoid bodies restricts the efficient and uniform delivery of GFs, leading to the formation of necrotic zones and hindered differentiation. To address this, we developed 2 types of gelatin microparticles (GelMPs) with distinct degradation rates for sequential delivery of GFs to enhance differentiation while preventing necrotic zones. In 2-dimensional culture, bone morphogenetic protein-4 (BMP4) and fibroblast growth factor 2 (FGF2) were identified as key proteins inducing iPSC differentiation into mesodermal cells and MSCs. The sequential presentation of these GFs was optimized for a 3-dimensional culture system by engineering fast-degrading GelMPs conjugated with BMP4 and slow-degrading GelMPs conjugated with FGF2. Our approach facilitated efficient iPSC differentiation into induced mesenchymal stem cells (iMSCs), as demonstrated by enhanced expression of mesodermal markers during the early stages of differentiation and MSC-specific markers at later stages. The resulting iMSCs exhibited characteristic surface markers (e.g., CD73, CD90, CD105, and CD44) and trilineage differentiation capability and were genetically stable. Compared to adult-derived MSCs, iMSCs showed superior proliferative capacity and reduced senescence, making them advantageous for cell therapy and regenerative medicine. This innovative approach of generating iMSCs has vast potential for therapeutic applications.
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Affiliation(s)
- Nityanand Prakash
- Department of Biomedical Engineering,
Dongguk University, Seoul 04620, Republic of Korea
| | - Young Cha
- Molecular Neurobiology Laboratory, McLean Hospital and Department of Psychiatry,
Harvard Medical School, Belmont, MA 02478, USA
| | - Won-Gun Koh
- Department of Chemical and Biomolecular Engineering,
Yonsei University, Seoul 03722, Republic of Korea
| | - Hansoo Park
- School of Integrative Engineering,
Chung-Ang University, Seoul 06911, Republic of Korea
| | - Alvin Bacero Bello
- Department of Biomedical Engineering,
Dongguk University, Seoul 04620, Republic of Korea
| | - Soo-Hong Lee
- Department of Biomedical Engineering,
Dongguk University, Seoul 04620, Republic of Korea
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Mulaudzi PE, Abrahamse H, Crous A. Impact of photobiomodulation on neural embryoid body formation from immortalized adipose-derived stem cells. Stem Cell Res Ther 2024; 15:489. [PMID: 39707453 PMCID: PMC11662703 DOI: 10.1186/s13287-024-04088-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2024] [Accepted: 12/03/2024] [Indexed: 12/23/2024] Open
Abstract
BACKGROUND Embryoid bodies (EBs) are three-dimensional (3D) multicellular cell aggregates that are derived from stem cell and play a pivotal role in regenerative medicine. They recapitulate many crucial aspects of the early stages of embryonic development and is the first step in the generation of various types of stem cells, including neuronal stem cells. Current methodologies for differentiating stem cells into neural embryoid bodies (NEBs) in vitro have advanced significantly, but they still have limitations which necessitate improvement. Photobiomodulation (PBM) a low powered light therapy is a non-invasive technique shown to promote stem cell proliferation and differentiation. METHODS This in vitro study elucidated the effects of photobiomodulation (PBM) on the differentiation of immortalized adipose-derived stem cells (iADSCs) into NEBs within a 3D cell culture environment. The study utilized PBM at wavelengths of 825 nm, 525 nm, and a combination of both, with fluences of 5 and 10 J/cm2. Morphology, viability, metabolic activity, and differentiation following PBM treatment was analysed. RESULTS The results revealed that the effects of photobiomodulation (PBM) are dose dependent. PBM, at 825 nm with a fluence of 10 J/cm2, significantly enhanced the size of neural embryoid bodies (NEBs), improved cell viability and proliferation, and reduced lactate dehydrogenase (LDH) levels, indicating minimal cell damage. Interestingly, the stem cell marker CD 44 was upregulated at 5 J/cm2 in all treatment groups at 24 and 96 hpi, CD105 increased with 825 nm at 10 J/cm2 at 24 hpi, which may be attributed to a heterogeneous cell population within the NEBs. Pax6 expression showed transient activation. Nestin was upregulated at 825 nm with 10 J/cm2 at 96 hpi, suggesting a promotion of neural precursor populations. GFAP an intermediate filament protein was upregulated at 825 nm at 10 J/cm2 at both 24 and 96 hpi. SOX2, a pluripotency marker, was expressed at 5 J/cm2 across all wavelengths. Neu N a neuronal nuclei marker was expressed at 5 J/cm2 in all treatments at 24 hpi and over time the expression was observed in all treatment groups at 10 J/cm2. CONCLUSION In conclusion, the application of PBM at 825 nm with a fluence of 10 J/cm2 during the differentiation of iADSCs into NEBs resulted in optimal differentiation. Notably, the neuronal marker Nestin was significantly upregulated, highlighting the potential of the PBM approach for enhancing neuronal differentiation its promising applications in regenerative medicine.
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Affiliation(s)
- Precious Earldom Mulaudzi
- Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein, 2028, South Africa
| | - Heidi Abrahamse
- Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein, 2028, South Africa
| | - Anine Crous
- Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein, 2028, South Africa.
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Komosa ER, Lin WH, Ogle BM. Toward robust and reproducible pluripotent stem cell expansion in bioprinted GelMA constructs. Int J Bioprint 2024; 11:363-381. [PMID: 40330989 PMCID: PMC12052315 DOI: 10.36922/ijb.4633] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/08/2025] Open
Abstract
Combining the technologies of 3D bioprinting and human induced pluripotent stem cells (hiPSCs) has allowed for the creation of tissues with organ-level function in the lab, a promising technique for disease modeling and regenerative medicine. Expanding the stem cells in bioprinted tissues prior to differentiation allows for high cell density, which is important for the formation of cell-cell junctions necessary for macroscale function upon differentiation. Yet, stem cell expansion, critical to successful in situ differentiation, depends heavily on the composition of the bioprinted scaffold. Here, we demonstrate how a common bioink component, gelatin methacryloyl (GelMA), varies depending on the vendor and degree of functionalization. We found that the vendor/GelMA production technique played a greater role in dictating the mechanical properties of the bioprinted constructs than the degree of functionalization, emphasizing the importance of reporting detailed characterization of GelMA scaffolds. Furthermore, the ability of singularized hiPSCs to survive and expand in GelMA scaffolds greatly varied across batches from different vendors and degrees of functionalization, where expansion correlated with the mechanical properties of the scaffold. Yet, we found that using a commercial cloning supplement could restore the ability of single hiPSCs to survive and expand across GelMA types, thus compensating for the varied mechanical properties of the scaffolds. These findings provide a practical guide for the expansion of hiPSCs in GelMA constructs with various mechanical properties as required for successful in situ differentiation.
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Affiliation(s)
- Elizabeth R. Komosa
- Department of Biomedical Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America
- Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, United States of America
| | - Wei-Han Lin
- Department of Biomedical Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America
- Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, United States of America
| | - Brenda M. Ogle
- Department of Biomedical Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America
- Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, United States of America
- Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota, United States of America
- Department of Pediatrics, Medical School, University of Minnesota, Minneapolis, Minnesota, United States of America
- Institute for Engineering in Medicine, University of Minnesota, Minneapolis, Minnesota, United States of America
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America
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Treschow AF, Vinggaard AM, Valente MJ. Standardization and optimization of the hiPSC-based PluriLum assay for detection of embryonic and developmental toxicants. Arch Toxicol 2024; 98:4107-4116. [PMID: 39365317 PMCID: PMC11496362 DOI: 10.1007/s00204-024-03870-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Accepted: 09/10/2024] [Indexed: 10/05/2024]
Abstract
New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data, while reducing turnover time and costs, and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay, a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes, we sought to introduce standardization steps to the protocol, improving the overall robustness of the PluriLum assay, as well as a shortening of the assay protocol. First, we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm, robust differentiation can be anticipated. In terms of reproducibility, exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data, compared to more reliable concentration-response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol, resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid, both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally, we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter, which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion, we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing.
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Affiliation(s)
- Andreas Frederik Treschow
- Cell Toxicology Team, National Food Institute, Technical University of Denmark, Kemitorvet B204, 2800 Kgs, Lyngby, Denmark.
| | - Anne Marie Vinggaard
- Cell Toxicology Team, National Food Institute, Technical University of Denmark, Kemitorvet B204, 2800 Kgs, Lyngby, Denmark
| | - Maria João Valente
- Cell Toxicology Team, National Food Institute, Technical University of Denmark, Kemitorvet B204, 2800 Kgs, Lyngby, Denmark
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Da Silva André G, Labouesse C. Mechanobiology of 3D cell confinement and extracellular crowding. Biophys Rev 2024; 16:833-849. [PMID: 39830117 PMCID: PMC11735831 DOI: 10.1007/s12551-024-01244-z] [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] [Subscribe] [Scholar Register] [Received: 07/27/2024] [Accepted: 09/30/2024] [Indexed: 01/22/2025] Open
Abstract
Cells and tissues are often under some level of confinement, imposed by the microenvironment and neighboring cells, meaning that there are limitations to cell size, volume changes, and fluid exchanges. 3D cell culture, increasingly used for both single cells and organoids, inherently impose levels of confinement absent in 2D systems. It is thus key to understand how different levels of confinement influences cell survival, cell function, and cell fate. It is well known that the mechanical properties of the microenvironment, such as stiffness and stress relaxation, are important in activating mechanosensitive pathways, and these are responsive to confinement conditions. In this review, we look at how low, intermediate, and high levels of confinement modulate the activation of known mechanobiology pathways, in single cells, organoids, and tumor spheroids, with a specific focus on 3D confinement in microwells, elastic, or viscoelastic scaffolds. In addition, a confining microenvironment can drastically limit cellular communication in both healthy and diseased tissues, due to extracellular crowding. We discuss potential implications of extracellular crowding on molecular transport, extracellular matrix deposition, and fluid transport. Understanding how cells sense and respond to various levels of confinement should inform the design of 3D engineered matrices that recapitulate the physical properties of tissues.
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Affiliation(s)
- Gabriela Da Silva André
- Macromolecular Engineering Laboratory, Department of Mechanical and Process Engineering, ETH Zurich, 8092 Zurich, Switzerland
| | - Céline Labouesse
- Macromolecular Engineering Laboratory, Department of Mechanical and Process Engineering, ETH Zurich, 8092 Zurich, Switzerland
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Cui LH, Noh JM, Kim DH, Seo HR, Joo HJ, Choi SC, Song MH, Kim KS, Huang LH, Na JE, Rhyu IJ, Qu XK, Lee KB, Lim DS. Nanotopography promotes cardiogenesis of pluripotent stem cell-derived embryoid bodies through focal adhesion kinase signaling. Biochem Biophys Res Commun 2024; 735:150796. [PMID: 39427377 DOI: 10.1016/j.bbrc.2024.150796] [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: 07/18/2024] [Revised: 09/20/2024] [Accepted: 10/07/2024] [Indexed: 10/22/2024]
Abstract
Controlling the microenvironment surrounding the pluripotent stem cells (PSCs) is a pivotal strategy for regulating cellular differentiation. Surface nanotopography is one of the key factors influencing the lineage-specific differentiation of PSCs. However, much of the underlying mechanism remains unknown. In this study, we focused on the effects of gradient nanotopography on the differentiation of embryoid bodies (EBs). EBs were cultured on three differently sized nanopillar surfaces (Large, 280-360; Medium, 200-280; Small, 120-200 nm) for spontaneous cardiomyocyte differentiation without chemical stimuli. The large nanotopography significantly promoted cardiogenesis, with increased expression of cardiac markers such as α-MHC, cTnT, and cTnI, and redistributed vinculin expression to the contact area. In addition, the small and medium nanotopographies also influenced EB differentiation, affecting both cardiogenesis and hematopoiesis to varying degrees. The phosphorylation of focal adhesion kinase (FAK) decreased in the EBs on the large nanotopography compared to that in the EBs cultured on the flat surface. The gradient nanotopography with 280-360 nm nanopillars is beneficial for the cardiogenesis of EBs in a FAK-dependent manner. This study provides valuable insights into controlling stem cell differentiation through nanotopographical cues, thereby advancing our understanding of the microenvironmental regulation in stem cell-based cardiac tissue engineering.
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Affiliation(s)
- Long-Hui Cui
- Department of Cardiology, Huadong Hospital Affiliated to Fudan University, Shanghai, China
| | - Ji-Min Noh
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea
| | - Dae Hwan Kim
- Department of Biomedical Engineering, College of Health Science, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea; BK21 Four R&E Center for Precision Public Health, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea
| | - Ha-Rim Seo
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea; Division of Drug Efficacy Evaluation, New Drug Development Center, Osong Medical Innovation Foundation, 123 Osongsaengmyeong-ro, Osong-eup, Heungdeok-gu, Cheonju-si, 28160, South Korea
| | - Hyung Joon Joo
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea
| | - Seung-Cheol Choi
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea; R&D Center for Companion Diagnosis, SOL Bio Corporation, Suite 510, 27, Seongsui-ro7-gil, Seongdong-gu, Seoul, 04780, South Korea
| | - Myeong-Hwa Song
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea
| | - Kyung-Seob Kim
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea
| | - Li-Hua Huang
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea
| | - Ji Eun Na
- Department of Anatomy, College of Medicine, Korea University, Seoul, 02841, South Korea
| | - Im Joo Rhyu
- Department of Anatomy, College of Medicine, Korea University, Seoul, 02841, South Korea
| | - Xin-Kai Qu
- Department of Cardiology, Huadong Hospital Affiliated to Fudan University, Shanghai, China.
| | - Kyu Back Lee
- Department of Biomedical Engineering, College of Health Science, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea.
| | - Do-Sun Lim
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, South Korea.
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Hyams NA, Kerr CM, Arhontoulis DC, Ruddy JM, Mei Y. Improving human cardiac organoid design using transcriptomics. Sci Rep 2024; 14:20147. [PMID: 39209865 PMCID: PMC11362591 DOI: 10.1038/s41598-024-61554-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2024] [Accepted: 05/07/2024] [Indexed: 09/04/2024] Open
Abstract
Cardiovascular disease (CVD) is the leading cause of death worldwide. To this end, human cardiac organoids (hCOs) have been developed for improved organotypic CVD modeling over conventional in vivo animal models. Utilizing human cells, hCOs hold great promise to bridge key gaps in CVD research pertaining to human-specific conditions. hCOs are multicellular 3D models which resemble heart structure and function. Varying hCOs fabrication techniques leads to functional and phenotypic differences. To investigate heterogeneity across hCO platforms, we performed a transcriptomic analysis utilizing bulk RNA-sequencing from four previously published unique hCO studies. We further compared selected hCOs to 2D and 3D hiPSC-derived cardiomyocytes (hiPSC-CMs), as well as fetal and adult human myocardium bulk RNA-sequencing samples. Upon investigation utilizing Principal Component Analysis, K-means clustering analysis of key genes, and further downstream analyses such as Gene Set Enrichment (GSEA), Gene Set Variation (GSVA), and GO term enrichment, we found that hCO fabrication method influences maturity and cellular heterogeneity across models. Thus, we propose that adjustment of fabrication method will result in an hCO with a defined maturity and transcriptomic profile to facilitate its specified applications, in turn maximizing its modeling potential.
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Affiliation(s)
- Nathaniel A Hyams
- Bioengineering Department, Clemson University, Clemson, SC, 29631, USA
| | - Charles M Kerr
- Molecular and Cellular Biology and Pathobiology Program, Medical University of South Carolina, Charleston, SC, 29425, USA
| | - Dimitrios C Arhontoulis
- Molecular and Cellular Biology and Pathobiology Program, Medical University of South Carolina, Charleston, SC, 29425, USA
| | - Jean Marie Ruddy
- Division of Vascular Surgery, Department of Surgery, Medical University of South Carolina, Charleston, SC, 29425, USA
| | - Ying Mei
- Bioengineering Department, Clemson University, Clemson, SC, 29631, USA.
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, 29425, USA.
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Bello AB, Canlas KKV, Kim D, Park H, Lee SH. Stepwise dual-release microparticles of BMP-4 and SCF in induced pluripotent stem cell spheroids enhance differentiation into hematopoietic stem cells. J Control Release 2024; 371:386-405. [PMID: 38844177 DOI: 10.1016/j.jconrel.2024.06.011] [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: 12/26/2023] [Revised: 06/03/2024] [Accepted: 06/03/2024] [Indexed: 06/11/2024]
Abstract
Recently, the formation of three-dimensional (3D) cell aggregates known as embryoid bodies (EBs) grown in media supplemented with HSC-specific morphogens has been utilized for the directed differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into clinically relevant hematopoietic stem cells (HSCs). However, delivering growth factors and nutrients have become ineffective in inducing synchronous differentiation of cells due to their 3D conformation. Moreover, irregularly sized EBs often lead to the formation of necrotic cores in larger EBs, impairing differentiation. Here, we developed two gelatin microparticles (GelMPs) with different release patterns and two HSC-related growth factors conjugated to them. Slow and fast releasing GelMPs were conjugated with bone morphogenic factor-4 (BMP-4) and stem cell factor (SCF), respectively. The sequential presentation of BMP-4 and SCF in GelMPs resulted in efficient and effective hematopoietic differentiation, shown by the enhanced gene and protein expression of several mesoderm and HSC-related markers, and the increased concentration of released HSC-related cytokines. In the present study, we were able to generate CD34+, CD133+, and FLT3+ cells with similar cellular and molecular morphology as the naïve HSCs that can produce colony units of different blood cells, in vitro.
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Affiliation(s)
- Alvin Bacero Bello
- Department of Biomedical Engineering, Dongguk University, Seoul 04620, Republic of Korea; School of Integrative Engineering, Chung-Ang University, Seoul 06911, Republic of Korea
| | | | - Deogil Kim
- Department of Biomedical Engineering, Dongguk University, Seoul 04620, Republic of Korea
| | - Hansoo Park
- School of Integrative Engineering, Chung-Ang University, Seoul 06911, Republic of Korea.
| | - Soo-Hong Lee
- Department of Biomedical Engineering, Dongguk University, Seoul 04620, Republic of Korea.
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Chen J, Kataoka O, Tsuchiya K, Oishi Y, Takao A, Huang YC, Komura H, Akiyama S, Itou R, Inui M, Enosawa S, Akutsu H, Komura M, Fuchimoto Y, Umezawa A. Automated xeno-free chondrogenic differentiation from human embryonic stem cells: Enhancing efficiency and ensuring high-quality mass production. Regen Ther 2024; 26:889-900. [PMID: 39822341 PMCID: PMC11735927 DOI: 10.1016/j.reth.2024.09.007] [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: 05/26/2024] [Revised: 08/22/2024] [Accepted: 09/23/2024] [Indexed: 01/19/2025] Open
Abstract
Introduction Repairing damaged cartilage poses significant challenges, particularly in cases of congenital cartilage defects such as microtia or congenital tracheal stenosis, or as a consequence of traumatic injury, as the regenerative potential of cartilage is inherently limited. Stem cell therapy and tissue engineering offer promising approaches to overcome these limitations in cartilage healing. However, the challenge lies in the size of cartilage-containing organs, which necessitates a large quantity of cells to fill the damaged areas. Therefore, pluripotent stem cells that can proliferate indefinitely are highly desirable as a cell source. This study aims to delineate the differentiation conditions for cartilage derived from human embryonic stem cells (ESCs) and to develop an automated cell culture system to facilitate mass production for therapeutic applications. Methods Cartilage cell sheets were derived from human ESCs (SEES2, clinical trial-compatible line) by forming embryoid bodies (EBs) with either conventional manual culture or a benchtop multi-pipetter and an automated medium exchange integrated cell incubator, using xeno-free media. Cell sheets were implanted into the subcutaneous tissue of immunodeficient NOG mice to obtain cartilage tissue. The properties of cartilage tissues were examined by histological staining and quantitative PCR analysis. Results We have optimized an efficient xeno-free system for cartilage production with the conventional culture method and successfully transitioned to an automated system. Differentiated cartilage was histologically uniform with cartilage-specific elasticity and strength. The cartilage tissues were stained by Alcian blue, safranin O, and toluidine blue, and quantitative PCR showed an increase in differentiation markers such as ACAN, COL2A1, and Vimentin. Automation significantly enhanced the efficiency of human ESC-derived chondrocyte differentiation. The number of constituent cells within EBs and the seeding density of EBs were identified as key factors influencing chondrogenic differentiation efficiency. By automating the process of chondrogenic differentiation, we achieved scalable production of chondrocytes. Conclusions By integrating the differentiation protocol with an automated cell culture system, there is potential to produce cartilage of sufficient size for clinical applications in humans. The resulting cartilage tissue holds promise for clinical use in repairing organs such as the trachea, joints, ears, and nose.
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Affiliation(s)
- JunLong Chen
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Department of Advanced Pediatric Medicine, Tohoku University School of Medicine, Sendai, Japan
- Division of Tissue Engineering, The University of Tokyo Hospital, Japan
| | - Oki Kataoka
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa, Japan
| | - Kazeto Tsuchiya
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Yoshie Oishi
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Ayumi Takao
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa, Japan
| | - Yen-Chih Huang
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Division of Tissue Engineering, The University of Tokyo Hospital, Japan
- Department of Tissue Stem Cell＆Dental Life Science, Graduate School of Medicine, The University of Tokyo, Japan
- Oral and Maxillofacial Surgery, Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Japan
| | - Hiroko Komura
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Division of Tissue Engineering, The University of Tokyo Hospital, Japan
- Department of Tissue Stem Cell＆Dental Life Science, Graduate School of Medicine, The University of Tokyo, Japan
| | - Saeko Akiyama
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Department of Advanced Pediatric Medicine, Tohoku University School of Medicine, Sendai, Japan
| | - Ren Itou
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa, Japan
| | - Masafumi Inui
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa, Japan
| | - Shin Enosawa
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Hidenori Akutsu
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
| | - Makoto Komura
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Division of Tissue Engineering, The University of Tokyo Hospital, Japan
- Department of Tissue Stem Cell＆Dental Life Science, Graduate School of Medicine, The University of Tokyo, Japan
| | - Yasushi Fuchimoto
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Department of Pediatric Surgery, International University of Health and Welfare School of Medicine, 852, Chiba, Japan
| | - Akihiro Umezawa
- Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, Tokyo, Japan
- Department of Advanced Pediatric Medicine, Tohoku University School of Medicine, Sendai, Japan
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Giolito MV, Bodoirat S, La Rosa T, Reslinger M, Guardia GDA, Mourtada J, Claret L, Joung A, Galante PAF, Penalva LOF, Plateroti M. Impact of the thyroid hormone T3 and its nuclear receptor TRα1 on colon cancer stem cell phenotypes and response to chemotherapies. Cell Death Dis 2024; 15:306. [PMID: 38693105 PMCID: PMC11063186 DOI: 10.1038/s41419-024-06690-x] [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: 11/12/2023] [Revised: 04/14/2024] [Accepted: 04/17/2024] [Indexed: 05/03/2024]
Abstract
Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com .
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MESH Headings
- Humans
- Neoplastic Stem Cells/metabolism
- Neoplastic Stem Cells/drug effects
- Neoplastic Stem Cells/pathology
- Fluorouracil/pharmacology
- Fluorouracil/therapeutic use
- Thyroid Hormone Receptors alpha/metabolism
- Thyroid Hormone Receptors alpha/genetics
- Caco-2 Cells
- Colonic Neoplasms/metabolism
- Colonic Neoplasms/drug therapy
- Colonic Neoplasms/pathology
- Colonic Neoplasms/genetics
- Spheroids, Cellular/drug effects
- Spheroids, Cellular/metabolism
- Spheroids, Cellular/pathology
- Triiodothyronine/pharmacology
- Leucovorin/pharmacology
- Leucovorin/therapeutic use
- Camptothecin/pharmacology
- Camptothecin/analogs & derivatives
- Camptothecin/therapeutic use
- Phenotype
- Antineoplastic Combined Chemotherapy Protocols/pharmacology
- Antineoplastic Combined Chemotherapy Protocols/therapeutic use
- Aldehyde Dehydrogenase 1 Family/metabolism
- Aldehyde Dehydrogenase 1 Family/genetics
- Drug Resistance, Neoplasm/drug effects
- Drug Resistance, Neoplasm/genetics
- Retinal Dehydrogenase/metabolism
- Retinal Dehydrogenase/genetics
- ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism
- ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics
- ATP Binding Cassette Transporter, Subfamily B/metabolism
- ATP Binding Cassette Transporter, Subfamily B/genetics
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Affiliation(s)
- Maria Virginia Giolito
- Université de Strasbourg, INSERM, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France
- Pole of Pharmacology and Therapeutics (FATH), Institut de Recherche Experimentale et Clinique (IREC), UCLouvain, Avenue Hippocrate 57, B1.57.04, B-1200, Brussels, Belgium
| | - Serguei Bodoirat
- Université de Strasbourg, INSERM, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France
| | - Theo La Rosa
- Stem-Cell and Brain Research Institute, U1208 INSERM, USC1361 INRA, 69675, Bron, France
| | - Mathieu Reslinger
- Université de Strasbourg, INSERM, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France
- Université de Strasbourg, CNRS, INSERM, IGBMC UMR 7104-UMR-S 1258, Illkirch, France
| | | | - Jana Mourtada
- Université de Strasbourg, INSERM, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France
| | - Leo Claret
- Université de Strasbourg, INSERM, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France
- Université de Strasbourg, CNRS, INSERM, IGBMC UMR 7104-UMR-S 1258, Illkirch, France
| | - Alain Joung
- Université de Strasbourg, INSERM, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France
- Laboratoire de Biologie Tumorale, Institut de Cancérologie Strasbourg Europe, Strasbourg, France
| | - Pedro A F Galante
- Centro de Oncologia Molecular, Hospital Sírio-Libanês, São Paulo, Brazil
| | - Luiz O F Penalva
- Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
| | - Michelina Plateroti
- Université de Strasbourg, INSERM, IRFAC/UMR-S1113, FMTS, 67200, Strasbourg, France.
- Université de Strasbourg, CNRS, INSERM, IGBMC UMR 7104-UMR-S 1258, Illkirch, France.
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11
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Zhang X, Wan J, Huang T, Tang P, Yang L, Bu X, Zhang W, Zhong L. Rapid and accurate identification of stem cell differentiation stages via SERS and convolutional neural networks. BIOMEDICAL OPTICS EXPRESS 2024; 15:2753-2766. [PMID: 38855654 PMCID: PMC11161375 DOI: 10.1364/boe.519093] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/17/2024] [Revised: 03/22/2024] [Accepted: 03/25/2024] [Indexed: 06/11/2024]
Abstract
Monitoring the transition of cell states during induced pluripotent stem cell (iPSC) differentiation is crucial for clinical medicine and basic research. However, both identification category and prediction accuracy need further improvement. Here, we propose a method combining surface-enhanced Raman spectroscopy (SERS) with convolutional neural networks (CNN) to precisely identify and distinguish cell states during stem cell differentiation. First, mitochondria-targeted probes were synthesized by combining AuNRs and mitochondrial localization signal (MLS) peptides to obtain effective and stable SERS spectra signals at various stages of cell differentiation. Then, the SERS spectra served as input datasets, and their distinctive features were learned and distinguished by CNN. As a result, rapid and accurate identification of six different cell states, including the embryoid body (EB) stage, was successfully achieved throughout the stem cell differentiation process with an impressive prediction accuracy of 98.5%. Furthermore, the impact of different spectral feature peaks on the identification results was investigated, which provides a valuable reference for selecting appropriate spectral bands to identify cell states. This is also beneficial for shortening the spectral acquisition region to enhance spectral acquisition speed. These results suggest the potential for SERS-CNN models in quality monitoring of stem cells, advancing the practical applications of stem cells.
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Affiliation(s)
- Xiao Zhang
- Key Laboratory of Photonics Technology for Integrated Sensing and Communication of Ministry of Education, Guangdong University of Technology, Guangzhou 510006, China
| | - Jianhui Wan
- Key Laboratory of Photonics Technology for Integrated Sensing and Communication of Ministry of Education, Guangdong University of Technology, Guangzhou 510006, China
| | - Tao Huang
- Key Laboratory of Photonics Technology for Integrated Sensing and Communication of Ministry of Education, Guangdong University of Technology, Guangzhou 510006, China
| | - Ping Tang
- Key Laboratory of Photonics Technology for Integrated Sensing and Communication of Ministry of Education, Guangdong University of Technology, Guangzhou 510006, China
- School of Physics and Optoelectronic Engineering, Guangdong University of Technology, Guangzhou 510006, China
| | - Liwei Yang
- Guangdong Provincial Key Laboratory of Nanophotonic Functional Materials and Devices, South China Normal University, Guangzhou 510006, China
| | - Xiaoya Bu
- Guangdong Provincial Key Laboratory of Nanophotonic Functional Materials and Devices, South China Normal University, Guangzhou 510006, China
| | - Weina Zhang
- Key Laboratory of Photonics Technology for Integrated Sensing and Communication of Ministry of Education, Guangdong University of Technology, Guangzhou 510006, China
| | - Liyun Zhong
- Key Laboratory of Photonics Technology for Integrated Sensing and Communication of Ministry of Education, Guangdong University of Technology, Guangzhou 510006, China
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12
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Blümke A, Simon J, Leber E, Scatena M, Giachelli CM. Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells. J Vis Exp 2024:10.3791/66527. [PMID: 38587386 PMCID: PMC11108805 DOI: 10.3791/66527] [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: 04/09/2024] Open
Abstract
This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.
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Affiliation(s)
- Alexander Blümke
- Department of Bioengineering, Department of Medicine, University of Washington; Department of Orthopedics and Trauma Surgery, Medical Faculty Mannheim, Heidelberg University
| | - Jessica Simon
- Department of Bioengineering, Department of Medicine, University of Washington
| | - Elizabeth Leber
- Department of Bioengineering, Department of Medicine, University of Washington
| | - Marta Scatena
- Department of Bioengineering, Department of Medicine, University of Washington
| | - Cecilia M Giachelli
- Department of Bioengineering, Department of Medicine, University of Washington;
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13
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Wu X, Zhang B, Chen K, Zhao J, Li Y, Li J, Liu C, He L, Fan T, Wang C, Li Y, Pei X, Li Y. Baffled-flow culture system enables the mass production of megakaryocytes from human embryonic stem cells by enhancing mitochondrial function. Cell Prolif 2023; 56:e13484. [PMID: 37088551 PMCID: PMC10693187 DOI: 10.1111/cpr.13484] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2022] [Revised: 03/06/2023] [Accepted: 04/12/2023] [Indexed: 04/25/2023] Open
Abstract
Human embryonic stem cells (hESCs) have become an ideal cell source for the ex vivo generation of megakaryocyte (MK) and platelet products for clinical applications. However, an ongoing challenge is to establish scalable culture systems to maximize the yield of stem cell-derived MKs that release platelets. We defined a specific dynamic 3D manufacturing system in a baffled-flow manner that could remarkably facilitate megakaryopoiesis and increase the yield of platelet-producing MKs from hESCs within a 12-day induction period. Additionally, an increased number of >16N ploidy MKs, proplatelets, and platelets were generated from induced cells harvested on Day 12 using the specific dynamic culture method. The specific dynamic culture method significantly enhanced endothelium-to-haematopoietic transition and early haematopoiesis. More importantly, MK fate was significantly facilitated in a specific dynamic manner during early haematopoiesis. Mechanistically, this dynamic culture significantly enhanced mitochondrial function via the oxidative phosphorylation pathway and caused differentiation skewing of hESCs toward megakaryopoiesis. This study can aid in the automatic and scalable production of MKs from stem cells using baffled-flow bioreactors and assist in the manufacturing of hESC-derived MK and platelet products.
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Affiliation(s)
- Xumin Wu
- School of PharmacyGuizhou UniversityGuiyangChina
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
| | - Bowen Zhang
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
- South China Research Center for Stem Cell & Regenerative Medicine, SCIBGuangzhouChina
| | - Keyi Chen
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
- College of Chemistry and Environmental ScienceHebei UniversityBaodingChina
| | - Jiahui Zhao
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
- School of Life ScienceHebei UniversityBaodingChina
| | - Yunxing Li
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
| | - Jisheng Li
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
| | - Chuanli Liu
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
| | - Lijuan He
- South China Research Center for Stem Cell & Regenerative Medicine, SCIBGuangzhouChina
| | - Tao Fan
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
| | - Chao Wang
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
| | - Yan Li
- School of PharmacyGuizhou UniversityGuiyangChina
| | - Xuetao Pei
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
- South China Research Center for Stem Cell & Regenerative Medicine, SCIBGuangzhouChina
| | - Yanhua Li
- Stem Cell and Regenerative Medicine LabBeijing Institute of Radiation MedicineBeijingChina
- South China Research Center for Stem Cell & Regenerative Medicine, SCIBGuangzhouChina
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14
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Carido M, Völkner M, Steinheuer LM, Wagner F, Kurth T, Dumler N, Ulusoy S, Wieneke S, Norniella AV, Golfieri C, Khattak S, Schönfelder B, Scamozzi M, Zoschke K, Canzler S, Hackermüller J, Ader M, Karl MO. Reliability of human retina organoid generation from hiPSC-derived neuroepithelial cysts. Front Cell Neurosci 2023; 17:1166641. [PMID: 37868194 PMCID: PMC10587494 DOI: 10.3389/fncel.2023.1166641] [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: 02/15/2023] [Accepted: 09/18/2023] [Indexed: 10/24/2023] Open
Abstract
The possible applications for human retinal organoids (HROs) derived from human induced pluripotent stem cells (hiPSC) rely on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare, and optimize organoid protocols are starting to be established, but are not yet complete. To advance this, we explored the efficiency and reliability of a differentiation method, called CYST protocol, that facilitates retina generation by forming neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines which reproducibly generated HROs. Histological and ultrastructural analyses indicate that HRO differentiation and maturation are regulated. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Although previous reports have shown that HROs in several other protocols contain a rather low number of cones, HROs from the CYST protocol are consistently richer in cones and with a comparable ratio of cones, rods, and Müller glia. To provide further insight into HRO cell composition, we studied single cell RNA sequencing data and applied CaSTLe, a transfer learning approach. Additionally, we devised a potential strategy to systematically evaluate different organoid protocols side-by-side through parallel differentiation from the same hiPSC batches: In an explorative study, the CYST protocol was compared to a conceptually different protocol based on the formation of cell aggregates from single hiPSCs. Comparing four hiPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the CYST protocol provided a higher HRO yield. So far, our data suggest that CYST-derived HROs remained stable up to at least day 200, while single hiPSC-derived HROs showed spontaneous pathologic changes by day 200. Overall, our data provide insights into the efficiency, reproducibility, and stability of the CYST protocol for generating HROs, which will be useful for further optimizing organoid systems, as well as for basic and translational research applications.
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Affiliation(s)
- Madalena Carido
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
| | - Manuela Völkner
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
- German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany
| | - Lisa Maria Steinheuer
- Department Computational Biology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany
- Department of Computer Science, Leipzig University, Leipzig, Germany
| | - Felix Wagner
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
| | - Thomas Kurth
- Center for Molecular and Cellular Bioengineering (CMCB), Technology Platform, Core Facility Electron Microscopy and Histology, TU Dresden, Dresden, Germany
| | - Natalie Dumler
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
| | - Selen Ulusoy
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
| | - Stephanie Wieneke
- German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany
| | | | - Cristina Golfieri
- German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany
| | - Shahryar Khattak
- Center for Molecular and Cellular Bioengineering (CMCB), Stem Cell Engineering Facility, TU Dresden, Dresden, Germany
| | - Bruno Schönfelder
- German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany
| | - Maria Scamozzi
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
| | - Katja Zoschke
- German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany
| | - Sebastian Canzler
- Department Computational Biology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany
| | - Jörg Hackermüller
- Department Computational Biology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany
- Department of Computer Science, Leipzig University, Leipzig, Germany
| | - Marius Ader
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
| | - Mike O Karl
- Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany
- German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany
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15
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Noh JM, Choi SC, Song MH, Kim KS, Jun S, Park JH, Kim JH, Kim K, Ko TH, Choi JI, Gim JA, Kim JH, Jang Y, Park Y, Na JE, Rhyu IJ, Lim DS. The Activation of the LIMK/Cofilin Signaling Pathway via Extracellular Matrix-Integrin Interactions Is Critical for the Generation of Mature and Vascularized Cardiac Organoids. Cells 2023; 12:2029. [PMID: 37626839 PMCID: PMC10453200 DOI: 10.3390/cells12162029] [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: 07/05/2023] [Revised: 08/02/2023] [Accepted: 08/08/2023] [Indexed: 08/27/2023] Open
Abstract
The generation of mature and vascularized human pluripotent stem cell-derived cardiac organoids (hPSC-COs) is necessary to ensure the validity of drug screening and disease modeling. This study investigates the effects of cellular aggregate (CA) stemness and self-organization on the generation of mature and vascularized hPSC-COs and elucidates the mechanisms underlying cardiac organoid (CO) maturation and vascularization. COs derived from 2-day-old CAs with high stemness (H-COs) and COs derived from 5-day-old CAs with low stemness (L-COs) were generated in a self-organized microenvironment via Wnt signaling induction. This study finds that H-COs exhibit ventricular, structural, metabolic, and functional cardiomyocyte maturation and vessel networks consisting of endothelial cells, smooth muscle cells, pericytes, and basement membranes compared to L-COs. Transcriptional profiling shows the upregulation of genes associated with cardiac maturation and vessel formation in H-COs compared with the genes in L-COs. Through experiments with LIMK inhibitors, the activation of ROCK-LIMK-pCofilin via ECM-integrin interactions leads to cardiomyocyte maturation and vessel formation in H-COs. Furthermore, the LIMK/Cofilin signaling pathway induces TGFβ/NODAL and PDGF pathway activation for the maturation and vascularization of H-COs. The study demonstrates for the first time that LIMK/Cofilin axis activation plays an important role in the generation of mature and vascularized COs.
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Affiliation(s)
- Ji-Min Noh
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
| | - Seung-Cheol Choi
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
- R&D Center for Companion Diagnostic, SOL Bio Corporation, Suite 510, 27, Seongsui-ro7-gil, Seongdong-gu, Seoul 04780, Republic of Korea
| | - Myeong-Hwa Song
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
| | - Kyung Seob Kim
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
| | - Seongmin Jun
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
| | - Jae Hyoung Park
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
| | - Ju Hyeon Kim
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
| | - Kyoungmi Kim
- Department of Physiology, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea;
| | - Tae Hee Ko
- Division of Cardiology, Department of Internal Medicine, Anam Hospital, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (T.H.K.); (J.-I.C.)
| | - Jong-Il Choi
- Division of Cardiology, Department of Internal Medicine, Anam Hospital, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (T.H.K.); (J.-I.C.)
| | - Jeong-An Gim
- Medical Science Research Center, Korea University Guro Hospital, 148, Gurodong-ro, Guro-gu, Seoul 08308, Republic of Korea;
| | - Jong-Hoon Kim
- Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea;
| | - Yongjun Jang
- Department of Biomedical Sciences, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (Y.J.); (Y.P.)
| | - Yongdoo Park
- Department of Biomedical Sciences, College of Medicine, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (Y.J.); (Y.P.)
| | - Ji Eun Na
- Department of Anatomy College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.E.N.); (I.J.R.)
| | - Im Joo Rhyu
- Department of Anatomy College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.E.N.); (I.J.R.)
| | - Do-Sun Lim
- Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, 73, Goryeodae-ro, Seongbuk-gu, Seoul 02841, Republic of Korea; (J.-M.N.); (S.-C.C.); (M.-H.S.); (K.S.K.); (S.J.); (J.H.P.); (J.H.K.)
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16
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Ietto G, Iori V, Gritti M, Inversini D, Costantino A, Izunza Barba S, Jiang ZG, Carcano G, Dalla Gasperina D, Pettinato G. Multicellular Liver Organoids: Generation and Importance of Diverse Specialized Cellular Components. Cells 2023; 12:1429. [PMID: 37408262 PMCID: PMC10217024 DOI: 10.3390/cells12101429] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 05/11/2023] [Accepted: 05/17/2023] [Indexed: 07/07/2023] Open
Abstract
Over 40,000 patients in the United States are estimated to suffer from end-stage liver disease and acute hepatic failure, for which liver transplantation is the only available therapy. Human primary hepatocytes (HPH) have not been employed as a therapeutic tool due to the difficulty in growing and expanding them in vitro, their sensitivity to cold temperatures, and tendency to dedifferentiate following two-dimensional culture. The differentiation of human-induced pluripotent stem cells (hiPSCs) into liver organoids (LO) has emerged as a potential alternative to orthotropic liver transplantation (OLT). However, several factors limit the efficiency of liver differentiation from hiPSCs, including a low proportion of differentiated cells capable of reaching a mature phenotype, the poor reproducibility of existing differentiation protocols, and insufficient long-term viability in vitro and in vivo. This review will analyze various methodologies being developed to improve hepatic differentiation from hiPSCs into liver organoids, paying particular attention to the use of endothelial cells as supportive cells for their further maturation. Here, we demonstrate why differentiated liver organoids can be used as a research tool for drug testing and disease modeling, or employed as a bridge for liver transplantation following liver failure.
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Affiliation(s)
- Giuseppe Ietto
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Valentina Iori
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Mattia Gritti
- Department of General Surgery, Humanitas Clinical and Research Center, Rozzano, 20089 Milan, Italy
| | - Davide Inversini
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Angelita Costantino
- Department of Drug and Health Sciences, University of Catania, 95124 Catania, Italy;
| | - Sofia Izunza Barba
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Z. Gordon Jiang
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Giulio Carcano
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Daniela Dalla Gasperina
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
- Department of Infectious Diseases, ASST-Sette Laghi, 21100 Varese, Italy
| | - Giuseppe Pettinato
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
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17
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Bahou WF, Marchenko N, Nesbitt NM. Metabolic Functions of Biliverdin IXβ Reductase in Redox-Regulated Hematopoietic Cell Fate. Antioxidants (Basel) 2023; 12:antiox12051058. [PMID: 37237924 DOI: 10.3390/antiox12051058] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Revised: 04/19/2023] [Accepted: 04/27/2023] [Indexed: 05/28/2023] Open
Abstract
Cytoprotective heme oxygenases derivatize heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin. Recent studies have implicated biliverdin IXβ reductase (BLVRB) in a redox-regulated mechanism of hematopoietic lineage fate restricted to megakaryocyte and erythroid development, a function distinct and non-overlapping from the BLVRA (biliverdin IXα reductase) homologue. In this review, we focus on recent progress in BLVRB biochemistry and genetics, highlighting human, murine, and cell-based studies that position BLVRB-regulated redox function (or ROS accumulation) as a developmentally tuned trigger that governs megakaryocyte/erythroid lineage fate arising from hematopoietic stem cells. BLVRB crystallographic and thermodynamic studies have elucidated critical determinants of substrate utilization, redox coupling and cytoprotection, and have established that inhibitors and substrates bind within the single-Rossmann fold. These advances provide unique opportunities for the development of BLVRB-selective redox inhibitors as novel cellular targets that retain potential for therapeutic applicability in hematopoietic (and other) disorders.
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Affiliation(s)
- Wadie F Bahou
- Department of Medicine, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA
| | - Natalia Marchenko
- Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA
| | - Natasha M Nesbitt
- Blood Cell Technologies, 25 Health Sciences Drive, Stony Brook, NY 11790, USA
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18
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Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment. Int J Mol Sci 2022; 23:ijms232415785. [PMID: 36555427 PMCID: PMC9779514 DOI: 10.3390/ijms232415785] [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: 10/23/2022] [Revised: 12/06/2022] [Accepted: 12/07/2022] [Indexed: 12/14/2022] Open
Abstract
Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.
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19
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Ho DLL, Lee S, Du J, Weiss JD, Tam T, Sinha S, Klinger D, Devine S, Hamfeldt A, Leng HT, Herrmann JE, He M, Fradkin LG, Tan TK, Standish D, Tomasello P, Traul D, Dianat N, Ladi R, Vicard Q, Katikireddy K, Skylar‐Scott MA. Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors. Adv Healthc Mater 2022; 11:e2201138. [PMID: 36314397 PMCID: PMC10234214 DOI: 10.1002/adhm.202201138] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Revised: 10/10/2022] [Indexed: 01/28/2023]
Abstract
Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.
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Affiliation(s)
- Debbie L. L. Ho
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Stacey Lee
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Jianyi Du
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | | | - Tony Tam
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Soham Sinha
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Danielle Klinger
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Sean Devine
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Art Hamfeldt
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Hope T. Leng
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Jessica E. Herrmann
- Department of BioengineeringStanford UniversityStanfordCA94305USA
- School of MedicineStanford UniversityStanfordCA94305USA
| | - Mengdi He
- Materials Science and EngineeringStanford UniversityStanfordCA94305USA
| | - Lee G. Fradkin
- Department of BioengineeringStanford UniversityStanfordCA94305USA
| | - Tze Kai Tan
- Institute of Stem Cell Biology and Regenerative MedicineStanford University School of MedicineStanfordCA94305USA
- Department of GeneticsStanford University School of MedicineStanfordCA94305USA
- Department of PathologyStanford University School of MedicineStanfordCA94305USA
| | - David Standish
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Peter Tomasello
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Donald Traul
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Noushin Dianat
- Sartorius Stedim France S.A.SZone Industrielle les PaludsAvenue de Jouques CS 71058Aubagne Cedex13781France
| | - Rukmini Ladi
- Sartorius Stedim North America Inc565 Johnson AvenueBohemiaNY11716USA
| | - Quentin Vicard
- Sartorius Stedim France S.A.SZone Industrielle les PaludsAvenue de Jouques CS 71058Aubagne Cedex13781France
| | | | - Mark A. Skylar‐Scott
- Department of BioengineeringStanford UniversityStanfordCA94305USA
- Basic Science and Engineering InitiativeChildren's Heart CenterStanford UniversityStanfordCA94305USA
- Chan Zuckerberg BiohubSan FranciscoCA94158USA
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20
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Abstract
An ensemble of in vitro cardiac tissue models has been developed over the past several decades to aid our understanding of complex cardiovascular disorders using a reductionist approach. These approaches often rely on recapitulating single or multiple clinically relevant end points in a dish indicative of the cardiac pathophysiology. The possibility to generate disease-relevant and patient-specific human induced pluripotent stem cells has further leveraged the utility of the cardiac models as screening tools at a large scale. To elucidate biological mechanisms in the cardiac models, it is critical to integrate physiological cues in form of biochemical, biophysical, and electromechanical stimuli to achieve desired tissue-like maturity for a robust phenotyping. Here, we review the latest advances in the directed stem cell differentiation approaches to derive a wide gamut of cardiovascular cell types, to allow customization in cardiac model systems, and to study diseased states in multiple cell types. We also highlight the recent progress in the development of several cardiovascular models, such as cardiac organoids, microtissues, engineered heart tissues, and microphysiological systems. We further expand our discussion on defining the context of use for the selection of currently available cardiac tissue models. Last, we discuss the limitations and challenges with the current state-of-the-art cardiac models and highlight future directions.
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Affiliation(s)
- Dilip Thomas
- Stanford Cardiovascular Institute
- Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305
| | - Suji Choi
- Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, John A. Paulson School of Engineering and Applied Sciences, Harvard University, Boston MA 02134
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115
| | - Christina Alamana
- Stanford Cardiovascular Institute
- Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305
| | - Kevin K. Parker
- Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, John A. Paulson School of Engineering and Applied Sciences, Harvard University, Boston MA 02134
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138
| | - Joseph C. Wu
- Stanford Cardiovascular Institute
- Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305
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21
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Afjeh-Dana E, Naserzadeh P, Moradi E, Hosseini N, Seifalian AM, Ashtari B. Stem Cell Differentiation into Cardiomyocytes: Current Methods and Emerging Approaches. Stem Cell Rev Rep 2022; 18:2566-2592. [PMID: 35508757 DOI: 10.1007/s12015-021-10280-1] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/05/2021] [Indexed: 12/26/2022]
Abstract
Cardiovascular diseases (CVDs) are globally known to be important causes of mortality and disabilities. Common treatment strategies for CVDs, such as pharmacological therapeutics impose serious challenges due to the failure of treatments for myocardial necrosis. By contrast, stem cells (SCs) based therapies are seen to be promising approaches to CVDs treatment. In such approaches, cardiomyocytes are differentiated from SCs. To fulfill SCs complete potential, the method should be appointed to generate cardiomyocytes with more mature structure and well-functioning operations. For heart repairing applications, a greatly scalable and medical-grade cardiomyocyte generation must be used. Nonetheless, there are some challenges such as immune rejection, arrhythmogenesis, tumorigenesis, and graft cell death potential. Herein, we discuss the types of potential SCs, and commonly used methods including embryoid bodies related techniques, co-culture, mechanical stimulation, and electrical stimulation and their applications, advantages and limitations in this field. An estimated 17.9 million people died from CVDs in 2019, representing 32 % of all global deaths. Of these deaths, 85 % were due to heart attack and stroke.
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Affiliation(s)
- Elham Afjeh-Dana
- Radiation Biology Research Centre, Iran University of Medical Sciences, Tehran, Iran
| | - Parvaneh Naserzadeh
- Radiation Biology Research Centre, Iran University of Medical Sciences, Tehran, Iran
| | - Elham Moradi
- Radiation Biology Research Centre, Iran University of Medical Sciences, Tehran, Iran.,Endocrine Research Center, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, Iran
| | - Nasrin Hosseini
- Neuroscience Research Centre, Iran University of Medical Sciences, Tehran, Iran.
| | - Alexander Marcus Seifalian
- Nanotechnology & Regenerative Medicine Commercialisation Centre (NanoRegMed Ltd), London BioScience Innovation Centre, London, UK
| | - Behnaz Ashtari
- Radiation Biology Research Centre, Iran University of Medical Sciences, Tehran, Iran. .,Endocrine Research Center, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, Iran. .,Cellular and Molecular Research Centre, Iran University of Medical Sciences, Tehran, Iran.
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22
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Oliveira NA, Sevim H. Dendritic cell differentiation from human induced pluripotent stem cells: challenges and progress. Stem Cells Dev 2022; 31:207-220. [PMID: 35316109 DOI: 10.1089/scd.2021.0305] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Dendritic cells (DCs) are the major antigen-presenting cells of the immune system responsible for initiating and coordinating immune responses. These abilities provide potential for several clinical applications, such as the development of immunogenic vaccines. However, difficulty in obtaining DCs from conventional sources, such as bone marrow (BM), peripheral blood (PBMC), and cord blood (CB), is a significantly hinders routine application. The use of human induced pluripotent stem cells (hiPSCs) is a valuable alternative for generating sufficient numbers of DCs to be used in basic and pre-clinical studies. Despite the many challenges that must be overcome to achieve an efficient protocol for obtaining the major DC types from hiPSCs, recent progress has been made. Here we review the current state of developing DCs from hiPSCs, as well as the key elements required to enable the routine use of hiPSC-derived DCs in pre-clinical and clinical assays.
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Affiliation(s)
- Nelio Aj Oliveira
- Jackson Laboratory - Farmington, 481263, Cell Engineering , Farmington, Connecticut, United States, 06032-2374;
| | - Handan Sevim
- Hacettepe Universitesi, 37515, Faculty of Science Department of Biology, Ankara, Ankara, Turkey;
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23
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Sağraç D, Şenkal S, Hayal TB, Şahin F, Çobandede Z, Doğan A. Surface coating materials regulates the attachment and differentiation of mouse embryonic stem cell derived embryoid bodies into mesoderm at culture conditions. Cytotechnology 2022; 74:293-307. [PMID: 35464166 PMCID: PMC8976036 DOI: 10.1007/s10616-022-00529-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Accepted: 02/11/2022] [Indexed: 11/03/2022] Open
Abstract
Abstract Pluripotent stem cells as a promising cell source with unlimited proliferation and differentiation capacity hold great promise for cell-based therapies in regenerative medicine. Establishment of appropriate culture conditions might enable the control of cellular fate decision in cell culture. Transfer of three-dimensional (3D) embryoid bodies to two-dimensional (2D) monolayer culture systems for initiation of cell differentiation and specialization requires an adaptation of cells which can be managed by extracellular matrix (ECM) materials. Here we compare the characteristics of four different cell culture coating materials and their effect on attachment and differentiation of cells spreading from mouse embryonic stem cell (mESC) derived embryoid bodies (EBs) in mesoderm inducing culture conditions. Atomic force microscope (AFM) and scanning electron microscope (SEM) analysis along with Water Contact Angle technique were used to analyze physical properties of ECM materials and to evaluate cellular behavior on surfaces. Cell migration and differentiation were performed initially by using mesoderm inducing culture conditions and then three germ layer specification conditions. We investigated properties of coating materials such as roughness and wettability control cell attachment, migration and differentiation of mESCs. Matrigel-Gelatin combination is suitable for cell attachment and migration of cells spreading from 3D EBs followed by transfer onto coated surfaces. Matrigel-Gelatin coating enhanced differentiation of cells into mesoderm like cells via EMT process. Our data demonstrated that the Matrigel-Gelatin combination as a cell culture coating matrix might serve as a suitable platform to transfer EBs for differentiation and might influence pluripotent stem cell fate decision into mesoderm and further mesoderm derivative cell populations. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s10616-022-00529-z.
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24
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Eilenberger C, Rothbauer M, Brandauer K, Spitz S, Ehmoser EK, Küpcü S, Ertl P. Screening for Best Neuronal-Glial Differentiation Protocols of Neuralizing Agents Using a Multi-Sized Microfluidic Embryoid Body Array. Pharmaceutics 2022; 14:pharmaceutics14020339. [PMID: 35214071 PMCID: PMC8878393 DOI: 10.3390/pharmaceutics14020339] [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: 12/21/2021] [Revised: 01/27/2022] [Accepted: 01/29/2022] [Indexed: 02/01/2023] Open
Abstract
Stem cell technology and embryonic stem cell models are of great interest in biomedical research since they provide deeper insights into, e.g., neurogenesis and early mammalian brain development. Despite their great scientific potential, the reliable establishment of three-dimensional embryoid bodies (EBs) remains a major challenge, and the current lack of standardization and comparability is still limiting a broader application and translation of stem cell technology. Among others, a vital aspect for the reliable formation of EBs is optimizing differentiation protocols since organized differentiation is influenced by soluble inducers and EB size. A microfluidic biochip array was employed to automate cell loading and optimize directed neuronal and astrocytic differentiation protocols using murine P19 embryoid bodies to facilitate reliable embryonic stem cell differentiation. Our gravity-driven microfluidic size-controlled embryoid body-on-a-chip system allows (a) the robust operation and cultivation of up to 90 EBs in parallel and (b) the reproducible generation of five increasing sizes ranging from 300 µm to 1000 µm diameters. A comparative study adds two differentiation-inducers such as retinoic acid and EC23 to size-controlled embryoid bodies to identify the optimal differentiation protocol. Our study revealed a 1.4 to 1.9-fold higher neuron and astrocyte expression in larger embryoid bodies (above 750 µm) over smaller-sized EBs (below 450 µm), thus highlighting the importance of EB size in the establishment of robust neurodevelopmental in vitro models.
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Affiliation(s)
- Christoph Eilenberger
- Faculty of Technical Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria; (K.B.); (S.S.); (P.E.)
- Correspondence: (C.E.); (M.R.)
| | - Mario Rothbauer
- Faculty of Technical Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria; (K.B.); (S.S.); (P.E.)
- Orthopedic Microsystems, Karl Chiari Lab for Orthopaedic Biology, Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
- Correspondence: (C.E.); (M.R.)
| | - Konstanze Brandauer
- Faculty of Technical Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria; (K.B.); (S.S.); (P.E.)
| | - Sarah Spitz
- Faculty of Technical Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria; (K.B.); (S.S.); (P.E.)
| | - Eva-Kathrin Ehmoser
- Department of Nanobiotechnology, Institute of Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria; (E.-K.E.); (S.K.)
| | - Seta Küpcü
- Department of Nanobiotechnology, Institute of Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria; (E.-K.E.); (S.K.)
| | - Peter Ertl
- Faculty of Technical Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria; (K.B.); (S.S.); (P.E.)
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25
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Poorna MR, Jayakumar R, Chen JP, Mony U. Hydrogels: A potential platform for induced pluripotent stem cell culture and differentiation. Colloids Surf B Biointerfaces 2021; 207:111991. [PMID: 34333302 DOI: 10.1016/j.colsurfb.2021.111991] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2021] [Revised: 07/16/2021] [Accepted: 07/18/2021] [Indexed: 01/02/2023]
Abstract
Induced pluripotent stem cells (iPSCs) can be used to generate desired types of cells that belong to the three germ layers (i.e., ectoderm, endoderm and mesoderm). These cells possess great potential in regenerative medicine. Before iPSCs are used in various biomedical applications, the existing xenogeneic culture methods must be improved to meet the technical standards of safety, cost effectiveness, and ease of handling. In addition to commonly used 2D substrates, a culture system that mimics the native cellular environment in tissues will be a good choice when culturing iPS cells and differentiating them into different lineages. Hydrogels are potential candidates that recapitulate the native complex three-dimensional microenvironment. They possess mechanical properties similar to those of many soft tissues. Moreover, hydrogels support iPSC adhesion, proliferation and differentiation to various cell types. They are xeno-free and cost-effective. In addition to other substrates, such as mouse embryonic fibroblast (MEF), Matrigel, and vitronectin, the use of hydrogel-based substrates for iPSC culture and differentiation may help generate large numbers of clinical-grade cells that can be used in potential clinical applications. This review mainly focuses on the use of hydrogels for the culture and differentiation of iPSCs into various cell types and their potential applications in regenerative medicine.
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Affiliation(s)
- M R Poorna
- Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi 682041, India
| | - R Jayakumar
- Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi 682041, India
| | - Jyh-Ping Chen
- Department of Chemical and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan, ROC; Department of Plastic and Reconstructive Surgery and Craniofacial Research Center, Chang Gung Memorial Hospital, Linkou, Kwei-San, Taoyuan 33305, Taiwan, ROC; Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33305, Taiwan, ROC.
| | - Ullas Mony
- Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi 682041, India; Department of Biochemistry, Centre of Molecular Medicine and Diagnostics (COMManD), Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, 600077, India.
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26
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Sen D, Voulgaropoulos A, Keung AJ. Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids. BMC Biotechnol 2021; 21:59. [PMID: 34641840 PMCID: PMC8507123 DOI: 10.1186/s12896-021-00718-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Accepted: 09/28/2021] [Indexed: 12/21/2022] Open
Abstract
Background Human cerebral organoids (hCO) are attractive systems due to their ability to model important brain regions and transcriptomics of early in vivo brain development. To date, they have been used to understand the effects of genetics and soluble factors on neurodevelopment. Interestingly, one of the main advantages of hCOs are that they provide three dimensionality that better mimics the in vivo environment; yet, despite this central feature it remains unclear how spatial and mechanical properties regulate hCO and neurodevelopment. While biophysical factors such as shape and mechanical forces are known to play crucial roles in stem cell differentiation, embryogenesis and neurodevelopment, much of this work investigated two dimensional systems or relied on correlative observations of native developing tissues in three dimensions. Using hCOs to establish links between spatial factors and neurodevelopment will require the use of new approaches and could reveal fundamental principles of brain organogenesis as well as improve hCOs as an experimental model. Results Here, we investigated the effects of early geometric confinements on transcriptomic changes during hCO differentiation. Using a custom and tunable agarose microwell platform we generated embryoid bodies (EB) of diverse shapes mimicking several structures from embryogenesis and neurodevelopment and then further differentiated those EBs to whole brain hCOs. Our results showed that the microwells did not have negative gross impacts on the ability of the hCOs to differentiate towards neural fates, and there were clear shape dependent effects on neural lineage specification. In particular we observed that non-spherical shapes showed signs of altered neurodevelopmental kinetics and favored the development of medial ganglionic eminence-associated brain regions and cell types over cortical regions. Transcriptomic analysis suggests these mechanotransducive effects may be mediated by integrin and Wnt signaling. Conclusions The findings presented here suggest a role for spatial factors in brain region specification during hCO development. Understanding these spatial patterning factors will not only improve understanding of in vivo development and differentiation, but also provide important handles with which to advance and improve control over human model systems for in vitro applications. Supplementary Information The online version contains supplementary material available at 10.1186/s12896-021-00718-2.
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Affiliation(s)
- Dilara Sen
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC, 27695-7905, USA
| | - Alexis Voulgaropoulos
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC, 27695-7905, USA
| | - Albert J Keung
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC, 27695-7905, USA.
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27
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Vajta G, Parmegiani L, Machaty Z, Chen WB, Yakovenko S. Back to the future: optimised microwell culture of individual human preimplantation stage embryos. J Assist Reprod Genet 2021; 38:2563-2574. [PMID: 33864207 PMCID: PMC8581087 DOI: 10.1007/s10815-021-02167-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2020] [Accepted: 03/22/2021] [Indexed: 02/01/2023] Open
Abstract
Although in vitro culture of human embryos is a crucial step in assisted reproduction, the lack of focused research hampers worldwide standardisation and consistent outcomes. Only 1.2% of research papers published in five leading journals in human reproduction in 2019 focused on in vitro culture conditions, creating the impression that the optimisation process has approached its limits. On the other hand, in vitro culture of mammalian embryos is based on old principles, while there is no consensus on basic issues as density, time, medium change, gas atmosphere and small technical details including the way of drop preparation. This opinion paper aims to highlight and analyse the slow advancement in this field and stimulate research for simple and affordable solutions to meet the current requirements. A possible way for advancement is discussed in detail. Selection of embryos with the highest developmental competence requires individual culture and modification of the widely used "drop under oil" approach. Current use of three-dimensional surfaces instead of large flat bottoms is restricted to time-lapse systems, but these wells are designed for optical clarity, not for the needs of embryos. The size and shape of the original microwells (Well of the Well; WOW) offer a practical and straightforward solution to combine the benefits of communal and individual incubation and improve the overall quality of cultured embryos.
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Affiliation(s)
- Gábor Vajta
- RVT Australia, Cairns, QLD 4870 Australia
- VitaVitro Biotech Co., Ltd., Shenzhen, China
| | | | - Zoltan Machaty
- Department of Animal Sciences, Purdue University, West Lafayette, IN USA
| | | | - Sergey Yakovenko
- Altravita IVF Clinic, Moscow, Russia
- Biophysics Department, Moscow State University, Moscow, Russia
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28
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Xie AW, Zacharias NA, Binder BYK, Murphy WL. Controlled aggregation enhances immunomodulatory potential of mesenchymal stromal cell aggregates. Stem Cells Transl Med 2021; 10:1184-1201. [PMID: 33818906 PMCID: PMC8284773 DOI: 10.1002/sctm.19-0414] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2019] [Revised: 02/04/2021] [Accepted: 03/08/2021] [Indexed: 02/06/2023] Open
Abstract
Human mesenchymal stromal cells (MSCs) are promising candidates for cell therapy due to their ease of isolation and expansion and their ability to secrete antiapoptotic, pro-angiogenic, and immunomodulatory factors. Three-dimensional (3D) aggregation "self-activates" MSCs to augment their pro-angiogenic and immunomodulatory potential, but the microenvironmental features and culture parameters that promote optimal MSC immunomodulatory function in 3D aggregates are poorly understood. Here, we generated MSC aggregates via three distinct methods and compared them with regard to their (a) aggregate structure and (b) immunomodulatory phenotype under resting conditions and in response to inflammatory stimulus. Methods associated with fast aggregation kinetics formed aggregates with higher cell packing density and reduced extracellular matrix (ECM) synthesis compared to those with slow aggregation kinetics. While all three methods of 3D aggregation enhanced MSC expression of immunomodulatory factors compared to two-dimensional culture, different aggregation methods modulated cells' temporal expression of these factors. A Design of Experiments approach, in which aggregate size and aggregation kinetics were systematically covaried, identified a significant effect of both parameters on MSCs' ability to regulate immune cells. Compared to small aggregates formed with fast kinetics, large aggregates with slow assembly kinetics were more effective at T-cell suppression and macrophage polarization toward anti-inflammatory phenotypes. Thus, culture parameters including aggregation method, kinetics, and aggregate size influence both the structural properties of aggregates and their paracrine immunomodulatory function. These findings underscore the utility of engineering strategies to control properties of 3D MSC aggregates, which may identify new avenues for optimizing the immunomodulatory function of MSC-based cell therapies.
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Affiliation(s)
- Angela W. Xie
- Department of Biomedical EngineeringUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | - Nicholas A. Zacharias
- Department of Biomedical EngineeringUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | - Bernard Y. K. Binder
- Department of Orthopedics and RehabilitationUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | - William L. Murphy
- Department of Biomedical EngineeringUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
- Department of Orthopedics and RehabilitationUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
- Department of Materials Science and EngineeringUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
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29
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Chen K, Zheng Y, Xue X, Liu Y, Resto Irizarry AM, Tang H, Fu J. Branching development of early post-implantation human embryonic-like tissues in 3D stem cell culture. Biomaterials 2021; 275:120898. [PMID: 34044259 PMCID: PMC8325636 DOI: 10.1016/j.biomaterials.2021.120898] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2020] [Revised: 04/22/2021] [Accepted: 05/13/2021] [Indexed: 12/15/2022]
Abstract
Human embryonic stem cells (hESCs) have the intrinsic capacity to self-organize and generate patterned tissues. In vitro models that coax hESCs to form embryonic-like structures by modulating physical environments and priming with chemical signals have become a powerful tool for dissecting the regulatory mechanisms underlying early human development. Here we present a 3D suspension culture system of hESCs that can generate post-implantation, pre-gastrulation embryonic-like tissues in an efficient and controllable manner. The efficiency of the development of asymmetric tissues, which mimic the post-implantation, pre-gastrulation amniotic sac, was about 50% in the 3D suspension culture. Quantitative imaging profiling and unsupervised trajectory analysis revealed that hESC aggregates first entered into a transitional stage expressing Brachyury (or T), before their development branched into different paths to develop into asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues, respectively. Moreover, the branching developmental trajectory of embryonic-like structures was affected by the initial cell seeding density or cluster size of hESCs. A higher percentage of amniotic-like tissues was observed under a small initial cell seeding density of hESCs. Conversely, a large initial cell seeding density of hESCs promoted the development of mesodermal-like tissues. Intermediate cell seeding densities of hESCs in the 3D suspension culture promoted the development of asymmetric embryonic-like tissues. Our results suggest that hESCs have the intrinsic capability to sense the initial cell population size, which in turn regulates their differentiation and self-organization into different embryonic-like tissues. Our 3D suspension culture thus provides a promising experimental tool to study the interplay between tissue topology and self-organization and progressive embryonic development using in vitro hESC-based models.
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Affiliation(s)
- Kejie Chen
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Yi Zheng
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Xufeng Xue
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Yue Liu
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA
| | | | - Huaijing Tang
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Jianping Fu
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.
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30
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Kim D, Lee SJ, Youn J, Hong H, Eom S, Kim DS. A deep and permeable nanofibrous oval-shaped microwell array for the stable formation of viable and functional spheroids. Biofabrication 2021; 13. [PMID: 34030141 DOI: 10.1088/1758-5090/ac044c] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Accepted: 05/24/2021] [Indexed: 12/26/2022]
Abstract
Despite the potential of a nanofibrous (NF) microwell array as a permeable microwell array to improve the viability and functions of spheroids, thanks to the superior permeability to both gases and solutes, there have still been difficulties regarding the stable formation of spheroids in the NF microwell array due to the low aspect ratio (AR) and the large interspacing between microwells. This study proposes a nanofibrous oval-shaped microwell array, named the NOVA microwell array, with both a high AR and a high well density, enabling us to not only collect cells in the microwell with a high cell seeding efficiency, but also to generate multiple viable and functional spheroids in a uniform and stable manner. To realize a deep NOVA microwell array with a high aspect ratio (AR = 0.9) and a high well density (494 wells cm-2), we developed a matched-mold thermoforming process for the fabrication of both size- and AR-controllable NOVA microwell arrays with various interspacing between microwells while maintaining the porous nature of the NF membrane. The human hepatocellular carcinoma (HepG2) cell spheroids cultured on the deep NOVA microwell array not only had uniform size and shape, with a spheroid circularity of 0.80 ± 0.03 at a cell seeding efficiency of 94.29 ± 9.55%, but also exhibited enhanced viability with a small fraction of dead cells and promoted functionality with increased albumin secretion, compared with the conventional impermeable microwell array. The superior characteristics of the deep NOVA microwell array, i.e. a high AR, a high well density, and a high permeability, pave the way to the production of various viable and functional spheroids and even organoids in a scalable manner.
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Affiliation(s)
- Dohui Kim
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77, Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea
| | - Seong Jin Lee
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77, Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea
| | - Jaeseung Youn
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77, Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea
| | - Hyeonjun Hong
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77, Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea
| | - Seongsu Eom
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77, Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea
| | - Dong Sung Kim
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77, Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea.,Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), 77, Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea.,Institute for Convergence Research and Education in Advanced Technology, Yonsei University, 50, Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea
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31
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Gao Y, Pu J. Differentiation and Application of Human Pluripotent Stem Cells Derived Cardiovascular Cells for Treatment of Heart Diseases: Promises and Challenges. Front Cell Dev Biol 2021; 9:658088. [PMID: 34055788 PMCID: PMC8149736 DOI: 10.3389/fcell.2021.658088] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Accepted: 03/25/2021] [Indexed: 12/15/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) are derived from human embryos (human embryonic stem cells) or reprogrammed from human somatic cells (human induced pluripotent stem cells). They can differentiate into cardiovascular cells, which have great potential as exogenous cell resources for restoring cardiac structure and function in patients with heart disease or heart failure. A variety of protocols have been developed to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain cardiovascular lineages with high differentiation efficiency. In this concise review, we summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their therapeutic application for treatment of cardiac diseases in large animal models, and discuss the challenges and limitations in the use of cardiac cells generated from hPSCs for a better clinical application of hPSC-based cardiac cell therapy.
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Affiliation(s)
- Yu Gao
- Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Jun Pu
- Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
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Vajta G, Parmegiani L, Machaty Z, Chen WB, Yakovenko S. Back to the future: optimised microwell culture of individual human preimplantation stage embryos. J Assist Reprod Genet 2021. [PMID: 33864207 DOI: 10.1007/s10815-021-02167-4.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/29/2022] Open
Abstract
Although in vitro culture of human embryos is a crucial step in assisted reproduction, the lack of focused research hampers worldwide standardisation and consistent outcomes. Only 1.2% of research papers published in five leading journals in human reproduction in 2019 focused on in vitro culture conditions, creating the impression that the optimisation process has approached its limits. On the other hand, in vitro culture of mammalian embryos is based on old principles, while there is no consensus on basic issues as density, time, medium change, gas atmosphere and small technical details including the way of drop preparation. This opinion paper aims to highlight and analyse the slow advancement in this field and stimulate research for simple and affordable solutions to meet the current requirements. A possible way for advancement is discussed in detail. Selection of embryos with the highest developmental competence requires individual culture and modification of the widely used "drop under oil" approach. Current use of three-dimensional surfaces instead of large flat bottoms is restricted to time-lapse systems, but these wells are designed for optical clarity, not for the needs of embryos. The size and shape of the original microwells (Well of the Well; WOW) offer a practical and straightforward solution to combine the benefits of communal and individual incubation and improve the overall quality of cultured embryos.
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Affiliation(s)
- Gábor Vajta
- RVT Australia, Cairns, QLD, 4870, Australia. .,VitaVitro Biotech Co., Ltd., Shenzhen, China.
| | | | - Zoltan Machaty
- Department of Animal Sciences, Purdue University, West Lafayette, IN, USA
| | | | - Sergey Yakovenko
- Altravita IVF Clinic, Moscow, Russia.,Biophysics Department, Moscow State University, Moscow, Russia
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33
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Borys BS, Dang T, So T, Rohani L, Revay T, Walsh T, Thompson M, Argiropoulos B, Rancourt DE, Jung S, Hashimura Y, Lee B, Kallos MS. Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors. Stem Cell Res Ther 2021; 12:55. [PMID: 33436078 PMCID: PMC7805206 DOI: 10.1186/s13287-020-02109-4] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Accepted: 12/21/2020] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. METHODS The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times. RESULTS CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function. CONCLUSIONS Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.
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Affiliation(s)
- Breanna S Borys
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
- Biomedical Engineering Graduate Program, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
- Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
| | - Tiffany Dang
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
- Biomedical Engineering Graduate Program, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
- Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
| | - Tania So
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
- Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
| | - Leili Rohani
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, T2N 4N1, Canada
| | - Tamas Revay
- Department of Medical Genetics, Alberta Health Services, Alberta Children's Hospital, 28 Oki Drive, Calgary, AB, T3B 6A8, Canada
| | - Tylor Walsh
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
- Biomedical Engineering Graduate Program, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
- Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada
| | - Madalynn Thompson
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, T2N 4N1, Canada
| | - Bob Argiropoulos
- Department of Medical Genetics, Alberta Health Services, Alberta Children's Hospital, 28 Oki Drive, Calgary, AB, T3B 6A8, Canada
| | - Derrick E Rancourt
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, T2N 4N1, Canada
| | - Sunghoon Jung
- PBS Biotech Inc, 1183 Calle Suerte, Camarillo, CA, 93012, USA
| | - Yas Hashimura
- PBS Biotech Inc, 1183 Calle Suerte, Camarillo, CA, 93012, USA
| | - Brian Lee
- PBS Biotech Inc, 1183 Calle Suerte, Camarillo, CA, 93012, USA
| | - Michael S Kallos
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada.
- Biomedical Engineering Graduate Program, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada.
- Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada.
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Kang SM, Kim D, Lee JH, Takayama S, Park JY. Engineered Microsystems for Spheroid and Organoid Studies. Adv Healthc Mater 2021; 10:e2001284. [PMID: 33185040 PMCID: PMC7855453 DOI: 10.1002/adhm.202001284] [Citation(s) in RCA: 63] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 10/01/2020] [Indexed: 01/09/2023]
Abstract
3D in vitro model systems such as spheroids and organoids provide an opportunity to extend the physiological understanding using recapitulated tissues that mimic physiological characteristics of in vivo microenvironments. Unlike 2D systems, 3D in vitro systems can bridge the gap between inadequate 2D cultures and the in vivo environments, providing novel insights on complex physiological mechanisms at various scales of organization, ranging from the cellular, tissue-, to organ-levels. To satisfy the ever-increasing need for highly complex and sophisticated systems, many 3D in vitro models with advanced microengineering techniques have been developed to answer diverse physiological questions. This review summarizes recent advances in engineered microsystems for the development of 3D in vitro model systems. The relationship between the underlying physics behind the microengineering techniques, and their ability to recapitulate distinct 3D cellular structures and functions of diverse types of tissues and organs are highlighted and discussed in detail. A number of 3D in vitro models and their engineering principles are also introduced. Finally, current limitations are summarized, and perspectives for future directions in guiding the development of 3D in vitro model systems using microengineering techniques are provided.
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Affiliation(s)
- Sung-Min Kang
- Department of Green Chemical Engineering, Sangmyung University, Cheonan, Chungnam, 31066, Republic of Korea
| | - Daehan Kim
- Department of Mechanical Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Ji-Hoon Lee
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine, Atlanta, GA, 30332, USA
- The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Shuichi Takayama
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine, Atlanta, GA, 30332, USA
- The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Joong Yull Park
- Department of Mechanical Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
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McKee C, Brown C, Bakshi S, Walker K, Govind CK, Chaudhry GR. Transcriptomic Analysis of Naïve Human Embryonic Stem Cells Cultured in Three-Dimensional PEG Scaffolds. Biomolecules 2020; 11:E21. [PMID: 33379237 PMCID: PMC7824559 DOI: 10.3390/biom11010021] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 12/09/2020] [Accepted: 12/24/2020] [Indexed: 12/21/2022] Open
Abstract
Naïve human embryonic stem cells (ESCs) are characterized by improved viability, proliferation, and differentiation capacity in comparison to traditionally derived primed human ESCs. However, currently used two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, altering morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naïve state characteristics of ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon the combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffold environment maintained the naïve state and supported the long-term growth of ESCs. RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown cells. Gene ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, extracellular matrix organization, and chromatin remodeling in 3-D grown cells. 3-D culture conditions also induced upregulation of genes associated with Wnt and focal adhesion signaling, while p53 signaling pathway associated genes were downregulated. Our findings, for the first time, provide insight into the possible mechanisms of self-renewal of naïve ESCs stimulated by the transduction of mechanical signals from the 3-D microenvironment.
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Affiliation(s)
- Christina McKee
- Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA; (C.M.); (C.B.); (S.B.); (K.W.); (C.K.G.)
- OU-WB Institute for Stem Cell and Regenerative Medicine, Rochester, MI 48309, USA
| | - Christina Brown
- Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA; (C.M.); (C.B.); (S.B.); (K.W.); (C.K.G.)
- OU-WB Institute for Stem Cell and Regenerative Medicine, Rochester, MI 48309, USA
| | - Shreeya Bakshi
- Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA; (C.M.); (C.B.); (S.B.); (K.W.); (C.K.G.)
- OU-WB Institute for Stem Cell and Regenerative Medicine, Rochester, MI 48309, USA
| | - Keegan Walker
- Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA; (C.M.); (C.B.); (S.B.); (K.W.); (C.K.G.)
- OU-WB Institute for Stem Cell and Regenerative Medicine, Rochester, MI 48309, USA
| | - Chhabi K. Govind
- Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA; (C.M.); (C.B.); (S.B.); (K.W.); (C.K.G.)
- OU-WB Institute for Stem Cell and Regenerative Medicine, Rochester, MI 48309, USA
| | - G. Rasul Chaudhry
- Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA; (C.M.); (C.B.); (S.B.); (K.W.); (C.K.G.)
- OU-WB Institute for Stem Cell and Regenerative Medicine, Rochester, MI 48309, USA
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Davaapil H, Shetty DK, Sinha S. Aortic "Disease-in-a-Dish": Mechanistic Insights and Drug Development Using iPSC-Based Disease Modeling. Front Cell Dev Biol 2020; 8:550504. [PMID: 33195187 PMCID: PMC7655792 DOI: 10.3389/fcell.2020.550504] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Accepted: 10/08/2020] [Indexed: 12/24/2022] Open
Abstract
Thoracic aortic diseases, whether sporadic or due to a genetic disorder such as Marfan syndrome, lack effective medical therapies, with limited translation of treatments that are highly successful in mouse models into the clinic. Patient-derived induced pluripotent stem cells (iPSCs) offer the opportunity to establish new human models of aortic diseases. Here we review the power and potential of these systems to identify cellular and molecular mechanisms underlying disease and discuss recent advances, such as gene editing, and smooth muscle cell embryonic lineage. In particular, we discuss the practical aspects of vascular smooth muscle cell derivation and characterization, and provide our personal insights into the challenges and limitations of this approach. Future applications, such as genotype-phenotype association, drug screening, and precision medicine are discussed. We propose that iPSC-derived aortic disease models could guide future clinical trials via “clinical-trials-in-a-dish”, thus paving the way for new and improved therapies for patients.
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Affiliation(s)
- Hongorzul Davaapil
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge, United Kingdom
| | - Deeti K Shetty
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge, United Kingdom
| | - Sanjay Sinha
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge, United Kingdom
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37
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Manzoor AA, Romita L, Hwang DK. A review on microwell and microfluidic geometric array fabrication techniques and its potential applications in cellular studies. CAN J CHEM ENG 2020. [DOI: 10.1002/cjce.23875] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Affiliation(s)
- Ahmad Ali Manzoor
- Department of Chemical Engineering Ryerson University Toronto Ontario Canada
- Keenan Research Centre for Biomedical Science St. Michael's Hospital Toronto Ontario Canada
- Institute for Biomedical Engineering Science and Technology (iBEST) A partnership between Ryerson University and St. Michael's Hospital Toronto Ontario Canada
| | - Lauren Romita
- Department of Chemical Engineering Ryerson University Toronto Ontario Canada
- Keenan Research Centre for Biomedical Science St. Michael's Hospital Toronto Ontario Canada
- Institute for Biomedical Engineering Science and Technology (iBEST) A partnership between Ryerson University and St. Michael's Hospital Toronto Ontario Canada
| | - Dae Kun Hwang
- Department of Chemical Engineering Ryerson University Toronto Ontario Canada
- Keenan Research Centre for Biomedical Science St. Michael's Hospital Toronto Ontario Canada
- Institute for Biomedical Engineering Science and Technology (iBEST) A partnership between Ryerson University and St. Michael's Hospital Toronto Ontario Canada
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38
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Zeevaert K, Elsafi Mabrouk MH, Wagner W, Goetzke R. Cell Mechanics in Embryoid Bodies. Cells 2020; 9:E2270. [PMID: 33050550 PMCID: PMC7599659 DOI: 10.3390/cells9102270] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Revised: 10/06/2020] [Accepted: 10/09/2020] [Indexed: 12/14/2022] Open
Abstract
Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells undergo a transition from rather homogeneous epithelial-like pluripotent stem cell colonies into a three-dimensional organization of various cell types with multifaceted cell-cell interactions and lumen formation-a process associated with repetitive epithelial-mesenchymal transitions. In the last few years, culture methods have further evolved to better control EB size, growth, cellular composition, and organization-e.g., by the addition of morphogens or different extracellular matrix molecules. There is a growing perception that the mechanical properties, cell mechanics, and cell signaling during EB development are also influenced by physical cues to better guide lineage specification; substrate elasticity and topography are relevant, as well as shear stress and mechanical strain. Epithelial structures outside and inside EBs support the integrity of the cell aggregates and counteract mechanical stress. Furthermore, hydrogels can be used to better control the organization and lineage-specific differentiation of EBs. In this review, we summarize how EB formation is accompanied by a variety of biomechanical parameters that need to be considered for the directed and reproducible self-organization of early cell fate decisions.
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Affiliation(s)
- Kira Zeevaert
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Mohamed H. Elsafi Mabrouk
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Wolfgang Wagner
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
| | - Roman Goetzke
- Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074 Aachen, Germany; (K.Z.); (M.H.E.M.)
- Institute for Biomedical Engineering–Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany
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Oss-Ronen L, Redden RA, Lelkes PI. Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity. Stem Cells Dev 2020; 29:1275-1284. [PMID: 32731794 DOI: 10.1089/scd.2020.0097] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A reduced Oct3/4 expression and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESC differentiation.
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Affiliation(s)
- Liat Oss-Ronen
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania, USA
| | - Robert A Redden
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania, USA
| | - Peter I Lelkes
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, Pennsylvania, USA
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Chen ACH, Lee KF, Yeung WSB, Lee YL. Human embryonic stem cells as an in vitro model for studying developmental origins of type 2 diabetes. World J Stem Cells 2020; 12:761-775. [PMID: 32952857 PMCID: PMC7477660 DOI: 10.4252/wjsc.v12.i8.761] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Revised: 04/28/2020] [Accepted: 06/14/2020] [Indexed: 02/06/2023] Open
Abstract
The developmental origins of health and diseases (DOHaD) is a concept stating that adverse intrauterine environments contribute to the health risks of offspring. Since the theory emerged more than 30 years ago, many epidemiological and animal studies have confirmed that in utero exposure to environmental insults, including hyperglycemia and chemicals, increased the risk of developing noncommunicable diseases (NCDs). These NCDs include metabolic syndrome, type 2 diabetes, and complications such as diabetic cardiomyopathy. Studying the effects of different environmental insults on early embryo development would aid in understanding the underlying mechanisms by which these insults promote NCD development. Embryonic stem cells (ESCs) have also been utilized by researchers to study the DOHaD. ESCs have pluripotent characteristics and can be differentiated into almost every cell lineage; therefore, they are excellent in vitro models for studying early developmental events. More importantly, human ESCs (hESCs) are the best alternative to human embryos for research because of ethical concerns. In this review, we will discuss different maternal conditions associated with DOHaD, focusing on the complications of maternal diabetes. Next, we will review the differentiation protocols developed to generate different cell lineages from hESCs. Additionally, we will review how hESCs are utilized as a model for research into the DOHaD. The effects of environmental insults on hESC differentiation and the possible involvement of epigenetic regulation will be discussed.
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Affiliation(s)
- Andy Chun-Hang Chen
- Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China
| | - Kai Fai Lee
- Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China
| | - William Shu Biu Yeung
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China
| | - Yin Lau Lee
- Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China.
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Lee SP, Chao SC, Chou MF, Huang SF, Dai NT, Wu GJ, Tsai CS, Loh SH, Tsai YT. Characterization of intracellular buffering power in human induced pluripotent stem cells and the loss of pluripotency is delayed by acidic stimulation and increase of NHE1 activity. J Cell Physiol 2020; 236:1515-1528. [PMID: 32841374 DOI: 10.1002/jcp.29959] [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: 04/21/2020] [Revised: 06/18/2020] [Accepted: 07/07/2020] [Indexed: 11/07/2022]
Abstract
The homeostasis of intracellular pH (pHi ) affects many cellular functions. Our previous study has established a functional and molecular model of the active pHi regulators in human induced pluripotent stem cells (hiPSCs). The aims of the present study were to further quantify passive pHi buffering power (β) and to investigate the effects of extracellular pH and Na+ -H+ exchanger 1 (NHE1) activity on pluripotency in hiPSCs. pHi was detected by microspectrofluorimetry with pH-sensitive dye-BCECF. Western blot, immunofluorescence staining, and flow cytometry were used to detect protein expression and pluripotency. Our study in hiPSCs showed that (a) the value of total (βtot ), intrinsic (βi ), and CO2 -dependent ( β C O 2 ) buffering power all increased while pHi increased; (b) during the spontaneous differentiation for 4 days, the β values of βtot and β C O 2 changed in a tendency of decrease, despite the absence of statistical significance; (c) an acidic cultured environment retained pluripotency and further upregulated expression and activity of NHE1 during spontaneous differentiation; (d) inhibition on NHE1 activity promoted the loss of pluripotency. In conclusion, we, for the first time, established a quantitative model of passive β during differentiation and demonstrated that maintenance of NHE1 at a higher level was of critical importance for pluripotency retention in hiPSCs.
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Affiliation(s)
- Shiao-Pieng Lee
- Department of Dentistry, School of Dentistry, Division of Oral and Maxillofacial Surgery, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan
| | - Shih-Chi Chao
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
| | - Mei-Fang Chou
- Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan
| | - Shu-Fu Huang
- Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan
| | - Niann-Tzyy Dai
- Department of Surgery, Division of Plastic and Reconstructive Surgery, Tri-Service General Hospital, Taipei, Taiwan
| | - Gwo-Jang Wu
- Department of Obstetrics and Gynecology, Tri-Service General Hospital, Taipei, Taiwan
| | - Chien-Sung Tsai
- Department of Surgery, Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
- Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan
- Institute of Pharmacy, I.M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Shih-Hurng Loh
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
- Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan
- Department of Surgery, Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Yi-Ting Tsai
- Department of Surgery, Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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Branco MA, Cabral JM, Diogo MM. From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges. Bioengineering (Basel) 2020; 7:E92. [PMID: 32785039 PMCID: PMC7552661 DOI: 10.3390/bioengineering7030092] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 07/30/2020] [Accepted: 08/06/2020] [Indexed: 12/19/2022] Open
Abstract
The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.
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Affiliation(s)
| | | | - Maria Margarida Diogo
- iBB-Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal; (M.A.B.); (J.M.S.C.)
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Yadav A, Seth B, Chaturvedi RK. Brain Organoids: Tiny Mirrors of Human Neurodevelopment and Neurological Disorders. Neuroscientist 2020; 27:388-426. [PMID: 32723210 DOI: 10.1177/1073858420943192] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Unravelling the complexity of the human brain is a challenging task. Nowadays, modern neurobiologists have developed 3D model systems called "brain organoids" to overcome the technical challenges in understanding human brain development and the limitations of animal models to study neurological diseases. Certainly like most model systems in neuroscience, brain organoids too have limitations, as these minuscule brains lack the complex neuronal circuitry required to begin the operational tasks of human brain. However, researchers are hopeful that future endeavors with these 3D brain tissues could provide mechanistic insights into the generation of circuit complexity as well as reproducible creation of different regions of the human brain. Herein, we have presented the contemporary state of brain organoids with special emphasis on their mode of generation and their utility in modelling neurological disorders, drug discovery, and clinical trials.
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Affiliation(s)
- Anuradha Yadav
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India.,Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
| | - Brashket Seth
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India.,Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
| | - Rajnish Kumar Chaturvedi
- Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India.,Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
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Green DW, Watson JA, Watson GS, Stamboulis A. Sequenced Somatic Cell Reprogramming and Differentiation Inside Nested Hydrogel Droplets. ACTA ACUST UNITED AC 2020; 4:e2000071. [PMID: 32597033 DOI: 10.1002/adbi.202000071] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2020] [Revised: 05/06/2020] [Indexed: 11/08/2022]
Abstract
The efficient genesis of pluripotent cells or therapeutic cells for regenerative medicine involves several external manipulations and conditioning protocols, which drives down clinical applicability. Automated programming of the genesis by microscale physical forces and chronological biochemistry can increase clinical success. The design and fabrication of nested polysaccharide droplets (millimeter-sized) with cell sustaining properties of natural tissues and intrinsic properties for time and space evolution of cell transformation signals between somatic cells, pluripotent cells and differentiated therapeutic cells in a swift and efficient manner without the need for laborious external manipulation are reported. Cells transform between phenotypic states by having single and double nested droplets constituted with extracellular matrix proteins and reprogramming, and differentiation factors infused chronologically across the droplet space. The cell transformation into germ layer cells and bone cells is successfully tested in vitro and in vivo and promotes the formation of new bone tissues. Thus, nested droplets with BMP-2 loaded guests synthesize mineralized bone tissue plates along the length of a cranial non-union bone defect at 4 weeks. The advantages of sequenced somatic cell reprogramming and differentiation inside an individual hydrogel module without external manipulation, promoted by formulating tissue mimetic physical, mechanical, and chemical microenvironments are shown.
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Affiliation(s)
- David W Green
- School of Metallurgy and Materials, Healthcare Technologies Institute, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
| | - Jolanta A Watson
- School of Science and Engineering, University of the Sunshine Coast, Fraser Coast, Hervey Bay, QLD, 4655, Australia
| | - Gregory S Watson
- School of Science and Engineering, University of the Sunshine Coast, Fraser Coast, Hervey Bay, QLD, 4655, Australia
| | - Artemis Stamboulis
- School of Metallurgy and Materials, Healthcare Technologies Institute, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
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Inoo K, Yamamoto M, Tabata Y. Preparation of cell aggregates incorporating gelatin hydrogel microspheres of sugar-responsive water solubilization. J Tissue Eng Regen Med 2020; 14:1050-1062. [PMID: 32478475 DOI: 10.1002/term.3076] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2020] [Revised: 05/01/2020] [Accepted: 05/11/2020] [Indexed: 12/18/2022]
Abstract
The objective of this study is to design hydrogel microspheres of a cell scaffold, which not only function as a scaffold to form cell aggregates of three-dimensional culture but also can disappear to release growth factors in the well-controlled manner by noncytotoxic stimulation in any timing. The hydrogel microspheres were prepared by a water-in-oil emulsion method from m-aminophenylboronic acid (APBA)-introduced gelatin (APBA-gelatin) with or without poly(vinyl alcohol) (PVA) mixing. Irrespective of the PVA concentration, the microspheres with the same diameter were prepared. The microspheres were water solubilized only by adding sorbitol of a sugar although the solubilization extent depended on the PVA concentration. When cocultured with the microspheres, mesenchymal stem cells formed cell aggregates homogeneously incorporating the microspheres. Upon adding sorbitol in the culture medium, mixed APBA-gelatin-PVA hydrogel microspheres disappeared with time in the cell aggregates. The microspheres containing basic fibroblast growth factor or bone morphogenetic protein-2 released the respective growth factor accompanied with the microspheres disappearance. It is concluded that the present microspheres of sugar-responsive water solubilization are promising scaffold of cell aggregates and have an ability to allow growth factors to be released in the cell aggregates when it is required.
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Affiliation(s)
- Kanako Inoo
- Laboratory of Biomaterials, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Masaya Yamamoto
- Laboratory of Biomaterials, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Yasuhiko Tabata
- Laboratory of Biomaterials, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
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Induced Pluripotent Stem Cells in Dental and Nondental Tissue Regeneration: A Review of an Unexploited Potential. Stem Cells Int 2020; 2020:1941629. [PMID: 32300365 PMCID: PMC7146092 DOI: 10.1155/2020/1941629] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2019] [Accepted: 03/06/2020] [Indexed: 12/16/2022] Open
Abstract
Cell-based therapies currently represent the state of art for tissue regenerative treatment approaches for various diseases and disorders. Induced pluripotent stem cells (iPSCs), reprogrammed from adult somatic cells, using vectors carrying definite transcription factors, have manifested a breakthrough in regenerative medicine, relying on their pluripotent nature and ease of generation in large amounts from various dental and nondental tissues. In addition to their potential applications in regenerative medicine and dentistry, iPSCs can also be used in disease modeling and drug testing for personalized medicine. The current review discusses various techniques for the production of iPSC-derived osteogenic and odontogenic progenitors, the therapeutic applications of iPSCs, and their regenerative potential in vivo and in vitro. Through the present review, we aim to explore the potential applications of iPSCs in dental and nondental tissue regeneration and to highlight different protocols used for the generation of different tissues and cell lines from iPSCs.
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Laco F, Lam ATL, Woo TL, Tong G, Ho V, Soong PL, Grishina E, Lin KH, Reuveny S, Oh SKW. Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor. Stem Cell Res Ther 2020; 11:118. [PMID: 32183888 PMCID: PMC7076930 DOI: 10.1186/s13287-020-01618-6] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 02/11/2020] [Accepted: 02/24/2020] [Indexed: 01/13/2023] Open
Abstract
Background The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. Methods Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. Results Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. Conclusion A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry. Supplementary information The online version of this article (10.1186/s13287-020-01618-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Filip Laco
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Alan Tin-Lun Lam
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore.
| | - Tsung-Liang Woo
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Gerine Tong
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Valerie Ho
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Poh-Loong Soong
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Elina Grishina
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Kun-Han Lin
- Ternion Biosciences, National Heart Centre of Singapore, Singapore, Singapore
| | - Shaul Reuveny
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore
| | - Steve Kah-Weng Oh
- Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore, 138668, Singapore.
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Jiang B, Yan L, Shamul JG, Hakun M, He X. Stem cell therapy of myocardial infarction: a promising opportunity in bioengineering. ADVANCED THERAPEUTICS 2020; 3:1900182. [PMID: 33665356 PMCID: PMC7928435 DOI: 10.1002/adtp.201900182] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Indexed: 02/06/2023]
Abstract
Myocardial infarction (MI) is a life-threatening disease resulting from irreversible death of cardiomyocytes (CMs) and weakening of the heart blood-pumping function. Stem cell-based therapies have been studied for MI treatment over the last two decades with promising outcome. In this review, we critically summarize the past work in this field to elucidate the advantages and disadvantages of treating MI using pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), adult stem cells, and cardiac progenitor cells. The main advantage of the latter is their cytokine production capability to modulate immune responses and control the progression of healing. However, human adult stem cells have very limited (if not 'no') capacity to differentiate into functional CMs in vitro or in vivo. In contrast, PSCs can be differentiated into functional CMs although the protocols for the cardiac differentiation of PSCs are mainly for adherent cells under 2D culture. Derivation of PSC-CMs in 3D, allowing for large-scale production of CMs via modulation of the Wnt/β-catenin signal pathway with defined chemicals and medium, may be desired for clinical translation. Furthermore, the technology of purification and maturation of the PSC-CMs may need further improvements to eliminate teratoma formation after in vivo implantation of the PSC-CMs for treating MI. In addition, in vitro derived PSC-CMs may have mechanical and electrical mismatch with the patient's cardiac tissue, which causes arrhythmia. This supports the use of PSC-derived cells committed to cardiac lineage without beating for implantation to treat MI. In this case, the PSC derived cells may utilize the mechanical, electrical, and chemical cues in the heart to further differentiate into mature/functional CMs in situ. Another major challenge facing stem cell therapy of MI is the low retention/survival of stem cells or their derivatives (e.g., PSC-CMs) in the heart for MI treatment after injection in vivo. This may be resolved by using biomaterials to engineer stem cells for reduced immunogenicity, immobilization of the cells in the heart, and increased integration with the host cardiac tissue. Biomaterials have also been applied in the derivation of CMs in vitro to increase the efficiency and maturation of differentiation. Collectively, a lot has been learned from the past failure of simply injecting intact stem cells or their derivatives in vivo for treating MI, and bioengineering stem cells with biomaterials is expected to be a valuable strategy for advancing stem cell therapy towards its widespread application for treating MI in the clinic.
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Affiliation(s)
- Bin Jiang
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Li Yan
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - James G Shamul
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Maxwell Hakun
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Xiaoming He
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
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49
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Guo NN, Liu LP, Zheng YW, Li YM. Inducing human induced pluripotent stem cell differentiation through embryoid bodies: A practical and stable approach. World J Stem Cells 2020; 12:25-34. [PMID: 32110273 PMCID: PMC7031760 DOI: 10.4252/wjsc.v12.i1.25] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/01/2019] [Revised: 09/30/2019] [Accepted: 12/13/2019] [Indexed: 02/06/2023] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use. They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research. However, significant limitations exist when using conventional flat culturing methods especially concerning cell expansion, differentiation efficiency, stability maintenance and multicellular 3D structure establishment, differentiation prediction. Embryoid bodies (EBs), the multicellular aggregates spontaneously generated from iPSCs in the suspension system, might help to address these issues. Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve, EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing, differentiation efficiency enhancement, ex vivo simulation, and organoid establishment. EBs can potentially also be used in early prediction of iPSC differentiation capability. To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs, critical factors including iPSC pluripotency maintenance, generation of uniform morphology using micro-pattern 3D culture systems, proper cellular density inoculation, and EB size control are discussed on the basis of both published data and our own laboratory experiences. Collectively, the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.
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Affiliation(s)
- Ning-Ning Guo
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Li-Ping Liu
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Yun-Wen Zheng
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, University of Tsukuba Faculty of Medicine, Tsukuba, Ibaraki 305-8575, Japan
- Yokohama City University School of Medicine, Yokohama, Kanagawa 234-0006, Japan
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, the University of Tokyo, Tokyo 108-8639, Japan.
| | - Yu-Mei Li
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
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Sung TC, Liu CH, Huang WL, Lee YC, Kumar SS, Chang Y, Ling QD, Hsu ST, Higuchi A. Efficient differentiation of human ES and iPS cells into cardiomyocytes on biomaterials under xeno-free conditions. Biomater Sci 2019; 7:5467-5481. [PMID: 31656967 DOI: 10.1039/c9bm00817a] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Current xeno-free and chemically defined methods for the differentiation of hPSCs (human pluripotent stem cells) into cardiomyocytes are not efficient and are sometimes not reproducible. Therefore, it is necessary to develop reliable and efficient methods for the differentiation of hPSCs into cardiomyocytes for future use in cardiovascular research related to drug discovery, cardiotoxicity screening, and disease modeling. We evaluated two representative differentiation methods that were reported previously, and we further developed original, more efficient methods for the differentiation of hPSCs into cardiomyocytes under xeno-free, chemically defined conditions. The developed protocol successively differentiated hPSCs into cardiomyocytes, approximately 90-97% of which expressed the cardiac marker cTnT, with beating speeds and sarcomere lengths that were similar to those of a healthy adult human heart. The optimal cell culture biomaterials for the cardiac differentiation of hPSCs were also evaluated using extracellular matrix-mimetic material-coated dishes. Synthemax II-coated and Laminin-521-coated dishes were found to be the most effective and efficient biomaterials for the cardiac differentiation of hPSCs according to the observation of hPSC-derived cardiomyocytes with high survival ratios, high beating colony numbers, a similar beating frequency to that of a healthy adult human heart, high purity levels (high cTnT expression) and longer sarcomere lengths similar to those of a healthy adult human heart.
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Affiliation(s)
- Tzu-Cheng Sung
- The Eye Hospital of Wenzhou Medical University, No. 270, Xueyuan Road, Wenzhou, Zhejiang 325027, China.
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