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

ABSTRACT: 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 ?M), 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 TGF? 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.

SUBMITTER: AlMuraikhi N

PROVIDER: S-EPMC6249887 | biostudies-other | 2018 Nov

REPOSITORIES: biostudies-other

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

AlMuraikhi Nihal N Ali Dalia D Alshanwani Aliah A Vishnubalaji Radhakrishnan R Manikandan Muthurangan M Atteya Muhammad M Siyal Abdulaziz A Alfayez Musaad M Aldahmash Abdullah A Kassem Moustapha M Alajez Nehad M NM Alajez Nehad M NM

Stem cell research & therapy 20181121 1

<h4>Background</h4>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.<h4>Methods</h4>We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiat ...[more]

PMID: 30463599

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Project description:: The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPAR?, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-? (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering.This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways controlling adipocyte and osteoblast differentiation of hMSCs. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies for tissue engineering, bone disease, obesity, and metabolic-disorders.

Project description:BackgroundDifferentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties.MethodsThrough the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers.ResultsThe cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization.ConclusionsMSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.

Project description:Skeletal stem cells (SSCs, or mesenchymal stromal cells typically referred to as mesenchymal stem cells from the bone marrow) are a dynamic progenitor population that can enter quiescence, self-renew or differentiate depending on regenerative demand and cues from their niche environment. However, ex vivo, in culture, they are grown typically on hard polystyrene surfaces, and this leads to rapid loss of the SSC phenotype. While materials are being developed that can control SSC growth and differentiation, very few examples of dynamic interfaces that reflect the plastic nature of the stem cells have, to date, been developed. Achieving such interfaces is challenging because of competing needs: growing SSCs require lower cell adhesion and intracellular tension while differentiation to, for example, bone-forming osteoblasts requires increased adhesion and intracellular tension. We previously reported a dynamic interface where the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD) was presented to the cells upon activation by user-added elastase that cleaved a bulky blocking group hiding RGD from the cells. This allowed for a growth phase while the blocking group was in place and the cells could only form smaller adhesions, followed by an osteoblast differentiation phase that was induced after elastase was added which triggered exposure of RGD and subsequent cell adhesion and contraction. Here, we aimed to develop an autonomous system where the surface is activated according to the need of the cell by using matrix metalloprotease (MMP) cleavable peptide sequences to remove the blocking group with the hypothesis that the SSCs would produce higher levels of MMP as the cells reached confluence. The current studies demonstrate that SSCs produce active MMP-2 that can cleave functional groups on a surface. We also demonstrate that SSCs can grow on the uncleaved surface and, with time, produce osteogenic marker proteins on the MMP-responsive surface. These studies demonstrate the concept for cell-controlled surfaces that can modulate adhesion and phenotype with significant implications for stem cell phenotype modulation.

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

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

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