Undifferentiated hESCs Research Articles

HLA-G Expression in Human Embryonic Stem Cells and Preimplantation Embryos

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells.

Effects of the physiochemical culture environment on the stemness and pluripotency of human embryonic stem cells

Both physical and chemical signals in the culture environment mediate stem cell fate. For research and the clinical application of stem cells, the development of defined and xeno-free culture conditions to maintain the undifferentiated growth of human embryonic stem cells (hESCs) is highly desirable. We evaluated the effects of different basal media, glucose concentrations, osmolarity, chemical compounds, and hypoxic conditions on hESC self-renewal using defined and xeno-free culture conditions and molecular characterization of the cultured hESCs. Our findings indicate that a combination of KnockOut™ DMEM basal medium, high glucose concentration, and osmolarity of 320-330 mOsm was optimal for maintaining the self-renewal of hESCs under defined, xeno-free conditions. In addition, specific lipids, retinol, and insulin further enhanced the growth of undifferentiated hESCs. Finally, hypoxic conditions increased proliferation and prevented the spontaneous differentiation of hESCs. Although the transcriptional expression of common genes associated with pluripotency was not affected by hypoxia, the translational expression of Oct4 increased significantly under hypoxic conditions. Our results indicated that hypoxia-induced regulation of cell signaling also involves the activation of the calcium and protein kinase C pathway, and several hypoxia signaling-related genes. We identified important nutrients and growth factors required for a defined, xeno-free culture condition to enable reasonable growth characteristics and to maintain the self-renewal of hESCs. Furthermore, we demonstrated that hypoxia supports hESC self-renewal in culture. The use of defined conditions for culturing hESCs will allow for better understanding of pluripotent stem cell regulation and provide more reproducible results.

In vitro and In silico Optogenetic Control of Differentiated Human Pluripotent Stem Cells

Purpose: Despite the use of a variety of differentiation protocols, no method exists to prospectively generate specific phenotypes of human embryonic stem cell (hESC)-derived cardiomyocytes (CM) such as pacemaker cells. We introduced light-activated channelrhodopsin-2 (ChR2) into hESC, and by using in vitro and in silico approaches, we were able to optogenetically synchronize hESC-CM, both experimentally and computationally.Methods: We experimentally introduced ChR2 coupled to yellow fluorescent protein (YFP) into undifferentiatedhESC via a lentiviral vector and tested for expression via PCR, flow cytometry (FC), and immunocytochemistry (ICC). hESCChR2+ were sorted, expanded, and tested for pluripotency. Via directed differentiation, wildtype hESC-CM and ChR2-CM were produced and subjected to both electrical and optical stimulation. Electrical, biochemical, and mechanical signals were then assessed by patch clamping, multielectrode arrays (MEAs), and video microscopy. To complement our in vitro approach with in silico analyses, we introduced ChR2 into an ionic cardiac cell model.Results: ChR2 was stably transduced into undifferentiated hESC and the resulting hESCChR2+ pluripotent line could be differentiated into CM, all confirmed by PCR, FC, ICC, and electrophysiological methods. Both WT-CM and ChR2-CM responded to traditional electrical stimulation and produced similar calcium and contractility features but only ChR2-CM could be synchronized by optical stimulation. In addition, by calibrating our ionic cell model with single cell action potential (AP) readings, we were able to virtually probe the impact of photostimulation stimulus amplitude, pulse width, and frequency on overall AP characteristics.Conclusions: Here we show for the first time that ChR2 can enable in vitro and in silico optical control of hESC-CM. The long-term application of optical stimulation could potentially lead to specific synchronous phenotypes of hESC-CM. This, in turn, would contribute significantly towards creating effective therapies for cardiovascular disease.

Functional Expression and Subcellular Localization of f-Channels in Native Human and hESC-Derived Cardiomyocytes

The Hyperpolarization-activated Cyclic-Nucleotide gated (HCN) genes encode for the alpha subunit of f-channel present in the heart. HCN4 is the predominant isoform in the sinoatrial node cells and, in the rabbit, it is localized into membrane caveolae, where the interaction with caveolin-3 (cav3) regulates f-current properties. HCN4 is abundant in undifferentiated human embryonic stem cells (hESC) and immature hESC-derived cardiomyocytes (hESC-CMs). Maturation is associated with modifications of f-channel functional properties. To date, no information is available on i) the subcellular localization of HCN4 and cav3 in hESC- and native human CMs ii) the functional consequences of their association on f-current properties. Confocal microscopy showed that HCN4 and cav3 colocalize in native human adult CMs. In the same cells, f-current was consistently recorded (70% cells), with a voltage of half maximal activation of −102 and −101 mV in atrial and ventricular CMs, respectively. Protein and mRNA for cav3 were not detected in undifferentiated hESC, but expression increased during maturation of hESC-CMs. HCN4 was highly expressed in hESC and d30 hESC-CMs, but decreased in d60 and d110 hESC-CMs. In the d110 cells, HCN4 appeared to be associated with cav3. Activation properties of f-current recorded from d110 hESC-CMs, resembled those measured in native atrial and ventricular CMs. Current activation occurred at more positive potentials in d60 hESC-CMs and native human fetal CMs. In native atrial CMs disruption of caveolae shifted f-current activation curve to more positive potentials. In conclusion, our data shows for the first time that HCN4 and cav3 associate in native human and hESC-derived CMs. Expression of cav3 and its association with ionic channels likely represents a crucial step of cardiac maturation, which may result in changes of cellular electrophysiological properties and modulation by endogenous signals. Correction et al.Biophysical JournalMay 04, 2011In Brief1079-Pos. Full-Text PDF Open Archive

Identification and Characterization of Adenovirus Early Region 1B-Associated Protein 5 as a Surface Marker on Undifferentiated Human Embryonic Stem Cells

Pluripotent human embryonic stem cells (hESCs) provide appropriate systems for developmental studies and prospective donor cell sources for regenerative medicine. Identification of surface markers specific to hESCs is a prerequisite for studying hESC biology and can be used to generate clinical-level donor cell preparations that are free from tumorigenic undifferentiated hESCs. We previously reported the generation of monoclonal antibodies that specifically recognize hESC surface antigens using a decoy immunization strategy. In this study, we show that monoclonal antibody 57-C11 recognizes a phosphorylated form of adenovirus early region 1B-associated protein 5 (E1B-AP5). E1B-AP5 is a nuclear RNA-binding protein, but we report that 57-C11-reactive E1B-AP5 is expressed on the surface of undifferentiated hESCs. In undifferentiated hESCs, 57-C11-reactive E1B-AP5 is localized to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, SOX2-, and NANOG-positive hESCs. In mixtures of undifferentiated hESCs and hESC-derived neurons, 57-C11 exclusively recognizes undifferentiated hESCs but not hESC-derived neuronal cells. Further, the expression of 57-C11-reactive E1B-AP5 decreases upon differentiation. Our results demonstrate that 57-C11-reactive E1B-AP5 is a novel surface molecule that is involved in the undifferentiated state of hESCs. As far as we know, this is the first report demonstrating that heterogeneous nuclear RNA-binding protein is expressed on the surface of undifferentiated hESCs.

Genomewide characterization of non-polyadenylated RNAs

BackgroundRNAs can be physically classified into poly(A)+ or poly(A)- transcripts according to the presence or absence of a poly(A) tail at their 3' ends. Current deep sequencing approaches largely depend on the enrichment of transcripts with a poly(A) tail, and therefore offer little insight into the nature and expression of transcripts that lack poly(A) tails.ResultsWe have used deep sequencing to explore the repertoire of both poly(A)+ and poly(A)- RNAs from HeLa cells and H9 human embryonic stem cells (hESCs). Using stringent criteria, we found that while the majority of transcripts are poly(A)+, a significant portion of transcripts are either poly(A)- or bimorphic, being found in both the poly(A)+ and poly(A)- populations. Further analyses revealed that many mRNAs may not contain classical long poly(A) tails and such messages are overrepresented in specific functional categories. In addition, we surprisingly found that a few excised introns accumulate in cells and thus constitute a new class of non-polyadenylated long non-coding RNAs. Finally, we have identified a specific subset of poly(A)- histone mRNAs, including two histone H1 variants, that are expressed in undifferentiated hESCs and are rapidly diminished upon differentiation; further, these same histone genes are induced upon reprogramming of fibroblasts to induced pluripotent stem cells.ConclusionsWe offer a rich source of data that allows a deeper exploration of the poly(A)- landscape of the eukaryotic transcriptome. The approach we present here also applies to the analysis of the poly(A)- transcriptomes of other organisms.

Hepatic differentiation of human embryonic stem cells in a 3D bioreactor environment

A number of articles addressing the hepatic differentiation of human embryonic stem cells (hESC) in conventional 2D culture systems have been published recently. However, the cell numbers and purity available through this culture method are limited. This study aims to investigate the hepatic differentiation of hESC in a dynamic 3D culture environment that supports cell differentiation and three-dimensional tissue reorganisation in vitro. Based on a perfused 3D-multicompartment bioreactor technology successfully used for primary human liver cell culture, a downscaled system (0.5ml cell volume) was developed to investigate the differentiation, maturation and function of the hESC-derived hepatic cells in vitro. Undifferentiated hESC were inoculated into a miniaturized 3D bioreactor and hepatic differentiation was induced by the sequential application of growth factors, hormones and cytokines. Metabolic parameters indicating metabolic activities (glucose, lactate) of the cells were measured daily. After 30 days the bioreactor was opened and samples for immunofluorescence staining and RNA analysis were taken.The time courses of glucose and lactate indicated stable cell metabolism over 30 days. Immunofluorescence analysis showed the expression of the biliary marker cytokeratin 19 (CK19), and CK18 as hepatocytic / biliary marker indicating hepatic differentiation. Both markers were localized predominantly at the peripheral areas of cell aggregates. These findings are consistent with the detected induction in the expression of albumin, AFP and cytochrome P450 2D6 in samples taken from the bioreactor following hepatic maturation of hESC compared to undifferentiated hESC. In conclusion, the results of the study suggest that the 3D-multicompartment bioreactor supports hepatic differentiation of hESC. Further studies are needed to increase the yield and maturation degree of the cells.

Involvement of neuropeptide Y and its Y1 and Y5 receptors in maintaining self‐renewal and proliferation of human embryonic stem cells

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self-renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long-term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder-free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY-mediated activation of AKT/protein kinase B and extracellular signal-regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY-induced activation of cAMP-response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self-renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno-free culture condition for the large-scale propagation of undifferentiated hESCs.

Human Embryonic Stem Cells Suffer from Centrosomal Amplification

Propagation of human embryonic stem cells (hESCs) in culture tends to alter karyotype, potentially limiting the prospective use of these cells in patients. The chromosomal instability of some malignancies is considered to be driven, at least in part, by centrosomal overamplification, perturbing balanced chromosome segregation. Here, we report, for the first time, that very high percentage of cultured hESCs has supernumerary centrosomes during mitosis. Supernumerary centrosomes were strictly associated with an undifferentiated hESC state and progressively disappeared on prolonged propagation in culture. Improved attachment to culture substratum and inhibition of CDK2 and Aurora A (key regulators of centrosomal metabolism) diminished the frequency of multicentrosomal mitoses. Thus, both attenuated cell attachment and deregulation of machinery controlling centrosome number contribute to centrosomal overamplification in hESCs. Linking the excessive number of centrosomes in mitoses to the ploidy indicated that both overduplication within a single cell cycle and mitotic failure contributed to generation of numerical centrosomal abnormalities in hESCs. Collectively, our data indicate that supernumerary centrosomes are a significant risk factor for chromosome instability in cultured hESCs and should be evaluated when new culture conditions are being implemented.

Epigenetic modification of retinoic acid-treated human embryonic stem cells

Epigenetic modification of the genome through DNA methylation is the key to maintaining the differentiated state of human embryonic stem cells (hESCs), and it must be reset during differentiation by retinoic acid (RA) treatment. A genome-wide methylation/gene expression assay was performed in order to identify epigenetic modifications of RA-treated hESCs. Between undifferentiated and RA-treated hESCs, 166 differentially methylated CpG sites and 2,013 differentially expressed genes were discovered. Combined analysis of methylation and expression data revealed that 19 genes (STAP2, VAMP8, C10orf26, WFIKKN1, ELF3, C1QTNF6, C10orf10, MRGPRF, ARSE, LSAMP, CENTD3, LDB2, POU5F1, GSPT2, THY1, ZNF574, MSX1, SCMH1, and RARB) were highly correlated with each other. The results provided in this study will facilitate future investigations into the interplay between DNA methylation and gene expression through further functional and biological studies.

Dysregulated Gene Expression During Hematopoietic Differentiation From Human Embryonic Stem Cells

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However, functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC, a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover, strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα), were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors, and may contribute to the block in definitive hematopoiesis from hESC.

Modeling Megakaryopoiesis and Leukemogenesis Using Human and Murine Embryonic Stem Cells

AbstractAbstract 2502Megakaryopoiesis involves the differentiation of progenitor cells into diploid megakaryocytes, which then undergo endomitosis and cytoplasmic maturation resulting in platelet release. Acute Megakaryoblastic Leukemia (AMKL) results when this process goes awry. Nearly one third of pediatric AMKL patients are infants who have the t (1;22) chromosomal translocation (t (1;22)-AMKL) resulting in fusion of the RNA Binding Motif 15 gene (RBM15) on chromosome 1 upstream of the transcriptional cofactor Megakaryoblastic Leukemia 1 gene (MKL1) on chromosome 22. Given that t (1;22)-AMKL primarily affects infants, the leukemia likely originates in utero when the hematopoietic system is in its embryonic stage.Our goal was to establish in vitro methods to study embryonic megakaryopoiesis and leukemogenesis using human and murine embryonic stem cells (hESCs and mESCs). Using the Invitrogen Gateway System, human MKL1 (hMKL1) and human RBM15-MKL1 (hRM) constructs were designed. H9-rtTA, a hESC line constitutively expressing the reverse transcriptional activator (rtTA) under the control of the Ubiquitin C promoter, were transduced with lentivirus harboring either hMKL1 or hRM with an IRES-GFP promoter under the control of the tetracycline responsive element (TRE) to derive doxycycline (dox) inducible cell lines. In the presence of dox, the rtTA binds to the TRE, causing transcriptional activation of the downstream transgene. hESC were co-cultured on murine OP-9 stromal cell layers with thrombopoietin to promote megakaryocytic differentiation. For construction of the inducible mESC lines, mESCs already containing the rtTA at the ROSA26 locus were electroporated with either murine MKL1 (mMKL1) or murine RBM15-MKL1 (mRM). Site-specific insertion of the transgenes using a cre recombinase and modified loxP sites placed mMKL1 or mRM under the control of a TRE. The inducible mMKL1 mESCs were differentiated into hematopoietic cells using embryoid body formation and subsequently co-cultured on OP-9s. The human and murine MKL1 and RM constructs were transiently transfected into 293FT cells and Western blotting was used to confirm their functionality. In the hESC studies, GFP expression was detected by flow cytometry 24 hrs after dox induction in both cell lines, with 54.7% (SD+3.1) GFP+ cells in hMKL1 transduced cells and 47.5% (SD+16.2) GFP+ cells in hRM transduced cells. hMKL1 protein was detected 24 hrs after dox induction; however, the hRM fusion protein was not detected in undifferentiated hESCs by Western blot or IP/IB after 6, 24, 48, 72, 96 hrs of dox exposure. Nearly one fourth of the cells transduced with hRM were GFP+ after only 6 hrs of doxycycline exposure. Interestingly, with continued dox exposure over six days, GFP expression in the undifferentiated hESCs declined, with a mean decrease in GFP expression of 14.5% in both cell lines. Successful incorporation of the vector carrying the hRM was confirmed by genomic PCR and resulted in dox-inducible expression of hRM mRNA detected by RT-PCR. Upon megakaryocytic differentiation, GFP and CD41 expression, detected by flow cytometry, occur in a mutually exclusive fashion in both MKL and RBM15-MKL1 transduced hESCs. In the mESC experiements, protein expression of mMKL1 in the undifferentiated cells was confirmed by Western blot 24, 48, and 72 hrs after dox addition. Expression of mRM protein in the undifferentiated mESCs was undetectable 6, 14, 24, 30, 48, or 72 hrs after dox addition. However, mRM mRNA was detected by qRT-PCR at 0, 6, 14, and 30 hours after dox treatment in the undifferentiated mESCs. Of note, transient transfection of undifferentiated mESCs with mRM resulted in protein expression by Western blot as early as 6 hrs after transfection. The fusion protein continued to be detected at 12 and 18 hrs after transfection, but by 24 hrs, it was no longer detectable. When differentiated, the mMKL1 mESC line no longer demonstrated inducible mMKL1 expression in the CD41+ (hematopoietic) population as determined by qRT-PCR. These findings confirm ongoing activity of the promoters in the undifferentiated hESC and mESC lines and indicate that the promoters may be silenced upon induction of hematopoietic differentiation of both hESC and mESC lines. In addition, the transient detection of RM after transfection into the mESCs suggests post-translational modification of the fusion protein likely results in its rapid degradation. Disclosures:No relevant conflicts of interest to declare.

Differentiation of Human Natural Killer Cells From Pluripotent Stem Cells In a Feeder Free System Amenable to Clinical Translation

AbstractAbstract 107Human natural killer (NK) cells are an attractive source of lymphocytes for adoptive immunotherapy. Although our understanding of natural killer cell biology continually grows, translating these concepts to the clinic has fallen behind. Currently, NK cell adoptive immunotherapy is only beneficial to a limited number of patients, particularly those with acute myelogenous leukemia (AML). In order to generate NK cells that are effective against a broader range of malignancies, our lab has focused on the generation of NK cells from human pluripotent stem cells. We have previously demonstrated the potency of NK cells derived from human embryonic stem cells (hESCs) both in vitro and in vivo. These previous studies utilized a stromal cell co-culture system to derive hematopoietic progenitor cells (CD34+CD45+ cells) from hESCs that can then produce CD45+CD56+ NK cells in a secondary culture system. More recently, we used a similar co-culture system to generate fully functional NK cells from several human induced pluripotent stem cell (iPSC) lines, which could provide NK cells on a patient-specific basis. Both hESC and iPSC-derived NK cells consist of a mature, homogenous population of cells expressing CD56, CD94, killer immunoglobulin-like receptors (KIRs), CD16, and the apoptosis-inducing ligands FasL and TRAIL. We now aim to convert this system into a completely defined stromal cell-free system. These studies will make this system more amenable to clinical scale up and allow us to better define elements essential for hematopoietic and NK cell development from human pluripotent stem cells. For example, hESC-derived NK cells would require minimal cell processing compared peripheral blood NK (PB-NK) cells that typically requires depletion of CD3+ and CD20+ cells to prevent GVH and passenger lymphocyte syndrome, respectively. Neither T cells nor B cells are present in our cultures, preventing these complications. Here, we have now used a spin embryoid body (“spin-EB”) approach potentially suitable for clinical scale-up. In these spin-EB cultures, defined numbers of undifferentiated hESCs or iPSCs are first aggregated in 96 well plates by centrifugation (3000 cells per well) in serum-free media containing only the cytokines SCF, BMP4, and VEGF. Under these stage 1 conditions, hematopoietic progenitor cells that express CD34, CD45, CD43, and CD31 develop and expand over 6–11 days. After this time, EBs are directly transferred (without dissociation or sorting) to Stage 2 cultures with EL08 stromal cells or stroma-free conditions in serum-free media containing NK cell-initiating cytokines (IL15, SCF, FLT3L, IL7, and IL3). We find that these Stage 2 cells acquire all the typical markers of mature NK cells (CD56, CD94, KIRs, etc). They also kill the CML target K562 cells at similar effector-to-target ratios as stromal-derived NK cells and PB-NK cells. Most notably, spin-EB-derived NK cells generated in entirely feeder free conditions also exhibit a typical NK cell phenotype and kill K562 targets. However, the NK cells derived in feeder-free Stage 2 conditions are less proliferative than those cultured in Stage 2 conditions on EL08 cells. Specifically, 12,000 undifferentiated hESCs or iPSCs give rise to 0.8–1 × 106 NK cells using EL08 in Stage 2 cultures compared to 0.4– 1 × 105 cells using the feeder-free Stage 2 conditions. Additionally, the efficiency of NK cell development from spin-EB cultures is not uniform: 58% of EBs produce NK cells in Stage 2 using EL08 stromal cells, and 25% of EBs produce NK cells in the completely feeder-free system. Based on these calculations, given 100% efficiency of this spin-EB NK cell developmental system, we estimate that 240,000 starting hESCs would provide enough NK cells to treat a single patient with a dose of 20 × 106 NK cells. Even at the lower efficiencies demonstrated here, one plate of undifferentiated hESCs or iPSCs (typically with 5–10 × 106 undifferentiated cells) could treat several patients. Ongoing work is focused on enhancing the percentage of hematopoietic progenitors obtained from the spin EB approach and expanding feeder-free NK cells with artificial antigen presenting cells in Stage 2 conditions. Disclosures:No relevant conflicts of interest to declare.

P136. Effects of mycoplasma elimination agent on cardiac differentiation of human embryonic stem cells

Mycoplasma contamination is a major problem in continuous cell culture. Various agents are used for the elimination of mycoplasma. However, their effects on cardiac differentiation potential of human embryonic stem cells (hESCs) are rarely known. In this study, we tried to evaluate the effects of mycoplasma elimination agent (plasmocin) on differentiation into cardiomyocytes (CMs) from hESCs. Undifferentiated hESCs were induced to differentiated into CMs in the presence of P/S and plasmocin (2.5, 5.0, 10.0 μg/ml). The expression of cardiac specific genes such as Nkx2.5, Tbx5, α-myosin heavy chain (MHC) and cardiac troponin (cTn) I was evaluated using immunocytochemistry and qPCR. After 14 days of differentiation, the beating cluster formation of hESCs was reduced in plasmocin-treated group. The expression of cardiac genes was down-regulated and the decrease of cTn I was significant in plasmocin-treated group. In this study, we demonstrated the effects of mycoplasma elimination agent on cardiac differentiation potential of hESCs. There results showed that addition of mycoplasma elimination agent reduced the differentiation potential of hESCs (Nos. 2010-0016819 and SC1150).

Teratomas from pluripotent stem cells: A clinical hurdle

Although basic research on human embryonic stem cells (hESCs) at the laboratory bench has progressed with enviable speed there has been little head way in terms of its clinical application. A look at the Internet however shows several stem cell clinics worldwide offering direct transplantation of undifferentiated hESCs to patients for the cure of a variety of diseases before bona fide evidence-based results can be demonstrated from large controlled studies. This raises concern because reliable protocols have to be first developed to resolve the three major hurdles delaying clinical trials such as inadequate cell numbers, immunorejection and tumorigenesis. Cell expansion methods using bioreactors, rotary culture and mitotic agents have now been developed to generate stem cell derivatives in large numbers. The problem of immunorejection can now be overcome with the development of indirect and direct reprogramming protocols to personalize tissues to patients (human induced pluripotent stem cells, hiPSCs; nuclear transfer stem cells, NTSCs; induced neuronal cells, iN). However, hESC, hiPSC, and NTSCs being pluripotent have the disadvantage of teratoma formation in vivo which has to be carefully addressed so as to provide safe stem cell based therapies to the patient. This review addresses the issue of tumorigenesis and discusses approaches by which this concern may be overcome and at the same time emphasizes the need to concurrently explore alternative stem cell sources that do not confer the disadvantages of pluripotency but are widely multipotent so as to yield safe desirable tissues for clinical application as soon as possible.

Red Ginseng Extract Facilitates the Early Differentiation of Human Embryonic Stem Cells into Mesendoderm Lineage

Human embryonic stem cells (hESCs) have capacities to self-renew and differentiate into all cell types in vitro. Red ginseng (RG) is known to have a wide range of pharmacological effects in vivo; however, the reports on its effects on hESCs are few. In this paper, we tried to demonstrate the effects of RG on the proliferation and differentiation of hESCs. Undifferentiated hESCs, embryoid bodies (EBs), and hESC-derived cardiac progenitors (CPs) were treated with RG extract at 0.125, 0.25, and 0.5 mg/mL. After treatment of undifferentiated hESCs from day 2 to day 6 of culture, BrdU labeling showed that RG treatment increased the proliferation of hESCs, and the expression of Oct4 and Nanog was increased in RG-treated group. To find out the effects of RG on early differentiation stage cells, EBs were treated with RG extract for 10 days and attached for further differentiation. Immunostaining for three germ layer markers showed that RG treatment increased the expressions of Brachyury and HNF3β on EBs. Also, RG treatment increased the expression of Brachyury in early-stage and of Nkx2.5 in late-stage hESC-derived CPs. These results demonstrate facilitating effects of RG extract on the proliferation and early differentiation of hESC.

Quantitative Comparison of Constitutive Promoters in Human ES cells

BackgroundConstitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking.Methodology/Principal FindingsWe have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies.Conclusion/SignificanceThe ACTB, EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75–80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs.

Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells.

Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.

Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex™ or Matrigel™. In addition we have validated an ascorbate free media capable of supporting CD30 low populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.

Expansion and characterization of human embryonic stem cell-derived osteoblast-like cells.

Human embryonic stem cells (hESCs) have the potential to serve as a repository of cells for the replacement of damaged or diseased tissues and organs. However, to use hESCs in clinically relevant scenarios, a large number of cells are likely to be required. The aim of this study was to demonstrate an alternative cell culture method to increase the quantity of osteoblast-like cells directly derived from hESCs (hESCs-OS). Undifferentiated hESCs were directly cultivated and serially passaged in osteogenic medium (hESC-OS), and exhibited similar expression patterns of osteoblast-related genes to osteoblast-like cells derived from mesenchymal stem cells derived from hESCs (hESCs-MSCs-OS) and human bone marrow stromal cells (hBMSCs-OS). In comparison to hESCs-MSCs-OS, the hESCs-OS required a shorter expansion time to generate a homogenous population of osteoblast-like cells that did not contain contaminating undifferentiated hESCs. Identification of human specific nuclear antigen (HuNu) in the newly formed bone in calvarial defects verified the role of the transplanted hESCs-OS as active bone forming cells in vivo. Taken together, this study suggests that osteoblast-like cells directly derived from hESCs have the potential to serve as an alternative source of osteoprogenitors for bone tissue engineering strategies.