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.     

Equivalence of conventionally-derived and parthenote-derived human embryonic stem cells

Background

As human embryonic stem cell (hESC) lines can be derived via multiple means, it is important to determine particular characteristics of individual lines that may dictate the applications to which they are best suited. The objective of this work was to determine points of equivalence and differences between conventionally-derived hESC and parthenote-derived hESC lines (phESC) in the undifferentiated state and during neural differentiation.

Methodology/Principal Findings

hESC and phESC were exposed to the same expansion conditions and subsequent neural and retinal pigmented epithelium (RPE) differentiation protocols. Growth rates and gross morphology were recorded during expansion. RTPCR for developmentally relevant genes and global DNA methylation profiling were used to compare gene expression and epigenetic characteristics. Parthenote lines proliferated more slowly than conventional hESC lines and yielded lower quantities of less mature differentiated cells in a neural progenitor cell (NPC) differentiation protocol. However, the cell lines performed similarly in a RPE differentiation protocol. The DNA methylation analysis showed similar general profiles, but the two cell types differed in methylation of imprinted genes. There were no major differences in gene expression between the lines before differentiation, but when differentiated into NPCs, the two cell types differed in expression of extracellular matrix (ECM) genes.

Conclusions/Significance

These data show that hESC and phESC are similar in the undifferentiated state, and both cell types are capable of differentiation along neural lineages. The differences between the cell types, in proliferation and extent of differentiation, may be linked, in part, to the observed differences in ECM synthesis and methylation of imprinted genes.     

No relationship between embryo morphology and successful derivation of human embryonic stem cell lines

Background

The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation.

Methods

We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines.

Results

Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line.

Conclusion

Even very poor quality embryos with few cells in the ICM can give origin to hESC lines.     

Albumin-associated lipids regulate human embryonic stem cell self-renewal

Background

Although human embryonic stem cells (hESCs) hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal.

Methodology/Principal Findings

In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect.

Conclusions/Significance

Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems.     

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

Rationale

Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.

Objective

We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.

Methods and Results

Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.

Conclusions

These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.     

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic,Induced Pluripotent and Adipose Stem Cells

Background

The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.

Methodology/Principal Findings

Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.

Conclusion/Significance

Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.     

Porphyrin Homeostasis Maintained by ABCG2 Regulates Self-Renewal of Embryonic Stem Cells

Background

Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined.

Methodology/Principal Findings

Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of γH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment.

Conclusions/Significance

The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and γH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells.     

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

Background

Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.

Results

The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Le^x and H type 2 antennae in sialylated complex-type N-glycans.

Conclusion

The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.     

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.     

Activin A is essential for Feeder‐free culture of human induced pluripotent stem cells

Feeder‐free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder‐free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA‐1‐60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA‐1‐60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder‐free culture of hiPS cells requires A or AC to maintain pluripotency. J. Cell. Biochem. 114: 584–588, 2013. © 2012 Wiley Periodicals, Inc.     

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

Background

Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro.

Methods and Findings

To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene.

Conclusion

VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.     

Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

Background

MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation.

Methodology/Principal Findings

SOLiD ultra-deep sequencing identified >10⁷ unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.

Conclusions/Significance

Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.     

Generation of lung epithelial-like tissue from human embryonic stem cells

Background

Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.

Methods

Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.

Results

Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and β-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay.

Conclusion

These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.