NR2F1 mediated down-regulation of osteoblast differentiation was rescued by bone morphogenetic protein-2 (BMP-2) in human MSC.

Endochondral ossification is the process by which long bones are formed; the process of long bone formation is regulated by numerous factors such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone Nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. Whole genome microarray data from our previous study revealed that most hHNR's are up-regulated during osteoblast differentiation in hMSCS. NR2F1 was among the highest expressed hHNR during osteogenesis, NR2F1 belongs to the steroid/thyroid hormone nuclear receptor superfamily. NR2F1 is designated as an orphan nuclear receptor because its ligands are unknown. NR2F1 plays a wide range of roles, including cell differentiation, cancer progression, and central and peripheral neurogenesis. Identifying signaling networks involved in osteoblast differentiation is important in orchestrating new therapeutic and clinical applications in bone biology. This study aimed to identify alterations in signaling networks mediated by NR2F1 in osteoblast differentiation. siRNA-mediated down-regulation of NR2F1 leads to impairment in the differentiation of hBMSC-TERT to osteoblast; gene-expression results confirmed the down-regulation of osteoblast markers such as RUNX2, ALPL, OSC, and BSP. Global whole gene expression analysis revealed that most down-regulated genes were associated with osteoblast differentiation (DDIT3, BMP2). Pathway analysis revealed prominent signaling pathways that were down-regulated, including the TGFß pathway and MAPK pathway. Functional studies on NR2F1 transfected cells, during osteoblast differentiation in combination with TGFß1 and BMP-2, showed that TGFß1 does not recover osteoblast differentiation, whereas BMP-2 rescues osteoblast differentiation in NR2F1 siRNA transfected cells. Thus, our results showed that BMP-2 could intervene in NR2F1 down-regulated signaling pathways to recover osteoblast differentiation.

In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells.

BACKGROUND: Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs) possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs) and adult dermal skin (hADSSCs) using explants cultures and were compared with bone marrow (hMSC-TERT) and adipose tissue-derived mesenchymal stem cells (hADMSCs) for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. RESULTS: Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC), with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA) was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. CONCLUSIONS: Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs represents a novel source of stem/stromal cells for tissue regeneration and the vascularization of engineered tissues. Moreover, the CD146 investigations suggested that the microenvironmental niche might contribute to direct stromal cells multipotency toward certain lineages, which warrants further investigation.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells.

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFß, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

Transgelin is a poor prognostic factor associated with advanced colorectal cancer (CRC) stage promoting tumor growth and migration in a TGFß-dependent manner.

Colorectal cancer (CRC) is the fourth most common cancer type globally. Investigating the signaling pathways that maintain cancer cell phenotype can identify new biomarkers for targeted therapy. Aberrant transforming growth factor-ß (TGFß) signaling has been implicated in CRC progression, however, the exact mechanism by which TGFß exerts its function is still being unraveled. Herein, we investigated TAGLN expression, prognostic value, and its regulation by TGFß in CRC. While TAGLN was generally found to be downregulated in CRC, elevated expression of TAGLN was associated with advanced CRC stage and predicted poor overall survival (hazard ratio (HR) = 1.8, log-rank test P-value = 0.014) and disease-free survival (HR = 1.6, log-rank test P-value = 0.046), hence implicating TAGLN as poor prognostic factor in CRC. Forced expression of TAGLN was associated with enhanced CRC cell proliferation, clonogenic growth, cell migration and in vivo tumor formation in immunocompromised mice, while targeted depletion of TAGLN exhibited opposing biological effects. Global gene expression profiling of TAGLN-overexpressing or TAGLN-deficient CRC cell lines revealed deregulation of multiple cancer-related genes and signaling pathways. Transmission electron microscopy (TEM) revealed ultrastructural changes due to loss of TAGLN, including disruption of actin cytoskeleton organization and aberrant actin filament distribution. Hierarchical clustering, principle component, and ingenuity pathway analyses revealed distinct molecular profile associated with TAGLNhigh CRC patients with remarkable activation of a number of mechanistic networks, including SMARCA4, TGFß1, and P38 MAPK. The P38 MAPK was the top predicted upstream regulator network promoting cell movement through regulation of several intermediate molecules, including TGFß1. Concordantly, functional categories associated with cellular movement and angiogenesis were also enriched in TAGLNhigh CRC, supporting a model for the molecular mechanisms linking TGFß-induced upregulation of TAGLN and CRC tumor progression and suggesting TAGLN as potential prognostic marker associated with advanced CRC pathological stage.

MicroRNA Expression Profiling on Paired Primary and Lymph Node Metastatic Breast Cancer Revealed Distinct microRNA Profile Associated With LNM.

Breast cancer (BC) is the foremost cause of cancer-related deaths in women. BC patients are oftentimes presented with lymph node metastasis (LNM), which increases their risk of recurrence. Compelling data have recently implicated microRNAs in promoting BC metastasis. Therefore, the identification of microRNA (miRNA)-based molecular signature associated with LNM could provide an opportunity for a more personalized treatment for BC patients with high risk of LNM. In current study, we performed comprehensive miRNA profiling in matched primary breast and LNM and identified 40 miRNAs, which were differentially expressed in LNM compared to primary tumors. The expression of 14 miRNAs (Up: hsa-miR-155-5p, hsa-miR-150-5p, hsa-miR-146a-5p, hsa-miR-142-5p and down: hsa-miR-200a-3p, hsa-miR-200b-3p, hsa-miR-200c-3p, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-214-3p, hsa-miR-141-3p, hsa-miR-127-3p, hsa-miR-125a-5p, and hsa-let-7c-5p) was subsequently validated in a second cohort of 32 breast and 32 matched LNM tumor tissues. Mechanistically, forced expression of hsa-miR-205-5p, or hsa-miR-214-3p epigenetically inhibited MDA-MB-231 cell proliferation, colony formation, and cell migration. Global gene expression profiling on MDA-MB-231 cells overexpressing hsa-miR-205-5p, or hsa-miR-214-3p in combination with in silico target prediction and ingenuity pathway analyses identified multiple bona fide targets for hsa-miR-205-5p, hsa-miR-214-3p affecting cellular proliferation and migration. Interestingly, interrogation of the expression levels of hsa-miR-205 and hsa-miR-214 in the METABRIC breast cancer dataset revealed significantly poor overall survival in patients with downregulated expression of miR-205 [HR = 0.75 (0.61-0.91)], p = 0.003 and hsa-miR-214 [HR = 0.74 (0.59-0.93) p = 0.008]. Our data unraveled the miRNA-transcriptional landscape associated with LNM and provide novel insight on the role of several miRNAs in promoting BC LNM, and suggest their potential utilization in the clinical management of BC patients.

Resveratrol inhibits adipocyte differentiation and cellular senescence of human bone marrow stromal stem cells.

Bone marrow adipose tissue (BMAT) is a unique adipose depot originating from bone marrow stromal stem cells (BMSCs) and regulates bone homeostasis and energy metabolism. An increased BMAT volume is observed in several conditions e.g. obesity, type 2 diabetes, osteoporosis and is known to be associated with bone fragility and increased risk for fracture. Therapeutic approaches to decrease the accumulation of BMAT are clinically relevant. In a screening experiment of natural compounds, we identified Resveratrol (RSV), a plant-derived antioxidant mediating biological effects via sirtuin- related mechanisms, to exert significant effects of BMAT formation. Thus, we examined in details the effects RSV on adipocytic and osteoblastic differentiation of tolermerized human BMSCs (hBMSC-TERT). RSV (1.0 µM) enhanced osteoblastic differentiation and inhibited adipocytic differentiation of hBMSC-TERT when compared with control and Sirtinol (Sirtuin inhibitor). Global gene expression profiling and western blot analysis revealed activation of a number of signaling pathways including focal adhesion kinase (FAK). Pharmacological inhibition of FAK using (PF-573228) and AKT inhibitor (LY-294002) (5µM), diminished RSV-induced osteoblast differentiation. In addition, RSV reduced the levels of senescence-associated secretory phenotype (SASP), gene markers associated with senescence (P53, P16, and P21), intracellular ROS levels and increased gene expression of enzymes protecting cells from oxidative damage (HMOX1 and SOD3). In vitro treatment of primary hBMSCs from aged patients characterized with high adipocytic and low osteoblastic differentiation ability with RSV, significantly enhanced osteoblast and decreased adipocyte formation when compared to hBMSCs from young donors. RSV targets hBMSCs and inhibits adipogenic differentiation and senescence-associated phenotype and thus a potential agent for treating conditions of increased BMAT formation.

MicroRNA-3148 acts as molecular switch promoting malignant transformation and adipocytic differentiation of immortalized human bone marrow stromal cells via direct targeting of the SMAD2/TGFß pathway.

MicroRNAs (miRs/miRNAs) play a key role in posttranscriptional regulation of gene expression and are implicated in a number of physiological and pathological conditions, including cellular malignant transformation. In the current study, we investigated the role of miR-3148 in regulating human stromal (mesenchymal) stem cell (hMSC) differentiation and transformation. Stable expression of miR-3148 in telomerized hMSC (hMSC-miR-3148) led to significant increase in in vitro adipocytic differentiation and suppression of osteoblastic differentiation. Concordantly, global gene expression profiling revealed significant enrichment in cholesterol biosynthesis pathway, and pathways related to enhanced cell movement and survival, whereas processes related to bone and connective tissue developments, cell death, apoptosis, and necrosis were downregulated. Global proteomic analysis using 2D-DIGE followed by mass spectrometry (MS) revealed significant changes in protein expression in hMSC-miR-3148 and enrichment in protein networks associated with carcinogenesis. Functional studies revealed that hMSC-miR-3148 exhibited enhanced in vitro cell proliferation, colony formation, migration, invasion, sphere formation, doxorubicin resistance, and increased active number of cells in S and G2/M cell cycle phases and formed sarcoma-like tumors with adipocyte infiltration when implanted into immunocompromised mice. SMAD2 was identified as bone fide gene target for miR-3148 using qRT-PCR, Western blotting, and UTR-based reporter assay. In agreement with our data, SMAD2 expression was downregulated in 47% of patients with soft tissue sarcoma. Bioinformatics analysis revealed that elevated miR-3148 expression correlates with poor prognosis in several human cancer types, including sarcoma. Our study identified miR-3148 as factor regulating hMSC differentiation and is involved in promoting malignant transformation of telomerized hMSC.

Author Correction: Convergence of TGFß and BMP signaling in regulating human bone marrow stromal cell differentiation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Convergence of TGFß and BMP signaling in regulating human bone marrow stromal cell differentiation.

Targeting regulatory signaling pathways that control human bone marrow stromal (skeletal or mesenchymal) stem cell (hBMSC) differentiation and lineage fate determination is gaining momentum in the regenerative medicine field. Therefore, to identify the central regulatory mechanism of osteoblast differentiation of hBMSCs, the molecular phenotypes of two clonal hBMSC lines exhibiting opposite in vivo phenotypes, namely, bone forming (hBMSC+bone) and non-bone forming (hBMSC-Bone) cells, were studied. Global transcriptome analysis revealed significant downregulation of several TGFß responsive genes, namely, TAGLN, TMP1, ACTA2, TGFß2, SMAD6, SMAD9, BMP2, and BMP4 in hBMSC-Bone cells and upregulation on SERPINB2 and NOG. Transcriptomic data was associated with marked reduction in SMAD2 protein phosphorylation, which thereby implies the inactivation of TGFß and BMP signaling in those cells. Concordantly, activation of TGFß signaling in hBMSC-Bone cells using either recombinant TGFß1 protein or knockdown of SERPINB2 TGFß-responsive gene partially restored their osteoblastic differentiation potential. Similarly, the activation of BMP signaling using exogenous BMP4 or via siRNA-mediated knockdown of NOG partially restored the differentiation phenotype of hBMSC-Bone cells. Concordantly, recombinant NOG impaired ex vivo osteoblastic differentiation of hBMSC+Bone cells, which was associated with SERBINB2 upregulation. Our data suggests the existence of reciprocal relationship between TGFB and BMP signaling that regulates hBMSC lineage commitment and differentiation, whilst provide a plausible strategy for generating osteoblastic committed cells from hBMSCs for clinical applications.

Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells.

BACKGROUND: Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. METHODS: A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. RESULTS: BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling. CONCLUSIONS: We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo.

Despite recent advances in cancer management and therapy, resistance to cytotoxic medications remains a major clinical challenge; hence, combination-based anti-cancer treatment regimens are currently gaining momentum. PTC-209 reduced BMI1 protein expression, while palbociclib inhibited CDK4, Rb, and pRbSer795 protein expression in MDA-MB-231 cells. PTC-209 and palbociclib exhibited dose-dependent cytotoxic effects against MDA-MB-231 (breast), HCT116 (colon), and PC-3 (prostate) models, which was more profound in the combination group. Transcriptome and pathway analyses revealed inhibition of insulin signaling, focal adhesion, DNA damage response, and Wnt/pluripotency signaling pathways as well as cell proliferation, and cellular movement functional categories by PTC-209. Transcriptome and pathway analyses revealed palbociclib to mainly affect cell cycle progression and survival. Upstream analysis identified several networks affected by PTC-209 (EZH2, IFNB1, TRIB3, EGFR, SREBF1, IL1A, ERG, TGFB1, MAX, MNT) and palbociclib (RABL6, MITF, RARA, TAL1, AREG, E2F3, FOXM1, ESR1, ERBB2, and E2F). PTC-209 and palbociclib reduced colony and sphere formation, cell migration, and cell viability, which was further enhanced in the combination group. Concordantly, combination of PTC-209 and palbociclib exhibited more profound effects on MDA-MB-231 tumor formation in vivo. Our data suggest concurrent targeting of BMI1 and CDK4/CDK6 might provide novel therapeutic opportunity for breast, colon, and prostate cancer.

Notch Signaling Inhibition by LY411575 Attenuates Osteoblast Differentiation and Decreased Ectopic Bone Formation Capacity of Human Skeletal (Mesenchymal) Stem Cells.

BACKGROUND: Chemical biology approaches using small molecule inhibitors targeting specific signaling pathways are useful tools to dissect the molecular mechanisms governing stem cell differentiation and for their possible use in therapeutic interventions. METHODS: Stem cell signaling small molecule library functional screen was performed employing human bone marrow skeletal (mesenchymal) stem cells (hBMSCs). Alkaline phosphatase (ALP) activity and formation of mineralized matrix visualized by Alizarin red staining were employed as markers for osteoblastic differentiation. Global gene expression profiling was conducted using the Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. Pathway analyses were conducted using the Ingenuity Pathway Analysis (IPA) tool. In vivo ectopic bone formation was performed using hBMSC mixed with hydroxyapatite-tricalcium phosphate granules that were implanted subcutaneously in 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius red staining were performed to identify bone formation in vivo. RESULTS: Among the tested molecules, LY411575, a potent γ-secretase and Notch signaling inhibitor, exhibited significant inhibitory effects on osteoblastic differentiation of hBMSCs manifested by reduced ALP activity, mineralized matrix formation, and decreased osteoblast-specific gene expression as well as in vivo ectopic bone formation. Global gene expression profiling of LY411575-treated cells revealed changes in multiple signaling pathways, including focal adhesion, insulin, TGFß, IL6, and Notch signaling, and decreased the expression of genes associated with functional categories of tissue development. Among the affected signaling networks were TGFß1, SPP1, and ERK regulatory networks. CONCLUSIONS: We identified γ-secretase inhibitor (LY411575) as a potent regulator of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with ectopic bone formation.

Neoplastic Transformation of Human Mesenchymal Stromal Cells Mediated via LIN28B.

Bone marrow stromal (Mesenchymal) stem cells (MSCs) are multipotent bone cells capable of differentiating into mesoderm-type cells, such as osteoblasts and adipocytes. Existing evidence suggests that transformation of MSCs gives rise to sarcoma. In order to identify the molecular mechanism leading to spontaneous transformation of human bone marrow MSCs (hBMSCs), we performed comprehensive microRNA (miRNA) and mRNA profiling in the transformed hBMSC-Tum line compared to the parental clone. As a result, we identified multiple dysregulated molecular networks associated with the hBMSC transformed phenotype. LIN28B was upregulated 177.0-fold in hBMSC-Tum, which was associated with marked reduction in LET-7 expression and upregulated expression of its target HMGA2. Targeted depletion of LIN28B or exogenous expression of LET-7b suppressed hBMSC-Tum proliferation, colony formation, and migration. On the other hand, forced expression of LIN28B promoted malignant transformation of parental hBMSC cells as shown by enhanced in vitro colony formation, doxorubicin resistance, and in vivo tumor formation in immunocompromised mice. Analysis of LIN28B and HMGA2 expression levels in cohorts from The Cancer Genome Atlas sarcoma dataset revealed a strong inverse-relationship between elevated expression and overall survival (OS) in 260 patients (p = 0.005) and disease-free survival (DFS) in 231 patients (p = 0.02), suggesting LIN28B and HMGA2 are important regulators of sarcoma biology. Our results highlight an important role for the LIN28B/LET-7 axis in human sarcoma pathogenesis and suggest that the therapeutic targeting of LIN28B may be relevant for patients with sarcoma.

Molecular profiling of ALDH1+ colorectal cancer stem cells reveals preferential activation of MAPK, FAK, and oxidative stress pro-survival signalling pathways.

Tumour heterogeneity leads to variable clinical response and inaccurate diagnostic and prognostic assessment. Cancer stem cells (CSCs) represent a subpopulation responsible for invasion, metastasis, therapeutic resistance, and recurrence in many human cancer types. However, the true identity of colorectal cancer (CRC) SCs remains elusive. Here, we aimed to characterize and define the gene expression portrait of CSCs in CRC-model SW403 cells. We found that ALDH+ positive cells are clonogenic and highly proliferative; their global gene expression profiling-based molecular signature revealed gene enrichment related to DNA damage, MAPK, FAK, oxidative stress response, and Wnt signalling. ALDH+ cells showed enhanced ROS stress resistance, whereas MAPK/FAK pathway pharmacologic inhibition limited their survival. Conversely, 5-fluorouracil increased the ALDH+ cell fraction among the SW403, HCT116 and SW620 CRC models. Notably, analysis of ALDH1A1 and POU5F1 expression levels in cohorts of 462 or 420 patients for overall (OS) or disease-free (DFS) survival, respectively, obtained from the Cancer Genome Atlas CRC dataset, revealed strong association between elevated expression and poor OS (p = 0.006) and poor DFS (p = 0.05), thus implicating ALDH1A1 and POU5F1 in CRC prognosis. Our data reveal distinct molecular signature of ALDH+ CSCs in CRC and suggest pathways relevant for successful targeted therapies and management of CRC.

Romidepsin Promotes Osteogenic and Adipocytic Differentiation of Human Mesenchymal Stem Cells through Inhibition of Histondeacetylase Activity.

Bone marrow mesenchymal stem cells (BMSCs) are adult multipotent stem cells that can differentiate into mesodermal lineage cells, including adipocytes and osteoblasts. However, the epigenetic mechanisms governing the lineage-specific commitment of BMSCs into adipocytes or osteoblasts are under investigation. Herein, we investigated the epigenetic effect of romidepsin, a small molecule dual inhibitor targeting HDAC1 and HDAC2 identified through an epigenetic library functional screen. BMSCs exposed to romidepsin (5 nM) exhibited enhanced adipocytic and osteoblastic differentiation. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis and osteogenesis in romidepsin-treated BMSCs during induction into adipocytes or osteoblasts, respectively. Pharmacological inhibition of FAK signaling during adipogenesis or inhibition of FAK or TGFß signaling during osteogenesis diminished the biological effects of romidepsin on BMSCs. The results of chromatin immunoprecipitation combined with quantitative polymerase chain reaction indicated a significant increase in H3K9Ac epigenetic markers in the promoter regions of peroxisome proliferator-activated receptor gamma (PPARγ) and KLF15 (related to adipogenesis) or SP7 (Osterix) and alkaline phosphatase (ALP) (related to osteogenesis) in romidepsin-treated BMSCs. Our data indicated that romidepsin is a novel in vitro modulator of adipocytic and osteoblastic differentiation of BMSCs.

ACI/EG eutectic mixture mediated synthesis, characterization and in vitro osteoblast differentiation assessment of spiropyrrolo[1,2-b]isoquinoline analogues.

An eco-friendly acetylcholine iodide-ethylene glycol (ACI/EG) deep eutectic mixture mediated green protocol has been developed for the synthesis of hitherto unexplored multi-functionalized linear tricyclic spiropyrrolo[1,2-b]isoquinoline analogues. The effects of the synthesized compounds on the osteoblast differentiation of hBMSC-TERT cell lines were investigated and promising results were observed with significant IC50 values. In addition, molecular modeling simulations were also performed with the 3D structure of BMP-2 to reveal binding interactions and orientations of highly potent spiropyrrolo[1,2-b]isoquinoline analogues.

Whole genome mRNA expression profiling revealed multiple deregulated pathways in stromal vascular fraction from erectile dysfunction patients.

Background: Stem-cell-based therapies have recently been explored in the field of erectile dysfunction (ED). However, the cellular and molecular phenotype of adipose derived stem cells (ADSCs) stromal vascular fraction (SVF) from ED patients remains largely unknown. Herein we compared the global gene expression profile in the SVF from ED patients and healthy individuals and identified altered signaling pathways between the two groups.Methods: Samples (2-5 g) of abdominal adipose tissue from ED patients (n = 6) and healthy individual controls (n = 3) undergoing elective cosmetic liposuction were collected. Immediately after removal, SVF was separated using Collagenase type I and type IV protocol. RNA was isolated and microarray experiments were conducted using the Agilent platform. Data were normalized and pathway analyses were performed using GeneSpring software.Results: Our data revealed multiple differentially expressed genes between the ED and control group. Hierarchical clustering based on differentially expressed mRNAs revealed clear separation of the two groups. The distribution of the top enriched pathways for the up-regulated genes indicated enrichment in inflammatory response and T-cell receptor signaling, while pathway analysis performed on the down-regulated genes revealed enrichment in mitogen-activated protein kinase, TGF-ß, senescence, FAK, adipogenesis, androgen receptor, and EGF-EGFR signaling pathways in SVF from ED patient.Conclusion: Our data revealed the existence of multiple altered signaling pathways in the SVF from ED patients, which could potentially play a role in the etiology of this disease. Therefore, therapeutic strategies targeting these pathways might provide novel therapeutic opportunity for ED patients.

Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells.

BACKGROUND: Better understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate. METHODS: We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite-tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo. RESULTS: We identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 µM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFß and insulin signaling, endochondral ossification, and focal adhesion. CONCLUSIONS: We identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.

TGFß1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton.

TGFß is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGFß1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGFß-mediated enhancement of lineage commitment, we compared the gene expression profiles of TGFß1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFßl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGFß1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFß1 and cytochalasin D. Our study demonstrates that TGFß1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.