Generation of human female reproductive tract epithelium from human embryonic stem cells

Background

Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT).

Methodology/Principal Findings

We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1⁺ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA).

Conclusions/Significance

These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.     

Xeno-Free Defined Conditions for Culture of Human Embryonic Stem Cells,Neural Stem Cells and Dopaminergic Neurons Derived from Them

Background

Human embryonic stem cells (hESCs) may provide an invaluable resource for regenerative medicine. To move hESCs towards the clinic it is important that cells with therapeutic potential be reproducibly generated under completely defined conditions.

Methodology/Principal Findings

Here we report a four-step scalable process that is readily transferable to a Good Manufacture Practice (GMP) facility for the production of functional dopaminergic neurons from hESCs for potential clinical uses. We show that each of the steps (propagation of ESC→generation of neural stem cells (NSC)→induction of dopaminergic precursors→maturation of dopaminergic neurons) could utilize xeno-free defined media and substrate, and that cells could be stored at intermediate stages in the process without losing their functional ability. Neurons generated by this process expressed midbrain and A9 dopaminergic markers and could be transplanted at an appropriate time point in development to survive after transplant.

Conclusions/Significance

hESCs and NSCs can be maintained in xeno-free defined media for a prolonged period of time while retaining their ability to differentiate into authentic dopaminergic neurons. Our defined medium system provides a path to a scalable GMP-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential screening applications.     

hESC Expansion and Stemness Are Independent of Connexin Forty-Three-Mediated Intercellular Communication between hESCs and hASC Feeder Cells

Background

Human embryonic stem cells (hESCs) are a promising and powerful source of cells for applications in regenerative medicine, tissue engineering, cell-based therapies, and drug discovery. Many researchers have employed conventional culture techniques using feeder cells to expand hESCs in significant numbers, although feeder-free culture techniques have recently been developed. In regard to stem cell expansion, gap junctional intercellular communication (GJIC) is thought to play an important role in hESC survival and differentiation. Indeed, it has been reported that hESC-hESC communication through connexin 43 (Cx43, one of the major gap junctional proteins) is crucial for the maintenance of hESC stemness during expansion. However, the role of GJIC between hESCs and feeder cells is unclear and has not yet been reported.

Methodology/Principal Findings

This study therefore examined whether a direct Cx43-mediated interaction between hESCs and human adipose-derived stem cells (hASCs) influences the maintenance of hESC stemness. Over 10 passages, hESCs cultured on a layer of Cx43-downregulated hASC feeder cells showed normal morphology, proliferation (colony growth), and stemness, as assessed by alkaline phosphatase (AP), OCT4 (POU5F1-Human gene Nomenclature Database), SOX2, and NANOG expression.

Conclusions/Significance

These results demonstrate that Cx43-mediated GJIC between hESCs and hASC feeder cells is not an important factor for the conservation of hESC stemness and expansion.     

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

Background

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

Methodology/Principal Findings

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

Conclusions/Significance

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

Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation

Background

Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1^G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1^G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1^G93A animals.

Methods/Principal Findings

Presymptomatic SOD1^G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1^G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1^G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1^G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.

Conclusions/Significance

These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.     

The ROCK Inhibitor Y-27632 Improves Recovery of Human Embryonic Stem Cells after Fluorescence-Activated Cell Sorting with Multiple Cell Surface Markers

Background

Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.

Methodology/Principal Findings

HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.

Conclusions/Significance

The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.     

Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions

Background

Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury.

Methodology and Principal Findings

The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis.

Conclusions/Significance

These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages.     

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

Background

Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.

Methodology/Principal Findings

We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.

Conclusions/Significance

Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.     

Identification by Automated Screening of a Small Molecule that Selectively Eliminates Neural Stem Cells Derived from hESCs but Not Dopamine Neurons

Background

We have previously described fundamental differences in the biology of stem cells as compared to other dividing cell populations. We reasoned therefore that a differential screen using US Food and Drug Administration (FDA)-approved compounds may identify either selective survival factors or specific toxins and may be useful for the therapeutically-driven manufacturing of cells in vitro and possibly in vivo.

Methodology/Principal Findings

In this study we report on optimized methods for feeder-free culture of hESCs and hESC-derived neural stem cells (NSCs) to facilitate automated screening. We show that we are able to measure ATP as an indicator of metabolic activity in an automated screening assay. With this optimized platform we screened a collection of FDA-approved drugs to identify compounds that have differential toxicity to hESCs and their neural derivatives. Nine compounds were identified to be specifically toxic for NSCs to a greater extent than for hESCs. Six of these initial hits were retested and verified by large-scale cell culture to determine dose-responsive NSC toxicity. One of the compounds retested, amiodarone HCL, was further tested for possible effects on postmitotic neurons, a likely target for transplant therapy. Amiodarone HCL was found to be selectively toxic to NSCs but not to differentiated neurons or glial cells. Treated and untreated NSCs and neurons were then interrogated with global gene expression analysis to explore the mechanisms of action of amiodarone HCl. The gene expression analysis suggests that activation of cell-type specific cationic channels may underlie the toxicity of the drug.

Conclusions/Significance

In conclusion, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in a Chemistry, Manufacture and Control protocol that can be safely used to deplete unwanted contaminating precursor cells from a differentiated cell product. Our results also suggest that such a strategy is rich in the potential of identifying lineage specific reagents and provides additional evidence for the utility of stem cells in screening and discovery paradigms.     

Generation of CCR5-defective CD34 cells from ZFN-driven stop codon-integrated mesenchymal stem cell clones

Backgrounds

Homozygous 32-bp deletion of the chemokine receptor 5 gene (CCR5) is associated with resistance to human immunodeficiency virus (HIV) infection, while heterozygosity delays HIV progression. Bone marrow transplantation (BMT) from a 32/32 donor has been shown to cure an HIV-infected patient. However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment. Zinc finger nuclease (ZFN) targeting is a powerful method for achieving genomic disruption at specific DNA sites of interest.

Results

Taking advantage of the self-renewal and plasticity properties of stem cells, in this study, we successfully generated isogenic and six-cell clones of bone marrow-derived mesenchymal stem cells that carry the stop codon of the CCR5 gene by using a ZFN-mediated homology-directed repair technique. These cells were expandable for more than 5 passages, and thus show potential to serve as an individual’s cell factory. When Oct4 was overexpressed, the mutated cells robustly converted to CD34+ progenitor cells.

Conclusion

We here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones. We believe that our approach will be beneficial in future HIV treatment.     

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.     

MHC mismatch inhibits neurogenesis and neuron maturation in stem cell allografts

Background

The role of histocompatibility and immune recognition in stem cell transplant therapy has been controversial, with many reports arguing that undifferentiated stem cells are protected from immune recognition due to the absence of major histocompatibility complex (MHC) markers. This argument is even more persuasive in transplantation into the central nervous system (CNS) where the graft rejection response is minimal.

Methodology/Principal Findings

In this study, we evaluate graft survival and neuron production in perfectly matched vs. strongly mismatched neural stem cells transplanted into the hippocampus in mice. Although allogeneic cells survive, we observe that MHC-mismatch decreases surviving cell numbers and strongly inhibits the differentiation and retention of graft-derived as well as endogenously produced new neurons. Immune suppression with cyclosporine-A did not improve outcome but non-steroidal anti-inflammatory drugs, indomethacin or rosiglitazone, were able to restore allogeneic neuron production, integration and retention to the level of syngeneic grafts.

Conclusions/Significance

These results suggest an important but unsuspected role for innate, rather than adaptive, immunity in the survival and function of MHC-mismatched cellular grafts in the CNS.     

miR-34a regulates mouse neural stem cell differentiation

Background

MicroRNAs (miRNAs or miRs) participate in the regulation of several biological processes, including cell differentiation. Recently, miR-34a has been implicated in the differentiation of monocyte-derived dendritic cells, human erythroleukemia cells, and mouse embryonic stem cells. In addition, members of the miR-34 family have been identified as direct p53 targets. However, the function of miR-34a in the control of the differentiation program of specific neural cell types remains largely unknown. Here, we investigated the role of miR-34a in regulating mouse neural stem (NS) cell differentiation.

Methodology/Principal Findings

miR-34a overexpression increased postmitotic neurons and neurite elongation of mouse NS cells, whereas anti-miR-34a had the opposite effect. SIRT1 was identified as a target of miR-34a, which may mediate the effect of miR-34a on neurite elongation. In addition, acetylation of p53 (Lys 379) and p53-DNA binding activity were increased and cell death unchanged after miR-34a overexpression, thus reinforcing the role of p53 during neural differentiation. Interestingly, in conditions where SIRT1 was activated by pharmacologic treatment with resveratrol, miR-34a promoted astrocytic differentiation, through a SIRT1-independent mechanism.

Conclusions

Our results provide new insight into the molecular mechanisms by which miR-34a modulates neural differentiation, suggesting that miR-34a is required for proper neuronal differentiation, in part, by targeting SIRT1 and modulating p53 activity.     

Baculovirus as an Ideal Radionuclide Reporter Gene Vector: A New Strategy for Monitoring the Fate of Human Stem Cells In Vivo

Purpose

Radionuclide reporter gene imaging holds promise for non-invasive monitoring of transplanted stem cells. Thus, the feasibility of utilizing recombinant baculoviruses carrying the sodium iodide symporter (NIS) reporter gene in monitoring stem cell therapy by radionuclide imaging was explored in this study.

Methods

Recombinant baculoviruses carrying NIS and green fluorescent protein (GFP) reporter genes (Bac-NIS and Bac-GFP) were constructed and used to infect human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and human umbilical cord blood mesenchymal stem cells (hUCB-MSCs). Infection efficiency, total fluorescence intensity and duration of transgene expression were determined by flow cytometry. Cytotoxicity/proliferative effects of baculovirus on hUCB-MSCs were assessed using CCK-8 assays. ¹²⁵I uptake and perchlorate inhibition assays were performed on Bac-NIS-infected hUCB-MSCs. Radionuclide imaging of mice transplanted with Bac-NIS-infected hUCB-MSCs was performed by NanoSPECT/CT imaging.

Results

Infection efficiencies of recombinant baculovirus in hESCs, hiPSCs and hUCB-MSCs increased with increasing MOIs (27.3%, 35.8% and 95.6%, respectively, at MOI = 800). Almost no cytotoxicity and only slight effects on hUCB-MSCs proliferation were observed. Obvious GFP expression (40.6%) remained at 8 days post-infection. The radioiodide was functionally accumulated by NIS gene products and specifically inhibited by perchlorate (ClO₄ ^-). Radioiodide uptake, peaking at 30 min and gradually decreasing over time, significantly correlated with hUCB-MSCs cell number (R² = 0.994). Finally, radionuclide imaging showed Bac-NIS-infected hUCB-MSCs effectively accumulated radioiodide in vivo, which gradually weakened over time.

Conclusion

Baculovirus as transgenic vector of radionuclide reporter gene imaging technology is a promising strategy for monitoring stem cell transplantation therapy.     

A novel xenogeneic co-culture system to examine neuronal differentiation capability of various adult human stem cells

Background

Targeted differentiation of stem cells is mainly achieved by the sequential administration of defined growth factors and cytokines, although these approaches are quite artificial, cost-intensive and time-consuming. We now present a simple xenogeneic rat brain co-culture system which supports neuronal differentiation of adult human stem cells under more in vivo-like conditions.

Methods and Findings

This system was applied to well-characterized stem cell populations isolated from human skin, parotid gland and pancreas. In addition to general multi-lineage differentiation potential, these cells tend to differentiate spontaneously into neuronal cell types in vitro and are thus ideal candidates for the introduced co-culture system. Consequently, after two days of co-culture up to 12% of the cells showed neuronal morphology and expressed corresponding markers on the mRNA and protein level. Additionally, growth factors with the ability to induce neuronal differentiation in stem cells could be found in the media supernatants of the co-cultures.

Conclusions

The co-culture system described here is suitable for testing neuronal differentiation capability of numerous types of stem cells. Especially in the case of human cells, it may be of clinical relevance for future cell-based therapeutic applications.     

Quantitative Comparison of Constitutive Promoters in Human ES cells

Background

Constitutive 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 Findings

We 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/Significance

The 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.     

Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

Background

Reprogrammed cells, including induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cells (NT-ESCs), are similar in many respects to natural embryonic stem cells (ESCs). However, previous studies have demonstrated that iPSCs retain a gene expression signature that is unique from that of ESCs, including differences in microRNA (miRNA) expression, while NT-ESCs are more faithfully reprogrammed cells and have better developmental potential compared with iPSCs.

Results

We focused on miRNA expression and explored the difference between ESCs and reprogrammed cells, especially ESCs and NT-ESCs. We also compared the distinct expression patterns among iPSCs, NT-ESCs and NT-iPSCs. The results demonstrated that reprogrammed cells (iPSCs and NT-ESCs) have unique miRNA expression patterns compared with ESCs. The comparison of differently reprogrammed cells (NT-ESCs, NT-iPSCs and iPSCs) suggests that several miRNAs have key roles in the distinct developmental potential of reprogrammed cells.

Conclusions

Our data suggest that miRNAs play a part in the difference between ESCs and reprogrammed cells, as well as between MEFs and pluripotent cells. The variation of miRNA expression in reprogrammed cells derived using different reprogramming strategies suggests different characteristics induced by nuclear transfer and iPSC generation, as well as different developmental potential among NT-ESCs, iPSCs and NT-iPSCs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-488) contains supplementary material, which is available to authorized users.