Defining Differentially Methylated Regions Specific for the Acquisition of Pluripotency and Maintenance in Human Pluripotent Stem Cells via Microarray

Background

Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appear to have a hypermethylated status compared with differentiated cells. However, the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally, differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.

Methodology

Here, we determined the DNA methylation profiles of 10 human cell lines, including 2 ESC lines, 4 virally derived iPSC lines, 2 episomally derived iPSC lines, and the 2 parental cell lines from which the iPSCs were derived using Illumina''s Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness, whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.

Conclusions

This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods, the corresponding somatic cells, and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine.     

DHP-Derivative and Low Oxygen Tension Effectively Induces Human Adipose Stromal Cell Reprogramming

Background and Methods

In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl)-derivative (DHP-d) to directly induce adipose tissue stromal cells (ATSC) to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1α and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC). Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions.

Conclusions/Significance

Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy.     

Direct Contact with Endoderm-Like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells

Rationale

Pluripotent stem cell–derived cardiac progenitor cells (CPCs) have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations.

Objective

Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells.

Method and Result

To test the hypothesis, we cocultured mouse embryonic stem (ES) cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1⁺ PDGFRa⁺ CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS) cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5⁺ and Isl1⁺ CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR⁺ PDGFRa⁺ CPCs from human ES cells.

Conclusions

Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.     

Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis

Introduction

Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.

Methods

A 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.

Results

After 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.

Conclusion

FLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.     

Differentiation of Human Pluripotent Stem Cells into Nephron Progenitor Cells in a Serum and Feeder Free System

Objectives

Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney, stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources, pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However, little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study, we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system.

Materials and Methods

We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak, intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR, real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility.

Results

After modification of culture period and concentration of exogenous factors, hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2, GDNF, HOXD11, WT1 and CITED1 in addition to OSR1, PAX2, SALL1 and EYA1. Moreover, NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular, approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems.

Conclusions

Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.     

Granulin-epithelin precursor is an oncofetal protein defining hepatic cancer stem cells

Background and Aims

Increasing evidence has suggested that hepatocellular carcinoma (HCC) might originate from a distinct subpopulation called cancer stem cells (CSCs), which are responsible for the limited efficacy of conventional therapies. We have previously demonstrated that granulin-epithelin precursor (GEP), a pluripotent growth factor, is upregulated in HCC but not in the adjacent non-tumor, and that GEP is a potential therapeutic target for HCC. Here, we characterized its expression pattern and stem cell properties in fetal and cancerous livers.

Methods

Protein expression of GEP in fetal and adult livers was examined in human and mouse models by immunohistochemical staining and flow cytometry. Liver cancer cell lines, isolated based on their GEP and/or ATP-dependent binding cassette (ABC) drug transporter ABCB5 expression, were evaluated for hepatic CSC properties in terms of colony formation, chemoresistance and tumorigenicity.

Results

We demonstrated that GEP was a hepatic oncofetal protein that expressed in the fetal livers, but not in the normal adult livers. Importantly, GEP+ fetal liver cells co-expressed the embryonic stem (ES) cell-related signaling molecules including β-catenin, Oct4, Nanog, Sox2 and DLK1, and also hepatic CSC-markers CD133, EpCAM and ABCB5. Phenotypic characterization in HCC clinical specimens and cell lines revealed that GEP+ cancer cells co-expressed these stem cell markers similarly as the GEP+ fetal liver cells. Furthermore, GEP was shown to regulate the expression of ES cell-related signaling molecules β-catenin, Oct4, Nanog, and Sox2. Isolated GEP^high cancer cells showed enhanced colony formation ability and chemoresistance when compared with the GEP_low counterparts. Co-expression of GEP and ABCB5 better defined the CSC populations with enhanced tumorigenic ability in immunocompromised mice.

Conclusions

Our findings demonstrate that GEP is a hepatic oncofetal protein regulating ES cell-related signaling molecules. Co-expression of GEP and ABCB5 further enriches a subpopulation with enhanced CSC properties. The current data provide new insight into the therapeutic strategy.     

Epigenetic Signatures Associated with Different Levels of Differentiation Potential in Human Stem Cells

Background

The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles.

Methodology/Principal Findings

to address this issue, we have investigated the levels of epigenetic regulation in well characterized populations of pluripotent embryonic stem cells (ESC) and multipotent adult stem cells (ASC) at the trancriptome, methylome, histone modification and microRNA levels. Differences in gene expression profiles allowed classification of stem cells into three separate populations including ESC, multipotent adult progenitor cells (MAPC) and mesenchymal stromal cells (MSC). The analysis of the PcG repressive marks, histone modifications and gene promoter methylation of differentiation and pluripotency genes demonstrated that stem cell populations with a wider differentiation potential (ESC and MAPC) showed stronger representation of epigenetic repressive marks in differentiation genes and that this epigenetic signature was progressively lost with restriction of stem cell potential. Our analysis of microRNA established specific microRNA signatures suggesting specific microRNAs involved in regulation of pluripotent and differentiation genes.

Conclusions/Significance

Our study leads us to propose a model where the level of epigenetic regulation, as a combination of DNA methylation and histone modification marks, at differentiation genes defines degrees of differentiation potential from progenitor and multipotent stem cells to pluripotent stem cells.     

Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach

Background

Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS) cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine.

Methodology/Principal Findings

In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT) and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES) cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5′-aza-2′-deoxycytidine) and cultured in human embryonic stem cell (ES) medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days.

Conclusion/Significance

Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.     

Fibroblast growth factor-10 promotes cardiomyocyte differentiation from embryonic and induced pluripotent stem cells

Background

The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.

Methodology/Principal Findings

We used mouse ES cells stably transfected with a cardiac-specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co-administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation.

Conclusion/Significance

FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.     

Generation,Purification and Transplantation of Photoreceptors Derived from Human Induced Pluripotent Stem Cells

Background

Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.

Methodology/Physical Findings

In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers.

Conclusions

This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.     

Membrane Potential Controls Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells

Background

Control of stem cell behavior is a crucial aspect of developmental biology and regenerative medicine. While the functional role of electrophysiology in stem cell biology is poorly understood, it has become clear that endogenous ion flows represent a powerful set of signals by means of which cell proliferation, differentiation, and migration can be controlled in regeneration and embryonic morphogenesis.

Methodology/Principal Findings

We examined the membrane potential (V_mem) changes exhibited by human mesenchymal stem cells (hMSCs) undergoing adipogenic (AD) and osteogenic (OS) differentiation, and uncovered a characteristic hyperpolarization of differentiated cells versus undifferentiated cells. Reversal of the progressive polarization via pharmacological modulation of transmembrane potential revealed that depolarization of hMSCs prevents differentiation. In contrast, treatment with hyperpolarizing reagents upregulated osteogenic markers.

Conclusions/Significance

Taken together, these data suggest that the endogenous hyperpolarization is a functional determinant of hMSC differentiation and is a tractable control point for modulating stem cell function.     

Genetically Matched Human iPS Cells Reveal that Propensity for Cartilage and Bone Differentiation Differs with Clones,not Cell Type of Origin

Background

For regenerative therapy using induced pluripotent stem cell (iPSC) technology, cell type of origin to be reprogrammed should be chosen based on accessibility and reprogramming efficiency. Some studies report that iPSCs exhibited a preference for differentiation into their original cell lineages, while others did not. Therefore, the type of cell which is most appropriate as a source for iPSCs needs to be clarified.

Methodology/Principal Findings

Genetically matched human iPSCs from different origins were generated using bone marrow stromal cells (BMSCs) and dermal fibroblasts (DFs) of the same donor, and global gene expression profile, DNA methylation status, and differentiation properties into the chondrogenic and osteogenic lineage of each clone were analyzed. Although genome-wide profiling of DNA methylation suggested tissue memory in iPSCs, genes expressed differentially in BMSCs and DFs were equally silenced in our bona fide iPSCs. After cell-autonomous and induced differentiation, each iPSC clone exhibited various differentiation properties, which did not correlate with cell-of-origin.

Conclusions/Significance

The reprogramming process may remove the difference between DFs and BMSCs at least for chondrogenic and osteogenic differentiation. Qualified and genetically matched human iPSC clone sets established in this study are valuable resources for further basic study of clonal differences.     

Gi-Coupled GPCR Signaling Controls the Formation and Organization of Human Pluripotent Colonies

Background

Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G_i-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies.

Methodology/Principal Findings

Specific and irreversible inhibition of G_i-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G_s-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells.

Conclusions/Significance

Experiments with pertussis toxin suggest that G_i signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G_i-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications.     

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

Introduction

Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.

Methods

An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.

Results

Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.

Conclusion

Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.