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.     

Derivation and characterization of three new human embryonic stem cell lines in Finland

Human embryonic stem cell (hESC) lines can be established from the preimplantation embryos. Due to their ability to differentiate into all three embryonic layers, hESC are of significant interest as a renewable source of cell material for different applications, especially for cell replacement therapy. Since the establishment of the first hESC lines in 1998, several studies have described the derivation and culture of new hESC lines using various derivation methods and culture conditions. Our group has currently established eight new hESC lines of which three of the latest ones are described in a more detailed way in this report. The described lines have been established using mechanical derivation methods for surplus bad quality embryos and culture conditions containing human foreskin fibroblast feeder cells and serum-free culture medium. All the new lines have a normal karyotype and typical hESC characteristics analyzed in vitro. The described hESC lines are available for research purposes upon request (www.regea.fi).     

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.     

Derivation, culture, and characterization of VUB hESC lines

In this report, we present the derivation and characterization of 15 hESC lines established at the Vrije Universiteit Brussel, Belgium in collaboration with the Universitair Ziekenhuis Brussel, Belgium, using surplus in vitro fertilization embryos and embryos carrying monogenic disorders donated for research. Four lines were derived from blastocyst-stage embryos presumed to be genetically normal, and 11 hESC lines were obtained from embryos shown to carry genetic mutations by preimplantation genetic diagnosis. All the lines express markers of pluripotency as determined by immunocytochemistry and RT-PCR, and formed teratomas when injected into SCID mice. All VUB hESC lines, except for VUB17, are reported in the European hESC registry and are available upon request after signing a Material Transfer Agreement from the VUB (contact person: Prof. Dr. Karen Sermon; Karen.Sermon@uzbrussel.be).     

High-resolution chromosomal microarray analysis of early-stage human embryonic stem cells reveals an association between X chromosome instability and skewed X inactivation

X chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell via a random process. Skewed XCI is relevant to many diseases, but the mechanism leading to it remains unclear. Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of blastocyst-stage embryos have provided an excellent model system for understanding XCI initiation and maintenance. Here, we derived hESC lines with random or skewed XCI patterns from poor-quality embryos and investigated the genome-wide copy number variation (CNV) and loss of heterozygosity (LOH) patterns at the early passages of these two groups of hESC lines. It was found that the average size of CNVs on the X chromosomes in the skewed group is twice as much as that in the random group. Moreover, the LOH regions of the skewed group covered the gene locus of either XIST or XACT, which are master long non-coding RNA (lncRNA) effectors of XCI in human pluripotent stem cells. In conclusion, our work has established an experimentally tractable hESC model for study of skewed XCI and revealed an association between X chromosome instability and skewed XCI.     

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.     

Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification

Realizing the potential of human embryonic stem cells (hESCs) in research and commercial applications requires generic protocols for culture, expansion and genetic modification that function between multiple lines. Here we describe a feeder-free hESC culture protocol that was tested in 13 independent hESC lines derived in five different laboratories. The procedure is based on Matrigel adaptation in mouse embryonic fibroblast conditioned medium (CM) followed by monolayer culture of hESC. When combined, these techniques provide a robust hESC culture platform, suitable for high-efficiency genetic modification via plasmid transfection (using lipofection or electroporation), siRNA knockdown and viral transduction. In contrast to other available protocols, it does not require optimization for individual lines. hESC transiently expressing ectopic genes are obtained within 9 d and stable transgenic lines within 3 weeks.     

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.     

The mode of lymphoblastoid cell death in response to gas phase cigarette smoke is dose-dependent

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.     

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.     

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.     

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.     

Role of the embryology laboratory in the human embryonic stem cell line derivation process

With the introduction of regenerative medicine and cell therapy programmes by means of human embryonic stem cells (hESC), several research centres have begun projects of derivation of hESC lines. In some stem cell banks, such as the Andalusian Stem Cell Bank, the law also permits the creation of these cell lines. Therefore, the recovery of cryopreserved embryos, their culture and the subsequent derivation to hESC lines requires a suitable embryology laboratory and specialized and highly qualified staff. Moreover, new techniques, from therapeutic nuclear transfer, need this type of laboratory and staff, too. Several International Associations have drawn up some guidelines for laboratories where embryos are manipulated and they reflect the physical space, the staff and the equipment needed in these kinds of laboratories. Nevertheless, we can see that these guidelines do not distinguish between IVF laboratories and other laboratories that obtain hESC lines, so it would be convenient to make a distinction. Following these guidelines, we have tried to draw up concurrent aspects applicable to areas of embryology within stem cell banks. So, the design and the specific implementation programmes for these areas and other research centres with this area but which do not use IVF techniques is vital to develop embryonic cell lines in optimum conditions for future therapeutic applications, although maybe it is rather premature to standardize this type of research.     

Generation of new human embryonic stem cell lines with diploid and triploid karyotypes

Human pluripotent embryonic stem cells (hESC) have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hESC lines, we present the derivation and characterization of five hESC lines on mouse embryonic fibroblast cells. Our stem cell lines are characterized by morphology, long-term expansion, and expression profiles of a number of specific markers, including TRA-1-60, TRA-1-81, alkaline phosphatase, connexin 43, OCT-4, NANOG, CXCR4, NODAL, LEFTY2, THY-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in vitro conditions. Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hESC, two of the cell lines carried a triploid karyotype.