Wnt/ß-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo.

Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/ß-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation (MKI67, PCNA) genes and the upregulation of genes for differentiation (CX43, KRT3) in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated ß-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/ß-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/ß-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.

A Novel Technique of Amniotic Membrane Preparation Mimicking Limbal Epithelial Crypts Enhances the Number of Progenitor Cells upon Expansion.

We aimed to investigate whether a novel technique of human amniotic membrane (HAM) preparation that mimics the crypts in the limbus enhances the number of progenitor cells cultured ex vivo. The HAMs were sutured on polyester membrane (1) standardly, to obtain a flat HAM surface, or (2) loosely, achieving the radial folding to mimic crypts in the limbus. Immunohistochemistry was used to demonstrate a higher number of cells positive for progenitor markers p63α (37.56 ± 3.34% vs. 62.53 ± 3.32%, p = 0.01) and SOX9 (35.53 ± 0.96% vs. 43.23 ± 2.32%, p = 0.04), proliferation marker Ki-67 (8.43 ± 0.38 % vs. 22.38 ± 1.95 %, p = 0.002) in the crypt-like HAMs vs. flat HAMs, while no difference was found for the quiescence marker CEBPD (22.99 ± 2.96% vs. 30.49 ± 3.33 %, p = 0.17). Most of the cells stained negative for the corneal epithelial differentiation marker KRT3/12, and some were positive for N-cadherin in the crypt-like structures, but there was no difference in staining for E-cadherin and CX43 in crypt-like HAMs vs. flat HAMs. This novel HAM preparation method enhanced the number of progenitor cells expanded in the crypt-like HAM compared to cultures on the conventional flat HAM.

Integrated differential DNA methylation and gene expression of formalin-fixed paraffin-embedded uveal melanoma specimens identifies genes associated with early metastasis and poor prognosis.

PURPOSE: Uveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival. METHODS: FFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976-1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as "Early metastasis" (metastases and death within 2-5 years after enucleation, n = 8), "Late metastasis" (metastases and death within 9-21 years after enucleation, n = 7) and "No metastasis" (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: DNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups ("Early", "Late", "No") nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in "Subset Early metastasis" vs "Subset No metastasis" and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in "Subset Early metastasis vs "Subset No metastasis" and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM. CONCLUSIONS: DNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.

Long-term myofibroblast persistence in the capsular bag contributes to the late spontaneous in-the-bag intraocular lens dislocation.

Late spontaneous in-the-bag intraocular lens (IOL) dislocation is a complication presenting 6 months or later after cataract surgery. We aimed to characterize the cells in the lens capsules (LCs) of 18 patients with spontaneous late in-the-bag IOL dislocation. Patients' average age was 82.6 ± 1.5 years (range 72-98), and most of them had pseudoexfoliation syndrome (PEX). Cells from the LCs were positive for myofibroblast (αSMA), proliferation (Ki-67, PCNA), early lens development/lens progenitor (SOX2, PAX6), chemokine receptor (CXCR4), and transmembrane (N-cadherin) markers, while negative for epithelial (E-cadherin) marker. Moreover, the cells produced abundant fibronectin, type I and type V collagen in the nearby extracellular matrix (ECM). During ex vivo cultivation of dislocated IOL-LCs in toto, the cells proliferated and likely migrated onto the IOL's anterior side. EdU proliferation assay confirmed the proliferation potential of the myofibroblasts (MFBs) in dislocated IOL-LCs. Primary cultured lens epithelial cells/MFBs isolated from the LC of dislocated IOLs could induce collagen matrix contraction and continuously proliferated, migrated, and induced ECM remodeling. Taken together, this indicates that long-lived MFBs of dislocated IOLs might contribute to the pathogenic mechanisms in late in-the-bag IOL dislocation.

Ex vivo 3D human corneal stroma model for Schnyder corneal dystrophy - role of autophagy in its pathogenesis and resolution.

INTRODUCTION: Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. METHODS: Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. RESULTS: CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. CONCLUSIONS: MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

Proliferative Cells Isolated from the Adult Human Peripheral Retina only Transiently Upregulate Key Retinal Markers upon Induced Differentiation.

Purpose/Aim: The adult human retina has limited regenerative potential, and severe injury will result in permanent damage. Lower vertebrates handle retinal injury by activating neural stem cells (NSCs) in the ciliary marginal zone (CMZ). Müller glia-like cells expressing markers of NSCs are also present in the peripheral retina (PR) of the adult human eye, leading to the hypothesis that a CMZ-like zone might exists also in humans. In order to shed further light on this hypothesis we investigated the in vitro differentiation potential of proliferative cells isolated from the adult human PR towards a retinal phenotype. MATERIALS AND METHODS: Proliferative cells were isolated from the peripheral retina of human eyes (n = 6) within 24 to 48 hours post mortem and further expanded for 2 or 3 passages before being differentiated for 1-3 weeks. Gene expression was analyzed by microarray and qRT-PCR analysis, while protein expression was identified by immunocytochemistry. RESULTS: A high density of cells co-staining with markers for progenitor cells and Müller glia was found in situ in the PR. Cells isolated from this region and cultured adherently showed fibrillary processes and were positive for the immature marker Nestin and the glial marker GFAP, while a few co-expressed PAX6. After 7 days of differentiation, there was a transient upregulation of early and mature photoreceptor markers, including NRL, CRX, RHO and RCVRN, as well as the Müller cell and retinal pigmented epithelium (RPE) marker CRALBP, and the early RPE marker MITF. However, the expression of all these markers dropped from Day 14 and onwards. CONCLUSIONS: Upon exposure of proliferating cells from the adult human PR to differentiating conditions in culture, there is a widespread change in morphology and gene expression, including the upregulation of key retinal markers. However, this upregulation is only transient and decreases after 14 days of differentiation.

Multicellular tumor spheroids of human uveal melanoma induce genes associated with anoikis resistance, lipogenesis, and SSXs.

PURPOSE: Uveal melanoma (UM) has a high propensity for metastatic spread, and approximately 40-50% of patients die of metastatic disease. Metastases can be found at the time of diagnosis but also several years after the tumor has been removed. The survival of disseminated cancer cells is known to be linked to anchorage independence, anoikis resistance, and an adaptive cellular metabolism. The cultivation of cancer cells as multicellular tumor spheroids (MCTS) by anchorage-independent growth enriches for a more aggressive phenotype. The present study examines the differential gene expression of adherent cell cultures, non-adherent MCTS cultures, and uncultured tumor biopsies from three patients with UM. We elucidate the biochemical differences between the culture conditions to find whether the culture of UM as non-adherent MCTS could be linked to an anchorage-independent and more aggressive phenotype, thus unravelling potential targets for treatment of UM dissemination. METHODS: The various culture conditions were evaluated with microarray analysis, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), RNAscope, immunohistochemistry (IHC), and transmission electron microscopy (TEM) followed by gene expression bioinformatics. RESULTS: The MCTS cultures displayed traits associated with anoikis resistance demonstrated by ANGPTL4 upregulation, and a shift toward a lipogenic profile by upregulation of ACOT1 (lipid metabolism), FADS1 (biosynthesis of unsaturated fatty acids), SC4MOL, DHCR7, LSS (cholesterol biosynthesis), OSBPL9 (intracellular lipid receptor), and PLIN2 (lipid storage). Additionally, the present study shows marked upregulation of synovial sarcoma X breakpoint proteins (SSXs), transcriptional repressors related to the Polycomb group (PcG) proteins that modulate epigenetic silencing of genes. CONCLUSIONS: The MCTS cultures displayed traits associated with anoikis resistance, a metabolic shift toward a lipogenic profile, and upregulation of SSXs, related to the PcG proteins.

The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women.

Inhibition of sclerostin, a glycoprotein secreted by osteocytes, offers a new therapeutic paradigm for treatment of osteoporosis (OP) through its critical role as Wnt/catenin signaling regulator. This study describes the epigenetic regulation of SOST expression in bone biopsies of postmenopausal women. We correlated serum sclerostin to bone mineral density (BMD), fractures, and bone remodeling parameters, and related these findings to epigenetic and genetic disease mechanisms. Serum sclerostin and bone remodeling biomarkers were measured in two postmenopausal groups: healthy (BMD T-score > -1) and established OP (BMD T-score < -2.5, with at least one low-energy fracture). Bone specimens were used to analyze SOST mRNAs, single nucleotide polymorphisms (SNPs), and DNA methylation changes. The SOST gene promoter region showed increased CpG methylation in OP patients (n = 4) compared to age and body mass index (BMI) balanced controls (n = 4) (80.5% versus 63.2%, p = 0.0001) with replication in independent cohorts (n = 27 and n = 36, respectively). Serum sclerostin and bone SOST mRNA expression correlated positively with age-adjusted and BMI-adjusted total hip BMD (r = 0.47 and r = 0.43, respectively; both p < 0.0005), and inversely to serum bone turnover markers. Five SNPs, one of which replicates in an independent population-based genomewide association study (GWAS), showed association with serum sclerostin or SOST mRNA levels under an additive model (p = 0.0016 to 0.0079). Genetic and epigenetic changes in SOST influence its bone mRNA expression and serum sclerostin levels in postmenopausal women. The observations suggest that increased SOST promoter methylation seen in OP is a compensatory counteracting mechanism, which lowers serum sclerostin concentrations and reduces inhibition of Wnt signaling in an attempt to promote bone formation.

Epigenetic regulation of nestin expression during neurogenic differentiation of adipose tissue stem cells.

Adipose-tissue-derived stem cells (ASCs) have received considerable attention due to their easy access, expansion potential, and differentiation capacity. ASCs are believed to have the potential to differentiate into neurons. However, the mechanisms by which this may occur remain largely unknown. Here, we show that culturing ASCs under active proliferation conditions greatly improves their propensity to differentiate toward osteogenic, adipogenic, and neurogenic lineages. Neurogenic-induced ASCs express early neurogenic genes as well as markers of mature neurons, including voltage-gated ion channels. Nestin, highly expressed in neural progenitors, is upregulated by mitogenic stimulation of ASCs, and as in neural progenitors, then repressed during neurogenic differentiation. Nestin gene (NES) expression under these conditions appears to be regulated by epigenetic mechanisms. The neural-specific, but not muscle-specific, enhancer regions of NES are DNA demethylated by mitogenic stimulation, and remethylated upon neurogenic differentiation. We observe dynamic changes in histone H3K4, H3K9, and H3K27 methylation on the NES locus before and during neurogenic differentiation that are consistent with epigenetic processes involved in the regulation of NES expression. We suggest that ASCs are epigenetically prepatterned to differentiate toward a neural lineage and that this prepatterning is enhanced by demethylation of critical NES enhancer elements upon mitogenic stimulation preceding neurogenic differentiation. Our findings provide molecular evidence that the differentiation repertoire of ASCs may extend beyond mesodermal lineages.

Histone H3 modifications associated with differentiation and long-term culture of mesenchymal adipose stem cells.

Long-term culture of mesenchymal stem cells leads to a loss of differentiation capacity, the molecular mechanism of which remains not understood. We show here that expansion of adipose stem cells (ASCs) to late passage (replicative senescence) is associated with promoter-specific and global changes in epigenetic histone modifications. In undifferentiated ASCs, inactive adipogenic and myogenic promoters are enriched in a repressive combination of trimethylated H3K4 (H3K4m3) and H3K27m3 in the absence of H3K9m3, a heterochromatin mark. Sequential chromatin immunoprecipitation assays indicate that H3K4m3 and H3K27m3 co-occupy a fraction of nucleosomes on some but not all lineage-specific promoters examined. However in cultured primary keratinocytes, adipogenic and myogenic promoters are enriched in trimethylated H3K4, K27, and K9, illustrating two distinct epigenetic states of inactive promoters related to potential for activation. H3K4m3 and H3K27m3 stably mark promoters during long-term ASC culture indicating that loss of differentiation capacity is not due to alterations in these histone modifications on these loci. Adipogenic differentiation in early passage results in H3K27 demethylation and H3K9 acetylation specifically on adipogenic promoters. On induction of differentiation in late passage, however, transcriptional upregulation is impaired, H3K27 trimethylation is maintained and H3K9 acetylation is inhibited on promoters. In addition, the polycomb proteins Ezh2 and Bmi1 are targeted to promoters. This correlates with global cellular Ezh2 increase and H3K9 deacetylation. Promoter targeting by Ezh2 and Bmi1 in late passage ASCs suggests the establishment of a polycomb-mediated epigenetic program aiming at repressing transcription.

High-resolution analysis of genetic stability of human adipose tissue stem cells cultured to senescence.

The potential use of human mesenchymal stem cells for therapeutic applications implies large scale in vitro culture, increasing the probability of genetic instability and transformation. We examine here the incidence of unbalanced and balanced chromosome rearrangements in polyclonal and single cell-derived cultures of human adipose stem cells to senescence. G-banding karyotyping of the polyclonal cultures shows a normal karyotype. In addition, high-resolution microarray-based comparative genomic hybridization analyses relative to uncultured adipose stem cells from the same donors reveal overall genomic stability in long-term (approximately 6 months) polyclonal and clonal culture. One adipose stem cell clone displayed minor deletions in gene-rich telomeric and sub-telomeric regions on three chromosomes in early passage. This however, was detected only in a sub-population of cells that was subsequently spontaneously eliminated from the culture. Apparent pericentromeric instabilities are also occasionally detected in specific chromosomes. Our results indicate that clonal chromosomal aberrations may arise transiently in early passage adipose stem cells (ASC) cultures. Nonetheless, incidence of these aberrations seems to be negligible in the majority of long-term ASC cultures, at least under the culture conditions used here.

Epigenetic Basis for the Differentiation Potential of Mesenchymal and Embryonic Stem Cells.

SUMMARY: Stem cells have the ability to self-renew, and give rise to one or more differentiated cell types. Embryonic stem cells can differentiate into all cell types of the body and have unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues. They have an extensive but finite lifespan and can differentiate into a more restricted range of cell types. Increasing evidence indicates that the multilineage differentiation ability of stem cells is defined by the potential for expression of developmentally regulated transcription factors and of lineage specification genes. Gene expression, or as emphasized here, the potential for gene expression, is largely controlled by epigenetic modifications of DNA (DNA methylation) and chromatin (such as post-translational histone modifications) in the regulatory regions of specific genes. Epigenetic modifications can also influence the timing of DNA replication. We highlight here how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through the mapping of DNA methylation profiles on differentiation-regulated promoters and at the genome-wide level, histone modifications, and transcription factor binding. Epigenetic marks on developmentally regulated and lineage specification genes in stem cells seem to define a state of pluripotency.

Programming the genome in embryonic and somatic stem cells.

In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state.

Dynamics of adipogenic promoter DNA methylation during clonal culture of human adipose stem cells to senescence.

BACKGROUND: Potential therapeutic use of mesenchymal stem cells (MSCs) is likely to require large-scale in vitro expansion of the cells before transplantation. MSCs from adipose tissue can be cultured extensively until senescence. However, little is known on the differentiation potential of adipose stem cells (ASCs) upon extended culture and on associated epigenetic alterations. We examined the adipogenic differentiation potential of clones of human ASCs in early passage culture and upon senescence, and determined whether senescence was associated with changes in adipogenic promoter DNA methylation. RESULTS: ASC clones cultured to senescence display reduced adipogenic differentiation capacity in vitro, on the basis of limited lipogenesis and reduced transcriptional upregulation of FABP4 and LPL, two adipogenic genes, while LEP and PPARG2 transcription remains unaffected. In undifferentiated senescent cells, PPARG2 and LPL expression is unaltered, whereas LEP and FABP4 transcript levels are increased but not in all clones. Bisulfite sequencing analysis of DNA methylation reveals overall relative stability of LEP, PPARG2, FABP4 and LPL promoter CpG methylation during senescence and upon differentiation. Mosaicism in methylation profiles is maintained between and within ASC clones, and any CpG-specific methylation change detected does not necessarily relate to differentiation potential. One exception to this contention is CpG No. 21 in the LEP promoter, whose senescence-related methylation may impair upregulation of the gene upon adipogenic stimulation. CONCLUSION: Senescent ASCs display reduced in vitro differentiation ability and transcriptional activation of adipogenic genes upon differentiation induction. These restrictions, however, cannot in general be attributed to specific changes in DNA methylation at adipogenic promoters. There also seems to be a correlation between CpGs that are hypomethylated and important transcription factor binding sites.

CpG methylation profiles of endothelial cell-specific gene promoter regions in adipose tissue stem cells suggest limited differentiation potential toward the endothelial cell lineage.

In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported, and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes, CD31 and CD144, in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells, freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes, marginal upregulation of CD31, and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however, no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters, which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage.

Stable CpG hypomethylation of adipogenic promoters in freshly isolated, cultured, and differentiated mesenchymal stem cells from adipose tissue.

Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells.

On the way to reprogramming cells to pluripotency using cell-free extracts.

The functional reprogramming of a differentiated cell to pluripotency may present beneficial applications in regenerative medicine. Somatic cell nuclear transfer may offer this possibility, but technical hurdles and ethical guidelines currently prevent application of this technology in several countries. As a result, alternative approaches are being developed for altering cell fate. Recent non-nuclear transfer-based approaches for reprogramming somatic cells are discussed as well as ways to enhance their differentiation potential. These approaches include the fusion of differentiated cells with embryonic stem cells and the use of extract from pluripotent cells to reprogramme differentiated cells into multipotent or pluripotent cells.

Epigenetic programming of mesenchymal stem cells from human adipose tissue.

Stromal stem cells identified in various adult mesenchymal tissues (commonly called mesenchymal stem cells [MSCs]) have in past years received more attention as a result of their potential interest as replacement cells in regenerative medicine. An abundant and easily accessible source of adult human MSCs are stem cells harvested from liposuction material. Similarly to bone marrow-derived MSCs, human adipose tissue-derived stem cells (ASCs) can give rise to a variety of cell types in vitro and in vivo; however, they have a propensity to differentiate into primarily mesodermal lineages. Even so, their capacity to differentiate into nonadipogenic mesodermal pathways seems to be restricted. Emerging DNA methylation profiles at adipogenic and nonadipogenic gene promoters in freshly isolated, cultured, or differentiated ASCs aim to provide an epigenetic explanation for this restrictive differentiation potential. A review of these studies indicates that human ASCs are epigenetically marked by mosaic hypomethylation of adipogenic promoters, whereas nonadipogenic lineage-specific promoters are hypermethylated. Surprisingly, in vitro differentiation toward various pathways maintains the overall methylation profiles of undifferentiated cells, raising the hypothesis that ASCs are at least epigenetically preprogrammed for adipogenesis. Novel attempts at reprogramming the epigenome of MSCs have been initiated to enhance the differentiation capacity of these cells.