Generation of universal and hypoimmunogenic human pluripotent stem cells

There is a need to store very large numbers of conventional human pluripotent stem cell (hPSC) lines for their off‐the‐shelf usage in stem cell therapy. Therefore, it is valuable to generate “universal” or “hypoimmunogenic” hPSCs with gene‐editing technology by knocking out or in immune‐related genes. A few universal or hypoimmunogenic hPSC lines should be enough to store for their off‐the‐shelf usage. Here, we overview and discuss how to prepare universal or hypoimmunogenic hPSCs and their disadvantages. β2‐Microglobulin‐knockout hPSCs did not harbour human leukocyte antigen (HLA)‐expressing class I cells but rather activated natural killer (NK) cells. To avoid NK cell and macrophage activities, homozygous hPSCs expressing a single allele of an HLA class I molecule, such as HLA‐C, were developed. Major HLA class I molecules were knocked out, and PD‐L1, HLA‐G and CD47 were knocked in hPSCs using CRISPR/Cas9 gene editing. These cells escaped activation of not only T cells but also NK cells and macrophages, generating universal hPSCs.     

Fine‐tuning of dual‐SMAD inhibition to differentiate human pluripotent stem cells into neural crest stem cells

ObjectivesThe derivation of neural crest stem cells (NCSCs) from human pluripotent stem cells (hPSCs) has been commonly induced by WNT activation in combination with dual‐SMAD inhibition.In this study, by fine‐tuning BMP signalling in the conventional dual‐SMAD inhibition, we sought to generate large numbers of NCSCs without WNT activation.Materials and methodsIn the absence of WNT activation, we modulated the level of BMP signalling in the dual‐SMAD inhibition system to identify conditions that efficiently drove the differentiation of hPSCs into NCSCs. We isolated two NCSC populations separately and characterized them in terms of global gene expression profiles and differentiation ability.ResultsOur modified dual‐SMAD inhibition containing a lower dose of BMP inhibitor than that of the conventional dual‐SMAD inhibition drove hPSCs into mainly NCSCs, which consisted of HNK⁺p75high and HNK⁺p75low cell populations. We showed that the p75high population formed spherical cell clumps, while the p75low cell population generated a 2D monolayer.We detected substantial differences in gene expression profiles between the two cell groups and showed that both p75high and p75low cells differentiated into mesenchymal stem cells (MSCs), while only p75high cells had the ability to become peripheral neurons.ConclusionsThis study will provide a framework for the generation and isolation of NCSC populations for effective cell therapy for peripheral neuropathies and MSC‐based cell therapy.     

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.     

Insulin resistance in diabetes: The promise of using induced pluripotent stem cell technology

Insulin resistance(IR)is associated with several metabolic disorders,including type 2 diabetes(T2D).The development of IR in insulin target tissues involves genetic and acquired factors.Persons at genetic risk for T2D tend to develop IR several years before glucose intolerance.Several rodent models for both IR and T2D are being used to study the disease pathogenesis;however,these models cannot recapitulate all the aspects of this complex disorder as seen in each individual.Human pluripotent stem cells(hPSCs)can overcome the hurdles faced with the classical mouse models for studying IR.Human induced pluripotent stem cells(hiPSCs)can be generated from the somatic cells of the patients without the need to destroy a human embryo.Therefore,patient-specific hiPSCs can generate cells genetically identical to IR individuals,which can help in distinguishing between genetic and acquired defects in insulin sensitivity.Combining the technologies of genome editing and hiPSCs may provide important information about the genetic factors underlying the development of different forms of IR.Further studies are required to fill the gaps in understanding the pathogenesis of IR and diabetes.In this review,we summarize the factors involved in the development of IR in the insulin-target tissues leading to diabetes.Also,we highlight the use of hPSCs to understand the mechanisms underlying the development of IR.     

Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.     

Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions

Cardiovascular progenitor cells (CVPCs) derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold great promise for the study of cardiovascular development and cell-based therapy of heart diseases, but their applications are challenged by the difficulties in their efficient generation and stable maintenance. This study aims to develop chemically defined systems for robust generation and stable propagation of hPSC-derived CVPCs by modulating the key early developmental pathways involved in human cardiovascular specification and CVPC self-renewal. Herein we report that a combination of bone morphogenetic protein 4 (BMP4), glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 and ascorbic acid is sufficient to rapidly convert monolayer-cultured hPSCs, including hESCs and hiPSCs, into homogeneous CVPCs in a chemically defined medium under feeder- and serum-free culture conditions. These CVPCs stably self-renewed under feeder- and serum-free conditions and expanded over 10⁷-fold when the differentiation-inducing signals from BMP, GSK3 and Activin/Nodal pathways were simultaneously eliminated. Furthermore, these CVPCs exhibited expected genome-wide molecular features of CVPCs, retained potentials to generate major cardiovascular lineages including cardiomyocytes, smooth muscle cells and endothelial cells in vitro, and were non-tumorigenic in vivo. Altogether, the established systems reported here permit efficient generation and stable maintenance of hPSC-derived CVPCs, which represent a powerful tool to study early embryonic cardiovascular development and provide a potentially safe source of cells for myocardial regenerative medicine.     

Stable propagation of human embryonic and induced pluripotent stem cells on decellularized human substrates

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have gained enormous interest as potential sources for regenerative biomedical therapies and model systems for studying early development. Traditionally, mouse embryonic fibroblasts have been used as a supportive feeder layer for the sustained propagation of hPSCs. However, the use of nonhuman‐derived feeders presents concerns about the possibility of xenogenic contamination, labor intensiveness, and variability in experimental results in hPSC cultures. Toward addressing some of these concerns, we report the propagation of three different hPSCs on feeder‐free extracellular matrix (ECM)‐based substrates derived from human fibroblasts. hPSCs propagated in this setting were indistinguishable by multiple criteria, including colony morphology, expression of pluripotency protein markers, trilineage in vitro differentiation, and gene expression patterns, from hPSCs cultured directly on a fibroblast feeder layer. Further, hPSCs maintained a normal karyotype when analyzed after 15 passages in this setting. Development of this ECM‐based culture system is a significant advance in hPSC propagation methods as it could serve as a critical component in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Dendritic cells derived from pluripotent stem cells:Potential of large scale production

Human pluripotent stem cells(hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells(DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension cultureof embryoid bodies(EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.     

Dendritic cells derived from pluripotent stem cells: Potential of large scale production简

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells (DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension culture of embryoid bodies (EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

Practical choice for robust and efficient differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the distinct advantage of being able to differentiate into cells of all three germ layers. Target cells or tissues derived from hPSCs have many uses such as drug screening, disease modeling, and transplantation therapy. There are currently a wide variety of differentiation methods available. However, most of the existing differentiation methods are unreliable, with uneven differentiation efficiency and poor reproducibility. At the same time, it is difficult to choose the optimal method when faced with so many differentiation schemes, and it is time-consuming and costly to explore a new differentiation approach. Thus, it is critical to design a robust and efficient method of differentiation. In this review article, we summarize a comprehensive approach in which hPSCs are differentiated into target cells or organoids including brain, liver, blood, melanocytes, and mesenchymal cells. This was accomplished by employing an embryoid body-based three-dimensional (3D) suspension culture system with multiple cells co-cultured. The method has high stable differentiation efficiency compared to the conventional 2D culture and can meet the requirements of clinical application. Additionally, ex vivo co-culture models might be able to constitute organoids that are highly similar or mimic human organs for potential organ transplantation in the future.     

Large‐scale generation of megakaryocytes from human embryonic stem cells using transgene‐free and stepwise defined suspension culture conditions

ObjectivesEx vivo engineered production of megakaryocytes (MKs) and platelets (PLTs) from human pluripotent stem cells is an alternative approach to solve shortage of donor‐donated PLTs in clinics and to provide induced PLTs for transfusion. However, low production yields are observed and the generation of clinically applicable MKs and PLTs from human pluripotent stem cells without genetic modifications still needs to be improved.Materials and MethodsWe defined an optimal, stepwise and completely xeno‐free culture protocol for the generation of MKs from human embryonic stem cells (hESCs). To generate MKs from hESCs on a large scale, we improved the monolayer induction manner to define three‐dimensional (3D) and sphere‐like differentiation systems for MKs by using a special polystyrene CellSTACK culture chamber.ResultsThe 3D manufacturing system could efficiently generate large numbers of MKs from hESCs within 16‐18 days of continuous culturing. Each CellSTACK culture chamber could collect on an average 3.4 × 10⁸ CD41⁺ MKs after a three‐stage orderly induction process. MKs obtained from hESCs via 3D induction showed significant secretion of IL‐8, thrombospondin‐1 and MMP9. The induced cells derived from hESCs in our culture system were shown to have the characteristics of MKs as well as the function to form proPLTs and release PLTs. Furthermore, we generated clinically applicable MKs from clinical‐grade hESC lines and confirmed the biosafety of these cells.ConclusionsWe developed a simple, stepwise, 3D and completely xeno‐free/feeder‐free/transgene‐free induction system for the generation of MKs from hESCs. hESC‐derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.     

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.