Application Of Pluripotent Stem Cells Research Articles

X-linked severe combined immunodeficiency (X-SCID) rats for xeno-transplantation and behavioral evaluation

BackgroundTo evaluate the in vivo function of human dopaminergic (DA) neurons, Parkinson's disease (PD) model rats made by the hemi-lateral injection of 6-hydroxydopamine (6-OHDA) are widely used as host animals. In the case of such xeno-transplantation, however, immunosuppression is needed for good survival of the grafted cells. New methodsIn order to determine whether human mature neurons can survive in X-linked severe combined immunodeficiency (X-SCID) rats without immunosuppression, we grafted human embryonic stem cell (ESC)-derived DA neurons into the striatum of X-SCID rats. We next treated the X-SCID rats with 6-OHDA and grafted mouse fetal DA neurons or human induced pluripotent stem cell (iPSC)-derived DA neurons to examine whether these rats can be used as PD model rats. ResultsX-SCID rats did not elicit immune responses against human ESC-derived DA neurons and consequently resulted in good survival of the cells without immunosuppression. Furthermore, 6-OHDA-lesioned X-SCID rats exhibited rotational behavior, which was recovered by grafting mouse fetal DA neurons or human iPSC-derived DA neurons. Comparison with existing methodsImmunosuppression by drugs such as Cyclosporine A requires daily injection, which is stressful for rats and moreover may cause renal or hepatic failure. Furthermore, blood levels of the drug may not be stable, which weakens the reliability of the data. ConclusionsOur results provide a more accessible and reliable method to evaluate the in vivo function of human DA neurons, potentially offering a pre-clinical study for the application of pluripotent stem cells.

Abstract 19831: In Vivo Molecular Imaging of Human Pluripotent Stem Cell-derived Cardiomyocytes in a Murine Myocardial Injury Model via a Safe Harbor Integration of a Reporter Gene

Background: Clinical application of pluripotent stem cells and their tissue specific derivatives will require in vivo monitoring of engraftment and systemic distribution following transplantation. Clinical translation of molecular imaging, employing reporter gene (RG) technology, requires site-specific transgene insertion at safe harbor loci, such as AAVS1. Hypothesis: Zinc finger nuclease (ZFN)-mediated integration of a RG into the AAVS1 locus in human embryonic and induced pluripotent stem cells (hESCs and iPSCs) will allow monitoring of pluripotent stem cell-derived cardiomyocytes (hCMs and iCMs) in host myocardium. Methods and Results: ZFN technology was used to integrate a triple fusion RG, containing firefly luciferase (BLI), red fluorescent protein (RFP) and herpes simplex virus thymidine kinase (PET), under regulation of a constitutive ubiquitin promoter, into the AAVS1 locus of hESCs and iPSCs. hESCs and iPSCs monoclonal lines were then differentiated into hCMs and iCMs, respectively, under chemically defined conditions using CHIR and IWR small molecules to modulate Wnt pathway activity. Co-localization of cardiac phenotype and striated morphology was readily observed (Fig. A). Site-specific integration was confirmed by southern blot analysis. RG expression by BLI was maintained successfully over 3 months in vitro. The hCMs (n=6) and iCMs (n=1) were transplanted into murine myocardial injury model. Four weeks post-injury, LVEF was better preserved in all cell-treated vs. PBS-treated mice (26.50 ± 2.325 (N=6) vs. 15.40 ± 4.600 (N=3), p<0.05). Robust in vivo expression of ZFN-edited BLI and PET signal of engraftment persisted for 4-weeks duration (Figs. B and C). Conclusion: This study demonstrates the successful use of ZFN-edited cells to confirm the engraftment of hCMs and iCMs. ZFN technology enables targeted site-specific insertion of RGs to allow clinical translation of molecular imaging in cell therapy.

Concise Review: Parthenote Stem Cells for Regenerative Medicine: Genetic, Epigenetic, and Developmental Features

Embryonic stem cells (ESCs) have the potential to provide unlimited cells and tissues for regenerative medicine. ESCs derived from fertilized embryos, however, will most likely be rejected by a patient's immune system unless appropriately immunomatched. Pluripotent stem cells (PSCs) genetically identical to a patient can now be established by reprogramming of somatic cells. However, practical applications of PSCs for personalized therapies are projected to be unfeasible because of the enormous cost and time required to produce clinical-grade cells for each patient. ESCs derived from parthenogenetic embryos (pESCs) that are homozygous for human leukocyte antigens may serve as an attractive alternative for immunomatched therapies for a large population of patients. In this study, we describe the biology and genetic nature of mammalian parthenogenesis and review potential advantages and limitations of pESCs for cell-based therapies.

Stem Cells, a Reservoir for Life

Stem Cells, a Reservoir for Life

Comprehensive Comparison Of Gene Expression, Genomic DNA Methylation, and In Vitro Hematopoietic Differentiation Among Many Human iPS and ES Cell Lines

ObjectiveHematopoietic differentiation from human induced pluripotent stem (iPS)/embryonic stem (ES) cell attracts much attention due to its huge potential for regenerative medicine. As indicated by some earlier papers, there is large variation in differentiation potential among pluripotent stem cell (PSC) lines, and this is one of major concerns in clinical application of PSCs. If it becomes possible to predict which PSC line has high differentiation potential without real differentiation experiment, it would greatly contribute to clinical application of PSCs. Although some papers reported about presence of epigenetic memories of parental somatic cells in iPS cells, the amount of the influence on differentiation potential remains to be known. Furthermore, especially in studies using human PSCs, genetic difference among individual donors of iPS/ES cells seems to be large, thus the study using many PSC lines from many donors is warranted. To address these issues, we planned to collect data of many iPS/ES cell lines on genome-wide gene expression and genomic DNA methylation, and differentiation potentials of individual lines, and identify the factors which affected difference in differentiation potential among PSC lines. The final goal of this study is to create data base about gene expression and DNA methylation profile and differentiation potentials of many PSC lines. We believe that this dataset will allow us to predict differentiation potentials of individual PSC lines, and accelerate clinical application of PSC lines in hematology field. MethodWe utilized 39 iPS/ES lines (iPS 35 lines, ES 4 lines) in this study. The iPS cell lines were derived from dermal fibroblast (n = 16), cord blood (n = 3), peripheral blood (n = 10), keratinocyte (n = 3), and dental pulp cell (n = 3), and were generated by retrovirus vector (n = 9), episomal vector (n = 25), and sendai virus vector (n = 1). The iPS cells were derived from 15 donors, and the ES cells were derived from 4 donors. We assessed hematopoietic differentiation potential by investigating hematopoietic differentiation efficiency for the first 15 days from start of differentiation, and colony forming potential of hematopoietic precursor cells (CD34+CD38-CD43+lineage marker- population) generated from PSC lines using semi-solid methylcellulose based-media. In addition, we collected genome-wide mRNA expression and DNA methylation profile of PSC lines, parental lines of iPS cells, hematopoietic precursor cells generated from PSCs by using mRNA microarray, genomic methylation beads array, and next generation sequencers, and analyzed correlation of these data with differentiation potentials of individual PSC lines. ResultWe have found that there is large variation in hematopoietic differentiation efficiency and colony forming ability as reported previously. Genome-wide investigation of gene expression and genomic DNA methylation revealed that expression of some genes or some factors were significantly correlated with hematopoietic differentiation efficiency or colony forming ability of hematopoietic precursor cells. Importantly, the factors affecting differentiation efficiency for first 15 days and those affecting colony-forming ability were absolutely different. More importantly, by combining several factors discovered in this analysis, we can predict hematopoietic differentiation potential of individual iPS/ES cell lines regardless of what parental cell lines iPS cells are derived or whether it is an iPS cell or ES cell. ConclusionFrom genome-wide analysis of gene expression and genomic DNA methylation, and hematopoietic differentiation experiments, we discovered the factors that were associated with difference in differentiation potential among PSC lines. Now, we are focusing on investigating molecular mechanisms by which the discovered factors are responsible for the difference in hematopoietic differentiation potentials among PSC lines. We believe that our findings will contribute not only to clinical application of hematopoietic cells generated from human PSCs, but also to further understanding of human developmental hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

A Comment on Pluripotent Stem Cells in Next-Generation Biomedical Theranostics

New developments in DNA sequencing platforms and the advancements in GWAS studies (genome-wide association studies) are changing the understanding of human pathologies. Such developments will ultimately result in a deeper understanding of how genomic variations contribute to diseases. Induced pluripotent stem cells (iPSCs) are currently entering clinical research phases, allowing the investigation of disease pathways and the identification of new targets and potentially druggable biomarkers. IPSCs can serve as a model for studying human diseases as they retain all the genetic information from a patient; iPSC-derived cells can be used as a tool for drug screening or discovery. In combination with next generation sequencing (NGS)-based and GWAS technologies, iPSCs have the potential to become a novel platform technology to predict adverse drug and off-target effects, and using such cell models to predict toxicity. In view of the arising concepts of regenerative theranostics, iPSCs and NGS technologies provide a powerful means to analyze the complexity of diseases on the molecular level and to better understand the processes that lead to pathobiology. To promote the widespread use of iPSC-based approaches in drug development it has to be shown that the cells can be reliably produced in the quantity, consistency and purity needed to meet pharmaceutical standards. Integrative genomics and genetic approaches have shown to be a useful tool in elucidating the complexity found in gene regulatory pathways. In this review, the application of pluripotent stem cells for the generation of next-generation theranostics and newer perspectives on iPSCs in modeling clinical diseases, are discussed.

Vascular research using human pluripotent stem cells and humoral factors [Review

Embryonic stem (ES) cells are pluripotent cells collected from the inner cell mass of blastocysts. Induced pluripotent stem cells exhibit characteristics and pluripotency similar to ES cells, even though they were generated from adult somatic cells. We have been investigating the vascular differentiation kinetics of human pluripotent stem cells (PSCs) and their application to human vascular research and clinical medicine. In this review, we present an overview of recent vascular research using human PSCs, focusing on the role of humoral factors and their receptors. We also discuss possible future application of human PSCs to translational research on human vascular disorders.

Activation of Notch Signaling Mediates Ex Vivo Expansion of Multilineage, Engrafting Murine Hematopoietic Progenitors From Embryonic Sources.

Abstract 2321An important goal in the application of pluripotent stem cells (PSC) for therapeutic purposes is the derivation of hematopoietic stem and progenitor cells (HSPC) capable of efficient engraftment in vivo. Fundamental to achieving this goal is improved understanding of key signal pathways required to establish, maintain and expand HSPCs from embryonic sources. Ex vivo activation of Notch signaling in mouse bone marrow and human cord blood-derived HSC can facilitate expansion of rapidly engrafting multilineage progenitors, which has recently been translated for therapeutic purposes. In contrast, similar expansion of engrafting progenitors has not been successful from PSC. This prompted us to evaluate whether embryonic-derived HSPC have capacity to respond to ligand-induced Notch signaling ex vivo, and whether Notch activation could promote expansion of engrafting progenitors from these embryonic sources. We have examined the effects of ex vivo activation of Notch receptors by immobilized, exogenous Notch ligands on highly enriched populations of embryonic HSC and HSC precursors (pre-HSC) at various developmental stages. We find that activation of Notch by the ligand Delta1 within HSC/pre-HSC isolated from embryonic aorta-gonad-mesonephros (AGM) promotes expansion of progenitors with erythromyeloid colony forming potential and T/B-lymphoid potential in vitro, with concurrent expression of surface phenotypes resembling fetal liver-stage HSC. Furthermore, Notch activation in embryonic HSPC also mediates expansion of progenitors with rapidly engrafting myeloid and lymphoid capacity in irradiated mouse models. Our results demonstrate that embryonic stage HSPC have capacity to expand in response to Notch activation, and thus further studies comparing AGM- and PSC-derived hematopoietic precursors are needed to elucidate differences that may account for failure to expand HSPC from PSC. Disclosures:Bernstein:Seattle Genetics, Inc.: Consultancy.

An ECM-based culture system for the generation and maintenance of xeno-free human iPS cells

Pluripotent stem cells (PSCs) including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) have emerged as a promising source for treating incurable diseases. Problems that urgently need to be resolved before the clinical application include avoiding potential xenopathogenic transmission and immune rejection that may be caused by the exposure of PSCs to animal-derived products. In addition, an efficient feeder cell-free culture condition would be required for reducing batch-to-batch variation and facilitating scale-up. Therefore, establishing an efficient xeno-free and extracelluar matrix-based culture system is a prerequisite for the clinical application of PSCs. In this study, by blocking protein kinase C and histone deacetylase activities, we formulated a medium that, in combination with vitronectin as an extracellular matrix, not only allows the long-term culture of hESCs and iPSCs but also efficiently generates xeno-free iPSCs. This xeno-free and feeder cell-free culture system would facilitate the clinical applications of both iPSC- and ESC-based cell therapies in the future.

Suspension Culture of Human Pluripotent Stem Cells in Controlled, Stirred Bioreactors

Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end, we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption, but in parallel functional integration of all probes mandatory for process monitoring, that is, for pO₂ and pH, experiments were performed in 100 mL culture volume in a "mini reactor platform" consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×10⁸ hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained, underlining practical utility of this new process. The presented data provide key steps toward scalable mass expansion of human iPS and ES cells thereby enabling translation of stem cell research to (pre)clinical application in relevant large animal models and valuable in vitro assays for drug development and validation as well.

Challenges to the clinical application of pluripotent stem cells: towards genomic and functional stability

Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and are pluripotent, retaining the ability to differentiate into all cell types in the body. As a renewable source of various types of human cells, hESCs hold great therapeutic potential. Although significant advances have been achieved in defining the conditions needed to differentiate hESCs into various types of biologically active cells, many challenges remain in the clinical development of hESC-based cell therapy, such as the immune rejection of allogeneic hESC-derived cells by recipients. Breakthroughs in the generation of induced pluripotent stem cells (iPSCs), which are reprogrammed from somatic cells with defined factors, raise the hope that autologous cells derived from patient-specific iPSCs can be transplanted without immune rejection. However, recent genomic studies have revealed epigenetic and genetic abnormalities associated with induced pluripotency, a risk of teratomas, and immunogenicity of some iPSC derivatives. These findings have raised safety concerns for iPSC-based therapy. Here, we review recent advances in understanding the genomic and functional stability of human pluripotent stem cells, current challenges to their clinical application and the progress that has been made to overcome these challenges.

Recent Progress on Chemical Biology of Pluripotent Stem Cell Selfrenewal, Reprogramming and Cardiomyogenesis

Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, hold great promise as a cell source for regenerative therapies to treat many major diseases characterized by an irreversible loss of functional tissues. However, the future clinical application of pluripotent stem cells faces a number of obstacles regarding the safety, efficiency and long-term benefits. Some of these challenges are being addressed by the chemical biology approach using small molecules. In this paper, we review the recent progress and patents on small molecules which promote pluripotent stem cell maintenance, reprogramming, and direct differentiation with a focus on cardiomyogenesis.

What can pluripotent stem cells teach us about neurodegenerative diseases?

Neurodegenerative diseases represent a growing public health challenge. Current medications treat symptoms, but none halt or retard neurodegeneration. The recent advent of pluripotent cell biology has opened new avenues for neurodegenerative disease research. The greatest potential for induced pluripotent cells derived from affected individuals is likely to be their utility for modeling and understanding the mechanisms underlying neurodegenerative processes, and for searching for new treatments, including cell replacement therapies. However, much work remains to be done before pluripotent cells can be used for preclinical and clinical applications. Here we discuss the challenges of generating specific neural cell subtypes from pluripotent stem cells, the use of pluripotent stem cells to model both cell-autonomous and non-cell-autonomous mechanisms of neurodegeneration, whether adult-onset neurodegeneration can be emulated in short-term cultures and the hurdles of cell replacement therapy. Progress in these four areas will substantially accelerate effective application of pluripotent stem cells.

Pluripotent stem cells: private obsession and public expectation.

In the 1960s it was shown that a rare type of tumour called a teratocarcinoma contains cells that are both pluripotent and self‐renewing (Kleinsmith & Pierce, 1964). Pluripotent means the capacity of an individual cell to give rise to all other cell types of the body and the germline. This property is normally restricted to a brief window in early development. Self‐renewal is the production of identical daughter cells while retaining the ability for differentiation. It is the defining feature of a stem cell. The study of teratocarcinoma stem cells led to particular culture conditions that allowed them to be propagated ex vivo without differentiation. In 1981 Martin Evans, Matt Kaufman and Gail Martin found that cells from early mouse embryos exposed to the same culture environment can suspend developmental progression and continue to multiply while remaining pluripotent (Evans & Kaufman, 1981; Martin, 1981). These are embryonic stem (ES) cells. I was completing my undergraduate studies in Oxford at that time and was fortunate to learn about this rather esoteric research from a wonderful teacher, Chris Graham. Chris also talked about parallel discoveries of growth factors and the provocative idea that they might regulate cell fate. I was captivated by the idea of understanding and controlling the pluripotent state » I was captivated by the idea of understanding and controlling the pluripotent state. « and that has been my obsession ever since. I went to Edinburgh to do a PhD on teratocarcinoma stem cells with Martin Hooper. There I was struck by an observation by a previous PhD student that the requirement of the stem cells for co‐culture with a feeder layer could be replaced for a short time by conditioned medium. To me this meant only one thing; a growth factor that could block differentiation. This was not …

Roadblocks en route to the clinical application of induced pluripotent stem cells

Since the first studies of human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs), the stem-cell field has been abuzz with the promise that these pluripotent populations will one day be a powerful therapeutic tool. Although it has been proposed that hiPSCs will supersede hESCs with respect to their research and/or clinical potential because of the ease of their derivation and the ability to create immunologically matched iPSCs for each individual patient, recent evidence suggests that iPSCs in fact have several underappreciated characteristics that might mean they are less suitable for clinical application. Continuing research is revealing the similarities, differences and deficiencies of various pluripotent stem-cell populations, and suggests that many years will pass before the clinical utility of hESCs and hiPSCs is realized. There are a plethora of ethical, logistical and technical roadblocks on the route to the clinical application of pluripotent stem cells, particularly of iPSCs. In this Essay, we discuss what we believe are important issues that should be considered when attempting to bring hiPSC-based technology to the clinic.

Nongenetic method for purifying stem cell–derived cardiomyocytes

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

Induced Pluripotent Stem Cells

Embryonic stem cells (ESCs) were first derived in the early 1980s simultaneously by 2 independent groups,1,2 and because of their plasticity and potentially unlimited capacity for self-renewal, they were predicted to transform research in mammalian development, genetics, stem cell biology, and regenerative medicine. However, it quickly became apparent that although ESCs would become an invaluable tool to study development and stem cell biology, translating their promise into the clinics would be problematic. Twenty-eight years after the first report describing mouse ESCs and 10 years after the successful derivation of human ESCs, the US Food and Drug Administration approved the first human ESC-related clinical trial for spinal cord injuries this year; however, it is now on its second clinical hold, and no subjects have yet been enrolled. Although this delay in translation to human trials is likely multifactorial, major obstacles remain with the clinical use of ESCs over ethical issues, oncogenic risk, and the fact that ESC derivatives for tissue repair involve the use of allogeneic cells that can lead to rejection of mismatched cellular grafts. The oncogenic risk associated with stem cell therapies is no longer just theoretical. This year, the first report of a patient who developed multiple tumors in his brain and spinal cord after receiving fetal neural stem cells was published.3 Thus, therapeutic application of pluripotent stem cells (PSCs), particularly in the cardiovascular field, requires further advancement and new approaches such as that described by Zwi et al4 in this issue of Circulation . Article see p 1513 Although how to minimize the oncogenic risk of PSCs continues to be an area of intense research, investigators have long sought to overcome the ethical and immunologic issues surrounding ESCs by creating autologous PSCs. Initial attempts to create these cells involved nuclear cloning technology in which …

A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells

An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation. We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632. Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.

Somatic cell dedifferentiation/reprogramming for regenerative medicine.

The concept of dedifferentiation or reprogramming of a somatic cell into a pluripotent embryonic stem cell-like cell (ES-like cell), which give rise to three germ layers and differentiate various cell types, opens a new era in stem cell biology and provides potential therapeutic modality in regenerative medicine. Here, we outline current dedifferentiation/reprogramming methods and their technical hurdles, and the safety and therapeutic applications of reprogrammed pluripotent stem cells in regenerative medicine. This review summarizes the concept and data of somatic cell nuclear transfer, fusion of somatic cells with ES cells, viral or non-viral transduction of pluripotency-related genes into somatic cells, introduction of extract (or proteins) of pluripotent cells into somatic cells. Dedifferentiated/reprogrammed ES-like cells could be a perfect genetic match (autologous or tailored pluripotent stem cells) for future applications. Further studies regarding technical refinements as well as mechanistic analysis of dedifferentiation induction and re-differentiation into specific cell types will provide us with the substantial application of pluripotent stem cells to therapeutic purposes.

A Highly Efficient Method to Differentiate Smooth Muscle Cells From Human Embryonic Stem Cells

To the Editor: The molecular mechanisms and the control of smooth muscle cell (SMC) differentiation have been extensively investigated because of its therapeutic potential.1 To date, different cell types have been used to study SMC differentiation, including a variety of mouse embryonic stem cells,2 adult stem cells,3,4 and others.5 Because several fundamental differences exist between mouse and human embryonic development,6 lack of a good model system to study human SMC differentiation has hampered the progress of translating SMC knowledge to novel clinical therapies. Human embryonic stem (hES) cells provide a valuable source of cells for studying human cell differentiation and developing therapeutic potentials in regenerative medicine. Since the initial report describing the derivation of hES cells,7 a variety of studies have established in vitro differentiation strategies to several lineages. Recently, it has been demonstrated that vascular progenitors derived from hES cells could be differentiated into endothelial cells and SMCs by endothelial …