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.     

AAV-mediated gene targeting methods for human cells

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10(-5) to 10(-2) per infected cell. These targeting frequencies are 1-4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ～10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.     

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.     

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.     

Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.     

Safeguarding clinical translation of pluripotent stem cells with suicide genes

The generation of human induced pluripotent stem cells (hiPSCs) opens a new avenue in regenerative medicine. However, transplantation of hiPSC-derived cells carries a risk of tumor formation by residual pluripotent stem cells. Numerous adaptive strategies have been developed to prevent or minimize adverse events and control the in vivo behavior of transplanted stem cells and their progeny. Among them, the application of suicide gene modifications, which is conceptually similar to cancer gene therapy, is considered an ideal means to control wayward stem cell progeny in vivo. In this review, the choices of vectors, promoters, and genes for use in suicide gene approaches for improving the safety of hiPSCs-based cell therapy are introduced and possible new strategies for improvements are discussed. Safety-enhancing strategies that can selectively ablate undifferentiated cells without inducing virus infection or insertional mutations may greatly aid in translating human pluripotent stem cells into cell therapies in the future.     

Transient characteristics of universal cells on human‐induced pluripotent stem cells and their differentiated cells derived from foetal stem cells with mixed donor sources

IntroductionIt is important to prepare ‘hypoimmunogenic’ or ‘universal’ human pluripotent stem cells (hPSCs) with gene‐editing technology by knocking out or in immune‐related genes, because only a few hypoimmunogenic or universal hPSC lines would be sufficient to store for their off‐the‐shelf use. However, these hypoimmunogenic or universal hPSCs prepared previously were all genetically edited, which makes laborious processes to check and evaluate no abnormal gene editing of hPSCs.MethodsUniversal human‐induced pluripotent stem cells (hiPSCs) were generated without gene editing, which were reprogrammed from foetal stem cells (human amniotic fluid stem cells) with mixing 2‐5 allogenic donors but not with single donor. We evaluated human leucocyte antigen (HLA)‐expressing class Ia and class II of our hiPSCs and their differentiated cells into embryoid bodies, cardiomyocytes and mesenchymal stem cells. We further evaluated immunogenic response of transient universal hiPSCs with allogenic mononuclear cells from survival rate and cytokine production, which were generated by the cells due to immunogenic reactions.ResultsOur universal hiPSCs during passages 10‐25 did not have immunogenic reaction from allogenic mononuclear cells even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Furthermore, the cells including the differentiated cells did not express HLA class Ia and class II. Cardiomyocytes differentiated from transient universal hiPSCs at passage 21‐22 survived and continued beating even after treatment with allogenic mononuclear cells.     

Fourier transform infrared microspectroscopy reveals unique phenotypes for human embryonic and induced pluripotent stem cell lines and their progeny

Fourier transform infrared (FTIR) microspectroscopy was employed to elucidate the macromolecular phenotype of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and their differentiated progeny. Undifferentiated hESCs and hiPSC lines were found to be not clearly distinguishable from each other. However, although both hESC and hiPSC variants appeared to undergo similar changes during differentiation in terms of cell surface antigens, the derived cell types from all cell lines could be discriminated using FTIR spectroscopy. We foresee a possible future role for FTIR microspectroscopy as a powerful and objective investigative and quality control tool in regenerative medicine. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Changes in human pluripotent stem cell gene expression after genotoxic stress exposures

Human pluripotent stem cells (hPSCs) represent heterogeneous populations, including induced pluripotent stem cells (iPSCs), endogenous plastic somatic cells, and embryonic stem cells (ESCs). Human ESCs are derived from the inner cell mass of the blastocyst, and they are characterized by the abilities to self-renew indefinitely, and to give rise to all cell types of embryonic lineage (pluripotency) under the guidance of the appropriate chemical, mechanical and environmental cues. The combination of these critical features is unique to hESCs, and set them apart from other human cells. The expectations are high to utilize hESCs for treating injuries and degenerative diseases; for modeling of complex illnesses and development; for screening and testing of pharmacological products; and for examining toxicity, mutagenicity, teratogenicity, and potential carcinogenic effects of a variety of environmental factors, including ionizing radiation (IR). Exposures to genotoxic stresses, such as background IR, are unavoidable; moreover, IR is widely used in diagnostic and therapeutic procedures in medicine on a routine basis. One of the key outcomes of cell exposures to IR is the change in gene expression, which may underlie the ultimate hESCs fate after such a stress. However, gaps in our knowledge about basic biology of hESCs impose a serious limitation to fully realize the potential of hESCs in practice. The purpose of this review is to examine the available evidence of alterations in gene expression in human pluripotent stem cells after genotoxic stress, and to discuss strategies for future research in this important area.     

Targeted transgene insertion into human chromosomes by adeno-associated virus vectors

Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences. Here we have used AAV vectors to introduce large (>1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.     

High-Fidelity Correction of Mutations at Multiple Chromosomal Positions by Adeno-Associated Virus Vectors

The gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. This is due in large part to the technical problems and/or inefficiency of conventional DNA transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. We recently described an alternative approach in which adeno-associated virus (AAV) vectors were used to modify homologous chromosomal sequences, and targeting rates close to 1% were observed at the single copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human cells (D. W. Russell and R. K. Hirata, Nat. Genet. 18:325-330, 1998). Here we report experiments in which we used a retroviral shuttle vector system to introduce and characterize target loci in human chromosomes, and demonstrate that AAV vectors can correct several types of mutations with high fidelity, independent of chromosomal position. The gene targeting rates varied depending on the type of mutation being corrected, implicating cellular mismatch recognition functions in the reaction. Since AAV vectors can efficiently deliver DNA to many cell types both in vivo and ex vivo, our results suggest that AAV-mediated gene targeting will have wide applicability, including therapeutic gene correction.     

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.