hESC-derived Endothelial Cells Research Articles

Identification of RNA-based cell-type markers for stem-cell manufacturing systems with a statistical scoring function

Cell-type biomarkers are useful in stem-cell manufacturing to monitor cell purity, quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. Emerging questions include which RNA transcripts can serve as biomarkers during stem cell culture and how to discover these biomarkers efficiently and precisely. We developed a scoring function system to identify RNA biomarkers with RNA-seq data for systems that have a limited number of cell types. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The first data set has RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. The validity of these findings was confirmed by the utilization of additional data sets and experimental procedures. We also used deep-learning-based prediction methods and simulated data to validate these discovered biomarkers.

Genome editing HLA alleles for a pilot immunocompatible hESC line in a Chinese hESC bank for cell therapies

Robust allogeneic immune reactions after transplantation impede the translational pace of human embryonic stem cells (hESCs)-based therapies. Selective genetic editing of human leucocyte antigen (HLA) molecules has been proposed to generate hESCs with immunocompatibility, which, however, has not been specifically designed for the Chinese population yet. Herein, we explored the possibility of customizing immunocompatible hESCs based on Chinese HLA typing characteristics. We generated an immunocompatible hESC line by disrupting HLA-B, HLA-C, and CIITA genes while retaining HLA-A*11:01 (HLA-A*11:01-retained, HLA-A11R ), which covers ~21% of the Chinese population. The immunocompatibility of HLA-A11R hESCs was verified by in vitro co-culture and confirmed in humanized mice with established human immunity. Moreover, we precisely knocked an inducible caspase-9 suicide cassette into HLA-A11R hESCs (iC9-HLA-A11R ) to promote safety. Compared with wide-type hESCs, HLA-A11R hESC-derived endothelial cells elicited much weaker immune responses to human HLA-A11+ T cells, while maintaining HLA-I molecule-mediated inhibitory signals to natural killer (NK) cells. Additionally, iC9-HLA-A11R hESCs could be induced to undergo apoptosis efficiently by AP1903. Both cell lines displayed genomic integrity and low risks of off-target effects. In conclusion,we customized a pilot immunocompatible hESC cell line based on Chinese HLA typing characteristics with safety insurance. This approach provides a basis for establishment of a universal HLA-AR bank of hESCs covering broad populations worldwide and may speed up the clinical application of hESC-based therapies.

Synthetic liver fibrotic niche extracts achieve in vitro hepatoblasts phenotype enhancement and expansion

SummaryIt is still a challenge for synthesizing ‘cellular niche-mimics’ in vitro with satisfactory reproducibility and fidelity to recreate the natural niche components (e.g., extracellular matrices and soluble factors) for stem cell cultivation. Inspired by the massive amplification of hepatic progenitor cells during liver fibrosis in vivo, here we optimized the in vitro liver fibrotic niches and subsequently harvested their bioactive ingredients as niche extracts (NEs). The fibrosis-relevant NE marginally outperformed Matrigel for phenotype maintenance of human embryonic stem cell (hESC)-derived hepatoblasts (HBs) and recapitulation of the pathological angiogenesis of hESC-derived endothelial cells both in 2D culture and 3D liver organoids. Finally, defined NE components (i.e., collagen III, IV, IL-17, IL-18 and M-CSF) were resolved by the quantitative proteomics which exhibited advantage over Matrigel for multi-passaged HB expansion. The pathology-relevant and tissue-specific NEs provide innovative and generalizable strategies for the discovery of optimal cellular niche and bioactive niche compositions.

Peripheral sensory neurons promote angiogenesis in neurovascular models derived from hESCs.

In the adult tissues, blood vessels traverse the body with neurons side by side; and share common signaling molecules. Developmental studies on animal models have shown that peripheral sensory neurons (PSNs) secrete angiogenic factors and endothelial cells (ECs) secrete neurotrophic factors which contribute to their coexistence, thereby forming the peripheral neurovascular (PNV) unit. Despite the large number of studies showing that innervation and vascularization complement each other, the interaction between human PSNs and ECs is still largely unknown. To study this interaction and to evaluate if PSNs affect angiogenesis, we derived both PSNs and ECs from human embryonic stem cells (hESCs) and developed a co-culture system. Seeding the two cell types together showed that PSNs induced endothelial morphogenesis with formation of vessel-like structures (VLSs). The PSN precursors, neural crest stem cells also induced VLS formation in the co-culture system; however, to a lesser extent. This sheds new light on the in vitro angiogenic potential of these cell types. PSNs derived from hESCs are powerful tools for studying development and disease as human PSNs are inaccessible for in vitro assays. Our novel approach, with optimized media condition allowed for integrating hESC-derived PSNs with hESC-derived ECs in three-dimensional (3D) collagen gel for creating a completely humanised PNV model. This preliminary model showed that innervation improves the development of vascularized channels in vitro, and provides insight to the development of innervated 3D models in future.

Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing.

Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.

EGFL7 Mediates BMP9-Induced Sprouting Angiogenesis of Endothelial Cells Derived from Human Embryonic Stem Cells.

SummaryHuman embryonic stem cells (hESCs) are instrumental in characterizing the molecular mechanisms of human vascular development and disease. Bone morphogenetic proteins (BMPs) play a pivotal role in cardiovascular development in mice, but their importance for vascular cells derived from hESCs has not yet been fully explored. Here, we demonstrate that BMP9 promotes, via its receptor ALK1 and SMAD1/5 activation, sprouting angiogenesis of hESC-derived endothelial cells. We show that the secreted angiogenic factor epidermal growth factor-like domain 7 (EGFL7) is a downstream target of BMP9-SMAD1/5-mediated signaling, and that EGFL7 promotes expansion of endothelium via interference with NOTCH signaling, activation of ERK, and remodeling of the extracellular matrix. CRISPR/Cas9-mediated deletion of EGFL7 highlights the critical role of EGFL7 in BMP9-induced endothelial sprouting and the promotion of angiogenesis. Our study illustrates the complex role of the BMP family in orchestrating hESC vascular development and endothelial sprouting.

Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

Like conventional transplants, immunosuppression is required to facilitate survival and function of human embryonic stem cell (hESC) derivatives after implantation into xenogeneic recipients. We have previously reported that T cells alone are sufficient to reject allogeneic murine ESC derivatives; and strategies that inhibit T-cell activation, including coreceptor and costimulation blockade, prevent hESC derivatives from being rejected. This study aimed to investigate, in addition to T cells, whether macrophages contribute to transplant rejection of hESC xenografts with nonobese diabetic (NOD)/SCID mice that lack functional T and B cells but have macrophages. We show that acute rejection against hESC-derived endothelial cells (hESC-ECs) was mediated, to some degree, by infiltrating macrophages that phagocytosed them. Transgenic expression of murine CD47 on cell surface of hESC-ECs mitigates macrophage-mediated phagocytosis and improves their survival after transplantation. Our results highlight that innate immune cells, such as macrophages, can reject hESC derivatives, raising concern against the use of NOD/SCID as transplant recipients for testing in vivo function of hESC-derived tissues. Augmenting CD47 signaling promotes survival and function of hESC derivatives after xenogeneic transplantation.-Leung, C. S., Li, J., Xu, F., Wong, A. S. L., Lui, K. O. Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

Directing human embryonic stem cells towards functional endothelial cells easily and without purification.

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

Signaling via PI3K/FOXO1A pathway modulates formation and survival of human embryonic stem cell-derived endothelial cells.

Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied phosphatidylinositol 3-kinase (PI3K)-Forkhead box O transcription factor 1A (FOXO1A) pathways during differentiation of hESC toward endothelial lineage and on proliferation, maturation, and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodeling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31(+) hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by small interfering RNA (siRNA) showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, while FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC and in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.

Functional Assessment of Hematopoietic Niche Cells Derived from Human Embryonic Stem Cells

To evaluate hematopoietic niche cell populations isolated from human embryonic stem cells (hESCs), we tested the ability of hESC-derived stromal lines to support CD34(+) umbilical cord blood (UCB)- and hESC-derived CD34(+)45(+) cells in long-term culture initiating cell (LTC-IC) assays. Specifically, these hematopoietic populations were cocultured with hESC-derived mesenchymal stromal cells (hESC-MSCs) and hESC-derived endothelial cells (hESC-ECs), and then assessed for their LTC-IC potential in comparison to coculture with bone marrow (BM)-derived MSCs and the mouse stromal line M2-10B4. We found that the hESC-derived stromal lines supported LTC-ICs from UCB similar to M2-10B4 cells and better than BM-MSCs. However, none of the stromal populations supported LTC-IC from hESC-derived CD34(+)45(+) cells. Engraftment data using the output from LTC-IC assays showed long-term repopulation (12 weeks) of NSG mice to correlate with LTC-IC support on a given stromal layer. Therefore, hESC-derived stromal lines can be used to efficiently evaluate putative hematopoietic stem/progenitor cells derived from hESCs or other cell sources.

Immunosuppressive Agents Modulate Function, Growth, and Survival of Cardiomyocytes and Endothelial Cells Derived from Human Embryonic Stem Cells

Cardiac cell replacement therapy by using human embryonic stem cell (hESC) derivatives remains a potential approach to regenerate myocardium. The major hurdles to clinical application of this technology are immunogenicity and post-transplantation cell death. Here we examined the effects of calcineurin-targeting immunosuppressants cyclosporine A (CsA) and FK506, as well as rapamycin and a selective inhibitor of calcineurin-binding downstream nuclear factor of activated T-cell (NFAT) transcription factor VIVIT on the proliferative activity, function, and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. As shown by automated microscopy, treatments with CsA, FK506, and rapamycin all decreased proliferation, reducing the percentage of hESC-CM and hESC-EC with the mitotic marker Ki67(+) by as much as 60% and 74%, respectively. Administration of the cell permeable analogue 11R-VIVIT protein did not modulate their proliferative activity. All immunosuppressants reversed the proapoptotic effect of chelerythrine in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT and mammalian target of rapamycin (mTOR) pathways in hESC-CM survival (using apoptotic marker caspase-3), whereas the protection was less obvious in hESC-EC exposed to H2O2. Immunosuppressants did not affect cell viability in hESC-EC. Our results show that immunosuppressants reduce proliferation, while offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressant therapy would be compatible with stem cell transplantation, but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given. The effects of NFAT-binding immunosuppressant molecules, which do not affect hESC-CM proliferation, may point the way forward for new classes of compounds better suited to cell implantation.

Therapeutic Efficacy of Human Embryonic Stem Cell–Derived Endothelial Cells in Humanized Mouse Models Harboring a Human Immune System

Allogeneic transplantation of human embryonic stem cell (hESC) derivatives has the potential to elicit the patient's immune response and lead to graft rejection. Although hESCs and their derivatives have been shown to have advantageous immune properties in vitro, such observations could not be determined experimentally in vivo because of ethical and technical constraints. However, the generation of humanized mice (hu-mice) harboring a human immune system has provided a tool to perform in vivo immunologic studies of human cells and tissues. Using this model, we sought to examine the therapeutic potential of hESC-derived endothelial cells, human embryonic fibroblasts, and cord blood-derived endothelial progenitor cells in a human immune system environment. All cell types transplanted in hu-mice showed significantly reduced cell survival during the first 14 days post-transplantation compared with that observed in immunodeficient mice. During this period, no observable therapeutic effects were detected in the hindlimb ischemic mouse models. After this point, the cells demonstrated improved survival and contributed to a long-term improvement in blood perfusion. All cell types showed reduced therapeutic efficacy in hu-mice compared with NOD scid IL2 receptor gamma chain knockout mice. Interestingly, the eventual improvement in blood flow caused by the hESC-derived endothelial cells in hu-mice was not much lower than that observed in NOD scid IL2 receptor gamma chain knockout mice. These findings suggest that hESC derivatives may be considered a good source for cell therapy and that hu-mice could be used as a preclinical in vivo animal model for the evaluation of therapeutic efficacy to predict the outcomes of human clinical trials.

Differentiation of human embryonic stem cells to endothelial cells via improved three-dimension approach

To establish an improved three-dimension (3D) and serum-free approach to differentiate human embryonic stem cells (hESCs) into endothelial cells, and detect the endothelial functions of the obtained cells. We cultured undifferentiated H9 human embryonic stem cell line in low-adhesion dishes to form embryonic bodies (EBs). After 12 days, EBs were harvested, re-suspended into rat tail collagen type I, and put into the incubator (37℃). After 30 minutes, EGM-2 culture medium was added to the solidified collagen, and the EBs were cultured for another 3 days to form embryonic body-sproutings (EB-sproutings). EB-sproutings were digested with 0.25% collagenase I and 0.56 U/ml Liberase Blendzyme for 20 minutes respectively, and the CD31(+) cells were sorted by FACS. The endothelial functions were tested by Dil-ac-LDL uptake assay and tube formation assay. This approach raised the efficiency of endothelial differentiation to 18%, and also avoided the contamination with animal materials. The obtained hESC-derived endothelial cells (hESC-ECs) had the similar pattern of surface biomarkers as human umbilical vein endothelial cells (HUVECs), and their endothelial functions were confirmed by the uptake of Dil-ac-LDL and the tube formation on Matrigel. The improved 3D approach can enhance the efficiency of differentiation from hESCs into endothelial cells. Furthermore, serum free differentiation system may be applied in future hESC-based therapies for various ischemic diseases.

APELIN promotes hematopoiesis from human embryonic stem cells

AbstractTranscriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein–coupled APELIN receptor (APLNR). APLNR-positive cells, identified by binding of the fluoresceinated peptide ligand, APELIN (APLN), or an anti-APLNR mAb, were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs), increased the expression of hematoendothelial genes in differentiating hESCs, and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore, APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells.

Effective Development of Hematopoietic Niche Cells From hESCs: hESC-Derived Stromal Cells Support LTC-ICs Better Than Bone Marrow-Derived Stromal Cells

Abstract 4804Quantification of putative hematopoietic stem cells (HSCs) in human bone marrow or cord blood is typically assessed by its potential to mediate long-term, multilineage engraftment when transplanted into immunodeficient murine recipients. However, in vitro surrogate assays are extremely attractive due to their relative ease of implementation, low cost and improved throughput of results. The long-term culture initiating cell (LTC-IC) assay serves this purpose by quantifying the ability of putative HSCs in a given population to be cultured for an extended period, typically 5 weeks. Previously, we have derived both mesenchymal stromal cells (MSCs) and endothelial cells (ECs) from human embryonic stem cells (hESCs). MSCs and ECs are important components of the osteogenic and vascular hematopoietic niches and hESC-derived stromal cells can provide autologous supporting cell populations for putative hESC-derived HSCs. Therefore, we evaluated if hESC-derived MSCs (hESC-MSCs) and hESC-derived ECs (hESC-ECs) would have improved ability to support LTC-IC activity of UCB and hESC-derived hematopoietic cells compared to MSCs isolated from human bone marrow (BM-MSCs) and a murine stromal line, M2-10B4. In the present study, we utilized LTC-IC culture of both hESC-derived CD34+CD45+ populations and UCB 34+ cells to test their LTC-IC potential on hESC-MSCs, hESC-ECs and a combination of the two hESC-derived populations. LTC-IC frequency and maintenance of input populations was assessed by survival/proliferation and preservation of colony forming cell (CFC) progenitors over 5 weeks. Initial studies showed all stromal layers were able to support short-term (3 week) culture of CFCs from CD34+ UCB. Total cell proliferation was found to be greatest with the hESC-derived stroma at 3 weeks. Additionally, hESC-MSCs and hESC-ECs yielded CFC comparable to M2-10B4 and significantly more than BM-MSCs. Co-culture of CD34+ UCB with a 1:1 mixture of hESC-MSCs and hESC-ECs yielded similar proliferation and CFC results to either stroma alone. After 5 weeks of culture to quantify LTC-ICs, both of the hESC-derived cells again supported expansion of UCB total cell numbers better than BM-MSCs and similar to M2-10B4. LTC-IC yield at 5 weeks was 85 ± 29 and 74 ± 12 per 103 input cells for hESC-MSC and hESC-EC, respectively, as compared to 15 ± 12 for BM-MSCs and 104 ± 56 for M2-10B4 co-culture. Combining the hESC-derived MSCs and ECs produced an LTC-IC yield of 54 ± 4. Next, we tested the ability of hESC-derived CD34+CD45+ cells to produce CFCs and LTC-ICs using co-culture with hESC-MSCs and M2-10B4. In short-term (2-3 week) assays, hESC-derived CD34+CD45+ cells declined in number to about 30% of the starting population on both stroma. However, between 3–5 weeks, cell number remained fairly constant on M2-10B4 while continuing to decrease on hESC-MSC. Interestingly, CFC yield for hESC-derived CD34+CD45+ cells at 2 weeks was 45 ± 43 per 103 input cells on M2-10B4, while co-culture with hESC-MSCs regularly failed to produce any CFC at this time. Between 3–5 weeks of culture no CFC or LTC-ICs were produced from hESC CD34+CD45+ using any of the stromal cells. Similar results were seen for hESC-derived CD34+CD43+ populations. Together, these results have two key findings. First, hESC-derived ECs and MSCs are effective in supporting LTC-ICs from UCB, and do so better than human BM-MSCs. Second, these same stromal cells do not support survival of hESC-derived LTC-ICs. In this manner, these results closely mimic studies that show limited long-term hematopoietic engraftment of hESC-derived cells when transplanted into immunodeficient mice. Therefore, this system can now be used to aid in identification of both hematopoietic niche elements and specific cell populations that mediate development and support of putative HSCs derived from hESCs and iPSCs. Disclosures:No relevant conflicts of interest to declare.

A system for treating ischemic disease using human embryonic stem cell-derived endothelial cells without direct incorporation

Despite studies on the use of human embryonic stem cell (hESC) derivatives to treat ischemic diseases, there are technical safety issues that have yet to be resolved. Herein, we sought to develop a method for using secreted angiogenic factors from hESC-derived endothelial cell derivatives (hESC-ECs), while avoiding direct cell incorporation, to reduce tumorigenesis or unidentified side effects of injected cells in vivo. Multicellular aggregation of hESC-ECs (MA-hESC-ECs) increases survivability, and encapsulation in Matrigel (EnMA-hESC-ECs) blocks the cells’ in vivo migration. To examine the therapeutic effects of EnMA-hESC-ECs, we implanted both forms of hESC-ECs in a mouse model of hindlimb ischemia. Treatment with EnMA-hESC-ECs suppressed limb loss and tissue damage with no noticeable side effects, such as tumorigenesis or teratoma formation, and the Matrigel implant could be easily removed after the procedure. Thus, MA and the encapsulation system are effective techniques for utilizing humoral factors secreted by hESC derivatives that aid in the survivability and safety. We expect these results to contribute to the thriving stem cell field by improving the bioavailability of hESC derivatives for regenerative medicine.

Lentiviral Transduction and Clonal Selection of hESCs with Endothelial‐Specific Transgenic Reporters

Generation of vascular endothelial cells (EC) from human embryonic stem cells (hESC) is a vital component of cell-based strategies for treatment of cardiovascular disease. Before hESC-derived ECs can be administered in therapeutic modalities, however, chemically defined culture conditions must be developed that reproducibly and robustly induce vascular differentiation. One approach to screening for culture conditions that support differentiation of hESCs to any cell type is their genetic modification with exogenous DNA sequence comprising a tissue-specific gene promoter driving reporters such as fluorescent protein or antibiotic drug resistance. The protocols herein provide instructions for the generation of clonal hESC lines containing a reporter transgene that is specifically expressed in vascular endothelial derivatives. Additionally, they demonstrate the methodology employed to assess vascular differentiation from clonal lines. Together, these protocols provide a solid foundation for study of vascular differentiation, and may also be applied, in principle, to studies of other specialized cell types derived from hESCs.

Functional Characterization and Expression Profiling of Human Induced Pluripotent Stem Cell- and Embryonic Stem Cell-Derived Endothelial Cells

With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the "distances" among hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and HUVECs. We further conclude that hiPSCs can differentiate into functional endothelial cells, but with limited expansion potential compared with hESC-ECs; thus, extensive studies should be performed to explore the cause and extent of such differences before clinical application of hiPSC-ECs can begin.

A Fibrin Patch-Based Enhanced Delivery of Human Embryonic Stem Cell-Derived Vascular Cell Transplantation in a Porcine Model of Postinfarction Left Ventricular Remodeling

It is unknown how to use human embryonic stem cell (hESC) to effectively treat hearts with postinfarction left ventricular (LV) remodeling. Using a porcine model of postinfarction LV remodeling, this study examined the functional improvement of enhanced delivery of combined transplantation of hESC-derived endothelial cells (ECs) and hESC-derived smooth muscle cells (SMCs) with a fibrin three-dimensional (3D) porous scaffold biomatrix. To facilitate tracking the transplanted cells, the hESCs were genetically modified to stably express green fluorescent protein and luciferase (GFP/Luc). Myocardial infarction (MI) was created by ligating the first diagonal coronary artery for 60 minutes followed by reperfusion. Two million each of GFP/Luc hESC-derived ECs and SMCs were seeded in the 3D porous biomatrix patch and applied to the region of ischemia/reperfusion for cell group (MI+P+C, n = 6), whereas biomatrix without cell (MI+P, n = 5), or saline only (MI, n = 5) were applied to control group hearts with same coronary artery ligation. Functional outcome (1 and 4 weeks follow-up) of stem cell transplantation was assessed by cardiac magnetic resonance imaging. The transplantation of hESC-derived vascular cells resulted in significant LV functional improvement. Significant engraftment of hESC-derived cells was confirmed by both in vivo and ex vivo bioluminescent imaging. The mechanism underlying the functional beneficial effects of cardiac progenitor transplantation is attributed to the increased neovascularization. These findings demonstrate a promising therapeutic potential of using these hESC-derived vascular cell types and the mode of patch delivery.

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an Id1-YFP hESC reporter line, we showed that TGFbeta inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.