Gene Expression In Stem Cells Research Articles

JAK2/IDH-mutant-driven myeloproliferative neoplasm is sensitive to combined targeted inhibition.

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype.

Effect of MTA and CEM on Mineralization-Associated Gene Expression in Stem Cells Derived from Apical Papilla.

Introduction:This study assessed the effect of mineral trioxide aggregate (MTA) and calcium-enriched mixture (CEM) cement on odontogenic differentiation and mineralization of stem cells. Methods and Materials:After confirmation of stemness and homogeneity of stem cells derived from apical papilla (SCAPs) using flow cytometry, the cells were exposed for 3 weeks to either osteogenic medium (OS) or CEM extract+OS (CEM+OS) or MTA extract in OS (MTA+OS) or DMEM based regular culture media (negative control). Relative expression of alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP), osteocalcin (OSC), and osterix (SP7) were measured at days 14 and 21 using RT-qPCR method. At the same time points Alizarin Red staining method was used to assess mineralization potential of SCAPS. Gene expression changes analysis were made automatically using REST® software and a P<0.05 was considered significant. Results: After 2 weeks of exposure, expression of all genes were between 3 and 52 times the expression of GADPH (all were upregulated except SP7 in the control, P<0.05). After 3 weeks, relative expressions of the genes: ALP, SP7, DSPP, and OSC were respectively 275.9, 528.3, 98.4, and 603.7 times the expression of GADPH in the control group (OS). These were respectively 17.405, 29.2, 11.8, and 6.5 in CEM+OS group, and 163.8, 119.7, 102.5, and 723.9 in MTA+OS group. All of these were confirmed as upregulated (P<0.05) except for ALP and OSC of DM+CEM group. After 2 weeks, alizarin red staining showed similar mineralized nodules in OS, MTA+OS, and CEM+OS. In third week, larger nodules were seen in MTA+OS and OS, but not in CEM+OS. Conclusion:After 2 weeks, gene expressions were almost comparable in OS, CEM+OS, and MTA+OS. After 3 weeks, OS and MTA+OS upregulated genes much greater than in 2nd week. However, upregulation in CEM+OS might not increase in 3rd week compared to those in 2nd week.

Statistical analysis of multi-dimensional, temporal gene expression of stem cells to elucidate colony size-dependent neural differentiation.

High throughput gene expression analysis using qPCR is commonly used to identify molecular markers of complex cellular processes. However, statistical analysis of multi-dimensional, temporal gene expression data is complicated by limited biological replicates and large number of measurements. Moreover, many available statistical tools for analysis of time series data assume that the data sequence is static and does not evolve over time. With this assumption, the parameters used to model the time series are fixed and thus, can be estimated by pooling data together. However, in many cases, dynamic processes of biological systems involve abrupt changes at unknown time points, making the assumption of stationary time series break down. We addressed this problem using a combination of statistical methods including hierarchical clustering, change point detection, and multiple testing. We applied this multi-step method to multi-dimensional, temporal gene expression data that resulted from our study of colony size-dependent neural cell differentiation of stem cells. The gene expression data were time series as the observations were recorded sequentially over time. Hierarchical clustering segregated the genes into three distinct clusters based on their temporal expression profiles; change point detection identified specific time points at which the entire dataset was divided into several homogenous subsets to allow a separate analysis of each subset; and multiple testing procedure identified the differentially expressed genes in each cluster within each subset of data. We established that our multi-step approach pinpoints specific sets of genes that underlie colony size-mediated neural differentiation of stem cells and demonstrated its advantages over conventional parametric and non-parametric tests that do not take into account temporal dynamics of the data. Importantly, our proposed approach is broadly applicable to any multivariate data sets of limited sample size from high throughput and high content screening such as in drug and biomarker discovery studies.

Zinc increases nestin but not vimentin gene expression in mouse mesenchymal stem cells

Zinc increases nestin but not vimentin gene expression in mouse mesenchymal stem cells

Recombinant human BMP-7 grafted poly(lactide-co-glycolide)/hydroxyapatite scaffolds via polydopamine for enhanced calvarial repair.

Poly(lactic-co-glycolic acid) (PLGA) and hydroxyapatite (HA) are considered potential osteoinductive materials because of their biodegradability and mineralization features. However, the hydrophobicity of scaffold surfaces is less supportive of cell attachment and proliferation because of poor wettability. The mode of binding of growth factors to the scaffold also affects cell differentiation into osteoblasts. The half-life of a growth factor in vivo can be increased by binding the factor to the scaffold surface. In this work, we prepared a porous PLGA/HA scaffold grafted with recombinant human bone morphogenic protein-7 (rhBMP-7) attached via polydopamine (pDA) for bone repair. The pDA coated PLGA/HA (pDA-PLGA/HA) scaffolds were characterized by energy dispersive X-ray analysis and Fourier-transform infrared spectroscopy. The microstructure and porosity of PLGA/HA scaffolds were analyzed by scanning electron microscopy and micro-computed tomography. The release profile of rhBMP-7 grafted onto the pDA-PLGA/HA (pDA-PLGA/HA/BMP-7) scaffolds was examined for 21 days. The attachment efficiency, cell proliferation rate, alkaline phosphatase activity, calcium deposition, and osteoblast-related gene expression of bone marrow-derived stem cells to PLGA/HA, pDA-PLGA/HA, and pDA-PLGA/HA/BMP-7 scaffolds were evaluated. To assess the ability of bone repair in vivo, scaffolds were implanted into critical-sized calvarial defects created in mice, and the in vivo tissue-engineered bone was monitored by micro-computed tomography and histology. In vivo experiments revealed rapid healing of the defects treated with the pDA-PLGA/HA/BMP-7 scaffolds compared with pDA-PLGA/HA and PLGA/HA scaffolds at week 8 post-surgery. These results collectively demonstrate that the rhBMP-7-immobilized PLGA/HA scaffold via pDA is a promising candidate for calvarial repair.

Combination of network pathway and epithelial-mesenchymal transition-related gene expression in gastric cancer and mesenchymal stem cells

Combination of network pathway and epithelial-mesenchymal transition-related gene expression in gastric cancer and mesenchymal stem cells

Intrinsic Immunity Shapes Viral Resistance of Stem Cells

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the invivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.

Tropism of liver epithelial cells toward hepatocellular carcinoma in vitro and in vivo with altering gene expression of cancer stem cells

BackgroundRat liver epithelial (RLE) cells could inhibit the proliferation and invasiveness of hepatoma cells in vitro. This study is to understand the tropism and the effect of RLE cells on mouse hepatoma cells both in vitro and in vivo. MethodsRLE cells were isolated from new-born rats and characterized their stem cell markers. Co-culture and HCC mouse model was established to detect therapeutic effect of RLE cells. ResultsRLE cells (including Thy-1+ RLE cells, Thy-1- RLE cells, RLE cells) displayed a selective tropism toward ML-1 hepatoma cells both in vitro and in vivo. They altered the gene expression of some cancer stem cell markers in the liver tumor. ConclusionLiver epithelial cells have a selective tropism toward HCC in vitro and in vivo. They could alter the gene expression of cancer stem cells.

Altered gene expression and metabolism in fetal umbilical cord mesenchymal stem cells correspond with differences in 5-month-old infant adiposity gain

The intrauterine period is a critical time wherein developmental exposure can influence risk for chronic disease including childhood obesity. Using umbilical cord-derived mesenchymal stem cells (uMSC) from offspring born to normal-weight and obese mothers, we tested the hypothesis that changes in infant body composition over the first 5 months of life correspond with differences in cellular metabolism and transcriptomic profiles at birth. Higher long-chain acylcarnitine concentrations, lipid transport gene expression, and indicators of oxidative stress in uMSC-adipocytes were related to higher adiposity at 5 months of age. In uMSC-myocytes, lower amino acid concentrations and global differential gene expression for myocyte growth, amino acid biosynthesis, and oxidative stress were related to lower infant percent fat-free mass at 5 months of age, particularly in offspring of obese mothers. This is the first evidence of human infant adipocyte- or myocyte-related alterations in cellular metabolic pathways that correspond with increased adiposity and lower fat-free mass in early infancy. These pathways might reflect the effects of an adverse maternal metabolic environment on the fetal metabolome and genome. Our findings suggest that programmed differences in infant stem cell metabolism correspond with differences in body composition in early life, a known contributor to obesity risk.

Brain plasticity, cognitive functions and neural stem cells: a pivotal role for the brain-specific neural master gene |-SRGAP2-FAM72-|.

Due to an aging society with an increased dementia-induced threat to higher cognitive functions, it has become imperative to understand the molecular and cellular events controlling the memory and learning processes in the brain. Here, we suggest that the novel master gene pair |-SRGAP2-FAM72-| (SLIT-ROBO Rho GTPase activating the protein 2, family with sequence similarity to 72) reveals a new dogma for the regulation of neural stem cell (NSC) gene expression and is a distinctive player in the control of human brain plasticity. Insight into the specific regulation of the brain-specific neural master gene |-SRGAP2-FAM72-| may essentially contribute to novel therapeutic approaches to restore or improve higher cognitive functions.

A Novel Notch–YAP Circuit Drives Stemness and Tumorigenesis in Embryonal Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), a cancer characterized by skeletal muscle features, is the most common soft-tissue sarcoma of childhood. While low- and intermediate-risk groups have seen improved outcomes, high-risk patients still face a 5-year survival rate of <30%, a statistic that has not changed in over 40 years. Understanding the biologic underpinnings of RMS is critical. The developmental pathways of Notch and YAP have been identified as potent but independent oncogenic signals that support the embryonal variant of RMS (eRMS). Here, the cross-talk between these pathways and the impact on eRMS tumorigenesis is reported. Using human eRMS cells grown as three-dimensional (3D) rhabdospheres, which enriches in stem cells, it was found that Notch signaling transcriptionally upregulates YAP1 gene expression and YAP activity. Reciprocally, YAP transcriptionally upregulates the Notch ligand genes JAG1 and DLL1 and the core Notch transcription factor RBPJ This bidirectional circuit boosts expression of key stem cell genes, including SOX2, which is functionally required for eRMS spheres. Silencing this circuit for therapeutic purposes may be challenging, because the inhibition of one node (e.g., pharmacologic Notch blockade) can be rescued by upregulation of another (constitutive YAP expression). Instead, dual inhibition of Notch and YAP is necessary. Finally, supporting the existence of this circuit beyond a model system, nuclear Notch and YAP protein expression are correlated in human eRMS tumors, and YAP suppression in vivo decreases Notch signaling and SOX2 expression.Implications: This study identifies a novel oncogenic signaling circuit driving eRMS stemness and tumorigenesis, and provides evidence and rationale for combination therapies co-targeting Notch and YAP. Mol Cancer Res; 15(12); 1777-91. ©2017 AACR.

JAM-C Identifies Src Family Kinase-Activated Leukemia-Initiating Cells and Predicts Poor Prognosis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells that acquire somatic mutations, leading to disease and clonogenic evolution. AML is characterized by accumulation of immature myeloid cells in the bone marrow and phenotypic cellular heterogeneity reflective of normal hematopoietic differentiation. Here, we show that JAM-C expression defines a subset of leukemic cells endowed with leukemia-initiating cell activity (LIC). Stratification of de novo AML patients at diagnosis based on JAM-C-expressing cells frequencies in the blood served as an independent prognostic marker for disease outcome. Using publicly available leukemic stem cell (LSC) gene expression profiles and gene expression data generated from JAM-C-expressing leukemic cells, we defined a single cell core gene expression signature correlated to JAM-C expression that reveals LSC heterogeneity. Finally, we demonstrated that JAM-C controls Src family kinase (SFK) activation in LSC and that LIC with exacerbated SFK activation was uniquely found within the JAM-C-expressing LSC compartment. Cancer Res; 77(23); 6627-40. ©2017 AACR.

In vitro characterization of CD133lo cancer stem cells in Retinoblastoma Y79 cell line

BackgroundRetinoblastoma (Rb), the most common childhood intraocular malignant tumor, is reported to have cancer stem cells (CSCs) similar to other tumors. Our previous investigation in primary tumors identified the small sized cells with low CD133 (Prominin-1) and high CD44 (Hyaluronic acid receptor) expression to be putative Rb CSCs using flow cytometry (FSClo/SSClo/CD133lo/CD44hi). With this preliminary data, we have now utilized a comprehensive approach of in vitro characterization of Y79 Rb cell line following CSC enrichment using CD133 surface marker and subsequent validation to confirm the functional properties of CSCs.MethodsThe cultured Rb Y79 cells were evaluated for surface markers by flow cytometry and CD133 sorted cells (CD133lo/CD133hi) were compared for CSC characteristics by size/percentage, cell cycle assay, colony formation assay, differentiation, Matrigel transwell invasion assay, cytotoxicity assay, gene expression using microarray and validation by semi-quantitative PCR.ResultsRb Y79 cell line shared the profile (CD133, CD90, CXCR4 and ABCB1) of primary tumors except for CD44 expression. The CD133lo cells (16.1 ± 0.2%) were FSClo/SSClo, predominantly within the G0/G1 phase, formed larger and higher number of colonies with ability to differentiate to CD133hi cells, exhibited increased invasive potential in a matrigel transwell assay (p < 0.05) and were resistant to Carboplatin treatment (p < 0.001) as compared to CD133hi cells. The CD133lo cells showed higher expression of several embryonic stem cell genes (HOXB2, HOXA9, SALL1, NANOG, OCT4, LEFTY), stem cells/progenitor genes (MSI2, BMI1, PROX1, ABCB1, ABCB5, ABCG2), and metastasis related gene- MACC1, when compared to the CD133hi cells.ConclusionsThis study validates the observation from our earlier primary tumor study that CSC properties in Rb Y79 cell line are endowed within the CD133lo population, evident by their characteristics- i.e. small sized, dormant in nature, increased colony forming ability, differentiation to CD133hi cells, higher invasiveness potential, drug resistance and primitive gene expression pattern. These findings provide a proof of concept for methodological characterization of the retinoblastoma CSCs with future implications for improved diagnostic and treatment strategies.

Reduction of pluripotent gene expression in murine embryonic stem cells exposed to mechanical loading or Cyclo RGD peptide

BackgroundSelf-renewal and differentiation of embryonic stem cells (ESCs) is directed by biological and/or physical cues that regulate multiple signaling cascades. We have previously shown that mESCs seeded in a type I collagen matrix demonstrate a loss of pluripotent marker expression and differentiate towards an osteogenic lineage. In this study, we examined if this effect was mediated in part through Arginylglycylaspartic acid (RGD) dependent integrin activity and/or mechano-transduction.ResultsThe results from this study suggest that mESC interaction with the local microenvironment through RGD dependent integrins play a role in the regulation of mESC core transcription factors (TF), Oct-4, Sox 2 and Nanog. Disruption of this interaction with a cyclic RGD peptide (cRGDfC) was sufficient to mimic the effect of a mechanical stimulus in terms of pluripotent gene expression, specifically, we observed that supplementation with cRGDfC, or mechanical stimulus, significantly influenced mESC pluripotency by down-regulating core transcription factors. Moreover, our results indicated that the presence of the cRGDfC peptide inhibited integrin expression and up-regulated early lineage markers (mesoderm and ectoderm) in a Leukemia inhibitory factor (LIF) dependent manner. When cRGDfC treated mESCs were injected in Severe combined immunodeficiency (SCID) mice, no tissue growth and/or teratoma formation was observed, suggesting that the process of mESC tumor formation in vivo is potentially dependent on integrin interaction.ConclusionsOverall, the disruption of cell-integrin interaction via cRGDfC peptide can mimic the effect of mechanical stimulation on mESC pluripotency gene expression and also inhibit the tumorigenic potential of mESCs in vivo.

The combination therapy of salinomycin and gefitinib using poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles for targeting both lung cancer stem cells and cancer cells.

PurposeLung cancer (LC) is the leading cause of cancer death worldwide. Evidences suggest that both LC cancer stem cells (CSCs) and cancer cells are supposed to be eliminated to achieve superior treatment effect against LC. Salinomycin could eradiate CSCs in various types of cancers, and gefitinib is a first-line therapy in LC. The purpose of the present study was to develop salinomycin-loaded nanoparticles (salinomycin-NPs) combined with gefitinib-loaded nanoparticles (gefitinib-NPs) to eradicate both LC CSCs and cancer cells.MethodsSalinomycin and gefitinib were encapsulated separately by poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles by the emulsion/solvent evaporation approach. The anti-LC activity of salinomycin-NPs and gefitinib-NPs was investigated.ResultsSalinomycin-NPs and gefitinib-NPs are of ~140 nm in size, high drug encapsulation efficacy and sustained release of drugs. CD133+ LC CSCs showed the characteristics of CSCs, including significantly enhanced stem cell gene expression, tumorsphere formation ability, and tumorigenicity in mice. Both salinomycin and salinomycin-NPs are capable of selectively inhibiting LC CSCs, as reflected by their enhanced cytotoxic effects toward CD133+ LC CSCs and ability to reduce tumorsphere formation in LC cell lines, whereas gefitinib and gefitinib-NPs could significantly inhibit LC cells. Salinomycin-NPs and salinomycin could reduce the population of LC CSCs in the tumors in vivo. It is noteworthy that salinomycin-NPs combined with gefitinib-NPs inhibited the growth of tumors more efficiently compared with salinomycin combined with gefitinib or single salinomycin-NPs or gefitinib-NPs.ConclusionSalinomycin-NPs combined with gefitinib-NPs represent a potential approach for LC by inhibiting both LC CSCs and cancer cells.

StemMapper: a curated gene expression database for stem cell lineage analysis.

Transcriptomic data have become a fundamental resource for stem cell (SC) biologists as well as for a wider research audience studying SC-related processes such as aging, embryonic development and prevalent diseases including cancer, diabetes and neurodegenerative diseases. Access and analysis of the growing amount of freely available transcriptomics datasets for SCs, however, are not trivial tasks. Here, we present StemMapper, a manually curated gene expression database and comprehensive resource for SC research, built on integrated data for different lineages of human and mouse SCs. It is based on careful selection, standardized processing and stringent quality control of relevant transcriptomics datasets to minimize artefacts, and includes currently over 960 transcriptomes covering a broad range of SC types. Each of the integrated datasets was individually inspected and manually curated. StemMapper's user-friendly interface enables fast querying, comparison, and interactive visualization of quality-controlled SC gene expression data in a comprehensive manner. A proof-of-principle analysis discovering novel putative astrocyte/neural SC lineage markers exemplifies the utility of the integrated data resource. We believe that StemMapper can open the way for new insights and advances in SC research by greatly simplifying the access and analysis of SC transcriptomic data. StemMapper is freely accessible at http://stemmapper.sysbiolab.eu.

Codelivery of salinomycin and docetaxel using poly(D,L-lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles to target both gastric cancer cells and cancer stem cells.

Cancer stem cells (CSCs) in gastric cancer (GC) have been established recently as key therapeutic targets for the successful treatment of GC. Emerging evidence suggests that both CSCs and cancer cells should be eradicated to achieve optimal therapeutic efficacy. In the present study, salinomycin, which has been reported to kill CSCs, was used in combination with docetaxel, a chemotherapeutic drug that is used as first-line therapy in GC, to eradicate both GC stem cells (SCs) and cancer cells. Salinomycin and docetaxel were loaded separately into poly(D,L-lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles of ∼140 nm with a narrow size distribution, high drug loading, and sustained drug release. GC SCs were isolated by magnetic-activated cell sorting on the basis of CD44 expression as the CSC phenotype. CD44 GC SCs showed the characteristics of CSCs, including increased SC gene expression, tumorsphere formation capacity, and tumorigenicity in nude mice. We found that both salinomycin and salinomycin-loaded nanoparticles (salinomycin-NPs) could selectively eradicate GC SCs, as reflected by reduced tumorsphere formation capacity and the frequency of CD44 GC cells, whereas docetaxel and docetaxel-loaded nanoparticles (docetaxel-NPs) could significantly eradicate GC cells. In nude mice bearing GC xenografts, salinomycin-NPs and salinomycin significantly decreased the intratumor population of GC SCs. Notably, salinomycin-NPs combined with docetaxel-NPs suppressed tumor growth more effectively than did salinomycin combined with docetaxel, single salinomycin-NPs, or docetaxel-NPs. Therefore, salinomycin-NPs combined with docetaxel-NPs represent a promising strategy for the treatment of GC by eradicating both GC SCs and cancer cells.

Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells.

The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col-GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt-related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG, PD98059 inhibits BSPII expression on Col-GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col-GAG and MC-GAG, however, PD98059 only inhibits mineralization on Col-GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c-Jun amino-terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Abstract: Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells on Collagen Glycosaminoglycan Scaffolds

Non-Mineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells INTRODUCTION: The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, we described a correlation between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of Smad1/5 in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished ERK1/2 activation when compared to non-mineralized scaffolds (Col-GAG). In this work, we utilize a canonical BMPR inhibitor, DMH1, and an inhibitor of the MEK/ERK cascade, PD98059, to characterize the necessity of each pathway for osteogenesis. METHODS: hMSCs were cultured in osteogenic media on Col-GAG or MC-GAG scaffolds. Scaffolds were untreated or treated with the DMH1 or PD98059 at 50 mM for 4 days, 14 days, 24 days, 4 weeks, and 8 weeks. Gene and protein expression were measured using quantitative RT-PCR and western blot analysis. Scaffolds were subjected to histochemical and micro-computed tomographic analyses. RESULTS: Inhibition of the BMPR signaling pathway inhibited Runx2 and BSPII gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG. In contrast, inhibition of the MEK/ERK axis downregulated BSPII expression on Col-GAG independent of Runx2 expression. While inhibition of the BMPR signaling pathway resulted in decreased mineralization on both Col-GAG and MC-GAG, inhibition of the MEK/ERK axis only affected mineralization on Col-GAG. When the mechanistic detais were evaluated in greater detail, inhibition of the BMPR pathway reduced both Smad1/5 phosphorylation and Runx2 protein expression on both MC-GAG and Col-GAG. Inhibition of the MEK/ERK axis downregulated phosphorylation of ERK1/2 and JNK1/2 without affecting Smad1/5 phosphorylation or Runx2 expression. CONCLUSION: Interactions between hMSCs and collagen-based materials result in mechanistic differences in osteogenesis. Activation of the canonical BMPR signaling is required for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

The Cohesin Complex Is Necessary for Epidermal Progenitor Cell Function through Maintenance of Self-Renewal Genes

Adult stem and progenitor cells are critical for replenishing lost tissue due to injury or normal turnover. How these cells maintain self-renewal and sustain the tissue they populate are areas of active investigation. Here, we show that the cohesin complex, which has previously been implicated in regulating chromosome segregation and gene expression, is necessary to promote epidermal stem and progenitor cell self-renewal through cell-autonomous mechanisms. Cohesin binds to genomic sites associated with open chromatin, including DNase-I-hypersensitive sites, RNA polymerase II, and histone marks such as H3K27ac and H3K4me3. Reduced cohesin expression results in spontaneous epidermal differentiation due to loss of open chromatin structure and expression of key self-renewal genes. Our results demonstrate a prominent role for cohesin in modulating chromatin structure to allow for enforcement of a stem and progenitor cell gene expression program.