Time Course Of Differentiation Research Articles

Transient promoter interactions modulate developmental gene activation

Transcriptional induction coincides with the formation of various chromatin topologies. Strong evidence supportsthat gene activation is accompanied by a general increase in promoter-enhancer interactions. However,it remains unclear how these topological changes are coordinated across time and space during transcriptional activation. Here, we combine chromatin conformation capture with transcription and chromatin profiling during an embryonic stem cell (ESC) differentiation time course to determine how 3D genome restructuring is related to transcriptional transitions. This approach allows us to identify distinct topological alterationsthat are associated with the magnitude of transcriptional induction. We detect transiently formed interactions and demonstrate by genetic deletions that associated distal regulatory elements (DREs), as well as appropriate formation and disruption of these interactions, can contribute to the transcriptional induction of linked genes. Together, our study links topological dynamics to the magnitude of transcriptional induction and detects an uncharacterized type of transcriptionally important DREs.

Differentiation of Spiral Ganglion Neurons from Human Dental Pulp Stem Cells: A Further Step towards Autologous Auditory Nerve Recovery.

The degeneration of spiral ganglion neurons (SGNs), which convey auditory signals from hair cells to the brain, can be a primary cause of sensorineural hearing loss (SNHL) or can occur secondary to hair cell loss. Emerging therapies for SNHL include the replacement of damaged SGNs using stem cell-derived otic neuronal progenitors (ONPs). However, the availability of renewable, accessible, and patient-matched sources of human stem cells is a prerequisite for successful replacement of the auditory nerve. In this study, we derived ONP and SGN-like cells by a reliable and reproducible stepwise guidance differentiation procedure of self-renewing human dental pulp stem cells (hDPSCs). This in vitro differentiation protocol relies on the modulation of BMP and TGFβ pathways using a free-floating 3D neurosphere method, followed by differentiation on a Geltrex-coated surface using two culture paradigms to modulate the major factors and pathways involved in early otic neurogenesis. Gene and protein expression analyses revealed efficient induction of a comprehensive panel of known ONP and SGN-like cell markers during the time course of hDPSCs differentiation. Atomic force microscopy revealed that hDPSC-derived SGN-like cells exhibit similar nanomechanical properties as their in vivo SGN counterparts. Furthermore, spiral ganglion neurons from newborn rats come in close contact with hDPSC-derived ONPs 5 days after co-culturing. Our data demonstrate the capability of hDPSCs to generate SGN-like neurons with specific lineage marker expression, bipolar morphology, and the nanomechanical characteristics of SGNs, suggesting that the neurons could be used for next-generation cochlear implants and/or inner ear cell-based strategies for SNHL.

Differentiation of SH-SY5Y neuroblastoma cells using retinoic acid and BDNF: a model for neuronal and synaptic differentiation in neurodegeneration

There has been much interest in the use of cell culture models of neurones, to avoid the animal welfare and cost issues of using primary and human-induced pluripotent stem cell (hiPSC)-derived neurones respectively. The human neuroblastoma cell line, SH-SY5Y, is extensively used in laboratories as they can be readily expanded, are of low cost and can be differentiated into neuronal-like cells. However, much debate remains as to their phenotype once differentiated, and their ability to recapitulate the physiology of bona fide neurones. Here, we characterise a differentiation protocol using retinoic acid and BDNF, which results in extensive neurite outgrowth/branching within 10 days, and expression of key neuronal and synaptic markers. We propose that these differentiated SH-SY5Y cells may be a useful substitute for primary or hiPSC-derived neurones for cell biology studies, in order to reduce costs and animal usage. We further propose that this characterised differentiation timecourse could be used as an in vitro model for neuronal differentiation, for proof-of principle studies on neurogenesis, e.g. relating to neurodegenerative diseases. Finally, we demonstrate profound changes in Tau phosphorylation during differentiation of these cells, suggesting that they should not be used for neurodegeneration studies in their undifferentiated state.

Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1μ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and μ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1μ non-muscle isoform protein expression before and during cardiac differentiation.

Active vitamin D increases myogenic differentiation in C2C12 cells via a vitamin D response element on the myogenin promoter.

Background: Skeletal muscle development during embryogenesis depends on proliferation of myoblasts followed by differentiation into myotubes/multinucleated myofibers. Vitamin D (VD) has been shown to affect these processes, but there is conflicting evidence within the current literature on the exact nature of these effects due to a lack of time course data. With 20%-40% of pregnant women worldwide being VD deficient, it is crucial that a clearer understanding of the impact of VD on myogenesis is gained. Methods: A detailed 8-day differentiation time course was used where C2C12 cells were differentiated in control media (2% horse serum) or with different concentrations of active VD, 1,25 (OH)2D3 (10-13M, 10-11M, 10-9M or 10-7M), and measurements were taken at 6 time points. DNA, creatine kinase and protein assays were carried out as well as quantitative PCR to determine expression of Myf5, MyoD, myogenin, MHC I, and MHC neonatal, MHC embryonic, MHC IIa, MHC IIx, and MHC IIb mRNAs. Transfections were carried out using one vector containing the myogenin promoter and another containing the same promoter with a 3 base mutation within a putative vitamin D response element (VDRE) to determine effects of 1,25 (OH)2D3 on myogenin transcription. Finally, a ChIP assay was performed to determine whether the VD receptor (VDR) binds to the putative VDRE. Results: 1,25(OH)2D3 caused an inhibition of proliferation and an increase in differentiation in C2C12 cells. Myf5, myogenin, MHC I, and MHC neonatal, MHC embryonic, MHC IIa, MHC IIx, and MHC IIb expression were all increased by 1,25(OH)2D3. Myotube size was also increased by VD. When the putative VDRE on the myogenin promoter was mutated, the increase in expression by VD was lost. ChIP analysis revealed that the VDR does bind to the putative VDRE on the myogenin promoter. Conclusion: Active VD directly increases myogenin transcription via a functional VDRE on the myogenin promoter, resulting in increased myogenic differentiation, increased expression of both the early and late MHC isoforms, and also increased myotube size. These results highlight the importance of VD status during pregnancy for normal myogenesis to occur, but further in vivo work is needed.

ARID4B loss leads to activated STAT1-dependent interferon pathway in mouse embryonic stem cells and during meso/endodermal differentiation.

Proper deactivation of the pluripotency network and activation of a lineage-specific gene expression program are critical for mouse embryonic stem cell (mESC) differentiation. This is achieved by the coordinated action of transcription and chromatin factors. Our previous work identified ARID4B as a critical chromatin factor for mesoderm and endoderm differentiation. As part of a histone deacetylase complex, ARID4B plays a role in transcriptional suppression of its direct targets. Here, we investigated the mechanism of ARID4B function in mESC differentiation by focusing on genes and pathways that are upregulated in its absence. We analyzed transcriptomic results of wild-type and arid4bΔ endoderm or mesoderm differentiated cells through integrative genomics viewer and ingenuity pathway analysis. We performed real-time quantitative polymerase chain reaction for selected genes. To understand pathway activation, we performed Western blot for candidate proteins during the time-course of differentiation. We also analyzed H3K4me3, H3K27me3 and H3K27Ac ChIP-seq results to understand changes in the chromatin environment. Interferon-related genes were activated in arid4bΔ mESCs and endoderm or mesoderm directed cells. Consistent with this, higher phosphorylated STAT1 levels were found in arid4bΔ mESCs while a related phosphorylated STAT3 was unchanged. Finally, we observed a significant increase in H3K4me3 around interferon-related distal gene regulatory regions with a combination of either upregulation of H3K27Ac level or downregulation of H3K27me3 level. These results provide evidence that ARID4B is involved in the suppression of interferon-related genes in mESCs and during meso/endoderm differentiation through modulation, mainly of H3K4me3. This regulation might be important for successful mESC differentiation.

Temporal Profiling of Epitranscriptomic Modulators during Osteogenic Differentiation of Human Embryonic Stem Cells.

Osteogenesis is modulated by multiple regulatory networks. Recent studies showed that RNA modifications and their reader, writer, and eraser (RWE) proteins are involved in regulating various biological processes. Few studies, however, were conducted to investigate the functions of RNA modifications and their RWE proteins in osteogenesis. By using LC-MS/MS in parallel-reaction monitoring (PRM) mode, we performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins throughout the entire time course of osteogenic differentiation in H9 human embryonic stem cells (ESCs). We found that approximately half of the 127 detected RWE proteins were down-regulated during osteogenic differentiation, and they included mainly proteins involved in RNA methylation and pseudouridylation. Protein-protein interaction (PPI) network analysis unveiled significant associations between the down-regulated epitranscriptomic RWE proteins and osteogenesis-related proteins. Gene set enrichment analysis (GSEA) of publicly available RNA-seq data obtained from osteogenesis imperfecta patients suggested a potential role of METTL1 in osteogenesis through the cytokine network. Together, this is the first targeted profiling of epitranscriptomic RWE proteins during osteogenic differentiation of human ESCs, and our work unveiled potential regulatory roles of these proteins in osteogenesis. LC-MS/MS data were deposited on ProteomeXchange (PXD039249).

Adult Neurogenesis Is Altered by Circadian Phase Shifts and the Duper Mutation in Female Syrian Hamsters.

Cell birth and survival in the adult hippocampus are regulated by a circadian clock. Rotating shift work and jet lag disrupt circadian rhythms and aggravate disease. Internal misalignment, a state in which abnormal phase relationships prevail between and within organs, is proposed to account for adverse effects of circadian disruption. This hypothesis has been difficult to test because phase shifts of the entraining cycle inevitably lead to transient desynchrony. Thus, it remains possible that phase shifts, regardless of internal desynchrony, account for adverse effects of circadian disruption and alter neurogenesis and cell fate. To address this question, we examined cell birth and differentiation in the duper Syrian hamster (Mesocricetus auratus), a Cry1-null mutant in which re-entrainment of locomotor rhythms is greatly accelerated. Adult females were subjected to alternating 8 h advances and delays at eight 16 d intervals. BrdU, a cell birth marker, was given midway through the experiment. Repeated phase shifts decreased the number of newborn non-neuronal cells in WT, but not in duper hamsters. The duper mutation increased the number of BrdU-IR cells that stained for NeuN, which marks neuronal differentiation. Immunocytochemical staining for proliferating cell nuclear antigen indicated no overall effect of genotype or repeated shifts on cell division rates after 131 days. Cell differentiation, assessed by doublecortin, was higher in duper hamsters but was not significantly altered by repeated phase shifts. Our results support the internal misalignment hypothesis and indicate that Cry1 regulates cell differentiation. Phase shifts may determine neuronal stem cell survival and time course of differentiation after cell birth. Figure created with BioRender.

A dataset of definitive endoderm and hepatocyte differentiations from human induced pluripotent stem cells

Hepatocytes are a major parenchymal cell type in the liver and play an essential role in liver function. Hepatocyte-like cells can be differentiated in vitro from induced pluripotent stem cells (iPSCs) via definitive endoderm (DE)-like cells and hepatoblast-like cells. Here, we explored the in vitro differentiation time-course of hepatocyte-like cells. We performed methylome and transcriptome analyses for hepatocyte-like cell differentiation. We also analyzed DE-like cell differentiation by methylome, transcriptome, chromatin accessibility, and GATA6 binding profiles, using finer time-course samples. In this manuscript, we provide a detailed description of the dataset and the technical validations. Our data may be valuable for the analysis of the molecular mechanisms underlying hepatocyte and DE differentiations.

Poly(acrylic acid)-grafted metal-organic framework carrying Mg ions for bone repair

In the field of biomedicine, metal-organic frameworks (MOFs) have been extensively studied. However, few studies have applied them to bone healing. In recent years, iron-based MOFs such as MIL-100(Fe) have been proven biocompatible and suitable for drug delivery applications. We used water-phase synthesis methods to prepare MIL-100(Fe) and NH2 functionalized MIL-100(Fe). We then performed post-synthesis modification to incorporate Magnesium (Mg) ions into the cages of the MIL-100(Fe) (i.e., Mg@MIL-100(Fe)). The poly(acrylic acid) (PAA) modification on the surface of Mg@MIL-100(Fe) (i.e., Mg@MIL-100(Fe)-PAA) prevented the Mg ions from leaking, thus significantly increasing the Mg loading. The cytotoxicity test of the osteoblast-like cell line MG-63 showed that Mg@MIL-100(Fe)-PAA was biocompatible and could slightly improve cell proliferation. Furthermore, the ALP assay showed that Mg@MIL-100(Fe)-PAA could alter the time course of cell differentiation, which could eventually accelerate the bone healing process. These findings revealed the potential of utilizing metal-organic frameworks as bone repairing materials.

Deep sequencing of short capped RNAs reveals novel families of noncoding RNAs.

In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3′ ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.

Induced pluripotent stem cell-derived smooth muscle cells to study cardiovascular calcification.

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date, only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs), which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture. There is an increasing, practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically, we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First, we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis, immunofluorescent staining, flow cytometry, and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust, well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs, and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation, we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions, with high potential for future applications in cardiovascular calcification research.

Transcriptomic analysis of Simpson Golabi Behmel syndrome cells during differentiation exhibit BAT-like function

High-throughput RNA sequencing of human Simpson-Golabi-Behmel syndrome cells (SGBS) was performed during the time-course of adipogenic differentiation at day 4 (D04), 6 (D06), 8 (D08), and 10 (D10) to characterize transcriptomic changes and to identify key patterns involved in adipogenesis and browning. In the comparisons, 932 and 384 overlapping transcripts were consistently up- and down-regulated, respectively. Combining the results of protein-protein interaction network analysis MCODE and CytoHubba, 55 up-regulated hub genes from four clusters and 9 down-regulated genes were identified. The up-regulated hub genes were mainly enriched in brown adipocyte differentiation, extracellular matrix organization, and valine, leucine, and isoleucine degradation. The enrichment of downregulated hub genes was related to NRF2 signalling and glutathione metabolism, indicating that oxidative stress also plays a role. Analysis of overlapping down-regulated genes, targets of transcription factors, revealed enrichment in the IL-18 signalling pathway, which is involved in browning process and extracellular matrix organization via actomyosin mechanics and integrin-extracellular matrix interactions. Finally, the comparison transcriptomic analysis with the gene signature reported by BATLAS and PROFAT web-based tools showed an increased percentage of the brown phenotype, confirming that differentiated SGBS cells at D06, D08, and D10 gradually acquire BAT-like function.

Single-cell sequencing reveals lineage-specific dynamic genetic regulation of gene expression during human cardiomyocyte differentiation.

Dynamic and temporally specific gene regulatory changes may underlie unexplained genetic associations with complex disease. During a dynamic process such as cellular differentiation, the overall cell type composition of a tissue (or an in vitro culture) and the gene regulatory profile of each cell can both experience significant changes over time. To identify these dynamic effects in high resolution, we collected single-cell RNA-sequencing data over a differentiation time course from induced pluripotent stem cells to cardiomyocytes, sampled at 7 unique time points in 19 human cell lines. We employed a flexible approach to map dynamic eQTLs whose effects vary significantly over the course of bifurcating differentiation trajectories, including many whose effects are specific to one of these two lineages. Our study design allowed us to distinguish true dynamic eQTLs affecting a specific cell lineage from expression changes driven by potentially non-genetic differences between cell lines such as cell composition. Additionally, we used the cell type profiles learned from single-cell data to deconvolve and re-analyze data from matched bulk RNA-seq samples. Using this approach, we were able to identify a large number of novel dynamic eQTLs in single cell data while also attributing dynamic effects in bulk to a particular lineage. Overall, we found that using single cell data to uncover dynamic eQTLs can provide new insight into the gene regulatory changes that occur among heterogeneous cell types during cardiomyocyte differentiation.

Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.

We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.

Myogenic Potential of Extracellular Matrix Derived from Decellularized Bovine Pericardium.

Skeletal muscles represent 40% of body mass and its native regenerative capacity can be permanently lost after a traumatic injury, congenital diseases, or tumor ablation. The absence of physiological regeneration can hinder muscle repair preventing normal muscle tissue functions. To date, tissue engineering (TE) represents one promising option for treating muscle injuries and wasting. In particular, hydrogels derived from the decellularized extracellular matrix (dECM) are widely investigated in tissue engineering applications thanks to their essential role in guiding muscle regeneration. In this work, the myogenic potential of dECM substrate, obtained from decellularized bovine pericardium (Tissuegraft Srl), was evaluated in vitro using C2C12 murine muscle cells. To assess myotubes formation, the width, length, and fusion indexes were measured during the differentiation time course. Additionally, the ability of dECM to support myogenesis was assessed by measuring the expression of specific myogenic markers: α-smooth muscle actin (α-sma), myogenin, and myosin heavy chain (MHC). The results obtained suggest that the dECM niche was able to support and enhance the myogenic potential of C2C12 cells in comparison of those grown on a plastic standard surface. Thus, the use of extracellular matrix proteins, as biomaterial supports, could represent a promising therapeutic strategy for skeletal muscle tissue engineering.

Interspecies chimeric conditions affect the developmental rate of human pluripotent stem cells

Human pluripotent stem cells hold significant promise for regenerative medicine. However, long differentiation protocols and immature characteristics of stem cell-derived cell types remain challenges to the development of many therapeutic applications. In contrast to the slow differentiation of human stem cells in vitro that mirrors a nine-month gestation period, mouse stem cells develop according to a much faster three-week gestation timeline. Here, we tested if co-differentiation with mouse pluripotent stem cells could accelerate the differentiation speed of human embryonic stem cells. Following a six-week RNA-sequencing time course of neural differentiation, we identified 929 human genes that were upregulated earlier and 535 genes that exhibited earlier peaked expression profiles in chimeric cell cultures than in human cell cultures alone. Genes with accelerated upregulation were significantly enriched in Gene Ontology terms associated with neurogenesis, neuron differentiation and maturation, and synapse signaling. Moreover, chimeric mixed samples correlated with in utero human embryonic samples earlier than human cells alone, and acceleration was dose-dependent on human-mouse co-culture ratios. The altered gene expression patterns and developmental rates described in this report have implications for accelerating human stem cell differentiation and the use of interspecies chimeric embryos in developing human organs for transplantation.

Abstract 15560: Induced Pluripotent Stem Cell (iPSC) Derived Smooth Muscle Cells as a Model to Study Vascular Calcification

Introduction: Vascular calcification is the top cause of mortality worldwide. Vascular smooth muscle cells (SMCs) are major contributors to vascular calcification. There is an urgent need for a large scale and inexhaustible source of functional SMCs to study mechanisms of cardiovascular calcification. Here, we describe a new platform utilizing iPSCs differentiated towards SMCs that can calcify, to model ectopic calcification and identify mechanisms of SMCs’ contribution in this pathological process. Methods: We differentiated human iPSCs towards an induced-SMC (iSMC) phenotype in a 10-day protocol. We verified successful iSMC differentiation through immunofluorescent staining for SMC marker proteins αSMA and MYH11, FACS using CD140b, and proteomics analysis on whole cell lysate at days 0, 3, 5, 7, and 10. A set of 19 canonical SMC proteins were used to generate a Pearson’s correlation over the differentiation time course. After differentiation, we cultured the iSMCs in osteogenic media (OM) for 14 days. Alizarin Red S staining performed on day 14 confirmed the presence of calcification. Three independent experiments were performed for all data analyses. Results: Typical SMC morphology with positive staining for αSMA and MYH11 was detected at day 10 by immunofluorescence (Fig 1A). FACS analysis on markers for iPSCs (TRA-1-60), ECs (CD31), and SMCs (CD140b) demonstrated that 93.9% of cells were positive for CD140b after 10 days of differentiation (Fig 1B, C). Time course proteomics showed a strong Pearson’s correlation with of iSMCs with SMC marker proteins (R = 0.89) by day 10 (Figure 1D). Calcification in osteogenic media was observed by day 14 in culture using Alizarin Red S staining (Fig 1E, F), with 4.8-fold higher calcification in OM as compared to normal media (NM). Conclusions: Investigated iSMCs have promising future applications in cardiovascular research and may serve as an inexhaustible resource for studying vascular SMC calcification.

DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity

Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6–8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

KLF3 Mediates Epidermal Differentiation through the Epigenomic Writer CBP.

SummaryImpairments in the differentiation process can lead to skin diseases that can afflict ∼20% of the population. Thus, it is of utmost importance to understand the factors that promote the differentiation process. Here we identify the transcription factor KLF3 as a regulator of epidermal differentiation. Knockdown of KLF3 results in reduced differentiation gene expression and increased cell cycle gene expression. Over half of KLF3's genomic binding sites occur at active enhancers. KLF3 binds to active enhancers proximal to differentiation genes that are dependent upon KLF3 for expression. KLF3's genomic binding sites also highly overlaps with CBP, a histone acetyltransferase necessary for activating enhancers. Depletion of KLF3 causes reduced CBP localization at enhancers proximal to differentiation gene clusters, which leads to loss of enhancer activation but not priming. Our results suggest that KLF3 is necessary to recruit CBP to activate enhancers and drive epidermal differentiation gene expression.