Vasiliy Galat

Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ESCs and iPSCs during Reprogramming and Time in Culture

Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety.

Recurrent Variations in DNA Methylation in Human Pluripotent Stem Cells and Their Differentiated Derivatives

Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.

Human embryonic stem cell lines with genetic disorders

A previous study described the establishment of human embryonic stem cell (ESC) lines from different sources of embryonic material, including morula, whole blastocyst and isolated inner cell mass. Using these methods, a repository of ESC lines has been established with different genetic abnormalities, which provides an unlimited source of disease cells in culture for undertaking research on the primary disturbances of the cellular processes in the genetically abnormal cells. ESC lines with genetic disorders were derived from the mutant embryos detected and avoided from transfer in the ongoing practice of preimplantation genetic diagnosis (PGD). The current repository contains 18 ESC lines with genetic disorders, including adrenoleukodystrophy, Duchenne and Becker muscular dystrophy, Fanconi anaemia, complementation group A, fragile-X syndrome, Huntington disease (three lines), Marfan syndrome, myotonic dystrophy (two lines), neurofibromatosis type I (five lines) and thalassaemia (two lines). These ESC lines are presently used for research purposes and may be available on request.

Restricted ethnic diversity in human embryonic stem cell lines

To the Editor: Human pluripotent stem cells (hPSCs) have the capacity to self-renew indefinitely and to differentiate into a wide array of cell types, which make them a potential source of essentially unlimited quantities of differentiated cells for basic and clinical research. The tremendous self-renewal of hPSCs might lead one to conclude that a small number of cell lines would be sufficient to meet all needs. However, it is becoming increasingly clear that the genetic background of human cell lines can have significant effects on experimental results. Although hundreds of associations between individual alleles and specific traits or diseases are discovered each year, a full understanding of the relationships between genetic variation and cellular or organismal phenotype is still remote1. The HapMap project2, along with many other studies, has made it clear that there are large differences in the allelic frequencies for many single-nucleotide polymorphisms (SNPs) among different ethnicities. Ethnicity can serve as a proxy for genetic variation to ensure diversity in genetic backgrounds in a study population. The availability of hPSCs from a variety of ethnic backgrounds would ensure the generalizability of results as well as increasing the likelihood that specific alleles or combinations of alleles of interest will be available for study. This is particularly important for the use of hPSCs and hPSC-derived cells for drug screening and toxicity studies. Several idiosyncratic drug effects have been attributed to specific genetic variations3, and the efficacy and toxicity of numerous drugs are presumed to be influenced by genetic factors. On the clinical side, hPSCs and their derivatives are potential sources for cell therapy, and recipients of cell transplants are more likely to find immunologic matches with donors who share their ethnic background. However, because most hPSC lines have been generated from de-identified material, the ethnic backgrounds of the donors are not known. We determined the ethnic origin of 47 human embryonic stem cell (hESC) lines (including 42 standard hESC lines and 5 parthenote hESC lines), 5 hiPSC lines and 58 non-pluripotent samples (19 blood and tissue samples and 39 primary cell lines). Although this study was not intended to be comprehensive, we included hESC lines derived in a variety of geographical locations in order to sample the spectrum of ethnic diversity present in hESC lines in general (Supplementary Table 1). Using genome-wide SNP genotyping and Bayesian analysis of population structure (BAPS; see Supplementary Methods), we determined that the ethnic origins of the hESC lines included in this study were quite restricted (Fig. 1, Supplementary Table 1). The large majority of hESC lines (43 of 47) were of European and East Asian ethnicity. The diversity of the hiPSC lines and nonpluripotent cells is also shown (Fig. 1, Supplementary Fig. 1). We note that the location of derivation of the hESC lines is not necessarily indicative of their ethnic origin. For instance, four of the five hESC lines described in one landmark publication4 were derived in the United States (Wisconsin) but reportedly5 came from blastocysts transported from Israel by collaborators. Our results on three of these lines indicate that they show a genetic profile similar to those of people from the region around Adygea (WA01), Tuscany (WA09) and Tuscany/Palestine (WA07), with indication of enzymatic activity3. We targeted residues Asp154, Glu155, Lys136 and Ser287 based on previous experiments showing increases in stability and/or enzymatic activity from mutations at these sites2,3. Mutations at Pro220 were tested based on a report of increased activity with alterations at this residue5; however, we were unable to find substitutions at Pro220 that increased activity or red-shift. The end result of this selection process was the variant RLuc/E155G/D162E/A164R/ L165I/M185V/Q235A/S287A, which we denote RLuc7-521. We used purified protein to assess the bioluminescence emission spectrum, enzymatic activity and quantum yield of the new variant (Supplementary Table 1). RLuc7-521’s signal was increased 1.6-fold on a photons per second per mole basis, and its emission spectra (Fig. 1a) showed a 40-nm red-shift compared to RLuc with both coelenterazine and the substrate analog coelenterazine-v. RLuc7-521’s quantum yield of 3.9 ± 0.1%, though higher than that of RLuc8.6535 (3.1 ± 0.2%), was lower than that of RLuc (5.3 ± 0.1%), indicating that further improvements may be achievable through additional mutagenesis. We tested RLuc7-521 as a mammalian reporter gene in cell culture and observed a nearly twofold improvement in light output compared to RLuc, and an almost identical intracellular stability (Fig. 1b). We also tested RLuc7-521 for small-animal imaging by injecting cells transiently transfected with RLuc or RLuc7-521 into mice. Western analysis of cells before injection showed equivalent levels of luciferase protein expression between conditions (Supplementary Fig. 2). Subsequent imaging of the mice showed a 3.3-fold increase in signal output for RLuc7-521 (Fig. 1c), a reflection of the improved light output of RLuc7-521 combined with reductions in signal attenuation by tissue for this red-shifted luciferase. In summary, RLuc7-521 is a seven-mutation variant of RLuc that shows a green-peaked (521-nm) emission spectrum, an identical intracellular stability, and at least a twofold increase in signal, with even greater signal gains in small-animal imaging due to decreased attenuation of its red-shifted photons. This new variant represents a direct replacement of Renilla luciferase for reporter-gene applications as it retains the temporal relationship between gene activation and luciferase activity while providing greater sensitivity.

Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines

Background The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. Methodology/Principal Findings Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. Conclusions/Significance Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.

Cancer hallmarks in induced pluripotent cells: New insights†

Studies are beginning to emerge that demonstrate intriguing differences between human‐induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Here, we investigated the expression of key members of the Nodal embryonic signaling pathway, critical to the maintenance of pluripotency in hESCs. Western blot and real‐time RT‐PCR analyses reveal slightly lower levels of Nodal (a TGF‐β family member) and Cripto‐1 (Nodals co‐receptor) and a dramatic decrease in Lefty (Nodals inhibitor and TGF‐β family member) in hiPSCs compared with hESCs. The noteworthy drop in hiPSCs Lefty expression correlated with an increase in the methylation of Lefty B CpG island. Based on these findings, we addressed a more fundamental question related to the consequences of epigenetically reprogramming hiPSCs, especially with respect to maintaining a stable ESC phenotype. A global comparative analysis of 365 microRNAs (miRs) in two hiPSC versus four hESC lines ultimately identified 10 highly expressed miRs in hiPCSs with >10‐fold difference, which have been shown to be cancer related. These data demonstrate cancer hallmarks expressed by hiPSCs, which will require further assessment for their impact on future therapies. J. Cell. Physiol. 225: 390–393, 2010.

Developmental potential of rat extraembryonic stem cells.

We have previously found that certain stem cells that are derived from rat blastocysts and named extraembryonic endoderm precursor (XEN-P) cells show a unique molecular signature sharing some of the characteristics of embryonic stem cells (ES), trophoblast stem cells (TS), and extraembryonic endoderm stem cells (XEN). These XEN-P cells are positive for AP, SSEA1, Oct4, and Rex1 markers similar to ES cells and also express signature markers of TS-eomesodermin (Eomes) and XEN-Gata6. Here we show that these cells integrate into the visceral and parietal extraembryonic endoderm lineages as well as into the inner cell mass (ICM), the primitive endoderm, and the polar and mural trophectoderm (TE) of cultured embryos. In addition, we find that the XEN-P cells colonize yolk sac and contribute to trophoblast lineages of postimplantation embryos following transfer to surrogate mothers. We also find that the XEN-P cell culture propagates by shedding cell clusters into the media in addition to typical expansion of colonies. Interestingly, the cell cultures exist as mixed populations of two interconvertible phenotypes of flat and round cells with preferential expression of stem cell markers Oct4 and SSEA1 in round cells. We believe these cells represent a metastable stage during ICM cellular segregation. These results are important for developing hypotheses of cell fate plasticity in the ICM and provide a model for the study of development and differentiation along the extraembryonic lineages.

A Study of Factors Affecting the Efficiency of Electrofusion of Enucleated Eggs and Cells-Donors of Nuclei

We studied the capacity of rabbit oocytes for electrofusion with morula blastomeres and fetal fibroblasts. The morula blastomeres fused with aging ooplasts more readily than the fetal fibroblasts: 92.9 versus 63.0%, p < 0.001. The fetal fibroblasts fused with young enucleated oocytes more efficiently than with the aging ones: 98.4 versus 63.0%, p < 0.001. Serum starvation of the fetal fibroblasts in DMEM medium for 7–14 days reduced their capacity for fusion with young ooplasts, as compared to that after starvation for 0–4 days: 67.2 versus 98.9%, p < 0.01). The increased time of starvation in an “impoverished” medium reduced the capacity of fetal fibroblasts with aging ooplasts as compared to the fibroblasts cultivated in the full medium and in the “impoverished” medium for one or two days: 64.5 versus 37.4%, p < 0.01. Hence, the efficiency of the fusion of the oocytes with nuclear donor cells depends on the age of the recipient oocyte, the origin of nuclear donor cells, and the conditions of cultivation.

Cytogenetic analysis of human somatic cell haploidization

Despite recent interest in the derivation of female and male gametes through somatic cell nuclear transfer, there is still insufficient data on chromosomal analysis of these gametes resulting from haploidization, especially involving a human nuclear donor and recipient oocytes. The objective of this study was to investigate the fidelity of chromosomal separation during haploidization of human cumulus cells by in-vitro matured human enucleated MII oocytes. A total of 129 oocytes were tested 4-7, 8-14, or 15-21 h after nuclear transfer (NT) followed by electro-stimulation, resulting in 71.3% activation efficiency on average. Haploidization was documented by the formation of two separate groups of chromosomes, originating from either polar body/pronucleus (PB/PN), or only 2PN, which were tested by 5-colour FISH, or DNA analysis for copy number of chromosomes 13, 16, 18, 21, 22 and X. Two PN were formed more frequently than PB/PN, irrespective of incubation time. In agreement with recent reports on mouse oocytes, as many as 90.2% of the resulting haploid sets tested showed abnormal chromosome segregation, suggesting unsuitability of the resulting artificial gametes for practical application at the present time.

Engineering Patient-Specific Valves Using Stem Cells Generated From Skin Biopsy Specimens

BACKGROUND Pediatric patients requiring valve replacement will likely require reoperations due to a progressive deterioration of valve durability and limited repair and growth potential. To address these concerns, we sought to generate a biologically active pulmonary valve using patient-specific valvular cells and decellularized human pulmonary valves. METHODS We generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblast cells. We then differentiated iPSCs to mesenchymal stem cells (iPCSs-MSCs) using culture conditions that favored an epithelial-to-mesenchymal transition. Next, decellularized human pulmonary heart valves were seeded with iPCS-MSCs using a combination of static and dynamic culture conditions and cultured up to 30 days. RESULTS The iPSCs-MSCs displayed cluster of differentiation CD105 and CD90 expression exceeding 90% after four passages and could differentiate into osteocytes, chondrocytes, and adipocytes (n = 4). Consistent with an MSC phenotype, iPSCs-MSCs lacked expression of CD45 and CD34. Compared with bone marrow MSCs, iPSCs-MSC proliferated more readily by twofold but maintained a gene expression profile exceeding 80% identical to bone marrow MSCs. In repopulated pulmonary valves compared with decellularized pulmonary valves, immunohistochemistry demonstrated increased cellularity, α-smooth muscle actin expression, and increased presence of extracellular matrix components, such as proteoglycans and glycosaminoglycans, suggesting sustained cell function and maturation. CONCLUSIONS Our results demonstrate the feasibility of constructing a biologically active human pulmonary valve using a sustainable and proliferative cell source. The bioactive pulmonary valve is expected to have advantages over existing valvular replacements, which will require further validation.