Differentiation Of Human Bone Marrow Research Articles

Oleanolic acid exerts bone anabolic effects via activation of osteoblastic 25-hydroxyvitamin D 1-alpha hydroxylase

Oleanolic acid (OA) is previously shown to exert bone protective effects in aged animals. However, its role in regulating osteoblastic vitamin D bioactivation, which is one of major causes of age-related bone loss, remains unclear. Our results revealed that treatment of OA significantly increased skeletal CYP27B1 expression and circulating 1,25(OH)2D3 in ovariectomized mice (p <0.01). Moreover, OA upregulated CYP27B1 protein expression and activity, as well as the vitamin D-responsive bone markers alkaline phosphatase (ALP) activity and osteopontin (OPN) protein expression, in human osteoblast-like MG-63 cells (p<0.05). CYP27B1 expression increased along with the osteoblastic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). CYP27B1 expression and cellular 1,25(OH)2D3 production were further potentiated by OA in cells at mature osteogenic stages. Notably, our study suggested that the osteogenic actions of OA were CYP27B1 dependent. In summary, the bone protective effects of OA were associated with the induction of CYP27B1 activity and expression in bone tissues and osteoblastic lineages. Hence, OA might be a potential approach for management of age-related bone loss.

Astragalus polysaccharide promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells by facilitating ANKFY1 expression through miR-760 inhibition.

Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay. The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs. APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression.

A pilot study: Nano-hydroxyapatite-PEG/PLA containing low dose rhBMP2 stimulates proliferation and osteogenic differentiation of human bone marrow derived mesenchymal stem cells.

Bone morphogenetic protein 2 (BMP2) can enhance posterolateral spinal fusion (PLSF). The minimum effective dose that may stimulate mesenchymal stem cells however remains unknown. Nano-hydroxyapatite (nHAp) polyethylene glycol (PEG)/polylactic acid (PLA) was combined with recombinant human BMP2 (rhBMP2). We in vitro evaluated proliferation, differentiation, and osteogenic genes of human bone marrow mesenchymal stem cells with 0.5, 1.0, and 3.0 μg/mL rhBMP2 doses in this study. In vitro experimental study was designed to proliferation by a real-time quantitative cell analysis system and the osteogenic differentiation by alkaline phosphatase (ALP) activity and osteogenic marker (Runx2, OPN, and OCN) gene expressions of human derived bone marrow mesenchymal stem cells (hBMMSCs). nHAp was produced by wet chemical process and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, and energy-dispersive x-ray spectroscopy. PEG/PLA polymer was produced at a 51:49 molar ratio. 0.5, 1.0, and 3.0 μg/mL rhBMP2 and nHAp was combined with the polymers. hBMMSCs were characterized by multipotency assays and surface markers were assessed by flow cytometer. The hBMMSC-rhBMP2 containing nHAp-PEG/PLA composite interaction was evaluated by transmission electron microscopy. Proliferative effect was evaluated by real-time proliferation analysis, and osteogenic capacity was evaluated by ALP activity assay and qPCR. hBMMSC proliferation in the 0.5 μg/mL rhBMP2 + nHAp-PEG/PLA and the 1.0 μg/mL rhBMP2 + nHAp-PEG/PLA groups were higher compared to control. 1.0 μg/mL rhBMP2 + nHAp-PEG/PLA and 3.0 μg/mL rhBMP2 + nHAp-PEG/PLA containing composites induced ALP activity on days 3 and 10. 0.5 μg/mL rhBMP2 + nHAp-PEG/PLA application stimulated Runx2 and OPN gene expressions. rhBMP2 + nHAp-PEG/PLA composites stimulate hBMMSC proliferation and differentiation. The nHAp-PEG/PLA composite with low dose of rhBMP2 may enhance bone formation in future clinical PLSF applications.

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

Hydroxyapatite-collagen- carboxylic carbon quantum dot composite loaded with chrysin supported the proliferation and differentiation of human bone marrow derived mesenchymal stem cells

Developing a bioactive scaffold with biocompatible material is a substantial approach to bone regeneration and functional healing. Hydroxyapatite (HAP) is the main component in bone formation as an inorganic component and regeneration due to its osteoconductive properties. In this study, we prepared a scaffold material composed of HAP and collagen (COL) cross-linked via carboxylic carbon quantum dots (CCQD) with a chrysin (CRN) molecule. CRN is a flavonoid that has been shown to encourage the bone development of bone marrow-derived mesenchymal stem cells. It is loaded for enhancing bone regeneration and HAP’s growth ability. XRD, FT-IR, SEM, and TEM analysis have characterized the prepared composites for their crystalline nature, functional behavior, and morphological evaluations. The HAP has retained its original crystalline lattice confirmed from XRD analysis in the prepared composites. The addition of CRN molecule has decreased the length of HAP rods from ∼932 nm to ∼459 nm, as confirmed by TEM images. The increased particle sizes have been observed for the prepared composites. It reaches the maximum at 938.0 nm for the final HAP/COL/CCQD/CRN composite, which was confirmed by particle size analysis. The in-vitro CRN release behavior shows that the CRN molecule has controlled release up to 23% for 48 h. The biocompatibility of prepared material was investigated and confirmed on human bone marrow-derived mesenchymal stem cells (hBMSCs). This examination has proven that the prepared material is good for bone cell regeneration. The material may apply for bone regeneration applications after in-vivo and clinical investigations.

Deubiquitinating Enzyme USP7 Is Required for Self-Renewal and Multipotency of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Ubiquitin-specific protease 7 (USP7) is highly expressed in a variety of malignant tumors. However, the role of USP7 in regulating self-renewal and differentiation of human bone marrow derived mesenchymal stromal cells (hBMSCs) remains unknown. Herein, we report that USP7 regulates self-renewal of hBMSCs and is required during the early stages of osteogenic, adipogenic, and chondrogenic differentiation of hBMSCs. USP7, a deubiquitinating enzyme (DUB), was found to be downregulated during hBMSC differentiation. Furthermore, USP7 is an upstream regulator of the self-renewal regulating proteins SOX2 and NANOG in hBMSCs. Moreover, we observed that SOX2 and NANOG are poly-ubiquitinated and their expression is downregulated in USP7-deficient hBMSCs. Overall, this study showed that USP7 is required for maintaining self-renewal and multipotency in cultured hBMSCs. Targeting USP7 might be a novel strategy to preserve the self-renewal capacity of hBMSCs intended for stem cell therapy.

Antibacterial wollastonite supported excellent proliferation and osteogenic differentiation of human bone marrow derived mesenchymal stromal cells

Biocompatibility and bacterial infections are the primary concerns associated with the current bone graft substitutes. The application of wollastonite-based scaffolds for bone tissue engineering becomes a novel subject of interest. In the present study, a single phasic wollastonite scaffold was synthesised using citric acid-based sol–gel combustion route. Its physicochemical characteristics, antibacterial properties as well as its biocompatibility and osteogenic induction effect on human bone marrow derived stromal cells (hBMSCs) are yet to be explored. The TGA/DTA, XRD and SEM/EDX confirmed the characteristics of wollastonite. The antibacterial test indicated wollastonite inhibition of 47.81% and 45.54% for gram-positive, Staphylococcus aureus and Staphylococcus epidermidis and 47.04% and 46.07% for gram-negative, Escherichia coli and Pseudomonas aeruginosa bacterial strains, respectively. The SEM micrographs demonstrated an excellent attachment of hBMSCs on wollastonite and comparable to commercial hydroxyapatite (cHA) scaffold. The alamar blue cell proliferation assay confirmed 1.7- and 1.8-fold significant increase in hBMSCs seeded on wollastonite and cHA scaffold, respectively, on day 14 as compared with day 1. The immunohistochemistry analysis on Type-I collagen (Col1) and Bone morphogenetic protein-2 (BMP2) expression on day 14 confirmed the osteogenic differentiation of hBMSCs seeded on wollastonite and comparable with cHA scaffold. In conclusion, wollastonite scaffold has a greater potential to substitute bone grafts in orthopaedic applications.

Effect of inorganic phosphate on migration and osteogenic differentiation of bone marrow mesenchymal stem cells

BackgroundPhosphate is the major ingredient of bone tissue, and is also an important component of commercial bone substitute materials, bone scaffolds, and implant surface coatings. With the dissolution of the bone substitute materials and the degradation by cells, local ion concentrations will change and affect bone tissue reconstruction. Bone marrow -derived mesenchymal stem cells (BM-MSCs) are main autologous cells to repair injured bone. When bone injure occurs, BM-MSCs migrate to the damaged area, differentiate into osteoblasts, and secrete bioactive factors to promote bone tissue repaired. This study aimed to investigate the effect of inorganic phosphate (Pi) at a series of concentration on migration and osteogenic differentiation of human bone marrow -derived mesenchymal stem cells(hBM-MSCs).MethodsThe culture of hBM-MSCs in mediums with different concentration of Pi from 2 mM to 10 mM were performed. HBM-MSCs migration were examined with transwell assays. HBM-MSCs proliferation were evaluated by cell counting kit-8 colorimetric method. Osteogenic genes expression were analyzed by real-time reverse transcriptase polymerase chain reaction. Mineralized nodules formation were demonstrated by Alizarin red staining.Result4–10 mM Pi could effectively promote the migration of hBM-MSCs at 12 h and 18 h. There was no significant difference in the migration number of hBM-MSCs in Pi culture mediums at a concentration of 6, 8, and10mM. 2–10 mM Pi could promote the proliferation of hBM-MSCs to varying degrees in the observation period, while 4–10 mM Pi could promote the osteogenic differentiation and mineralization of hBM-MSCs.ConclusionThe findings in our study showed 4-10 mM Pi could promote the migration, osteogenic differentiation, and mineralization of hBM-MSCs.

2019 Most Influential Bioelectromagnetics Journal Paper Award

The Bioelectromagnetics Society announces the 2019 Most Influential Bioelectromagnetics Journal Paper by citation from among primary research articles published between January 2014 and December 2018 to the authors: Alessia Ongaro, Agnese Pellati, Leila Bagheri, Cinzia Fortini, Stefania Setti, and Monica De Mattei. For their paper: Ongaro A, Pellati A, Bagheri L, Fortini C, Setti S, and De Mattei M. 2014. Pulsed electromagnetic fields stimulate osteogenic differentiation in human bone marrow and adipose tissue derived mesenchymal stem cells. Bioelectromagnetics 35: 426–436. Each of the authors received a certificate of the award and monetary prize from the Bioelectromagnetics Society, Frederick, Maryland, USA in June 2020. The Most Influential Bioelectromagnetics Journal Paper Award was established by the Bioelectromagnetics Society in 2007 to recognize scholarly contributions to the scientific community, and to acknowledge and foster ongoing excellence in scientific discovery and achievement in its field of research endeavor. A listing of past award papers are given in Table 1 below.

Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow-/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia.

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O2) and hypoxic (5% O2) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

Effects of glycosaminoglycan supplementation in the chondrogenic differentiation of bone marrow- and synovial- derived mesenchymal stem/stromal cells on 3D-extruded poly (ε-caprolactone) scaffolds

The lack of effective and long-term treatments for articular cartilage defects has increased the interest for innovative tissue engineering strategies. Such approaches, combining cells, biomaterial matrices and external biochemical/physical cues, hold promise for generating fully functional cartilage tissue. Herein, this study aims at exploring the use of the major cartilage glycosaminoglycans (GAGs), chondroitin sulfate (CS) and hyaluronic acid (HA), as external biochemical cues to promote the chondrogenic differentiation of human bone marrow- and synovium-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) on custom-made 3 D porous poly (ε-caprolactone) (PCL) scaffolds. The culture conditions, namely the chondrogenic medium and hypoxic environment (5% O2 tension), were firstly optimized by culturing hBMSCs on PCL scaffolds without GAG supplementation. For both MSC sources, GAG supplemented media, particularly with HA, promoted significantly cartilage-like extracellular matrix (ECM) production (higher sulfated GAG amounts) and chondrogenic gene expression. Remarkably, in contrast to tissues generated using hBMSCs, the hSMSC-based constructs showed decreased expression of hypertrophic marker COL X. Histological, immunohistochemical and transmission electron microscopy (TEM) analysis confirmed the presence of typical articular cartilage ECM components (GAGs, aggrecan, collagen fibers) in all the tissue constructs produced. Overall, our results highlight the potential of integrating GAG supplementation, hSMSCs and customizable 3 D scaffolds toward the fabrication of bioengineered cartilage tissue substitutes with reduced hypertrophy.

Effect of histone demethylase KDM5A on the odontogenic differentiation of human dental pulp cells

ABSTRACT Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells in response to exogenous stimuli. Histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. Previous studies have shown that histone methyltransferases (HMTs) and histone demethylases (HDMs) are crucial for the osteogenic differentiation of human bone marrow, adipose tissue, and tooth tissue. However, little is known about the role of histone methylation in hDPC differentiation. Here, the expression levels of HMTs and HDMs were profiled in hDPCs undergoing odontogenic induction. Among several differentially expressed enzymes, HDM KDM5A demonstrated significantly enhanced expression during cytodifferentiation. Furthermore, KDM5A expression increased during early passages and in a time-dependent manner during odontogenic induction. Using a shRNA-expressing lentivirus, KDM5A was knocked down in hDPCs. KDM5A depletion resulted in greater alkaline phosphatase activity and more mineral deposition formation. Meanwhile, the expression levels of the odontogenic markers DMP1, DSPP, OSX, and OCN were increased by KDM5A knockdown. As a histone demethylase specific for tri- and dimethylated histone H3 at lysine 4 (H3K4me3/me2), KDM5A deficiency led to a significant increment in total H3K4me3 levels, whereas no significant difference was found for H3K4 me2. H3K4me3 levels on the promoters of the odontogenic markers increased after KDM5A knockdown in hDPCs. These results demonstrated that KDM5A is present in hDPCs and inhibits the odontogenic differentiation potentiality of hDPCs by removing H3K4me3 from specific gene promoters, suggesting that KDM5A-dependent histone demethylation may play an important role in reparative dentinogenesis.

3D printing of step-gradient nanocomposite hydrogels for controlled cell migration

In this study, we report the step-gradient nanocomposite (NC) hydrogel generated easily by spatial connection of different nanocomposite hydrogel pastes varying in the concentrations of nanomaterials with the aid of a 3D printing technique. The prepared 3D printed gradient NC hydrogel has self-adhesive properties and is used to direct the migration of fibroblast cells towards the higher concentration of biopolymer-coated silica-based nanomaterials (NMs) within the 3D network of the hydrogel. Furthermore, we demonstrate the potential application of our gradient NC hydrogel in migration and subsequent enhanced osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBM MSC). The osteogenic differentiation of hBM MSC is achieved in the absence of osteogenic differentiation medium due to the silica-based NMs. The increase in the NM content in the gradient construct promotes hBM MSC migration and results in higher Ca2+ deposition.

Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells.

Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as β-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.

The Effect of Alisma canaliculatum and Polyporus umbellatus Extracts on Adipogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Stem cells

Objective This study investigated the effect of purified medical herb extracts such as Alisma canaliculatum and Polyporus umbellatus on adipogenic differentiation of human bone marrow derived mesenchymal stromal stem cells (hBMSCs). Methods Two different medical herb were extracted using hot distilled water. The optimal concentration of extracts were fixed at 100 ng/ml by means of cell viability and cytotoxic assay. To test the adipogenic differentiation ability of extracts, we induced the adipogenesis of hBMSCs for 21 days. At day 5, the cell was harvested to check mRNA and protein expression level of adipogenic related factors. The efficacy of lipid droplet formation was evaluated using the oil-red O staining method at days 21. Results Two different medical herb extracts have no toxicity on hBMSCs. And two different medical herb extracts significantly decreased formation of lipid droplet compared with control groups in hBMSCs. The A. canaliculatum extract group showed the lowest mRNA and protein expression level of adipossgenic related transcription factors. This data suggests that extract of A. canaliculatum and P. umbellata decrease the adipogenic differentiation of hBMSCs. Conclusions Our findings indicate that water-extract of A. canaliculatum and P. umbellata will be useful therapeutic reagents for prevention of obesity related disease such as diabetes, hyperlipidemia, coronary artery disease, and osteoporosis. Keywords: hBMSCs, Adipogenic differentiation, Alisma canaliculatum, Polyporus umbellata

Metformin promotes differentiation of human bone marrow derived mesenchymal stem cells into osteoblast via GSK3β inhibition.

Metformin, a common and first-line drug for diabetes mellitus, is widely used in the world. Recently, many studies have documented that osteogenesis could be mediated by metformin. However, the specific mechanism by which metformin affects osteogenesis has not been clearly identified. Therefore, the aim of this study is to evaluate the role of GSK3β in metformin-induced osteogenic differentiation of mesenchymal stem cells (MSCs). Osteoblast-marker genes, including Col-1, OCN, and RUNX2, were measured by RT-PCR in differentiated MSCs treated with Metformin. Osteogenic differentiation viability was measured by Alkaline phosphatase (ALP) assays and Alizarin Red Staining. The expression of GSK3β, β-catenin and AMPK were measured by Western blotting in MSCs treated with metformin. We found that metformin at 100 μM significantly promoted osteogenic differentiation of human mesenchymal stem cells (hBMSCs). Next, we showed that GSK3β and Wnt signaling pathway are involved in metformin-induced osteogenic differentiation of hBMSCs. Furthermore, osteogenic differentiation of hBMSCs induced by metformin could be eliminated by inhibiting phosphorylation of GSK3β. The data suggested that metformin promoted the osteoblast differentiation of MSCs by, at least partly, inhibiting GSK3β activity. Additionally, we also found that AMPK plays an essential role in the inhibition of GSK3β by metformin.

Differential Expression of PPARγ and CHOP‐10 During Adipogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) hold great promise for cell‐based therapy and regenerative medicine owing to their multilineage differentiation potential that ranges from osteogenic to cardiac lineages. Osteogenic and adipogenic differentiation of MSCs in particular is of great interest since any imbalance that perturbs the regulation of osteogenesis and adipogenesis is typically associated with diseases such as obesity, osteoporosis, and other age‐related diseases. In our project, we sought to profile the gene expression pattern of key osteogenic and adipogenic transcription factors during differentiation using time course experiments. Adipogenesis was induced in passage 4 human bone marrow derived mesenchymal stem cells (BM‐MSCs) seeded with a density of 6579 cells/cm2 and RNA was collected at Day 0 (control), Day 3, Day 7, and Day 14 after differentiation to analyze the expression level of master adipogenic transcription factor peroxisome proliferator activated receptor‐γ (PPARγ) and negative adipogenic factor CHOP‐10. Our preliminary data suggests that compared to control (Day 0, undifferentiated) samples, PPARγ expression increases about 20‐fold, 22‐fold, and 168‐fold at Day 3, 7, 14 respectively. In contrast, expression of CHOP‐10, which is a negative regulator of adipogenesis, is upregulated approximately 2.4‐fold at Day 3, then downregulated 1.13‐fold at Day 7, and again increased 1.1‐fold at Day 14 respectively. Future work will compare these results and expression pattern of other adipogenic transcription factors to MSC differentiation in a 3D culture system.Support or Funding InformationFunding for these projects are supported by Wright State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-146b is down-regulated during the chondrogenic differentiation of human bone marrow derived skeletal stem cells and up-regulated in osteoarthritis

Articular cartilage injury can result in chondrocyte loss and diminishment of specialised extracellular matrix, which can progress to an osteoarthritic (OA) phenotype. Stem cells have emerged as a favourable approach for articular cartilage regeneration. Identification of miRNAs which influence stem cell fate offers new approaches for application of miRNAs to regenerate articular cartilage. Skeletal stem cells (SSCs) isolated from human bone marrow were cultured as high density micromass’ using TGF-β3 to induce chondrogenesis. qPCR and TaqMan qPCR were used to assess chondrogenic gene and miRNA expression. Target prediction algorithms identified potential targets of miR-146b. Transient transfection with miR-146b mimic and western blotting was used to analyse SOX5. Human OA articular chondrocytes were examined for miR-146b expression. Chondrogenic differentiation of human bone marrow derived SSCs resulted in significant down-regulation of miR-146b. Gain of miR-146b function resulted in down-regulation of SOX5. MiR-146b expression was up-regulated in OA chondrocytes. These findings demonstrate the functional role of miR-146b in the chondrogenic differentiation of human bone marrow derived SSCs. MiR-146b may play a role in the pathophysiology of OA. Application of miR-146b combined with stem cell therapy could enhance regeneration of cartilaginous tissue and serve as a potential therapeutic target in the treatment of OA.

Extracellular matrix mimicking scaffold promotes osteogenic stem cell differentiation: A new approach inosteoporosis research.

Osteoporosis is a common metabolic disease, with mesenchymal stem cells discussed to play an important role in its pathomechanism. For in vitro osteoporosis studies, selection of adequate culture conditions is mandatory so as to preserve cell properties as far as possible. A suitable cell culture surface would ideally provide reproducible experimental conditions by resembling those in-vivo. Generating an improved growth surface for osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs). We modified electrospun gelatine meshes with hydroxyapatite nanopowder. The potential beneficial impact of the ensuing culture conditions were evaluated by cultivating and comparing the growth of cells from osteoporotic and non-osteoporotic donors on either hydroxyapatite-gelatine (HA) meshes, pure gelatine meshes, or 2D standard tissue culture surfaces. After 21 days of differentiation, cells grown on pure or HA-gelatine meshes showed significantly higher mineralization levels compared to cells cultured in standard conditions. The amount of mineralization varied considerably in hBMSC cultures of individual patients but showed no significant difference between stem cells obtained from osteoporotic or non-osteoporotic donors. Overall, these results indicate that the use of HA-gelatine meshes as growth surfaces may serve as a valuable tool for cultivation and differentiation of mesenchymal stem cells along the osteogenic lineage, facilitating future research on osteoporosis and related issues.

Icaritin, a novel plant-derived osteoinductive agent, enhances the osteogenic differentiation of human bone marrow- and human adipose tissue-derived mesenchymal stem cells.

For the treatment of diseases affecting bones using bone regenerative medicine, there is an urgent need to develop safe, inexpensive drugs that can strongly induce bone formation. In the present study, we systematically investigated the effects of icaritin, a metabolic product of icariin, on the osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells (hBMSCs) and human adipose tissue‑derived stem cells (hADSCs) invitro. After treatment with icaritin at concentrations of 10‑8-10‑5M, hBMSCs and hADSCs were examined for alkaline phosphatase activity, osteocalcin (OC) secretion, matrix mineralization and expression levels of bone‑related mRNA and proteins. Data showed that icaritin at concentrations 10‑7-10‑5M significantly increased alkaline phosphatase activity, OC secretion at different time points, and calcium deposition at day21. In addition, icaritin upregulated the mRNA expression of genes for bone morphogenetic proteins (BMP‑2,‑4and‑7), bone transcription factors (Runx2 and Dlx5) and bone matrix proteins (ALP, OC and Col‑1). Moreover, icaritin increased the protein levels of BMPs, Runx2 and OC, as detected by western blot analysis. These findings suggest that icaritin enhances the osteogenic differentiation of hBMSCS and hADSCs. Icaritin exerts its potent osteogenic effect possibly by directly stimulating the production of BMPs. Although the osteogenic activity of icaritin invitro was inferior to that of rhBMP‑2, icaritin displayed better results than icariin. Moreover, the low cost, simple extraction procedure, and an abundance of icaritin make it appealing as a bone regenerative medicine.