Early Differentiation Genes Research Articles

Temporal Gene Expression and DNA Methylation during Embryonic Stem Cell Derivation.

Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. ConclusionOur investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.

Dual Role of Act1 in Keratinocyte Differentiation and Host Defense: TRAF3IP2 Silencing Alters Keratinocyte Differentiation and Inhibits IL-17 Responses

TRAF3IP2 is a candidate psoriasis susceptibility gene encoding Act1, an adaptor protein with ubiquitin ligase activity that couples the IL-17 receptor to downstream signaling pathways. We investigated the role of Act1 in keratinocyte responses to IL-17 using a tetracycline inducible short hairpin RNA targeting TRAF3IP2. Tetracycline exposure for 7 days effectively silenced TRAF3IP2 mRNA and Act1 protein, resulting in 761 genes with significant changes in expression (495 down, 266 up; >1.5-fold, P < 0.05). Gene ontology analysis showed that genes affected by TRAF3IP2 silencing are involved in epidermal differentiation, with early differentiation genes (KRT1, KRT10, DSC1, DSG1) being down-regulated and late differentiation genes (SPRR2, SPRR3, LCE3) being up-regulated. AP1 binding sites were enriched upstream of genes up-regulated by TRAF3IP2 silencing. Correspondingly, nuclear expression of FosB and Fra1 was increased in TRAF3IP2-silenced cells. Many genes involved in host defense were induced by IL-17 in a TRAF3IP2-dependent fashion. Inflammatory differentiation conditions (serum addition for 4 days postconfluence) markedly amplified these IL-17 responses and increased basal levels and TRAF3IP2 silencing-dependent up-regulation of multiple late differentiation genes. These findings suggest that TRAF3IP2 may alter both epidermal homeostasis and keratinocyte defense responses to influence psoriasis risk.

Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes.

The mammalian cochlea is a highly specialized organ within the inner ear. Sensory hair cells (HC) in the cochlea detect and transduce sound waves into electrical impulses that are sent to the brain. Studies of the molecular pathways regulating HC formation are hindered by the very sparse nature of HCs, where only ~3300 are found within an entire mouse cochlea. Current cell lines mimic certain aspects of HCs but lack terminal HC marker expression. Here we successfully “pseudo-immortalized” cochlear progenitor cells using the “conditional reprogramming” technique. These cells, termed “Conditionally Reprogrammed Otic Stem Cells” (CR-OSC), are able to bypass the senescence inherent to cochlear progenitor cells without genetic alterations, allowing for the generation of over 15 million cells from a single cochlea. These cells can be differentiated and up-regulate both early and terminal differentiation genes associated with HCs, including the terminal HC differentiation marker prestin. CR-OSCs also respond to known HC cues, including upregulation of HC genes in response to Atoh1 overexpression, and upregulation of prestin expression after thyroid hormone application. Overall, we describe the creation of a HC line capable of regulated expression of HC genes that can easily be recreated in any laboratory from any mouse of interest.

EphB4/ephrinB2 Interaction Mediate Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Spreading and Osteogenic Differentiation

Background and Objective: Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment.Recent studies have indicated interaction between acute leukemia cells and MSCs has a major role in cancer progression and resistance to treatment.Our previous study found that EphB4 receptor was over expressed in CML-Blast Crisis (BC) patients and resistant cell lines. Furthermore, we performed the experiment to prove that aberrant over expressed of EphB4 play an important role to change characterize of Imatinib-resistant in chronic myeloid leukemia cells. However,the contribution of over expressed of EphB4 molecules in leukemia cells to change MSCs function remains to be determined. Therefore,we hypothesis that the change of EphB4/ephrinB2 molecules on leukemia cells might play an important role to transform characterize of MSCs through direct contact, which finally support to leukemia progression and disruption of normal hematopoiesis in microenvironment of the bone marrow.Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or patients' BM and cultures in CyagenBone marrow culture medium at 37 °C, 5% CO2 incubator. EphrinB2(2.628±0.2303 n=3; P<0.05), ALP\\RUNX2 (early osteogenesis differentiation genes)(6.430±0.1343, n=3P<0.001; 4.948±0.1418,n=3P<0.001)were over expressed in MSCs (CML patients)in contrast to normal human MSC by QRT-PCR. After osteogenic induced for 2 weeks,MSCs from CML-initial patient showed significantly higher osteogenic differentiation (Osteogenesis Score 4.5P<0.01) and protein (later period osteogenesis differentiation)(2.1669%±0.1443, n=3P<0.001)was overexpressed in MSCs (CML patients) in contrast to normal human MSC(0.2993%±0.1612n=3) by western blot. In functional spreading assay, cultured MSC (CML patient) exposed to EphB4-Fc (5 μg/mL) were significantly rounder (p<0.001) and smaller (p<0.001) as demonstrated with F-actin staining, compared to control and human-Fc.No difference inmorphology was observed when MSC were cultured in the presence of ephrin-B2-Fc.Incubation of MSC with signaling pathway-specific inhibitors before the spreading assay, the PI3Kinase pathway (LY294002) (p<0.001),not the Src kinase pathway (PP2), inhibited MSCs (CML) attachment and spreading. The results revealed that the PI3Kinase pathway was activated upon ephrinB2 reverse signaling in response to EphB4-Fc to promote the contraction and rounding up of MSC. Activation of ephrin-B2 molecules expressed by MSCs (CML)by EphB4-Fc(5 μg/mL), human-Fc(5 μg/mL) or blank control, which cultured in osteogenic media for 2 weeks, more mineral in the presence of EphB4-Fc was visualized by Alizarin Red staining compared to the control human-Fc.Furthermore, after co-cultured respectively with K562-R or K562-R-EPHB4-SH for 72h,ALP\\RUNX2 in MSCs (CML) were increased significantly in K562-R group(15.544±2.647; 6.378±2.0775 n=3)compared to K562-R-EPHB4-SH group (6.014±1.19273; 4.045±2.0273 n=3) and control group by QRT-PCR. However, ALP\\RUNX2 in normal MSC was unaffected when respectively co-cultured with K562-R(0.741±2.2121; 0.129±0.9194 n=3) or K562-R-EPHB4-SH(0.171±2.062; 0.232±2.0474 n=3) for 72h. The co-cultured assays were also performed using transwells. ALP\\RUNX2 in MSCs (CML) were no difference in K562-R group (6.28±1.7875; 4.232±2.4533 n=3) compared to K562-R-EPHB4-SH group (6.107±2.158; 4.139±1.9727 n=3).Conclusion: Our study illustrated that the change of EphB4/ephrinB2 molecules on leukemia cells may transform MSCs functional spreading and osteogenic differentiation through direct contact involved in PI3Kinase pathway. DisclosuresNo relevant conflicts of interest to declare.

Long-distance effects of inflammation on differentiation of adult spinal cord neural stem/progenitor cells

Studies in multiple sclerosis have demonstrated that normal-appearing white matter can harbor pathological changes. Here we investigated the effects of neuroinflammation, modeled by experimental autoimmune encephalomyelitis (EAE) on neural stem/progenitor cells (NPCs) located distally to inflammatory foci. We observed that EAE-derived NPCs had a lower capacity to differentiate into oligodendrocytes and an increased neuronal differentiation than control NPCs. This finding was corroborated with changes in gene expression of early differentiation genes. We conclude that inflammation has a long range effect on the NPCs in the diseased central nervous system, reaching NPC populations outside the lesion sites.

Transcriptomic Profiling and H3K27me3 Distribution Reveal Both Demethylase-Dependent and Independent Regulation of Developmental Gene Transcription in Cell Differentiation.

The removal of histone H3 trimethylation at lysine residue 27 (H3K27me3) plays a critical role in the transcriptional initiation of developmental genes. The H3K27me3-specific KDM6 demethylases JMJD3 and UTX are responsible for the transcriptional initiation of various developmental genes, but some genes are expressed in a KDM6 demethylase-independent manner. To address the role of H3K27me3 in the retinoic acid (RA)-induced differentiation of the human carcinoma NCCIT cell line, we inhibited JMJD3 and UTX using the H3K27me3 demethylase inhibitor GSK-J4. The commitment of JMJD3/UTX-inhibited cells to a specific fate was delayed, and transcriptome profiling also revealed the differential expression of genes related to cell fate specification in demethylase-inactivated cells; the expression levels of RA metabolism and HOX family genes significantly decreased. We observed a weak correlation between H3K27me3 enrichment and transcriptional repression in the control and JMJD/UTX-inhibited cells, except for a few sets of developmental genes that are indispensable for cell fate specification. Taken together, these results provide the H3K27me3 landscape of a differentiating cell line and suggest that both demethylase-dependent and demethylase-independent transcriptional regulation play a role in early differentiation and developmental gene expression activated by H3K27me3 demethylation.

Early differential gene expression profiling of harvest-induced senescence in detached Arabidopsis plants

After a series of stresses, detached plant organs such as leafy vegetables and cut flowers begin to appear declining in quality and then finally senescence. Comprehending, plants’ response to multiple stresses may result in new opportunities to extend the shelf life of postharvest. We investigated physiological responses of Arabidopsis plants after harvest and analyzed global gene transcription in dark-stored detached Arabidopsis plants leaves (DSD). Detached darkened plants of Arabidopsis were stored for 12 h in airtight boxes. Multiple stresses caused a distinguished decrease in chlorophyll, protein content and premature senescence of leaves. The microarray analysis revealed that 852 transcripts were upregulated and 1004 transcripts were downregulated, respectively, more than twofold. A gene ontology test and biological process analysis suggested that activated genes were mostly associated with regulation of transcription, secondary metabolism, response to water deprivation, signal transduction, and other stress responses. Meanwhile, genes that were downregulated were involved in protein biosynthesis, protein folding, lipid catabolism, ribosome biogenesis and assembly, ATP binding, and photosynthesis. Gene expression analysis data suggested that the leaves of detached Arabidopsis plants responded to integrated stresses by regulating diverse gene expression in leaves.

Adaptation of a Bioinformatics Microarray Analysis Workflow for a Toxicogenomic Study in Rainbow Trout.

Sex steroids play a key role in triggering sex differentiation in fish, the use of exogenous hormone treatment leading to partial or complete sex reversal. This phenomenon has attracted attention since the discovery that even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find out mechanisms of actions (MOA) and identify biomarkers related to the toxic action of a compound. However, high throughput data interpretation relies on statistical analysis, species genomic resources, and bioinformatics tools. The goals of this study are to improve the knowledge of feminisation in fish, by the analysis of molecular responses in the gonads of rainbow trout fry after chronic exposure to several doses (0.01, 0.1, 1 and 10 μg/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. We successfully adapted a bioinformatics microarray analysis workflow elaborated on human data to a toxicogenomic study using rainbow trout, a fish species lacking accurate functional annotation and genomic resources. The workflow allowed to obtain lists of genes supposed to be enriched in true positive differentially expressed genes (DEGs), which were subjected to over-representation analysis methods (ORA). Several pathways and ontologies, mostly related to cell division and metabolism, sexual reproduction and steroid production, were found significantly enriched in our analyses. Moreover, two sets of potential ovotestis biomarkers were selected using several criteria. The first group displayed specific potential biomarkers belonging to pathways/ontologies highlighted in the experiment. Among them, the early ovarian differentiation gene foxl2a was overexpressed. The second group, which was highly sensitive but not specific, included the DEGs presenting the highest fold change and lowest p-value of the statistical workflow output. The methodology can be generalized to other (non-model) species and various types of microarray platforms.

Dynamic Networking for Epidermal Differentiation

In this issue of Developmental Cell, Lopez-Pajares et al. (2015) identify regulatory networks that are controlled by the transcriptional regulators MAF:MAFB. These networks play a crucial and previously unrecognized role in epidermal differentiation, functioning both to repress progenitor genes and to activate early and late differentiation genes.

Structural and biochemical changes underlying a keratoderma-like phenotype in mice lacking suprabasal AP1 transcription factor function.

Epidermal keratinocyte differentiation on the body surface is a carefully choreographed process that leads to assembly of a barrier that is essential for life. Perturbation of keratinocyte differentiation leads to disease. Activator protein 1 (AP1) transcription factors are key controllers of this process. We have shown that inhibiting AP1 transcription factor activity in the suprabasal murine epidermis, by expression of dominant-negative c-jun (TAM67), produces a phenotype type that resembles human keratoderma. However, little is understood regarding the structural and molecular changes that drive this phenotype. In the present study we show that TAM67-positive epidermis displays altered cornified envelope, filaggrin-type keratohyalin granule, keratin filament, desmosome formation and lamellar body secretion leading to reduced barrier integrity. To understand the molecular changes underlying this process, we performed proteomic and RNA array analysis. Proteomic study of the corneocyte cross-linked proteome reveals a reduction in incorporation of cutaneous keratins, filaggrin, filaggrin2, late cornified envelope precursor proteins, hair keratins and hair keratin-associated proteins. This is coupled with increased incorporation of desmosome linker, small proline-rich, S100, transglutaminase and inflammation-associated proteins. Incorporation of most cutaneous keratins (Krt1, Krt5 and Krt10) is reduced, but incorporation of hyperproliferation-associated epidermal keratins (Krt6a, Krt6b and Krt16) is increased. RNA array analysis reveals reduced expression of mRNA encoding differentiation-associated cutaneous keratins, hair keratins and associated proteins, late cornified envelope precursors and filaggrin-related proteins; and increased expression of mRNA encoding small proline-rich proteins, protease inhibitors (serpins), S100 proteins, defensins and hyperproliferation-associated keratins. These findings suggest that AP1 factor inactivation in the suprabasal epidermal layers reduces expression of AP1 factor-responsive genes expressed in late differentiation and is associated with a compensatory increase in expression of early differentiation genes.

Transdifferentiation of human periodontal ligament stem cells into pancreatic cell lineage.

Human periodontal ligament-derived stem cells (PDLSCs) demonstrate self-renewal capacity and multilineage differentiation potential. In this study, we investigated the transdifferentiation potential of human PDLSCs into pancreatic islet cells. To form three-dimensional (3D) clusters, PDLSCs were cultured in Matrigel with media containing differentiation-inducing agents. We found that after 6 days in culture, PDLSCs underwent morphological changes resembling pancreatic islet-like cell clusters (ICCs). The morphological characteristics of PDLSC-derived ICCs were further assessed using scanning electron microscopy analysis. Using reverse transcription-polymerase chain reaction analysis, we found that pluripotency genes were downregulated, whereas early endoderm and pancreatic differentiation genes were upregulated, in PDLSC-derived ICCs compared with undifferentiated PDLSCs. Furthermore, we found that PDLSC-derived ICCs were capable of secreting insulin in response to high concentrations of glucose, validating their functional differentiation into islet cells. Finally, we also performed dithizone staining, as well as immunofluorescence assays and fluorescence-activated cell sorting analysis for pancreatic differentiation markers, to confirm the differentiation status of PDLSC-derived ICCs. These results demonstrate that PDLSCs can transdifferentiate into functional pancreatic islet-like cells and provide a novel, alternative cell population for pancreatic repair.

"Father knows best?".

Following fertilization, activation of a complex developmental program requires the differential expression of key genes. In most metazoa, the prevailing view is that early differential gene expression occurs primarily through post-transcriptional regulation of maternally deposited products in the oocyte. Two novel studies published from the Rajewsky laboratory in this issue of The EMBO Journal add significantly to the emerging notion that this process may be more complex than previously appreciated.

Identification of differentiation-stage specific markers that define the ex vivo osteoblastic phenotype

The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity. For the clinical use of human skeletal (stromal, mesenchymal) stem cells (hMSC) in therapy, there is also a need to identify a set of gene markers that predict in vivo bone forming capacity. Thus, we used RNA sequencing to examine changes in expression for a set of skeletally-related genes across 8 time points between 0 and 12days of ex vivo OB differentiation of hMSC. We identified 123 genes showing significant temporal expression change. Hierarchical clustering and Pearson's correlation generated 4 groups of genes: early stage differentiation genes (peak expression: 0–24h, n=28) which were enriched for extracellular matrix organisation, e.g. COL1A1, LOX, and SERPINH1; middle stage differentiating genes (peak expression days: 3 and 6, n=20) which were enriched for extracellular matrix/skeletal system development e.g. BMP4, CYP24A1, and TGFBR2; and late stage differentiation genes (peak expression days: 9 and 12, n=27) which were enriched for bone development/osteoblast differentiation, e.g. BMP2 and IGF2. In addition, we identified 13 genes with bimodal temporal expression (2 peaks of expression: days 0 and 12) including VEGFA, PDGFA and FGF2. We examined the specificity of the 123 genes' expression in skeletal tissues and thus propose a set of ex vivo differentiation-stage-specific markers (n=21). In an independent analysis, we identified a subset of genes (n=20, e.g. ELN, COL11A1, BMP4) to predict the bone forming capacity of hMSC and another set (n=20, e.g. IGF2, TGFB2, SMAD3) associated with the ex vivo phenotype of hMSC obtained from osteoporotic patients.

Effect of nano- and micro-scale topological features on alignment of muscle cells and commitment of myogenic differentiation

Skeletal muscle injury can lead to severe motor deficits that adversely affect movement and quality of life. Current surgical treatments for skeletal muscle are hindered by the poor formation of organized myotube bundles at the wound site. Tissue-engineered skeletal muscle constructs to date have been unable to generate high degrees of myotube density and alignment. Generating a suitable in vitro tissue-engineered skeletal muscle construct requires the design of a scaffold that recapitulates the structural combination of nanoscale collagen fibrils and aligned microscale basal lamina tracks present in the native extracellular matrix (ECM). We hypothesized that a 3D aligned tubular porous scaffold containing aligned nanofibers inside the pores can mimic the native muscle tissue environment. We constructed a laminar section of the hypothesized scaffold with aligned chitosan-PCL nanofibers arranged co-axially with the aligned microscale chitosan scaffold bands to mimic the required myogenic environment. A 6-day study of C2C12 mouse myoblast cells cultured on this hybrid scaffold indicated that the nanofibers and scaffold bands in the scaffold played a synergetic role in directing cell orientation, interaction, migration and organization. Our results showed that aligned nanofibers mediated cell alignment and the aligned scaffold bands induced the formation of a more compact assembly of myotube cells as compared to various control substrates including chitosan films, nanofibers, and chitosan bands. The expression levels of both early and late-stage myogenic differentiation genes associated with myogenin and myosin heavy chain, respectively, were higher on the hybrid substrate than on control substrates. Our study suggests that the combination of nano and microscale topological features in the ECM can direct myogenic differentiation, and the hybrid material has the potential to improve the outcome of skeletal tissue engineering.

PPARβ Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation

Epigenetic post-transcriptional modifications of histone tails are thought to help in coordinating gene expression during development. An epigenetic signature is set in pluripotent cells and interpreted later at the onset of differentiation. In pluripotent cells, epigenetic marks normally associated with active genes (H3K4me3) and with silent genes (H3K27me3) atypically co-occupy chromatin regions surrounding the promoters of important developmental genes. However, it is unclear how these epigenetic marks are recognized when cell differentiation starts and what precise role they play. Here, we report the essential role of the nuclear receptor peroxisome proliferator-activated receptor β (PPARβ, NR1C2) in Xenopus laevis early development. By combining loss-of-function approaches, large throughput transcript expression analysis by the mean of RNA-seq and intensive chromatin immunoprecipitation experiments, we unveil an important cooperation between epigenetic marks and PPARβ. During Xenopus laevis gastrulation PPARβ recognizes H3K27me3 marks that have been deposited earlier at the pluripotent stage to activate early differentiation genes. Thus, PPARβis the first identified transcription factor that interprets an epigenetic signature of pluripotency, in vivo, during embryonic development. This work paves the way for a better mechanistic understanding of how the activation of hundreds of genes is coordinated during early development.

Risedronate Positively Affects Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Bisphosphonates are widely used for the treatment of bone pathologies, mainly due to their ability to inhibit osteoclastic activity and thus bone resorption. Yet, their potential effect on bone formation is unclear. Our aim was to determine whether risedronate can affect osteoblastic differentiation of mesenchymal stromal cells (MSC). We evaluated the effect of the risedronate, which is a bisphosphonate widely used in clinical settings, on the differentiation of precursor cells of osteoblasts and adipocytes. Thus, MSC from human bone marrow were induced to differentiate into osteoblasts or adipocytes in the presence or absence of two risedronate concentrations (10⁻⁸ M and 10⁻⁹ M). Risedronate increased the levels of osteogenic markers including the extracellular matrix mineralization on cells induced to osteoblasts. On the other hand, such bisphosphonates did not have significant effects on the pparγ2 and lpl adipogenic genes or the fat vesicle accumulation on the cells induced into adipocytes. In addition, it increased the expression of early osteogenic differentiation genes like runx2 on MSC not induced to differentiate. Risedronate concentrations used favor the osteogenic differentiation and the MSC commitment towards osteoblasts. This suggests the possibility of using this bisphosphonate in cyclic bone treatments with anabolic drugs, enhancing the effect of the latter via a previous MSC commitment to preosteoblasts.

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets

Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.

Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni.

The outcome of infection in the host snail Biomphalaria glabrata with the digenean parasite Schistosoma mansoni is determined by the initial molecular interplay occurring between them. The mechanisms by which schistosomes evade snail immune recognition to ensure survival are not fully understood, but one possibility is that the snail internal defence system is manipulated by the schistosome enabling the parasite to establish infection. This study provides novel insights into the nature of schistosome resistance and susceptibility in B. glabrata at the transcriptomic level by simultaneously comparing gene expression in haemocytes from parasite-exposed and control groups of both schistosome-resistant and schistosome-susceptible strains, 2 h post exposure to S. mansoni miracidia, using an novel 5K cDNA microarray. Differences in gene expression, including those for immune/stress response, signal transduction and matrix/adhesion genes were identified between the two snail strains and tests for asymmetric distributions of gene function also identified immune-related gene expression in resistant snails, but not in susceptible. Gene set enrichment analysis revealed that genes involved in mitochondrial electron transport, ubiquinone biosynthesis and electron carrier activity were consistently up-regulated in resistant snails but down-regulated in susceptible. This supports the hypothesis that schistosome-resistant snails recognize schistosomes and mount an appropriate defence response, while in schistosome-susceptible snails the parasite suppresses this defence response, early in infection.

Analysis of osteoblastic gene expression in the early human mesenchymal cell response to a chemically modified implant surface: an in vitro study

The effect of a chemical modification of the SLA surface (SLActive surface) on human bone marrow-derived mesenchymal cells (hMSCs) on; (1) adhesion, (2) proliferation and (3) early transcriptional control of osteogenic differentiation was investigated. We are based on the hypothesis that expression patterns of genes responsible for osteogenesis might be dependent on the characteristics of the implant surface. hMSCs were allowed to grow on smooth (SMO-control), SLA and SLActive implant surfaces (chemically modified). Cell attachment and proliferation were assessed at 3 and 24 h using a MTT dye reduction assay. At 24 h of culture, DNA microarray analysis examined alterations in early gene expression using a human osteogenesis gene array, including 109 cDNAs in quadruplicates of major regulatory genes for osteogenesis. Initial attachment and proliferation were found to be significantly reduced. Nineteen genes were significantly upregulated when hMSCs were cultured on the SLA surfaces and 27 genes were significantly upregulated when hMSCs were cultured on the SLActive surfaces. Upregulated genes control cell differentiation, signal transduction, cell cycle regulation, angiogenesis, cell adhesion and extracellular matrix and bone formation. Chemical modification decreases further cell attachment and proliferation and upregulates early osteoblastic differentiation genes. Hence, a microenvironment is created around chemically modified implants that may enhance osseointegration.

Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation

The differentiation, maintenance, and repair of skeletal muscle is controlled by interactions between genetically determined transcriptional programs regulated by myogenic transcription factors and environmental cues activated by growth factors and hormones. Signaling through the insulin-like growth factor 1 (IGF1) receptor by locally produced IGF2 defines one such pathway that is critical for normal muscle growth and for regeneration after injury. IGF2 gene and protein expression are induced as early events in muscle differentiation, but the responsible molecular mechanisms are unknown. Here we characterize a distal DNA element within the imprinted mouse Igf2-H19 locus with properties of a muscle transcriptional enhancer. We find that this region undergoes a transition to open chromatin during differentiation, whereas adjacent chromatin remains closed, and that it interacts in differentiating muscle nuclei but not in mesenchymal precursor cells with the Igf2 gene found more than 100 kb away, suggesting that chromatin looping or sliding to bring the enhancer in proximity to Igf2 promoters is also an early event in muscle differentiation. Because this element directly stimulates the transcriptional activity of an Igf2 promoter-reporter gene in differentiating myoblasts, our results indicate that we have identified a bona fide distal transcriptional enhancer that supports Igf2 gene activation in skeletal muscle cells. Because this DNA element is conserved in the human IGF2-H19 locus, our results further suggest that its muscle enhancer function also is conserved among different mammalian species.