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Narozna M, Latham MC, Gorbsky GJ. Origin of Chromosome 12 Trisomy Surge in Human Induced Pluripotent Stem Cells (iPSCs). BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2024.12.02.626470. [PMID: 39677655 PMCID: PMC11642788 DOI: 10.1101/2024.12.02.626470] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
Cultured pluripotent stem cells are unique in being the only fully diploid immortal human cell lines. However, during continued culture, they acquire significant chromosome abnormalities. Chromosome 12 trisomy is the most common whole-chromosome abnormality found during culture of human induced pluripotent stem cells (iPSCs). The conventional paradigm is that trisomy 12 occurs very rarely but provides a proliferative advantage, enabling these cells to outcompete the diploid. Here, we challenge this prevailing model by demonstrating that trisomy 12 arises simultaneously in a very high percentage of diploid cells. Using a single cell line that reproducibly undergoes transition from diploid to trisomy 12, we found that proliferation differences alone do not account for the rapid dominance of trisomic cells. Through careful mapping by fluorescent in-situ hybridization, we identified critical transition passages where trisomic cells first appeared and swiftly gained dominance. Remarkably, single trisomic cells repeatedly emerged de novo from diploid parents. Delving deeper, we discovered an extremely high incidence of chromosome 12 anaphase bridging exclusively during transition passages, along with overrepresentation of chromosome 12 chromatids in micronuclei. These micronuclei fail to replicate during S phase. Subsequently, when these micronucleated cells enter mitosis they contain an unreplicated chromosome 12 chromatids. We also found that nearly 20% of the shorter p arms of chromosome 12 but not the longer q arms exhibited loss of subtelomeric repeats during transition passages. Chromosome 12p arms were exclusively responsible for the bridging observed in anaphase cells. Our findings unveil a novel mechanism of whole-chromosome instability in human stem cells, where chromosome 12p arm-specific segregation errors occur simultaneously in a high percentage of cells. The slight yet significant growth advantage of trisomy 12 cells allows them to persist and eventually dominate the population. Our findings detailing this novel interpretation of the origin of chromosome instability in cultured of human stem cells may have broad implications for understanding the genesis of aneuploidy across diverse biological systems.
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Affiliation(s)
- Maria Narozna
- Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Megan C. Latham
- Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Gary J. Gorbsky
- Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
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Li Z, Zhou D. Acrylate-Based PEG Hydrogels with Ultrafast Biodegradability for 3D Cell Culture. Biomacromolecules 2024; 25:6195-6202. [PMID: 39136362 DOI: 10.1021/acs.biomac.4c01051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/10/2024]
Abstract
Poly(ethylene glycol) (PEG)-based hydrogels are particularly challenging to degrade, which hinders efficient cell harvesting within the gel matrix. Here, highly branched copolymers of PEG methyl ether acrylate (PEGMA) and disulfide diacrylate (DSDA) (PEG-DS) with short primary chains and multiple pendent vinyl groups were synthesized by a "vinyl oligomer combination" approach. PEG-DS readily cross-links with thiolated gelatin (Gel-SH) to form hydrogels. Results demonstrate that shortening the primary chains of PEG-DS significantly enhances the viability of bone marrow mesenchymal stem cells (BMSCs) by up to 193.2%. Importantly, DS junctions can be easily cleaved into short primary chains using dithiothreitol (DTT), triggering ultrafast degradation of PEG-DS/Gel-SH hydrogels within 2 min under mild conditions and release of the encapsulated BMSCs. This study establishes a novel strategy to enhance the degradation of acrylate-based PEG hydrogels for three-dimensional (3D) cell culture and harvesting. These findings expand the potential applications of such hydrogels in various biomedical fields.
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Affiliation(s)
- Zhili Li
- School of Chemical Engineering and Technology, Xi'an Jiaotong University, Xi'an 710049, China
| | - Dezhong Zhou
- School of Chemical Engineering and Technology, Xi'an Jiaotong University, Xi'an 710049, China
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Algorta A, Artigas R, Rial A, Benavides U, Maisonnave J, Yaneselli K. Morphologic, Proliferative, and Cytogenetic Changes during In Vitro Propagation of Cat Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells. Animals (Basel) 2024; 14:2408. [PMID: 39199942 PMCID: PMC11350862 DOI: 10.3390/ani14162408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2024] [Revised: 07/12/2024] [Accepted: 07/18/2024] [Indexed: 09/01/2024] Open
Abstract
Stem cell therapy in cat patients needs a high quantity of mesenchymal stromal/stem cells (MSCs) requiring in vitro propagation under culture conditions which may potentially impact cellular characteristics and genetic stability. This study aimed to assess the in vitro characteristics and cytogenetic stability of cat adipose tissue-derived MSCs (cAT-MSCs). For this purpose, morphological features, clonogenic potential, and proliferative capacity of cAT-MSCs were assessed at passages 2 (P2), P4, and P6. Multipotency and immunophenotype were evaluated. Cytogenetic analyses were conducted up to P6. The cAT-MSCs exhibited a spindle-shaped morphology in early passages. The doubling time increased from 2.5 days at P2 to 9.4 at P4 and 10.5 at P6, accompanied by the observation of nuclear abnormalities such as cluster formation, karyorrhexis, karyolysis, and a decline in the mitotic index at P4. Cells demonstrated multipotency capacity and were CD45-, CD90+, and CD44+. Metaphase analysis at P2 and P4 revealed some indications of structural instability such as gaps, breaks, deletions, duplications, and early chromatid segregation, but these alterations did not show an increase across passages. In conclusion, cAT-MSCs decreased their proliferative capacity after P4, accompanied by morphological alterations and signs of structural instability.
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Affiliation(s)
- Agustina Algorta
- Unidad de Inmunología e Inmunoterapia, Departamento de Patobiología, Facultad de Veterinaria, Universidad de la República (UdelaR), Montevideo 13000, Uruguay; (U.B.); (J.M.)
| | - Rody Artigas
- Unidad de Genética y Mejoramiento Animal, Departamento de Producción Animal y Salud de los Sistemas Productivos, Facultad de Veterinaria, Universidad de la República (UdelaR), Montevideo 13000, Uruguay;
| | - Analía Rial
- Departamento de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo 11600, Uruguay;
| | - Uruguaysito Benavides
- Unidad de Inmunología e Inmunoterapia, Departamento de Patobiología, Facultad de Veterinaria, Universidad de la República (UdelaR), Montevideo 13000, Uruguay; (U.B.); (J.M.)
| | - Jacqueline Maisonnave
- Unidad de Inmunología e Inmunoterapia, Departamento de Patobiología, Facultad de Veterinaria, Universidad de la República (UdelaR), Montevideo 13000, Uruguay; (U.B.); (J.M.)
| | - Kevin Yaneselli
- Unidad de Inmunología e Inmunoterapia, Departamento de Patobiología, Facultad de Veterinaria, Universidad de la República (UdelaR), Montevideo 13000, Uruguay; (U.B.); (J.M.)
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Smith EJ, Beaumont RE, Dudhia J, Guest DJ. Equine Embryonic Stem Cell-Derived Tenocytes are Insensitive to a Combination of Inflammatory Cytokines and Have Distinct Molecular Responses Compared to Primary Tenocytes. Stem Cell Rev Rep 2024; 20:1040-1059. [PMID: 38396222 PMCID: PMC11087315 DOI: 10.1007/s12015-024-10693-8] [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] [Accepted: 02/06/2024] [Indexed: 02/25/2024]
Abstract
Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1β stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing.
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Affiliation(s)
- Emily J Smith
- Department of Clinical Sciences and Services, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK.
| | - Ross E Beaumont
- Department of Clinical Sciences and Services, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK
| | - Jayesh Dudhia
- Department of Clinical Sciences and Services, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK
| | - Deborah J Guest
- Department of Clinical Sciences and Services, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK.
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Krivec N, Ghosh MS, Spits C. Gains of 20q11.21 in human pluripotent stem cells: Insights from cancer research. Stem Cell Reports 2024; 19:11-27. [PMID: 38157850 PMCID: PMC10828824 DOI: 10.1016/j.stemcr.2023.11.013] [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: 09/08/2023] [Revised: 11/28/2023] [Accepted: 11/28/2023] [Indexed: 01/03/2024] Open
Abstract
The genetic abnormalities observed in hPSC cultures worldwide have been suggested to pose an important hurdle in their safe use in regenerative medicine due to the possibility of oncogenic transformation by mutant cells in the patient posttransplantation. One of the best-characterized genetic lesions in hPSCs is the gain of 20q11.21, found in 20% of hPSC lines worldwide, and strikingly, also amplified in 20% of human cancers. In this review, we have curated the existing knowledge on the incidence of this mutation in hPSCs and cancer, explored the significance of chromosome 20q11.21 amplification in cancer progression, and reviewed the oncogenic role of the genes in the smallest common region of gain, to shed light on the significance of this mutation in hPSC-based cell therapy. Lastly, we discuss the state-of-the-art strategies devised to detect aneuploidies in hPSC cultures, avoid genetic changes in vitro cultures of hPSCs, and strategies to eliminate genetically abnormal cells from culture.
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Affiliation(s)
- Nuša Krivec
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - Manjusha S Ghosh
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - Claudia Spits
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium.
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Meshrkey F, Scheulin KM, Littlejohn CM, Stabach J, Saikia B, Thorat V, Huang Y, LaFramboise T, Lesnefsky EJ, Rao RR, West FD, Iyer S. Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential. Stem Cell Res Ther 2023; 14:320. [PMID: 37936209 PMCID: PMC10631039 DOI: 10.1186/s13287-023-03546-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Accepted: 10/25/2023] [Indexed: 11/09/2023] Open
Abstract
BACKGROUND Human mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype. METHODS hiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer. RESULTS Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential. CONCLUSION Overall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.
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Affiliation(s)
- Fibi Meshrkey
- Department of Biological Sciences, J. William Fulbright College of Arts and Sciences, University of Arkansas, Science and Engineering 601, Fayetteville, AR, 72701, USA
- Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA
- Department of Histology and Cell Biology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
| | - Kelly M Scheulin
- Regenerative Bioscience Center, University of Georgia, Athens, GA, USA
- Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA
- Neuroscience Program, Biomedical and Health Sciences Institute, University of Georgia, Athens, GA, USA
| | - Christopher M Littlejohn
- Regenerative Bioscience Center, University of Georgia, Athens, GA, USA
- Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA
| | - Joshua Stabach
- Department of Biological Sciences, J. William Fulbright College of Arts and Sciences, University of Arkansas, Science and Engineering 601, Fayetteville, AR, 72701, USA
| | - Bibhuti Saikia
- Department of Biological Sciences, J. William Fulbright College of Arts and Sciences, University of Arkansas, Science and Engineering 601, Fayetteville, AR, 72701, USA
| | - Vedant Thorat
- Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH, USA
| | - Yimin Huang
- Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH, USA
| | - Thomas LaFramboise
- Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH, USA
| | - Edward J Lesnefsky
- Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, USA
- Cardiology Section Medical Service, McGuire Veterans Affairs Medical Center, Richmond, VA, USA
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA
- Division of Cardiology, Department of Internal Medicine, Pauley Heart Center, Virginia Commonwealth University, Richmond, VA, USA
| | - Raj R Rao
- Department of Biomedical Engineering, College of Engineering, University of Arkansas, Fayetteville, AR, USA
| | - Franklin D West
- Regenerative Bioscience Center, University of Georgia, Athens, GA, USA
- Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA
- Neuroscience Program, Biomedical and Health Sciences Institute, University of Georgia, Athens, GA, USA
| | - Shilpa Iyer
- Department of Biological Sciences, J. William Fulbright College of Arts and Sciences, University of Arkansas, Science and Engineering 601, Fayetteville, AR, 72701, USA.
- Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA.
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Hao J, Chen Y, Zhu M, Zhao Y, Zhang K, Xu X. Spatial-Temporal Heterogeneity in Large Three-Dimensional Nanofibrillar Cellulose Hydrogel for Human Pluripotent Stem Cell Culture. Gels 2023; 9:gels9040324. [PMID: 37102936 PMCID: PMC10138276 DOI: 10.3390/gels9040324] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2023] [Revised: 04/03/2023] [Accepted: 04/06/2023] [Indexed: 04/28/2023] Open
Abstract
One approach to cell expansion is to use large hydrogel for growing a large number of cells. Nanofibrillar cellulose (NFC) hydrogel has been used for human induced pluripotent stem cell (hiPSCs) expansion. However, little is known about the status of hiPSCs at the single cell level inside large NFC hydrogel during culture. To understand the effect of NFC hydrogel property on temporal-spatial heterogeneity, hiPSCs were cultured in 0.8 wt% NFC hydrogel with different thicknesses with the top surface exposed to the culture medium. The prepared hydrogel exhibits less restriction in mass transfer due to the presence of macropores and micropores interconnecting the macropores. More than 85% of cells at different depths survive after 5 days of culture inside 3.5 mm thick hydrogel. Biological compositions at different zones inside the NFC gel were examined over time at a single-cell level. A dramatic concentration gradient of growth factors estimated in the simulation along 3.5 mm NFC hydrogel could be a reason for the spatial-temporal heterogeneity in protein secondary structure and protein glycosylation and pluripotency loss at the bottom zone. pH change caused by the lactic acid accumulation over time leads to changes in cellulose charge and growth factor potential, probably another reason for the heterogeneity in biochemical compositions. This study may help to develop optimal conditions for producing high-quality hiPSCs in large nanofibrillar cellulose hydrogel at scale.
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Affiliation(s)
- Jin Hao
- Biochemical Engineering Research Center, Anhui University of Technology, Ma'anshan 243002, China
- School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan 243002, China
| | - Ying Chen
- Biochemical Engineering Research Center, Anhui University of Technology, Ma'anshan 243002, China
- School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan 243002, China
| | - Mingjian Zhu
- Biochemical Engineering Research Center, Anhui University of Technology, Ma'anshan 243002, China
- School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan 243002, China
| | - Yingqing Zhao
- Biochemical Engineering Research Center, Anhui University of Technology, Ma'anshan 243002, China
- School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan 243002, China
| | - Kai Zhang
- Biochemical Engineering Research Center, Anhui University of Technology, Ma'anshan 243002, China
- School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan 243002, China
| | - Xia Xu
- Biochemical Engineering Research Center, Anhui University of Technology, Ma'anshan 243002, China
- School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan 243002, China
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Algorta A, Artigas R, Rial A, Brandl S, Rodellar C, Benavides U, Maisonnave J, Yaneselli K. Isolation and characterization of feline dental pulp stem cells. J Feline Med Surg 2023; 25:1098612X221150625. [PMID: 36745130 PMCID: PMC10812064 DOI: 10.1177/1098612x221150625] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
OBJECTIVES The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability. METHODS Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P2, P4 and P6, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P2, and cells were immunophenotyped in P4 by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P2, P4 and P6. RESULTS The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P2 to P6. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45-, CD90+ and CD44+. Structural and numerical cytogenetic aberrations were observed in P2-P4. CONCLUSIONS AND RELEVANCE In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.
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Affiliation(s)
- Agustina Algorta
- Immunology and Immunotherapy Unit, Department of Patobiology, Veterinary Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
- Odontostomatology Service, Veterinary Hospital Clinical Department, Veterinary Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
| | - Rody Artigas
- Animal Genetics and Improvement Unit, Department of Animal Production and Health Production Systems, Veterinary Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
| | - Analía Rial
- Department of Biotechnology Development, Hygiene Institute, Medical Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
| | - Scott Brandl
- Microbiology Unit, Department of Pathobiology, Veterinary Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
| | - Clementina Rodellar
- LAGENBIO, Veterinary Faculty, Food and Agriculture Institute of Aragón-IA2, University of Zaragoza-CITA, Zaragoza, Spain
| | - Uruguaysito Benavides
- Immunology and Immunotherapy Unit, Department of Patobiology, Veterinary Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
| | - Jacqueline Maisonnave
- Immunology and Immunotherapy Unit, Department of Patobiology, Veterinary Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
| | - Kevin Yaneselli
- Immunology and Immunotherapy Unit, Department of Patobiology, Veterinary Faculty, University of the Republic (UdelaR), Montevideo, Uruguay
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A novel feature for monitoring the enzymatic harvesting process of adherent cell cultures based on lens-free imaging. Sci Rep 2022; 12:22202. [PMID: 36564377 PMCID: PMC9789138 DOI: 10.1038/s41598-022-22561-x] [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: 05/18/2022] [Accepted: 10/17/2022] [Indexed: 12/24/2022] Open
Abstract
Adherent cell cultures are often dissociated from their culture vessel (and each other) through enzymatic harvesting, where the detachment response is monitored by an operator. However, this approach is lacking standardisation and reproducibility, and prolonged exposure or too high concentrations can affect the cell's viability and differentiation potential. Quantitative monitoring systems are required to characterise the cell detachment response and objectively determine the optimal time-point to inhibit the enzymatic reaction. State-of-the-art methodologies rely on bulky imaging systems and/or features (e.g. circularity) that lack robustness. In this study, lens-free imaging (LFI) technology was used to develop a novel cell detachment feature. Seven different donors were cultured and subsequently harvested with a (diluted) enzymatic harvesting solution after 3, 5 and 7 days of culture. Cell detachment was captured with the LFI set-up over a period of 20 min (every 20 s) and by optimising the reconstruction of the LFI intensity images, a new feature could be identified. Bright regions in the intensity image were identified as detaching cells and using image analysis, a method was developed to automatically extract this feature, defined as the percentage of detached cell regions. Next, the method was quantitatively and qualitatively validated on a diverse set of images. Average absolute error values of 1.49%, 1.34% and 1.97% were obtained for medium to high density and overconfluent cultures, respectively. The detachment response was quantified for all conditions and the optimal time for enzyme inhibition was reached when approximately 92.5% of the cells were detached. On average, inhibition times of 9.6-11.1 and 16.2-17.2 min were obtained for medium to high density and overconfluent cultures, respectively. In general, overconfluent cultures detached much slower, while their detachment rate was also decreased by the diluted harvesting solution. Moreover, several donors exhibited similar trends in cell detachment behaviour, with two clear outliers. Using the novel feature, measurements can be performed with an increased robustness, while the compact LFI design could pave the way for in situ monitoring in a variety of culture vessels, including bioreactors.
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Lam ATL, Ho V, Vassilev S, Reuveny S, Oh SKW. An allied reprogramming, selection, expansion and differentiation platform for creating hiPSC on microcarriers. Cell Prolif 2022; 55:e13256. [PMID: 36574589 PMCID: PMC9357361 DOI: 10.1111/cpr.13256] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 04/19/2022] [Accepted: 04/28/2022] [Indexed: 12/30/2022] Open
Abstract
OBJECTIVES Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges. MATERIALS AND METHODS Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures. RESULTS Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50-fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β-catenin, E-cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ-layers, cardiomyocytes and haematopoietic stem cells were further demonstrated. CONCLUSIONS Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.
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Affiliation(s)
- Alan Tin Lun Lam
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Valerie Ho
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Svetlan Vassilev
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Shaul Reuveny
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
| | - Steve Kah Weng Oh
- Stem Cell Bioprocessing, Bioprocessing Technology InstituteAgency for Science, Technology and ResearchSingaporeRepublic of Singapore
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DuBose CO, Daum JR, Sansam CL, Gorbsky GJ. Dynamic Features of Chromosomal Instability during Culture of Induced Pluripotent Stem Cells. Genes (Basel) 2022; 13:genes13071157. [PMID: 35885940 PMCID: PMC9318709 DOI: 10.3390/genes13071157] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2022] [Revised: 06/15/2022] [Accepted: 06/21/2022] [Indexed: 02/04/2023] Open
Abstract
Induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine. By reprogramming a patient′s own cells, immunological rejection can be avoided during transplantation. For expansion and gene editing, iPSCs are grown in artificial culture for extended times. Culture affords potential danger for the accumulation of genetic aberrations. To study these, two induced pluripotent stem (iPS) cell lines were cultured and periodically analyzed using advanced optical mapping to detect and classify chromosome numerical and segmental changes that included deletions, insertions, balanced translocations and inversions. In one of the lines, a population trisomic for chromosome 12 gained dominance over a small number of passages. This appearance and dominance of the culture by chromosome 12 trisomic cells was tracked through intermediate passages by the analysis of chromosome spreads. Mathematical modeling suggested that the proliferation rates of diploid versus trisomic cells could not account for the rapid dominance of the trisomic population. In addition, optical mapping revealed hundreds of structural variations distinct from those generally found within the human population. Many of these structural variants were detected in samples obtained early in the culturing process and were maintained in late passage samples, while others were acquired over the course of culturing.
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Affiliation(s)
- Casey O. DuBose
- Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; (C.O.D.); (J.R.D.)
| | - John R. Daum
- Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; (C.O.D.); (J.R.D.)
| | - Christopher L. Sansam
- Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; (C.O.D.); (J.R.D.)
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
| | - Gary J. Gorbsky
- Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; (C.O.D.); (J.R.D.)
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
- Correspondence:
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12
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Molina-Ruiz FJ, Introna C, Bombau G, Galofre M, Canals JM. Standardization of Cell Culture Conditions and Routine Genomic Screening under a Quality Management System Leads to Reduced Genomic Instability in hPSCs. Cells 2022; 11:cells11131984. [PMID: 35805069 PMCID: PMC9265327 DOI: 10.3390/cells11131984] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2022] [Revised: 06/11/2022] [Accepted: 06/13/2022] [Indexed: 01/27/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) have generated unprecedented interest in the scientific community, given their potential applications in regenerative medicine, disease modeling, toxicology and drug screening. However, hPSCs are prone to acquire genomic alterations in vitro, mainly due to suboptimal culture conditions and inappropriate routines to monitor genome integrity. This poses a challenge to both the safety of clinical applications and the reliability of basic and translational hPSC research. In this study, we aim to investigate if the implementation of a Quality Management System (QMS) such as ISO9001:2015 to ensure reproducible and standardized cell culture conditions and genomic screening strategies can decrease the prevalence of genomic alterations affecting hPSCs used for research applications. To this aim, we performed a retrospective analysis of G-banding karyotype and Comparative Genomic Hybridization array (aCGH) data generated by our group over a 5-year span of different hESC and hiPSC cultures. This work demonstrates that application of a QMS to standardize cell culture conditions and genomic monitoring routines leads to a striking improvement of genomic stability in hPSCs cultured in vitro, as evidenced by a reduced probability of potentially pathogenic chromosomal aberrations and subchromosomal genomic alterations. These results support the need to implement QMS in academic laboratories performing hPSC research.
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Affiliation(s)
- Francisco J. Molina-Ruiz
- Laboratory of Stem Cells and Regenerative Medicine, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain; (F.J.M.-R.); (C.I.); (G.B.); (M.G.)
- Creatio, Production and Validation Center of Advanced Therapies, Faculty of Medicine and Health Science, University of Barcelona, 08036 Barcelona, Spain
- Institute of Neurosciences, University of Barcelona, 08036 Barcelona, Spain
- Networked Biomedical Research Centre for Neurodegenerative Disorders (CIBERNED), 08036 Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain
| | - Clelia Introna
- Laboratory of Stem Cells and Regenerative Medicine, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain; (F.J.M.-R.); (C.I.); (G.B.); (M.G.)
- Creatio, Production and Validation Center of Advanced Therapies, Faculty of Medicine and Health Science, University of Barcelona, 08036 Barcelona, Spain
- Institute of Neurosciences, University of Barcelona, 08036 Barcelona, Spain
- Networked Biomedical Research Centre for Neurodegenerative Disorders (CIBERNED), 08036 Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain
| | - Georgina Bombau
- Laboratory of Stem Cells and Regenerative Medicine, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain; (F.J.M.-R.); (C.I.); (G.B.); (M.G.)
- Creatio, Production and Validation Center of Advanced Therapies, Faculty of Medicine and Health Science, University of Barcelona, 08036 Barcelona, Spain
- Institute of Neurosciences, University of Barcelona, 08036 Barcelona, Spain
- Networked Biomedical Research Centre for Neurodegenerative Disorders (CIBERNED), 08036 Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain
| | - Mireia Galofre
- Laboratory of Stem Cells and Regenerative Medicine, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain; (F.J.M.-R.); (C.I.); (G.B.); (M.G.)
- Creatio, Production and Validation Center of Advanced Therapies, Faculty of Medicine and Health Science, University of Barcelona, 08036 Barcelona, Spain
- Institute of Neurosciences, University of Barcelona, 08036 Barcelona, Spain
- Networked Biomedical Research Centre for Neurodegenerative Disorders (CIBERNED), 08036 Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain
| | - Josep M. Canals
- Laboratory of Stem Cells and Regenerative Medicine, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain; (F.J.M.-R.); (C.I.); (G.B.); (M.G.)
- Creatio, Production and Validation Center of Advanced Therapies, Faculty of Medicine and Health Science, University of Barcelona, 08036 Barcelona, Spain
- Institute of Neurosciences, University of Barcelona, 08036 Barcelona, Spain
- Networked Biomedical Research Centre for Neurodegenerative Disorders (CIBERNED), 08036 Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain
- Correspondence: ; Tel.: +34-934-035-288
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13
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Picerno A, Stasi A, Franzin R, Curci C, di Bari I, Gesualdo L, Sallustio F. Why stem/progenitor cells lose their regenerative potential. World J Stem Cells 2021; 13:1714-1732. [PMID: 34909119 PMCID: PMC8641024 DOI: 10.4252/wjsc.v13.i11.1714] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Revised: 05/26/2021] [Accepted: 10/31/2021] [Indexed: 02/06/2023] Open
Abstract
Nowadays, it is clear that adult stem cells, also called as tissue stem cells, play a central role to repair and maintain the tissue in which they reside by their self-renewal ability and capacity of differentiating into distinct and specialized cells. As stem cells age, their renewal ability declines and their capacity to maintain organ homeostasis and regeneration is impaired. From a molecular perspective, these changes in stem cells properties can be due to several types of cell intrinsic injury and DNA aberrant alteration (i.e epigenomic profile) as well as changes in the tissue microenviroment, both into the niche and by systemic circulating factors. Strikingly, it has been suggested that aging-induced deterioration of stem cell functions may play a key role in the pathophysiology of the various aging-associated disorders. Therefore, understanding how resident stem cell age and affects near and distant tissues is fundamental. Here, we examine the current knowledge about aging mechanisms in several kinds of adult stem cells under physiological and pathological conditions and the principal aging-related changes in number, function and phenotype that determine the loss of tissue renewal properties. Furthermore, we examine the possible cell rejuvenation strategies. Stem cell rejuvenation may reverse the aging phenotype and the discovery of effective methods for inducing and differentiating pluripotent stem cells for cell replacement therapies could open up new possibilities for treating age-related diseases.
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Affiliation(s)
- Angela Picerno
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Alessandra Stasi
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Rossana Franzin
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Claudia Curci
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Ighli di Bari
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Loreto Gesualdo
- Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Bari 70124, Italy
| | - Fabio Sallustio
- Department of Interdisciplinary Medicine, University of Bari "Aldo Moro", Bari 70124, Italy
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14
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de Souza AF, Bressan FF, Pieri NCG, Botigelli RC, Revay T, Haddad SK, Covas DT, Ramos ES, King WA, Meirelles FV. Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients. Cells 2021; 10:cells10113099. [PMID: 34831322 PMCID: PMC8624672 DOI: 10.3390/cells10113099] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Revised: 11/02/2021] [Accepted: 11/04/2021] [Indexed: 12/31/2022] Open
Abstract
Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.
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Affiliation(s)
- Aline Fernanda de Souza
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (F.F.B.); (N.C.G.P.); (R.C.B.)
- Department of Biomedical Sciences, Ontario Veterinary College (OVC), University of Guelph, Guelph, ON N1G 2W1, Canada;
- Correspondence: (A.F.d.S.); (F.V.M.)
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (F.F.B.); (N.C.G.P.); (R.C.B.)
| | - Naira Caroline Godoy Pieri
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (F.F.B.); (N.C.G.P.); (R.C.B.)
| | - Ramon Cesar Botigelli
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (F.F.B.); (N.C.G.P.); (R.C.B.)
- Department of Pharmacology, Institute of Biosciences (IBB), São Paulo State University (UNESP), Botucatu 18618-689, Brazil
| | - Tamas Revay
- Department Alberta Children’s Hospital Research Institute (ACHRI), University of Calgary, Calgary, AB T2N 4N1, Canada;
| | - Simone Kashima Haddad
- Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, Brazil; (S.K.H.); (D.T.C.)
| | - Dimas Tadeu Covas
- Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14051-060, Brazil; (S.K.H.); (D.T.C.)
| | - Ester Silveira Ramos
- Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14049-900, Brazil;
| | - Willian Allan King
- Department of Biomedical Sciences, Ontario Veterinary College (OVC), University of Guelph, Guelph, ON N1G 2W1, Canada;
| | - Flavio Vieira Meirelles
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga 13635-000, Brazil; (F.F.B.); (N.C.G.P.); (R.C.B.)
- Correspondence: (A.F.d.S.); (F.V.M.)
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15
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van Essen M, Riepsaame J, Jacob J. CRISPR-Cas Gene Perturbation and Editing in Human Induced Pluripotent Stem Cells. CRISPR J 2021; 4:634-655. [PMID: 34582693 DOI: 10.1089/crispr.2021.0063] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Directing the fates of human pluripotent stem cells (hPSC) to generate a multitude of differentiated cell types allows the study of the genetic regulation of human development and disease. The translational potential of hPSC is maximized by exploiting CRISPR to silence or activate genes with spatial and temporal precision permanently or reversibly. Here, we summarize the increasingly refined and diverse CRISPR toolkit for the latter forms of gene perturbation in hPSC and their downstream applications. We discuss newer methods to install edits efficiently with single nucleotide resolution and describe pooled CRISPR screens as a powerful means of unbiased discovery of genes associated with a phenotype of interest. Last, we discuss the potential of these combined technologies in the treatment of hitherto intractable human diseases and the challenges to their implementation in the clinic.
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Affiliation(s)
- Max van Essen
- Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; and University of Oxford, Oxford, United Kingdom
| | - Joey Riepsaame
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - John Jacob
- Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; and University of Oxford, Oxford, United Kingdom
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16
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Riggs MJ, Sheridan SD, Rao RR. ARHGDIA Confers Selective Advantage to Dissociated Human Pluripotent Stem Cells. Stem Cells Dev 2021; 30:705-713. [PMID: 34036793 PMCID: PMC8309423 DOI: 10.1089/scd.2021.0079] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) have generated significant interest in the scientific community based on their potential applications in regenerative medicine. However, numerous research groups have reported a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. Work from our laboratory and others has demonstrated that amplification of chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during clinical use. In this study, we focus on trisomy 17 and a list of candidate genes with increased gene expression to hypothesize that overexpressing 17q25 located ARHGDIA will confer selective advantage to hPSCs. HPSC lines overexpressing ARHGDIA exhibited culture dominance in co-cultures of overexpression lines with nonoverexpression lines. Furthermore, during low-density seeding, we demonstrate increased clonality of our ARHGDIA lines against matched controls. A striking observation is that we could reduce this selective advantage by varying the hPSC culture conditions with the addition of ROCK inhibitor (ROCKi). This work is unique in (1) demonstrating a novel gene that confers selective advantage to hPSCs when overexpressed and may help explain a common trisomy dominance, (2) providing a selection model for studying culture conditions that reduce the appearance of genomically altered hPSCs, and (3) aiding in elucidation of a mechanism that may act as a molecular switch during culture adaptation.
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Affiliation(s)
- Marion J Riggs
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, USA.,Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.,Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA
| | - Steven D Sheridan
- Center for Quantitative Health, Center for Genomic Medicine and Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts, USA.,Department of Psychiatry, Harvard Medical School, Boston, Massachusetts, USA
| | - Raj R Rao
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, USA.,Department of Biomedical Engineering, College of Engineering, University of Arkansas, Fayetteville, Arkansas, USA
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17
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Skorik C, Mullin NK, Shi M, Zhang Y, Hunter P, Tang Y, Hilton B, Schlaeger TM. Xeno-Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin-521. ACTA ACUST UNITED AC 2021; 52:e103. [PMID: 31977148 DOI: 10.1002/cpsc.103] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Translating human induced pluripotent stem cell (hiPSC)-derived cells and tissues into the clinic requires streamlined and reliable production of clinical-grade hiPSCs. This article describes an entirely animal component-free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno-free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component-free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP-grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP-grade human laminin substrate is compatible with a number of xeno-free or chemically defined media (some available as cGMP-grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent-derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolating peripheral blood mononuclear cells using CPT tubes Support Protocol 1: Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1 Basic Protocol 2: PBMC expansion in an animal-free erythroblast expansion medium containing autologous serum Basic Protocol 3: Reprogramming of expanded PBMCs with Sendai viral reprogramming particles Alternate Protocol: Reprogramming of expanded PBMCs with episomal plasmids Basic Protocol 4: Picking, expanding, and cryopreserving hiPSC clones Support Protocol 2: Testing Sendai virus kit-reprogrammed hiPSC for absence of Sendai viral RNA Support Protocol 3: Testing Epi5 kit-reprogrammed hiPSC for absence of episomal plasmid DNA Support Protocol 4: Assessing the undifferentiated state of human pluripotent stem cell cultures by multi-color immunofluorescent staining and confocal imaging Support Protocol 5: Coating plates with extracellular matrices to support hiPSC attachment and expansion.
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Affiliation(s)
- Christian Skorik
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Stemcell Technologies, Cambridge, Massachusetts
| | - Nathaniel K Mullin
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Michael Shi
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,School of Medicine, Case Western Reserve University, Cleveland, Ohio
| | - Yosra Zhang
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Stemcell Technologies, Cambridge, Massachusetts
| | - Phoebe Hunter
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts
| | - Yang Tang
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts
| | - Brianna Hilton
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts
| | - Thorsten M Schlaeger
- Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.,Harvard Stem Cell Institute, Boston, Massachusetts.,Harvard Medical School, Boston, Massachusetts
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18
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Li C, Mills Z, Zheng Z. Novel cell sources for bone regeneration. MedComm (Beijing) 2021; 2:145-174. [PMID: 34766140 PMCID: PMC8491221 DOI: 10.1002/mco2.51] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 12/03/2020] [Accepted: 12/09/2020] [Indexed: 01/09/2023] Open
Abstract
A plethora of both acute and chronic conditions, including traumatic, degenerative, malignant, or congenital disorders, commonly induce bone disorders often associated with severe persisting pain and limited mobility. Over 1 million surgical procedures involving bone excision, bone grafting, and fracture repair are performed each year in the U.S. alone, resulting in immense levels of public health challenges and corresponding financial burdens. Unfortunately, the innate self-healing capacity of bone is often inadequate for larger defects over a critical size. Moreover, as direct transplantation of committed osteoblasts is hindered by deficient cell availability, limited cell spreading, and poor survivability, an urgent need for novel cell sources for bone regeneration is concurrent. Thanks to the development in stem cell biology and cell reprogramming technology, many multipotent and pluripotent cells that manifest promising osteogenic potential are considered the regenerative remedy for bone defects. Considering these cells' investigation is still in its relative infancy, each of them offers their own particular challenges that must be conquered before the large-scale clinical application.
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Affiliation(s)
- Chenshuang Li
- Department of Orthodontics, School of Dental MedicineUniversity of PennsylvaniaPhiladelphiaPennsylvaniaUSA
| | - Zane Mills
- College of DentistryUniversity of OklahomaOklahoma CityOklahomaUSA
| | - Zhong Zheng
- Division of Growth and Development, School of DentistryUniversity of CaliforniaLos AngelesCaliforniaUSA
- Department of Surgery, David Geffen School of MedicineUniversity of CaliforniaLos AngelesCaliforniaUSA
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19
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Sustained intrinsic WNT and BMP4 activation impairs hESC differentiation to definitive endoderm and drives the cells towards extra-embryonic mesoderm. Sci Rep 2021; 11:8242. [PMID: 33859268 PMCID: PMC8050086 DOI: 10.1038/s41598-021-87547-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2020] [Accepted: 03/31/2021] [Indexed: 12/13/2022] Open
Abstract
We identified a human embryonic stem cell subline that fails to respond to the differentiation cues needed to obtain endoderm derivatives, differentiating instead into extra-embryonic mesoderm. RNA-sequencing analysis showed that the subline has hyperactivation of the WNT and BMP4 signalling. Modulation of these pathways with small molecules confirmed them as the cause of the differentiation impairment. While activation of WNT and BMP4 in control cells resulted in a loss of endoderm differentiation and induction of extra-embryonic mesoderm markers, inhibition of these pathways in the subline restored its ability to differentiate. Karyotyping and exome sequencing analysis did not identify any changes in the genome that could account for the pathway deregulation. These findings add to the increasing evidence that different responses of stem cell lines to differentiation protocols are based on genetic and epigenetic factors, inherent to the line or acquired during cell culture.
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20
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Carvalho VS, Rissino JD, Nagamachi CY, Pieczarka JC, Noronha RCR. Isolation and establishment of skin-derived and mesenchymal cells from south American bat Artibeus planirostris (Chiroptera - Phyllostomidae). Tissue Cell 2021; 71:101507. [PMID: 33592503 DOI: 10.1016/j.tice.2021.101507] [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: 11/12/2020] [Revised: 02/02/2021] [Accepted: 02/03/2021] [Indexed: 11/24/2022]
Abstract
Animal models represent a crucial tool for biological research, so the establishment of new cultures is fundamental for the discovery of new therapies and the understanding of mechanisms of cell development in the most diverse animals. Here, we report the successful establishment of two new primary cell cultures derived from a South American bat (Artibeus planirostris). The establishment of a new bat culture can help in the investigation of new zoonoses since bats have been proposed as carriers of these diseases. We evaluated the chromosomal stability of cells from different passages. Primary cultures were collected from ear tissues and bone marrow of A. planirostris. Cultures were expanded, and osteogenic and adipogenic inductions were conducted for 21 days. For osteogenic differentiation, the medium was supplemented with 0.1 μM dexamethasone, 3 mM β-glycerophosphate, and 10 μM L-ascorbic acid 2-phosphate. For adipogenic differentiation, the medium was supplemented with 5 μM rosiglitazone, 0.4 μM insulin, 0.1 mM indomethacin, and 0.1 μM dexamethasone. After the induction period, the cells were stained with Alizarin Red to assess osteogenic differentiation and Oil Red O to assess adipogenic differentiation. We observed the appearance of lipid droplets in adipocytes and the extracellular deposition of calcium matrix by osteocytes, indicating that bone marrow-derived cells and skin-derived cells of A. planirostris could successfully differentiate into these lineages. Also, the number of chromosomes remained stable for both primary cultures during passages 2, 4, 6, and 8.
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Affiliation(s)
- Vinícius S Carvalho
- Laboratório De Citogenética, Centro De Estudos Avançados Em Biodiversidade, Instituto De Ciências Biológicas, Universidade Federal Do Pará, Campus Guamá, Rua Augusto Corrêa, nº 01, Guamá, CEP 66075-900, Belém, Pará, Brazil
| | - Jorge D Rissino
- Laboratório De Citogenética, Centro De Estudos Avançados Em Biodiversidade, Instituto De Ciências Biológicas, Universidade Federal Do Pará, Campus Guamá, Rua Augusto Corrêa, nº 01, Guamá, CEP 66075-900, Belém, Pará, Brazil
| | - Cleusa Y Nagamachi
- Laboratório De Citogenética, Centro De Estudos Avançados Em Biodiversidade, Instituto De Ciências Biológicas, Universidade Federal Do Pará, Campus Guamá, Rua Augusto Corrêa, nº 01, Guamá, CEP 66075-900, Belém, Pará, Brazil
| | - Julio C Pieczarka
- Laboratório De Citogenética, Centro De Estudos Avançados Em Biodiversidade, Instituto De Ciências Biológicas, Universidade Federal Do Pará, Campus Guamá, Rua Augusto Corrêa, nº 01, Guamá, CEP 66075-900, Belém, Pará, Brazil
| | - Renata C R Noronha
- Laboratório De Citogenética, Centro De Estudos Avançados Em Biodiversidade, Instituto De Ciências Biológicas, Universidade Federal Do Pará, Campus Guamá, Rua Augusto Corrêa, nº 01, Guamá, CEP 66075-900, Belém, Pará, Brazil.
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Carpenedo RL, Kwon SY, Tanner RM, Yockell-Lelièvre J, Choey C, Doré C, Ho M, Stewart DJ, Perkins TJ, Stanford WL. Transcriptomically Guided Mesendoderm Induction of Human Pluripotent Stem Cells Using a Systematically Defined Culture Scheme. Stem Cell Reports 2020; 13:1111-1125. [PMID: 31813826 PMCID: PMC6915803 DOI: 10.1016/j.stemcr.2019.11.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2019] [Revised: 11/03/2019] [Accepted: 11/05/2019] [Indexed: 01/11/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.
Single-cell seeding with BMP4/Activin A treatment supports hPSC mesendoderm induction The mesendoderm protocol is amenable to mesoderm and endoderm lineage differentiation Mesoderm/endoderm formation was enhanced in basal conditions in vitro and in vivo
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Affiliation(s)
- Richard L Carpenedo
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Ottawa Institute of Systems Biology, Ottawa, ON, Canada.
| | - Sarah Y Kwon
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - R Matthew Tanner
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Ottawa Institute of Systems Biology, Ottawa, ON, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Julien Yockell-Lelièvre
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Chandarong Choey
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Carole Doré
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Mirabelle Ho
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Duncan J Stewart
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Sinclair Centre for Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Theodore J Perkins
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Ottawa Bioinformatics Core Facility, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada
| | - William L Stanford
- The Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Ottawa Institute of Systems Biology, Ottawa, ON, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada; Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada.
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22
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Nowak-Imialek M, Wunderlich S, Herrmann D, Breitschuh-Leibling S, Gohring G, Petersen B, Klein S, Baulain U, Lucas-Hahn A, Martin U, Niemann H. In Vitro and In Vivo Interspecies Chimera Assay Using Early Pig Embryos. Cell Reprogram 2020; 22:118-133. [PMID: 32429746 DOI: 10.1089/cell.2019.0107] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Chimeric pigs harboring organs derived from human stem cells are promising for patient-specific regenerative therapies. Induced pluripotent stem cells (iPSCs) can contribute to all cell types of the fetus, including germline after injection into embryos. However, ethical concerns prohibit testing human iPSCs in chimera assays. Here, we evaluated porcine embryos as hosts for an interspecies chimera assay using iPSCs from either cynomolgus monkeys (cyiPSCs) or mouse (miPSCs). To establish an in vitro culture system compatible for cyiPSCs and porcine embryos, we determined blastocyst development in eight different stem cell media. The highest developmental rates of blastocysts were achieved in Knockout Dulbecco's modified Eagle's medium with 20% knockout serum replacement. We found that cyiPSCs injected into porcine embryos survived in vitro and were mostly located in the trophectoderm (TE). Instead, when miPSCs were injected into porcine embryos, the cells rapidly proliferated. The behavior of chimeras developed in vitro was recapitulated in vivo; cyiPSCs were observed in the TE, but not in the porcine epiblast. However, when miPSCs were injected into in vivo derived porcine embryos, mouse cells were found in both, the epiblast and TE. These results demonstrate that porcine embryos could be useful for evaluating the interspecies chimera-forming ability of iPSCs from different species.
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Affiliation(s)
- Monika Nowak-Imialek
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany.,REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Stephanie Wunderlich
- Leibniz Research Laboratories for Biotechnology and Artificial Organs-LEBAO, Hannover Medical School, Hannover, Germany
| | - Doris Herrmann
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany
| | | | - Gudrun Gohring
- Department of Human Genetics, Hannover Medical School, Hannover, Germany
| | - Björn Petersen
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany
| | - Sabine Klein
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany
| | - Ulrich Baulain
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany
| | - Andrea Lucas-Hahn
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany
| | - Ulrich Martin
- REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.,Leibniz Research Laboratories for Biotechnology and Artificial Organs-LEBAO, Hannover Medical School, Hannover, Germany
| | - Heiner Niemann
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt, Germany.,REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
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23
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Paterson YZ, Cribbs A, Espenel M, Smith EJ, Henson FMD, Guest DJ. Genome-wide transcriptome analysis reveals equine embryonic stem cell-derived tenocytes resemble fetal, not adult tenocytes. Stem Cell Res Ther 2020; 11:184. [PMID: 32430075 PMCID: PMC7238619 DOI: 10.1186/s13287-020-01692-w] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2020] [Revised: 04/21/2020] [Accepted: 04/28/2020] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND Tendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic. METHODS Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture were compared using RNA sequencing. RESULTS No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome-wide transcriptomic analysis using RNA sequencing revealed that ESC-derived tenocytes' transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this study adds to the growing evidence that monolayer cultured cells' gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed. CONCLUSION The information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.
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Affiliation(s)
- Y. Z. Paterson
- Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES UK
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - A. Cribbs
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, OX3 7LD UK
| | - M. Espenel
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - E. J. Smith
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - F. M. D. Henson
- Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES UK
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
| | - D. J. Guest
- Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU UK
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24
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He Q, Liao Y, Zhang J, Yao X, Zhou W, Hong Y, Ouyang H. "All-in-One" Gel System for Whole Procedure of Stem-Cell Amplification and Tissue Engineering. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2020; 16:e1906539. [PMID: 32141227 DOI: 10.1002/smll.201906539] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 01/20/2020] [Indexed: 06/10/2023]
Abstract
Microsphere (MS)-based systems provides great advantages for cell expansion and transplantation due to their high surface-to-volume ratio and biomimetic environment. However, a MS-based system that includes cell attachment, proliferation, passage, harvest, cryopreservation, and tissue engineering together has not been realized yet. An "all-in-one" gel MS-based system is established for human adipose-derived mesenchymal stem cells (hADSCs), realizing real 3D culture with enhanced expansion efficiency and simplified serial cell culture operations, and construction of macrotissues with uniform cell distribution and specific function. A 3D digital light-processing technology is developed to fabricate gel MSs in an effective way. The printed MSs present a suitable environment with rough surface architecture and the mechanical properties of soft tissues, leading to high cell viability, attachment, proliferation, activity, and differentiation potential. Further, convenient standard operation procedures, including cell passage, detachment, and cryopreservation, are established for cell culture on the gel MSs. Finally, hADSCs-loaded gel MSs form macrotissues through a "bottom-up" approach, which demonstrates the potential applications for tissue engineering. These findings exhibit the feasibility and beauty of "all-in-one" stem cell culture and tissue engineering system.
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Affiliation(s)
- Qiulin He
- Department of Orthopaedic Surgery, Second Affiliated Hospital and Zhejiang University-University of Edinburgh Institute and School of Basic Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Dr. Li Dak Sum and Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, 310058, China
| | - Youguo Liao
- Dr. Li Dak Sum and Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Guangxi Collaborative Innovation Center for Biomedicine, Guangxi Medical University, Nanning, 530021, China
| | - Jingwei Zhang
- Dr. Li Dak Sum and Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, 310058, China
| | - Xudong Yao
- Department of Orthopaedic Surgery, Second Affiliated Hospital and Zhejiang University-University of Edinburgh Institute and School of Basic Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Dr. Li Dak Sum and Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, 310058, China
| | - Wenyan Zhou
- Department of Orthopaedic Surgery, Second Affiliated Hospital and Zhejiang University-University of Edinburgh Institute and School of Basic Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Dr. Li Dak Sum and Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, 310058, China
| | - Yi Hong
- Department of Orthopaedic Surgery, Second Affiliated Hospital and Zhejiang University-University of Edinburgh Institute and School of Basic Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Dr. Li Dak Sum and Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, 310058, China
| | - Hongwei Ouyang
- Department of Orthopaedic Surgery, Second Affiliated Hospital and Zhejiang University-University of Edinburgh Institute and School of Basic Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Dr. Li Dak Sum and Yip Yio Chin Center for Stem Cells and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Key Laboratory of Tissue Engineering and Regenerative Medicine of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou, 310058, China
- Department of Sports Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China
- China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, 310058, China
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25
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Steichen C, Hannoun Z, Luce E, Hauet T, Dubart-Kupperschmitt A. Genomic integrity of human induced pluripotent stem cells: Reprogramming, differentiation and applications. World J Stem Cells 2019; 11:729-747. [PMID: 31692979 PMCID: PMC6828592 DOI: 10.4252/wjsc.v11.i10.729] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2019] [Revised: 06/13/2019] [Accepted: 07/30/2019] [Indexed: 02/06/2023] Open
Abstract
Ten years after the initial generation of induced pluripotent stem cells (hiPSCs) from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest. Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.
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Affiliation(s)
- Clara Steichen
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers F-86021, France
- Université de Poitiers, Faculté de Médecine et Pharmacie, Bâtiment D1, 6 rue de la milétrie, TSA 51115, 86073 Poitiers Cedex 9, France
| | - Zara Hannoun
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94800, France
- UMR_S1193, Université Paris-Saclay, Hôpital Paul Brousse, Villejuif F-94800, France
- The Jenner Institute, University of Oxford, Oxford OX3 7DQ, United Kingdom
| | - Eléanor Luce
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94800, France
- UMR_S1193, Université Paris-Saclay, Hôpital Paul Brousse, Villejuif F-94800, France
- Département Hospitalo-Universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
| | - Thierry Hauet
- INSERM U1082 IRTOMIT, CHU de Poitiers, Poitiers F-86021, France
- Université de Poitiers, Faculté de Médecine et Pharmacie, Bâtiment D1, 6 rue de la milétrie, TSA 51115, 86073 Poitiers Cedex 9, France
- Service de Biochimie, Pôle Biospharm, CHU de Poitiers, Poitiers F-86021, France
- Fédération Hospitalo-Universitaire SUPORT, CHU de Poitiers, Poitiers F-86021, France
| | - Anne Dubart-Kupperschmitt
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94800, France
- UMR_S1193, Université Paris-Saclay, Hôpital Paul Brousse, Villejuif F-94800, France
- Département Hospitalo-Universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
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26
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Markouli C, Couvreu De Deckersberg E, Regin M, Nguyen HT, Zambelli F, Keller A, Dziedzicka D, De Kock J, Tilleman L, Van Nieuwerburgh F, Franceschini L, Sermon K, Geens M, Spits C. Gain of 20q11.21 in Human Pluripotent Stem Cells Impairs TGF-β-Dependent Neuroectodermal Commitment. Stem Cell Reports 2019; 13:163-176. [PMID: 31178415 PMCID: PMC6627003 DOI: 10.1016/j.stemcr.2019.05.005] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2018] [Revised: 05/06/2019] [Accepted: 05/06/2019] [Indexed: 02/06/2023] Open
Abstract
Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-β- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic.
Bcl-xL overexpression drives the transcriptomic profile of 20q11.21 mutant lines 20q11.21 mutant lines downregulate CHCHD2, a known TGF-β pathway modulator Mutant lines differentially express genes involved in TGF-β and SMAD signaling Mutant lines show impaired ectoderm commitment due to TGF-β signaling deregulation
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Affiliation(s)
- C Markouli
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - E Couvreu De Deckersberg
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - M Regin
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - H T Nguyen
- Center for Molecular Biology, Institute of Research and Development, Duy Tan University, K7/25 Quang Trung, Danang 550000, Vietnam
| | - F Zambelli
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium; Clínica EUGIN, Travessera de les Corts 322, 08029 Barcelona, Spain
| | - A Keller
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - D Dziedzicka
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - J De Kock
- Department of In Vitro Toxicology & Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - L Tilleman
- Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium
| | - F Van Nieuwerburgh
- Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium
| | - L Franceschini
- Laboratory of Molecular & Cellular Therapy, Department of Immunology - Physiology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - K Sermon
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - M Geens
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium
| | - C Spits
- Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Laarbeeklaan 103, 1090 Brussels, Belgium.
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27
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Addressing Variability and Heterogeneity of Induced Pluripotent Stem Cell-Derived Cardiomyocytes. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1212:1-29. [DOI: 10.1007/5584_2019_350] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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28
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Attwood SW, Edel MJ. iPS-Cell Technology and the Problem of Genetic Instability-Can It Ever Be Safe for Clinical Use? J Clin Med 2019; 8:E288. [PMID: 30823421 PMCID: PMC6462964 DOI: 10.3390/jcm8030288] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 02/23/2019] [Accepted: 02/25/2019] [Indexed: 12/20/2022] Open
Abstract
The use of induced Pluripotent Stem Cells (iPSC) as a source of autologous tissues shows great promise in regenerative medicine. Nevertheless, several major challenges remain to be addressed before iPSC-derived cells can be used in therapy, and experience of their clinical use is extremely limited. In this review, the factors affecting the safe translation of iPSC to the clinic are considered, together with an account of efforts being made to overcome these issues. The review draws upon experiences with pluripotent stem-cell therapeutics, including clinical trials involving human embryonic stem cells and the widely transplanted mesenchymal stem cells. The discussion covers concerns relating to: (i) the reprogramming process; (ii) the detection and removal of incompletely differentiated and pluripotent cells from the resulting medicinal products; and (iii) genomic and epigenetic changes, and the evolutionary and selective processes occurring during culture expansion, associated with production of iPSC-therapeutics. In addition, (iv) methods for the practical culture-at-scale and standardization required for routine clinical use are considered. Finally, (v) the potential of iPSC in the treatment of human disease is evaluated in the light of what is known about the reprogramming process, the behavior of cells in culture, and the performance of iPSC in pre-clinical studies.
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Affiliation(s)
- Stephen W Attwood
- Department of Life Sciences, The Natural History Museum, London SW7 5BD, UK.
| | - Michael J Edel
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK.
- Control of Pluripotency Laboratory, Department of Physiological Sciences I, Faculty of Medicine, University of Barcelona, Hospital Clinic, Casanova 143, 08036 Barcelona, Spain.
- Victor Chang Cardiac Research Institute, Sydney, NSW 2145, Australia.
- Harry Perkins Research Institute, Fiona Stanley Hospital, University of Western Australia, PO Box 404, Bull Creek, Western Australia 6149, Australia.
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29
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Singh AM. An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells. Front Cell Dev Biol 2019; 7:11. [PMID: 30766873 PMCID: PMC6365467 DOI: 10.3389/fcell.2019.00011] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2018] [Accepted: 01/18/2019] [Indexed: 01/09/2023] Open
Abstract
Genomic manipulation of human pluripotent stem cells (hPSCs) has become essential to introduce genetic modifications and transgenes, and develop reporter lines. One of the major bottlenecks in gene editing is at the stage of single-cell cloning, which is thought to be variable across hPSC lines and is substantially reduced following a transfection. Due to the difficulty of performing fluorescent-assisted cell sorting (FACS) for single-cell isolation of hPSCs, previous approaches rely on manual colony picking, which is both time-consuming and labor-intensive. In this protocol, I provide a method for utilizing FACS to generate single-cell clones of hPSCs with efficiencies approaching 40% within 7–10 days. This can be achieved by sorting cells onto a feeder layer of MEFs in a stem cell defined medium with KSR and a Rock inhibitor, as early as 1–2 days following a transfection, streamlining the gene editing process. The approach described here provides a fundamental method for all researchers utilizing hPSCs for scientific studies.
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Affiliation(s)
- Amar M Singh
- Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, University of Georgia, Athens, GA, United States
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30
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Henry MP, Hawkins JR, Boyle J, Bridger JM. The Genomic Health of Human Pluripotent Stem Cells: Genomic Instability and the Consequences on Nuclear Organization. Front Genet 2019; 9:623. [PMID: 30719030 PMCID: PMC6348275 DOI: 10.3389/fgene.2018.00623] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2018] [Accepted: 11/23/2018] [Indexed: 12/11/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) are increasingly used for cell-based regenerative therapies worldwide, with embryonic and induced pluripotent stem cells as potential treatments for debilitating and chronic conditions, such as age-related macular degeneration, Parkinson's disease, spinal cord injuries, and type 1 diabetes. However, with the level of genomic anomalies stem cells generate in culture, their safety may be in question. Specifically, hPSCs frequently acquire chromosomal abnormalities, often with gains or losses of whole chromosomes. This review discusses how important it is to efficiently and sensitively detect hPSC aneuploidies, to understand how these aneuploidies arise, consider the consequences for the cell, and indeed the individual to whom aneuploid cells may be administered.
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Affiliation(s)
- Marianne P Henry
- Advanced Therapies Division, National Institute for Biological Standards and Control, Potters Bar, United Kingdom.,Laboratory of Nuclear and Genomic Health, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, London, United Kingdom
| | - J Ross Hawkins
- Advanced Therapies Division, National Institute for Biological Standards and Control, Potters Bar, United Kingdom
| | - Jennifer Boyle
- Advanced Therapies Division, National Institute for Biological Standards and Control, Potters Bar, United Kingdom
| | - Joanna M Bridger
- Laboratory of Nuclear and Genomic Health, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, London, United Kingdom
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31
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Abstract
The retina is a very fine and layered neural tissue, which vitally depends on the preservation of cells, structure, connectivity and vasculature to maintain vision. There is an urgent need to find technical and biological solutions to major challenges associated with functional replacement of retinal cells. The major unmet challenges include generating sufficient numbers of specific cell types, achieving functional integration of transplanted cells, especially photoreceptors, and surgical delivery of retinal cells or tissue without triggering immune responses, inflammation and/or remodeling. The advances of regenerative medicine enabled generation of three-dimensional tissues (organoids), partially recreating the anatomical structure, biological complexity and physiology of several tissues, which are important targets for stem cell replacement therapies. Derivation of retinal tissue in a dish creates new opportunities for cell replacement therapies of blindness and addresses the need to preserve retinal architecture to restore vision. Retinal cell therapies aimed at preserving and improving vision have achieved many improvements in the past ten years. Retinal organoid technologies provide a number of solutions to technical and biological challenges associated with functional replacement of retinal cells to achieve long-term vision restoration. Our review summarizes the progress in cell therapies of retina, with focus on human pluripotent stem cell-derived retinal tissue, and critically evaluates the potential of retinal organoid approaches to solve a major unmet clinical need—retinal repair and vision restoration in conditions caused by retinal degeneration and traumatic ocular injuries. We also analyze obstacles in commercialization of retinal organoid technology for clinical application.
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32
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Cantley W, Du C, Lomoio S, DePalma T, Peirent E, Kleinknecht D, Hunter M, Tang-Schomer M, Tesco G, Kaplan DL. Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells. ACS Biomater Sci Eng 2018; 4:4278-4288. [PMID: 33304995 PMCID: PMC7725274 DOI: 10.1021/acsbiomaterials.8b00622] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Three-dimensional in vitro cell culture models, particularly for the central nervous system, allow for the exploration of mechanisms of organ development, cellular interactions, and disease progression within defined environments. Here we describe the development and characterization of three-dimensional tissue models that promote the differentiation and long-term survival of functional neural networks. These tissue cultures show diverse cell populations including neurons and glial cells (astrocytes) interacting in 3D with spontaneous neural activity confirmed through electrophysiological recordings and calcium imaging over at least 8 months. This approach allows for the direct integration of pluripotent stem cells into the 3D construct bypassing early neural differentiation steps (embryoid bodies and neural rosettes), which streamlines the process while also providing a system that can be manipulated to support a variety of experimental applications. This tissue model has been tested in stem cells derived from healthy individuals as well as Alzheimer's and Parkinson's disease patients, with similar growth and gene expression responses indicating potential use in the modeling of disease states related to neurodegenerative diseases.
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Affiliation(s)
- William Cantley
- Department of Cell, Molecular and Developmental Biology, Sackler School, Tufts University, Boston, MA
| | - Chuang Du
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Selene Lomoio
- Department of Neuroscience, Sackler School, Tufts University, Boston, MA
| | - Thomas DePalma
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Emily Peirent
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | | | - Martin Hunter
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Min Tang-Schomer
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Giuseppina Tesco
- Department of Neuroscience, Sackler School, Tufts University, Boston, MA
| | - David L Kaplan
- Department of Biomedical Engineering, Tufts University, Medford, MA
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33
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Chen YH, Pruett-Miller SM. Improving single-cell cloning workflow for gene editing in human pluripotent stem cells. Stem Cell Res 2018; 31:186-192. [PMID: 30099335 DOI: 10.1016/j.scr.2018.08.003] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2018] [Revised: 07/27/2018] [Accepted: 08/02/2018] [Indexed: 01/01/2023] Open
Abstract
The availability of human pluripotent stem cells (hPSCs) and progress in genome engineering technology have altered the way we approach scientific research and drug development screens. Unfortunately, the procedures for genome editing of hPSCs often subject cells to harsh conditions that compromise viability: a major problem that is compounded by the innate challenge of single-cell culture. Here we describe a generally applicable workflow that supports single-cell cloning and expansion of hPSCs after genome editing and single-cell sorting. Stem-Flex and RevitaCell supplement, in combination with Geltrex or Vitronectin (VN), promote reliable single-cell growth in a feeder-free and defined environment. Characterization of final genome-edited clones reveals that pluripotency and normal karyotype are retained following this single-cell culture protocol. This time-efficient and simplified culture method paves the way for high-throughput hPSC culture and will be valuable for both basic research and clinical applications.
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Affiliation(s)
- Yi-Hsien Chen
- Washington University School of Medicine, Department of Genetics, St. Louis 63110, USA; Genome Engineering and iPSC Center, USA.
| | - Shondra M Pruett-Miller
- St. Jude Children's Research Hospital, Department of Cell & Molecular Biology, Memphis 38105, USA; Center for Advanced Genome Engineering, USA.
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34
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Park TS, Zimmerlin L, Evans-Moses R, Zambidis ET. Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Naïve-like State with Improved Multilineage Differentiation Potency. J Vis Exp 2018. [PMID: 29939183 DOI: 10.3791/57921] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Naïve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a naïve-like pluripotency with improved functionality. This chemical naïve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3β, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently naïve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. naïve-like states are outlined.
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Affiliation(s)
- Tea Soon Park
- Department of Oncology, Division of Pediatric Oncology and Institute for Cell Engineering, Johns Hopkins School of Medicine
| | - Ludovic Zimmerlin
- Department of Oncology, Division of Pediatric Oncology and Institute for Cell Engineering, Johns Hopkins School of Medicine;
| | - Rebecca Evans-Moses
- Department of Oncology, Division of Pediatric Oncology and Institute for Cell Engineering, Johns Hopkins School of Medicine
| | - Elias T Zambidis
- Department of Oncology, Division of Pediatric Oncology and Institute for Cell Engineering, Johns Hopkins School of Medicine;
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35
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Shroff G. A review on stem cell therapy for multiple sclerosis: special focus on human embryonic stem cells. Stem Cells Cloning 2018; 11:1-11. [PMID: 29483778 PMCID: PMC5813951 DOI: 10.2147/sccaa.s135415] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Multiple sclerosis (MS), a complex disorder of the central nervous system (CNS), is characterized with axonal loss underlying long-term progressive disability. Currently available therapies for its management are able to slow down the progression but fail to treat it completely. Moreover, these therapies are associated with major CNS and cardiovascular adverse events, and prolonged use of these treatments may cause life-threatening diseases. Recent research has shown that cellular therapies hold a potential for CNS repair and may be able to provide protection from inflammatory damage caused after injury. Human embryonic stem cell (hESC) transplantation is one of the promising cell therapies; hESCs play an important role in remyelination and help in preventing demylenation of the axons. In this study, an overview of the current knowledge about the unique properties of hESC and their comparison with other cell therapies has been presented for the treatment of patients with MS.
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Affiliation(s)
- Geeta Shroff
- Department of Stem Cell Therapy, Nutech Mediworld, New Delhi, India
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36
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Chen X, Harkness L, Jia Z, Prowse A, Monteiro MJ, Gray PP. Methods for Expansion of Three-Dimensional Cultures of Human Embryonic Stem Cells Using a Thermoresponsive Polymer. Tissue Eng Part C Methods 2017; 24:146-157. [PMID: 29239281 DOI: 10.1089/ten.tec.2017.0331] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.
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Affiliation(s)
- Xiaoli Chen
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Linda Harkness
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Zhongfan Jia
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Andrew Prowse
- 2 The Garvan Institute of Medical Research , Sydney, Australia
| | - Michael J Monteiro
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
| | - Peter P Gray
- 1 Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland , Brisbane, Australia
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37
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Sachs PC, Mollica PA, Bruno RD. Tissue specific microenvironments: a key tool for tissue engineering and regenerative medicine. J Biol Eng 2017; 11:34. [PMID: 29177006 PMCID: PMC5688702 DOI: 10.1186/s13036-017-0077-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2017] [Accepted: 08/24/2017] [Indexed: 12/12/2022] Open
Abstract
The accumulated evidence points to the microenvironment as the primary mediator of cellular fate determination. Comprised of parenchymal cells, stromal cells, structural extracellular matrix proteins, and signaling molecules, the microenvironment is a complex and synergistic edifice that varies tissue to tissue. Furthermore, it has become increasingly clear that the microenvironment plays crucial roles in the establishment and progression of diseases such as cardiovascular disease, neurodegeneration, cancer, and ageing. Here we review the historical perspectives on the microenvironment, and how it has directed current explorations in tissue engineering. By thoroughly understanding the role of the microenvironment, we can begin to correctly manipulate it to prevent and cure diseases through regenerative medicine techniques.
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Affiliation(s)
- Patrick C Sachs
- Medical Diagnostic and Translational Sciences, College of Health Science, Old Dominion University, Norfolk, VA 23529 USA
| | - Peter A Mollica
- Medical Diagnostic and Translational Sciences, College of Health Science, Old Dominion University, Norfolk, VA 23529 USA
| | - Robert D Bruno
- Medical Diagnostic and Translational Sciences, College of Health Science, Old Dominion University, Norfolk, VA 23529 USA
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38
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Peterson SE, Garitaonandia I, Loring JF. The tumorigenic potential of pluripotent stem cells: What can we do to minimize it? Bioessays 2017; 38 Suppl 1:S86-95. [PMID: 27417126 DOI: 10.1002/bies.201670915] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Revised: 02/05/2016] [Accepted: 02/09/2016] [Indexed: 12/14/2022]
Abstract
Human pluripotent stem cells (hPSCs) have the potential to fundamentally change the way that we go about treating and understanding human disease. Despite this extraordinary potential, these cells also have an innate capability to form tumors in immunocompromised individuals when they are introduced in their pluripotent state. Although current therapeutic strategies involve transplantation of only differentiated hPSC derivatives, there is still a concern that transplanted cell populations could contain a small percentage of cells that are not fully differentiated. In addition, these cells have been frequently reported to acquire genetic alterations that, in some cases, are associated with certain types of human cancers. Here, we try to separate the panic from reality and rationally evaluate the true tumorigenic potential of these cells. We also discuss a recent study examining the effect of culture conditions on the genetic integrity of hPSCs. Finally, we present a set of sensible guidelines for minimizing the tumorigenic potential of hPSC-derived cells. © 2016 The Authors. Inside the Cell published by Wiley Periodicals, Inc.
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Affiliation(s)
- Suzanne E Peterson
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.,Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, California, USA
| | - Ibon Garitaonandia
- Department of Neurogenetics, International Stem Cell Corporation, Oceanside, CA, USA
| | - Jeanne F Loring
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.,Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, California, USA
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39
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Martin U. Genome stability of programmed stem cell products. Adv Drug Deliv Rev 2017; 120:108-117. [PMID: 28917518 DOI: 10.1016/j.addr.2017.09.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Revised: 08/31/2017] [Accepted: 09/07/2017] [Indexed: 01/23/2023]
Abstract
Inherited and acquired genomic abnormalities are known to cause genetic diseases and contribute to cancer formation. Recent studies demonstrated a substantial mutational load in mouse and human embryonic and induced pluripotent stem cells (ESCs and iPSCs). Single nucleotide variants, copy number variations, and larger chromosomal abnormalities may influence the differentiation capacity of pluripotent stem cells and the functionality of their derivatives in disease modeling and drug screening, and are considered a serious risk for cellular therapies based on ESC or iPSC derivatives. This review discusses the types and origins of different genetic abnormalities in pluripotent stem cells, methods for their detection, and the mechanisms of development and enrichment during reprogramming and culture expansion.
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Affiliation(s)
- Ulrich Martin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH Cluster of Excellence, German Center for Lung Research, Hannover Medical School, Germany.
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40
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Abstract
A central challenge in sequencing single-cell genomes is the accurate determination of point mutations, phasing of these mutations, and identifying copy number variations with few assumptions. Ideally, this is accomplished under as low sequencing coverage as possible. Here we report our attempt to meet these goals with a novel library construction and library amplification methodology. In our approach, single-cell genomic DNA is first fragmented with saturated transposition to make a primary library that uniformly covers the whole genome by short fragments. The library is then amplified by a carefully optimized PCR protocol in a uniform and synchronized fashion for next-generation sequencing. Each step of the protocol can be quantitatively characterized. Our shallow sequencing data show that the library is tightly distributed and is useful for the determination of copy number variations.
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41
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Baek JA, Seol HW, Jung J, Kim HS, Oh SK, Choi YM. Clean-Up Human Embryonic Stem Cell Lines Using Humanized Culture Condition. Tissue Eng Regen Med 2017; 14:453-464. [PMID: 30603501 DOI: 10.1007/s13770-017-0053-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2016] [Revised: 03/19/2017] [Accepted: 03/21/2017] [Indexed: 11/28/2022] Open
Abstract
Human embryonic stem cell (hESC) culture system has been changing culture conditions from conventional to xeno-free for therapeutic cell applications, and N-glycolylneuraminic acid (Neu5Gc) could be a useful indicator of xenogeneic contaminations in hESCs because human cells can no longer produce it genetically. We set up the humanized culture condition using commercially available humanized materials and two different adaptation methods: sequential or direct. SNUhES4 and H1 hESC lines, previously established in conventional culture conditions, were maintained using the humanized culture condition and were examined for the presence of Neu5Gc. The hESCs showed the same morphology and character as those of the conventional culture condition. Moreover, they were negative for Neu5Gc within two passages without loss of pluripotency. This study suggested that this method can effectively cleanse previously established hESC lines, bringing them one step closer to being clinical-grade hESCs.
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Affiliation(s)
- Jin Ah Baek
- 1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 71, Ihwajang-gil, Jongno-gu, Seoul, 03087 Korea
| | - Hye Won Seol
- 1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 71, Ihwajang-gil, Jongno-gu, Seoul, 03087 Korea
| | - Juwon Jung
- 1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 71, Ihwajang-gil, Jongno-gu, Seoul, 03087 Korea
| | - Hee Sun Kim
- 1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 71, Ihwajang-gil, Jongno-gu, Seoul, 03087 Korea.,2Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 101, Daehak-ro, Jongno-gu, Seoul, 03080 Korea
| | - Sun Kyung Oh
- 1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 71, Ihwajang-gil, Jongno-gu, Seoul, 03087 Korea
| | - Young Min Choi
- 1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 71, Ihwajang-gil, Jongno-gu, Seoul, 03087 Korea.,2Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 101, Daehak-ro, Jongno-gu, Seoul, 03080 Korea
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42
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Coelho de Oliveira VC, Silva dos Santos D, Vairo L, Kasai Brunswick TH, Pimentel LAS, Carvalho AB, Campos de Carvalho AC, Goldenberg RCDS. Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells. Exp Ther Med 2017; 13:1779-1788. [PMID: 28565767 PMCID: PMC5443186 DOI: 10.3892/etm.2017.4195] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Accepted: 01/13/2017] [Indexed: 02/05/2023] Open
Abstract
The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific 'undifferentiation' markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-β1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.
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Affiliation(s)
| | - Danúbia Silva dos Santos
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
| | - Leandro Vairo
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
| | - Tais Hanae Kasai Brunswick
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
| | | | - Adriana Bastos Carvalho
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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43
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M Lee Y, Zampieri BL, Scott-McKean JJ, Johnson MW, Costa ACS. Generation of Integration-Free Induced Pluripotent Stem Cells from Urine-Derived Cells Isolated from Individuals with Down Syndrome. Stem Cells Transl Med 2017; 6:1465-1476. [PMID: 28371411 PMCID: PMC5689751 DOI: 10.1002/sctm.16-0128] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Accepted: 02/20/2017] [Indexed: 01/19/2023] Open
Abstract
Down syndrome (DS) is a genetic disorder caused by trisomy 21 (T21). Over the past two decades, the use of mouse models has led to significant advances in the understanding of mechanisms underlying various phenotypic features and comorbidities secondary to T21 and even informed the design of clinical trials aimed at enhancing the cognitive abilities of persons with DS. In spite of its success, this approach has been plagued by all the typical limitations of rodent modeling of human disorders and diseases. Recently, several laboratories have succeeded in producing T21 human induced pluripotent stem cells (T21-iPSCs) from individuals with DS, which is emerging as a promising complementary tool for the study of DS. Here, we describe the method by which we generated 10 T21-iPSC lines from epithelial cells in urine samples, presumably from kidney epithelial origin, using nonintegrating episomal vectors. We also show that these iPSCs maintain chromosomal stability for well over 20 passages and are more sensitive to proteotoxic stress than euploid iPSCs. Furthermore, these iPSC lines can be differentiated into glutamatergic neurons and cardiomyocytes. By culturing urine-derived cells and maximizing the efficiency of episomal vector transfection, we have been able to generate iPSCs noninvasively and effectively from participants with DS in an ongoing clinical trial, and thus address most shortcomings of previously generated T21-iPSC lines. These techniques should extend the application of iPSCs in modeling DS and other neurodevelopmental and neurodegenerative disorders, and may lead to future human cell-based platforms for high-throughput drug screening. Stem Cells Translational Medicine 2017;6:1465-1476.
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Affiliation(s)
- Young M Lee
- Division of Pediatric Neurology, Department of Pediatrics
| | | | | | - Mark W Johnson
- Division of Pediatric Neurology, Department of Pediatrics
| | - Alberto C S Costa
- Division of Pediatric Neurology, Department of Pediatrics.,Department of Psychiatry, Case Western Reserve University, Cleveland, Ohio, USA
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44
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Mesenchymal Stem and Progenitor Cells in Regeneration: Tissue Specificity and Regenerative Potential. Stem Cells Int 2017; 2017:5173732. [PMID: 28286525 PMCID: PMC5327785 DOI: 10.1155/2017/5173732] [Citation(s) in RCA: 127] [Impact Index Per Article: 15.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Accepted: 12/07/2016] [Indexed: 12/15/2022] Open
Abstract
It has always been an ambitious goal in medicine to repair or replace morbid tissues for regaining the organ functionality. This challenge has recently gained momentum through considerable progress in understanding the biological concept of the regenerative potential of stem cells. Routine therapeutic procedures are about to shift towards the use of biological and molecular armamentarium. The potential use of embryonic stem cells and invention of induced pluripotent stem cells raised hope for clinical regenerative purposes; however, the use of these interventions for regenerative therapy showed its dark side, as many health concerns and ethical issues arose in terms of using these cells in clinical applications. In this regard, adult stem cells climbed up to the top list of regenerative tools and mesenchymal stem cells (MSC) showed promise for regenerative cell therapy with a rather limited level of risk. MSC have been successfully isolated from various human tissues and they have been shown to offer the possibility to establish novel therapeutic interventions for a variety of hard-to-noncurable diseases. There have been many elegant studies investigating the impact of MSC in regenerative medicine. This review provides compact information on the role of stem cells, in particular, MSC in regeneration.
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45
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Abstract
The ability of human pluripotent stem cells (hPSCs) to proliferate indefinitely in culture while maintaining their pluripotent properties makes them a powerful tool for use in research, and provides tremendous potential for diagnostic testing, and therapeutic application. Success in these areas, however, is dependent on the ability to effectively expand them in long-term culture while preserving their distinct nature. Contained in this chapter are detailed protocols for the feeder-independent culture and expansion of hPSCs using mTeSR1 medium and Matrigel matrix, and guidelines for the successful transfer of those cells to alternative platforms. These protocols have been used widely by laboratories around the world to successfully expand hPSCs for long-term culture while maintaining their undifferentiated, pluripotent state.
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Affiliation(s)
- Jennifer L Brehm
- WiCell Research Institute, 504 S. Rosa Rd., Suite 101, Madison, WI, 53719, USA
| | - Tenneille E Ludwig
- WiCell Research Institute, 504 S. Rosa Rd., Suite 101, Madison, WI, 53719, USA.
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46
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Kyriakides O, Halliwell JA, Andrews PW. Acquired Genetic and Epigenetic Variation in Human Pluripotent Stem Cells. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2017; 163:187-206. [PMID: 29071402 DOI: 10.1007/10_2017_22] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Human pluripotent stem cells (hPSCs) can acquire non-random genomic variation during culture. Some of these changes are common in tumours and confer a selective growth advantage in culture. Additionally, there is evidence that reprogramming of human induced pluripotent stem cells (hiPSCs) introduces mutations. This poses a challenge to both the safety of clinical applications and the reliability of basic research using hPSCs carrying genomic variation. A number of methods are available for monitoring the genomic integrity of hPSCs, and a balance between practicality and sensitivity must be considered in choosing the appropriate methods for each use of hPSCs. Adjusting protocols by which hPSCs are derived and cultured is an evolving process that is important in minimising acquired genomic variation. Assessing genetic variation for its potential impact is becoming increasingly important as techniques to detect genome-wide variation improve.
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Affiliation(s)
- O Kyriakides
- Centre for Stem Cell Biology, Department Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK
| | - J A Halliwell
- Centre for Stem Cell Biology, Department Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK
| | - P W Andrews
- Centre for Stem Cell Biology, Department Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.
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Yedid N, Kalma Y, Malcov M, Amit A, Kariv R, Caspi M, Rosin-Arbesfeld R, Ben-Yosef D. The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells. BMC Cancer 2016; 16:952. [PMID: 28010732 PMCID: PMC5180406 DOI: 10.1186/s12885-016-2809-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2015] [Accepted: 09/23/2016] [Indexed: 12/14/2022] Open
Abstract
Background Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs. Methods Two FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) β-catenin expression was analyzed by Western blot analysis; 2) Wnt-β-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of β-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed. Results We have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced β-catenin/TCF-mediated activity. Additionally, β-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in β-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population. Conclusions Our results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2809-9) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Nofar Yedid
- Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel.,Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel
| | - Yael Kalma
- Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel
| | - Mira Malcov
- Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel
| | - Ami Amit
- Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel
| | - Revital Kariv
- Departmant of Gastroenterology, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel
| | - Michal Caspi
- Department of Clinical Microbiology and Immunology, Tel-Aviv University, Tel Aviv, Israel
| | - Rina Rosin-Arbesfeld
- Department of Clinical Microbiology and Immunology, Tel-Aviv University, Tel Aviv, Israel
| | - Dalit Ben-Yosef
- Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel. .,Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel.
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Shroff G, Vatsa D. Cell Viability of Human Embryonic Stem Cells Stored for a Period of 9 Years. EXP CLIN TRANSPLANT 2016; 15:344-349. [PMID: 27938317 DOI: 10.6002/ect.2016.0097] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
OBJECTIVES Human embryonic stem cells are pluripotent cell lines usually derived from human blastocysts. Their potential critically depends on long-term proliferative capacity, developmental potential after prolonged culture, and karyotypic stability. Cell viability is an important parameter for assessing cell sample quality. Here, we elaborate the stored human embryonic stem cell lines' viability in a ready to use form for a period of 9 years (from 2007 to 2015). MATERIALS AND METHODS Spare pre implantation stage in vitro fertilized ovum-derived cell lines were cultured in suitable media. Thereafter, they were centrifuged at 1000 revolutions/min over 5 minutes, and pellets were suspended in normal saline. Next, they were tested for viability from storage at -20°C. After being allowed to thaw slowly, the cells were stained with propidium iodide and analyzed using flow cytometry. Images of cells were taken at ×40 and ×100 magnification. RESULTS At ×100 magnification, cell population size ranged from 0.2 to 2 μm. The percentage of live cells was more than 95% throughout the 9 years. Cells frozen in 2015 showed cell viability of 96.8%. CONCLUSIONS We observed high cell viability in our cell lines for 9 years. Human embryonic stem cell lines in a ready-to-use form can be preserved for long-term purposes. Thus, they could be made available globally.
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Abstract
There is no good science in bad models. Cell culture is especially prone to artifacts. A number of novel cell culture technologies have become more broadly available in the 21st century, which allow overcoming limitations of traditional culture and are more physiologically relevant. These include the use of stem-cell derived human cells, cocultures of different cell types, scaffolds and extracellular matrices, perfusion platforms (such as microfluidics), 3D culture, organ-on-chip technologies, tissue architecture, and organ functionality. The physiological relevance of such models is further enhanced by the measurement of biomarkers (e.g., key events of pathways), organ specific functionality, and more comprehensive assessment cell responses by high-content methods. These approaches are still rarely combined to create microphysiological systems. The complexity of the combination of these technologies can generate results closer to the in vivo situation but increases the number of parameters to control, bringing some new challenges. In fact, we do not argue that all cell culture needs to be that sophisticated. The efforts taken are determined by the purpose of our experiments and tests. If only a very specific molecular target to cell response is of interest, a very simple model, which reflects this, might be much more suited to allow standardization and high-throughput. However, the less defined the end point of interest and cellular response are, the better we should approximate organ- or tissue-like culture conditions to make physiological responses more probable. Besides these technologic advances, important progress in the quality assurance and reporting on cell cultures as well as the validation of cellular test systems brings the utility of cell cultures to a new level. The advancement and broader implementation of Good Cell Culture Practice (GCCP) is key here. In toxicology, this is a major prerequisite for meaningful and reliable results, ultimately supporting risk assessment and product development decisions.
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Affiliation(s)
- David Pamies
- Center for Alternatives to Animal Testing (CAAT), Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland 21205, United States
| | - Thomas Hartung
- Center for Alternatives to Animal Testing (CAAT), Johns Hopkins Bloomberg School of Public Health , Baltimore, Maryland 21205, United States.,CAAT-Europe, University of Konstanz , 78464 Konstanz, Germany
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Mantle JL, Min L, Lee KH. Minimum Transendothelial Electrical Resistance Thresholds for the Study of Small and Large Molecule Drug Transport in a Human in Vitro Blood-Brain Barrier Model. Mol Pharm 2016; 13:4191-4198. [PMID: 27934481 DOI: 10.1021/acs.molpharmaceut.6b00818] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm2. By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.
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Affiliation(s)
- Jennifer L Mantle
- Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware , 15 Innovation Way, Newark, Delaware 19711, United States
| | - Lie Min
- Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware , 15 Innovation Way, Newark, Delaware 19711, United States
| | - Kelvin H Lee
- Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware , 15 Innovation Way, Newark, Delaware 19711, United States
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