-
ELife Mar 2015Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development...
Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease.
Topics: Cell Culture Techniques; Cell Differentiation; Cell Line; Cells, Cultured; Embryonic Stem Cells; Endoderm; Gene Expression Profiling; Humans; Induced Pluripotent Stem Cells; Lung; Microscopy, Confocal; Microscopy, Electron, Transmission; Organogenesis; Organoids; Pluripotent Stem Cells; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular; Tissue Engineering
PubMed: 25803487
DOI: 10.7554/eLife.05098 -
Development (Cambridge, England) Jan 2019Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids....
Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.
Topics: Cell Differentiation; Cell Line; Cell Proliferation; Cell Shape; Ependymoglial Cells; Humans; Interneurons; Models, Biological; Neural Stem Cells; Organoids; Pluripotent Stem Cells; Reproducibility of Results; Retina; Retinal Ganglion Cells; Synapses; Tomography, Optical Coherence
PubMed: 30567931
DOI: 10.1242/dev.171686 -
Nature Biotechnology Aug 2017Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients....
Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8 T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.
Topics: Animals; Female; Graft Rejection; HLA Antigens; Humans; Killer Cells, Natural; Mice; Pluripotent Stem Cells; Transplants
PubMed: 28504668
DOI: 10.1038/nbt.3860 -
Current Protocols in Human Genetics Oct 2015Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under...
Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal-derived components to help reveal the precise underlying mechanisms. Here we describe our latest, cost-effective and efficient methodology for the culture of hPS cells in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non-enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection-based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell-derived cardiomyocytes, cryopreservation, and thawing.
Topics: Cell Culture Techniques; Cell Differentiation; Flow Cytometry; Humans; Immunophenotyping; Induced Pluripotent Stem Cells; Myocytes, Cardiac; Phenotype; Pluripotent Stem Cells
PubMed: 26439715
DOI: 10.1002/0471142905.hg2103s87 -
Stem Cell Research & Therapy Jun 2019Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In...
BACKGROUND
Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.
METHODS
iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.
RESULTS
In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.
CONCLUSIONS
Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.
Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Embryo Transfer; Embryoid Bodies; Fibroblasts; Humans; Immunohistochemistry; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Mice; Pericytes; Pluripotent Stem Cells; Swine
PubMed: 31248457
DOI: 10.1186/s13287-019-1303-0 -
Cell Stem Cell Jul 2019Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in... (Review)
Review
Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.
Topics: Cell Differentiation; Cellular Reprogramming; Embryonic Development; Embryonic Stem Cells; Gene Expression Regulation, Developmental; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells; Regenerative Medicine; Transcriptome
PubMed: 31271751
DOI: 10.1016/j.stem.2019.06.010 -
Stem Cell Reports Aug 2023
Topics: Pluripotent Stem Cells; Induced Pluripotent Stem Cells; Cell Differentiation; Cell- and Tissue-Based Therapy; Stem Cell Transplantation
PubMed: 37557071
DOI: 10.1016/j.stemcr.2023.06.010 -
Cells Oct 2021Pluripotent stem cells, having long been considered the fountain of youth, have caught the attention of many researchers from diverse backgrounds due to their capacity... (Review)
Review
Pluripotent stem cells, having long been considered the fountain of youth, have caught the attention of many researchers from diverse backgrounds due to their capacity for unlimited self-renewal and potential to differentiate into all cell types. Over the past 15 years, the advanced development of induced pluripotent stem cells (iPSCs) has displayed an unparalleled potential for regenerative medicine, cell-based therapies, modeling human diseases in culture, and drug discovery. The transcription factor quartet (Oct4, Sox2, Klf4, and c-Myc) reprograms highly differentiated somatic cells back to a pluripotent state recapitulated embryonic stem cells (ESCs) in different aspects, including gene expression profile, epigenetic signature, and functional pluripotency. With the prior fruitful studies in SCNT and cell fusion experiments, iPSC finds its place and implicates that the differentiated somatic epigenome retains plasticity for re-gaining the pluripotency and further stretchability to reach a totipotency-like state. These achievements have revolutionized the concept and created a new avenue in biomedical sciences for clinical applications. With the advent of 15 years' progress-making after iPSC discovery, this review is focused on how the current concept is established by revisiting those essential landmark studies and summarizing its current biomedical applications status to facilitate the new era entry of regenerative therapy.
Topics: Animals; Cell Lineage; Cellular Reprogramming; Clinical Trials as Topic; Epigenesis, Genetic; Germ Cells; Humans; Pluripotent Stem Cells
PubMed: 34831113
DOI: 10.3390/cells10112888 -
Cells Oct 2020Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells... (Review)
Review
Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells undergo a transition from rather homogeneous epithelial-like pluripotent stem cell colonies into a three-dimensional organization of various cell types with multifaceted cell-cell interactions and lumen formation-a process associated with repetitive epithelial-mesenchymal transitions. In the last few years, culture methods have further evolved to better control EB size, growth, cellular composition, and organization-e.g., by the addition of morphogens or different extracellular matrix molecules. There is a growing perception that the mechanical properties, cell mechanics, and cell signaling during EB development are also influenced by physical cues to better guide lineage specification; substrate elasticity and topography are relevant, as well as shear stress and mechanical strain. Epithelial structures outside and inside EBs support the integrity of the cell aggregates and counteract mechanical stress. Furthermore, hydrogels can be used to better control the organization and lineage-specific differentiation of EBs. In this review, we summarize how EB formation is accompanied by a variety of biomechanical parameters that need to be considered for the directed and reproducible self-organization of early cell fate decisions.
Topics: Biomechanical Phenomena; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Embryoid Bodies; Embryonic Development; Humans; Pluripotent Stem Cells
PubMed: 33050550
DOI: 10.3390/cells9102270 -
International Journal of Molecular... Apr 2019The generation of immune cells from human pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) has been of keen interest to regenerative... (Review)
Review
The generation of immune cells from human pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) has been of keen interest to regenerative medicine. Pluripotent stem cell-derived immune cells such as natural killer cells, macrophages, and lymphoid cells, especially T cells, can be used in immune cell therapy to treat incurable cancers. Moreover, since the advent of chimeric antigen receptor (CAR) technology, the success of CAR-T cells in the clinic has galvanized new efforts to harness the power of CAR technology to generate CAR-engineered immune cells from pluripotent stem cells. This review provides a summary of pluripotent stem cell-derived immune cells and CAR technology, together with perspectives on combining pluripotent stem-cell derived immune cells and CAR engineering to pave a new way for developing next generation immune cell therapy.
Topics: Animals; Cell Differentiation; Humans; Immunotherapy; Killer Cells, Natural; Pluripotent Stem Cells; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Stem Cell Transplantation; Stem Cells; T-Lymphocytes
PubMed: 31013813
DOI: 10.3390/ijms20081825