Petr Dvorak

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb–3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells.

Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF‐2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF‐2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular‐mass isoforms of FGF‐2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF‐2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 → FGFR3 → FGFR4 → FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF‐2, extracellular signal‐regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF‐2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF‐2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF‐2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self‐renewal or differentiation of hESCs.

A complex role for FGF-2 in self-renewal, survival, and adhesion of human embryonic stem cells.

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor‐2 (FGF‐2), which activates FGF receptors (FGFRs) and stimulates the mitogen‐activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin‐like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF‐2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF‐2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation‐related genes and downregulation of stem cell genes. Thus, exogenous FGF‐2 reinforced the pluripotency maintenance program of intracrine FGF‐2 signaling. Consistent with this hypothesis, expression of endogenous FGF‐2 decreased during hESC differentiation and FGF‐2 knockdown‐induced hESC differentiation. In addition, FGF‐2 signaling via FGFR2 activated MAPK kinase/extracellular signal‐regulated kinase and AKT kinases, protected hESC from stress‐induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self‐renewal, cell survival, and adhesion by exogenous and endogenous FGF‐2 may synergize to maintain the undifferentiated growth of hESCs. STEM CELLS 2009;27:1847–1857

Comparative study of mouse and human feeder cells for human embryonic stem cells

Various types of feeder cells have been adopted for the culture of human embryonic stem cells (hESCs) to improve their attachment and provide them with stemness-supporting factors. However, feeder cells differ in their capacity to support the growth of undifferentiated hESCs. Here, we compared the expression and secretion of four well-established regulators of hESC pluripotency and/or differentiation among five lines of human foreskin fibroblasts and primary mouse embryonic fibroblasts throughout a standard hESC culture procedure. We found that human and mouse feeder cells secreted comparable levels of TGF beta 1. However, mouse feeder cells secreted larger quantities of activin A than human feeder cells. Conversely, FGF-2, which was produced by human feeder cells, could not be detected in culture media from mouse feeder cells. The quantity of BMP-4 was at about the level of detectability in media from all feeder cell types, although BMP-4 dimers were present in all feeder cells. Production of TGF beta 1, activin A, and FGF-2 varied considerably among the human-derived feeder cell lines. Low- and high-producing human feeder cells as well as mouse feeder cells were evaluated for their ability to support the undifferentiated growth of hESCs. We found that a significantly lower proportion of hESCs maintained on human feeder cell types expressed SSEA3, an undifferentiated cell marker. Moreover, SSEA3 expression and thus the pluripotent hESC compartment could be partially rescued by addition of activin A. Cumulatively, these results suggest that the ability of a feeder layer to promote the undifferentiated growth of hESCs is attributable to its characteristic growth factor production.

MicroRNAs Regulate p21Waf1/Cip1 Protein Expression and the DNA Damage Response in Human Embryonic Stem Cells

Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53‐p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESC‐specific miRNAs such as those of the miR‐302 family, miR‐371‐372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC‐enriched miRNA family miR‐302 (miR‐302a, miR‐302b, miR‐302c, and miR‐302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR‐302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage. STEM CELLS2012;30:1362–1372

Fibroblast growth factor signaling in embryonic and cancer stem cells.

The fibroblast growth factor 2 (FGF‐2) pathway is one of the most significant regulators of human embryonic stem cell (hESC) self‐renewal and cancer cell tumorigenesis. Here we summarize recent data on the effects of FGF‐2 and its receptors on hESCs and leukemic stem/progenitor cells. Also, we discuss the similarities of these findings with stem cell renewal and differentiation phenotypes.

Autopsy and clinical context in deceased patients with implanted pacemakers and defibrillators: intracardiac findings near their leads and electrodes

AIMS To evaluate intracardiac findings near leads and causes of death in pacemaker/defibrillator patients. METHODS AND RESULTS Special autopsy was performed on 78 patients deceased in a hospital. Age at death was 77.9 +/- 10.0, implantation-death interval 4.0 +/- 3.3 years, ventricular leads n = 78, and atrial leads n = 21. Thrombi along leads in brachiocephalic vein/upper caval vein (BV/UCV) were found in 22 (7), in right atrium (RA) in 11 (8), and in right ventricle (RV) in 11 cases. Bipolar lead rings were fixed by fibrous tissue in 43 (4) cases. Connective tissue bridges and tunnels were found in BV/UCV in 44 (13), in RA in 17 (15), and in RV in 68 cases, with a length of 0.2-12.0 cm. Right ventricular leads in tricuspidal orifice were fixed by fibrous tissue in 11 and penetrating chordae in 25 cases. Main causes of death were: heart failure in 35, pulmonary embolism in 9, and myocardial infarction in 11 cases. CONCLUSION We have found (i) thrombi on ventricular/atrial leads in 33/48%, (ii) bipolar lead rings fixed by fibrous tissue in 68/22%, (iii) connective tissue bridges or tunnels in ventricle/atrium in 87/71%, and (iv) ventricular leads fixed to valve or penetrating chordae in 46% of patients. We do recommend caution when extracting leads.

Points to consider in the development of seed stocks of pluripotent stem cells for clinical applications: International Stem Cell Banking Initiative (ISCBI)

In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes [1], which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications. The term ‘seed stock’ is used here to describe those cryopreserved stocks of cells established early in the passage history of a pluripotent stem cell line in the lab that derived the line or a stem cell bank, hereafter called the ‘repository’.

Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

Human pluripotent stem cells (hPSCs), namely, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs).