Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression

Rationale

Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.

Method and Result

We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30–70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7–8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95–98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5−10×10⁵ VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.

Conclusion

We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.     

Polymeric fibrous matrices for substrate-mediated human embryonic stem cell lineage differentiation

Chitosan-based fibrous matrices are prepared to mimic the ECM architecture and elucidate substrate-mediated hESC differentiation due to topographical scale and anisotropy without exogenic morphogens. Fibrous matrices support fewer pluripotent hESCs than films but enable topography-mediated hESC differentiation. Matrices composed of 400 nm and 1.1 μm diameter fibers support increased expression of neural markers indicative of ectodermal commitment while matrices of 200 nm diameter fibers increase expression of osteogenic and hepatic markers indicative of endodermal and mesodermal commitment. The fibrous-mediated hESC differentiation highlights the significant implication of tailored ECM-like substrates for hESC-based therapies.     

CD marker expression profiles of human embryonic stem cells and their neural derivatives,determined using flow-cytometric analysis,reveal a novel CD marker for exclusion of pluripotent stem cells

Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation.     

Differential Processing of Amyloid-�� Precursor Protein Directs Human Embryonic Stem Cell Proliferation and Differentiation into Neuronal Precursor Cells

The amyloid-β precursor protein (AβPP) is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-β (Aβ) protein, is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease, Down syndrome, and head injury. We recently reported that this protein, normally associated with neurodegenerative conditions, is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AβPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-β, which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AβPP cleavage by β-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression, an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AβPPα, which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AβPP is normally required for embryonic neurogenesis.The amyloid-β precursor protein (AβPP)⁵ is a ubiquitously expressed transmembrane protein whose cleavage product, the amyloid-β (Aβ) protein, is deposited in amyloid plaques in the aged brain, following head injury, and in the neurodegenerative conditions of Alzheimer disease (AD) and Down syndrome (DS). AβPP has structural similarity to growth factors (1) and modulates several important neurotrophic functions, including neuritogenesis, synaptogenesis, and synaptic plasticity (2). The function of AβPP during early embryogenesis and neurogenesis has not been well described.AβPP is processed by at least two pathways, the non-amyloidogenic and amyloidogenic pathways. Non-amyloidogenic processing of AβPP yields secreted AβPPα (sAβPPα), the secreted extracellular domain of AβPP that acts as a growth factor for many cell types and promotes neuritogenesis (3). Amyloidogenic processing of AβPP releases sAβPPβ, the AβPP intracellular domain, and Aβ proteins. The Aβ protein has both neurotoxic and neurotrophic properties (4) dependent on the differentiation state of the neuron; Aβ is neurotoxic to differentiating neurons via a mechanism involving differentiation-associated increases in the phosphorylation of the microtubule-associated protein tau (5) but neurotrophic to undifferentiated embryonic neurons. Evidence supporting a neurotrophic function for Aβ during development include its neurogenic activity toward rat neural stem cells (4–6). Consistent with these data, two studies have demonstrated increased hippocampal neurogenesis in young transgenic mice overexpressing human APP_Sw,Ind (7, 8).Recently we reported that human embryonic stem cells (hESCs) express AβPP and that both the stemness of the cells and the pregnancy-associated hormone human chorionic gonadotropin alter AβPP expression (9). These results suggest a functional role for AβPP during early human embryogenesis. To further investigate the function of AβPP and its cleavage products during early embryonic neurogenesis, we examined the expression and processing of this protein and its role in proliferation and differentiation of hESCs into neural precursor cells (NPCs). We found that amyloidogenic processing of AβPP promotes hESC proliferation whereas non-amyloidogenic processing induces hESC differentiation into NPCs. These data reveal an important function for AβPP during early human embryonic neurogenesis. Our data imply that any dysregulation in AβPP processing that leads to altered sAβPPα/Aβ production could result in aberrant neurogenesis as reported in the AD and DS brains.     

Generation of human female reproductive tract epithelium from human embryonic stem cells

Background

Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT).

Methodology/Principal Findings

We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1⁺ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA).

Conclusions/Significance

These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.     

Hypoxic preconditioning‐induced autophagy enhances survival of engrafted endothelial progenitor cells in ischaemic limb

Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell‐based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning‐induced autophagy on survival of the engrafted EPCs. Autophagy of CD34⁺VEGFR‐2⁺ EPCs isolated from rat bone marrow increased after treatment with 1% O₂. The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3‐methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O₂ for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler‐based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.     

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30^Hi-GCTM-2^Hi hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30^Neg-GCTM-2^Neg). In a further study, pluripotent stem cell-free samples of differentiated TG30^Neg-GCTM-2^Neg cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30^Hi-GCTM-2^Hi hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.     

Comparative study of hematopoietic differentiation between human embryonic stem cell lines

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34(+) or CD34(+)CD45(+) hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34(+) precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34(+) hematopoietic precursors generated in vitro versus in vivo.     

hESC Expansion and Stemness Are Independent of Connexin Forty-Three-Mediated Intercellular Communication between hESCs and hASC Feeder Cells

Background

Human embryonic stem cells (hESCs) are a promising and powerful source of cells for applications in regenerative medicine, tissue engineering, cell-based therapies, and drug discovery. Many researchers have employed conventional culture techniques using feeder cells to expand hESCs in significant numbers, although feeder-free culture techniques have recently been developed. In regard to stem cell expansion, gap junctional intercellular communication (GJIC) is thought to play an important role in hESC survival and differentiation. Indeed, it has been reported that hESC-hESC communication through connexin 43 (Cx43, one of the major gap junctional proteins) is crucial for the maintenance of hESC stemness during expansion. However, the role of GJIC between hESCs and feeder cells is unclear and has not yet been reported.

Methodology/Principal Findings

This study therefore examined whether a direct Cx43-mediated interaction between hESCs and human adipose-derived stem cells (hASCs) influences the maintenance of hESC stemness. Over 10 passages, hESCs cultured on a layer of Cx43-downregulated hASC feeder cells showed normal morphology, proliferation (colony growth), and stemness, as assessed by alkaline phosphatase (AP), OCT4 (POU5F1-Human gene Nomenclature Database), SOX2, and NANOG expression.

Conclusions/Significance

These results demonstrate that Cx43-mediated GJIC between hESCs and hASC feeder cells is not an important factor for the conservation of hESC stemness and expansion.     

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.     

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Quantitative Comparison of the Membrane Proteomes of Self-renewing and Differentiating Human Embryonic Stem Cells

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase ζ (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.Human embryonic stem cells (hESCs)¹ are stem cells derived from the blastocyst inner cell mass. They are pluripotent; thus they are able to differentiate into any human cell type. The self-renewal capacity and pluripotency make hESCs an ideal system to study the processes of cell development and differentiation. Moreover hESC research is highly relevant for regenerative medicine, which aims at replacing or restoring tissue damaged by disease or injury through transplantation of functional hESCs (1,2). However, factors responsible for maintaining the undifferentiated and pluripotent nature of hESCs are still largely unknown. Before hESCs can be used for transplantation into the human body, reliable and reproducible protocols for differentiating them into specific cell types are needed. To create such protocols we need to develop a thorough understanding of the mechanisms maintaining the undifferentiated pluripotent nature of hESCs and those guiding their differentiation into specific lineages.A number of factors involved in the maintenance of pluripotency have been described over the last few years (3). It has also been demonstrated that overexpression of some of these factors in somatic cells is sufficient to turn them into pluripotent stem cells very similar to hESCs (4–8). However, it is apparent that the processes occurring during such transformation are extremely complex. A large number of factors and pathways are involved in maintaining the pluripotent state and regulating self-renewal and differentiation. The process of specific hESC differentiation into distinct cell types is even less understood. Most current attempts to directionally differentiate hESCs are based on sequential application of empirically selected growth factors and consequent selection for markers expressed in the target cell types (9). A more systematic approach is needed to improve our understanding of the pathways that control the conversion of precursors into specific cell types, progressing toward the goal of reproducing these processes in vitro for the generation of functional cells and tissues for transplantation.Comprehensive quantitative analysis of the hESC proteome would mean an important advance in understanding the nature of “stemness,” pluripotency, and differentiation. Several studies targeting various aspects of the hESC proteome have already been reported (for reviews, see Refs. 10 and 11). The task, however, is so enormous that further detailed analysis and novel strategies are necessary and will be of high interest and importance. In this regard, MS-based quantitative proteomics and in particular stable isotope labeling by amino acids in cell culture (SILAC) may greatly facilitate the process of defining the mechanisms of hESC self-renewal and differentiation. With SILAC, the entire proteome of a given cell population is metabolically labeled by heavy, non-radioactive isotopic variants of amino acids, thus making it distinguishable by MS analysis (12). Thereafter two or more distinctly SILAC-labeled cell populations can be mixed and analyzed in one MS experiment that allows accurate quantitation of proteins from the different cellular states (13). This versatile strategy has been demonstrated to be very useful for comprehensive characterization of complex biological phenomena (14–21) including in-depth comparison of signaling pathways to identify control points determining cell fate of adult mesenchymal stem cells (22).Here we report a procedure for complete SILAC labeling of human ES cells. We show that these SILAC-encoded hESCs have preserved self-renewing undifferentiated status as well as pluripotent capabilities based on analysis of known markers. In addition, we further compared the overall proteomes and phosphoproteomes of SILAC-labeled hESCs and equivalent cells grown under conventional culture conditions. We next compared the membrane proteomes of undifferentiated and differentiated hESCs in a quantitative manner. Our analysis identified 811 membrane proteins, which to our knowledge is the largest data set of ES cell membrane proteome. This study also revealed 23 membrane proteins with large changes in their expression levels during the differentiation. Six of those cell surface molecules displayed more than 3-fold higher levels in the self-renewing cells, whereas the remaining 17 were identified as more abundant in the differentiated population. These may be useful as specific hESC markers for the corresponding ES cell state and help to shed light on the mechanisms for self-renewal and differentiation.