Murine Bone Marrow Mesenchymal Stem Research Articles

Ginsenoside Re promotes proliferation of murine bone marrow mesenchymal stem cells in vitro through estrogen-like action.

Ginsenoside Re (GS-Re) is a major saponin monomer found in Panax ginseng Meyer. It has been shown to exhibit a wide range of biological and pharmacological activities. This study aimed to investigate the effect of GS-Re on the proliferation of murine bone marrow-derived MSCs in vitro and to assess whether its effect is dependent on the estrogen receptor-mediated signal transduction. CFU colony formation assay, cell counting, and colorimetric MTT test were employed to examine effects of GS-Re on the in vitro proliferation of MSCs and the mechanisms of the underlying effect were detected by flow cytometric analysis, immunofluorescence staining for BrdU, and Western blotting. GS-Re dose-dependently promoted the in vitro proliferation of murine bone marrow-derived MSCs over a range of concentrations of 0.5 ~ 20µmol/L, and this effect approached the maximal level at 10µmol/L. Increases in the expression level of phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2) were observed in the passaged MSCs treated with 10µmol/L of GS-Re. These effects of GS-Re on the MSCs were significantly counteracted by the addition of ICI 182, 780 (an estrogen receptor antagonist) to the culture media. We concluded that GS-Re is able to exert a proliferation-promoting effect on murine bone marrow-derived mesenchymal stem cells in vitro, and its action is involved in the estrogen receptor-mediated signaling.

Cooling‐promoted myogenic differentiation of murine bone marrow mesenchymal stem cells through TRPM8 activation in vitro

AbstractTRPM8 agonist has been reported to promote osteogenic differentiation of mesenchymal stem cells (MSCs), therefore we evaluated whether cooling‐induced activation of TRPM8 promotes myogenic differentiation of MSCs. We used 5‐azacytidine as a myogenic differentiation inducer in murine bone marrow‐derived MSCs. Addition of menthol, a TRPM8 agonist, to the differentiation induction medium significantly, increased the percentage of MyoD‐positive cells, a specific marker of myogenic differentiation. We performed intracellular Ca2+ imaging experiments using fura‐2 to confirm TRPM8 activation by cooling stimulation. The results confirmed that intracellular Ca2+ concentration ([Ca2+]i) increases due to TRPM8 activation, and TRPM8 antagonist inhibits increase in [Ca2+]i at medium temperatures below 19°C. We also examined the effect of cooling exposure time on myogenic differentiation of MSCs using an external cooling stimulus set at 17°C. The results showed that 60 min of cooling had an acceleratory effect on differentiation (2.18 ± 0.27 times). We observed that the TRPM8 antagonist counteracted the differentiation‐promoting effect of the cooling. These results suggest that TRPM8 might modulate the multiple differentiation pathways of MSCs, and that cooling is an effective way of activating TRPM8, which regulates MSCs differentiation in vitro.

Comparison of the efficacy of bone morphogenetic protein-4 on in vitro differentiation of murine adipose and bone marrow mesenchymal stem cells into primordial germ cells.

Background and purpose:In vitro development of functional gametes from pluripotent stem cells is a promising prospect to treat infertility. Mesenchymal stem cells with a high degree of plasticity and less tumorigenicity are a reliable source of stem cells for the generation of gametes. The present study aimed to compare the differentiation potential in the mesenchymal stem cells that are derived from bone marrow (BMDMSCs) and adipose tissue derived mesenchymal stem cells (AD-MSCs) into germ cells in a culture medium containing bone morphogenic protein-4 (BMP-4).Experimental approach:In this study, MSCs were isolated from both bone marrow and adipose tissue of murine samples. To further verify the nature of the harvested stem cells, their multipotency and surface marker were examined. The identified stem cells were cultured in a medium supplemented with 0 and 25 ng/mL of BMP-4 for 4 days. Flow cytometry analysis, immunofluorescence staining, and real RT-PCR were used to assess the expression levels in germ cell-specific biomarkers (Mvh, Dazl, Stra8, and Scp3).Findings/Results:CD44+, CD45-, CD31-, BMD-MSCs, and AD-MSCs showed to be capable of differentiating to osteo-adipogenic lineages. The flow cytometry, immunofluorescence, and RT-PCR results indicated that early germ cell markers (Mvh and Dazl) were expressed in both types of cells but they were significantly higher in BMD-MSCs than AD-MSCs.Conclusion and implications:Based on our results, the addition of exogenous BMP4 to the culture medium could differentiate BMD-MSCs and AD-MSCs into primordial germ cells, but it is inadequate to further develop into late germ cells in vitro. Moreover, the results revealed that, although AD-MSCs were easier to collect and had faster growth and proliferation rates than BMD-MSCs, the BMD-MSCs were better capable of differentiation into primordial germ cells. They may serve to be considered a more suitable source of MSC for in vitro generation of gametes than AD-MSCs.

Improved cell seeding efficiency and cell distribution in porous hydroxyapatite scaffolds by semi-dynamic method.

Tissue engineering is a promising technique for the repair of bone defects. An efficient and homogeneous distribution of cell seeding into scaffold is a crucial but challenging step in the technique. Murine bone marrow mesenchymal stem cells were seeded into porous hydroxyapatite scaffolds of two morphologies by three methods: static seeding, semi-dynamic seeding, or dynamic perfusion seeding. Seeding efficiency, survival, distribution, and proliferation were quantitatively evaluated. To investigate the performance of the three seeding methods for larger/thicker scaffolds as well as batch seeding of numerous scaffolds, three scaffolds were stacked to form assemblies, and seeding efficiencies and cell distribution were analyzed. The semi-dynamic seeding and static seeding methods produced significantly higher seeding efficiencies, vitalities, and proliferation than did the dynamic perfusion seeding. On the other hand, the semi-dynamic seeding and dynamic perfusion seeding methods resulted in more homogeneous cell distribution than did the static seeding. For stacked scaffold assemblies, the semi-dynamic seeding method also created superior seeding efficiency and longitudinal cell distribution homogeneity. The semi-dynamic seeding method combines the high seeding efficiency of static seeding and satisfactory distribution homogeneity of dynamic seeding while circumventing their disadvantages. It may contribute to improved outcomes of bone tissue engineering.

Small molecule-mediated modulation of ubiquitination and neddylation improves HSC function ex vivo.

Hematopoietic stem cells (HSCs) are particularly characterized by their quiescence and self-renewal. Cell cycle regulators tightly control quiescence and self-renewal capacity. Studies suggest that modulation of ubiquitination and neddylation could contribute to HSC function via cyclin-dependent kinase inhibitors (CDKIs). S-phase kinase-associated protein 2 (SKP2) is responsible for ubiquitin-mediated proteolysis of CDKIs. Here, we modulated overall neddylation and SKP2-associated ubiquitination in HSCs by using SKP2-C25, an SKP2 inhibitor, and MLN4924 (Pevonedistat) as an inhibitor of the NEDD8 system. Treatments of SKP2-C25 and MLN4924 increased both murine and human stem and progenitor cell (HSPC) compartments. This is associated with the improved quiescence of murine HSC by upregulation of p27 and p57 CDKIs. A colony-forming unit assay showed an enhanced in vitro self-renewal potential post inhibition of ubiquitination and neddylation. In addition, MLN4924 triggered the mobilization of bone marrow HSPCs to peripheral blood. Intriguingly, MLN4924 treatment could decrease the proliferation of murine bone marrow mesenchymal stem cells or endothelial cells. These findings shed light on the contribution of SKP2, and associated ubiquitination and neddylation in HSC maintenance, self-renewal, and expansion.

G Protein-Coupled Estrogen Receptor Mediates Cell Proliferation through the cAMP/PKA/CREB Pathway in Murine Bone Marrow Mesenchymal Stem Cells.

Estrogen is an important hormone to regulate skeletal physiology via estrogen receptors. The traditional estrogen receptors are ascribed to two nuclear estrogen receptors (ERs), ERα and ERβ. Moreover, G protein-coupled estrogen receptor-1 (GPER-1) was reported as a membrane receptor for estrogen in recent years. However, whether GPER-1 regulated osteogenic cell biology on skeletal system is still unclear. GPER-1 is expressed in growth plate abundantly before puberty but decreased abruptly since the very late stage of puberty in humans. It indicates GPER-1 might play an important role in skeletal growth regulation. GPER-1 expression has been confirmed in osteoblasts, osteocytes and chondrocytes, but its expression in mesenchymal stem cells (MSCs) has not been confirmed. In this study, we hypothesized that GPER-1 is expressed in bone MSCs (BMSC) and enhances BMSC proliferation. The cultured tibiae of neonatal rat and murine BMSCs were tested in our study. GPER-1-specific agonist (G-1) and antagonist (G-15), and GPER-1 siRNA (siGPER-1) were used to evaluate the downstream signaling pathway and cell proliferation. Our results revealed BrdU-positive cell counts were higher in cultured tibiae in the G-1 group. The G-1 also enhanced the cell viability and proliferation, whereas G-15 and siGPER-1 reduced these activities. The cAMP and phosphorylation of CREB were enhanced by G-1 but inhibited by G-15. We further demonstrated that GPER-1 mediates BMSC proliferation via the cAMP/PKA/p-CREB pathway and subsequently upregulates cell cycle regulators, cyclin D1/cyclin-dependent kinase (CDK) 6 and cyclin E1/CDK2 complex. The present study is the first to report that GPER-1 mediates BMSC proliferation. This finding indicates that GPER-1 mediated signaling positively regulates BMSC proliferation and may provide novel insights into addressing estrogen-mediated bone development.

53. Biomimetic laser-etched titanium promotes gene expression of early bone markers

BACKGROUND CONTEXT Surface properties are a key factor in how bone interacts with an implant material. Accordingly, surface modification of materials can increase the prevalence of fast and stable fixation at the bone-implant interface. In particular, laser etching can be used to create surface pores designed to mimic osteoclast resorption pits. PURPOSE The aim of this study was to evaluate the osteogenic potential of smooth titanium (TS), polyether-ether-ketone (PEEK), and laser-etched titanium (TL) by comparing the capabilities of each substrate to promote bone-relevant gene expression. STUDY DESIGN/SETTING In vitro cell culture study. PATIENT SAMPLE Not applicable. OUTCOME MEASURES Cell viability was measured using a viability assay on day 1 and 7 for all materials. Gene expression was analyzed at day 1 and 7 using two-step RT-qPCR for the following markers of bone formation: alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), and RUNX2. METHODS Discs, 15 mm in diameter, of TS, PEEK, and TL were used to culture murine bone marrow mesenchymal stem cells (BM-MSCs). Cells were also cultured on tissue culture polystyrene (TCPS). Substrates were seeded with BM-MSCs at a density of ∼56×103 cells/cm2. Substrates were compared at each time point for gene expression and cell viability. RESULTS TL exhibited a higher cell count than TS and PEEK over 7 days, though significantly less than the TCPS at all time points. By D7, TCPS, TL, PEEK, and TS cultures resulted in the proliferation of (383±16, 162±7, 122±22, 47±45) x103 cells, respectively. The TL cell count was significantly (p CONCLUSIONS This study suggests that biomimetic patterning of titanium enhances the cellular response compared to titanium. Biomimetic, laser-etched titanium exhibited significantly higher cell counts and early bone markers than both smooth titanium and PEEK over 7 days. Further research using an animal model is required to assess the osseointegration potential of laser-etched titanium in vivo. FDA DEVICE/DRUG STATUS SINTROS (Globus Medical) (Approved for this indication)

67. 3D-printed porous biomimetic titanium promotes osteogenic gene expression in murine mesenchymal stem cells

BACKGROUND CONTEXT Titanium is a commonly used implant material in orthopedics due to its biocompatibility with bone. Nonetheless, bone resorption caused by stress shielding is a concern. It has been reported that 3D-printing can be used to impart porosity that mimics the structure of trabecular bone, which could create a more osteogenic environment. PURPOSE In this study, we compared the capabilities of smooth titanium (TS), polyether-ether-ketone (PEEK), and 3D-printed titanium (T3) to promote expression of bone-relevant genes. STUDY DESIGN/SETTING In vitro cell culture study. PATIENT SAMPLE Not applicable. OUTCOME MEASURES Cell count was measured using a cell viability assay on day 1, 7, and 14 for all materials. Gene expression was analyzed at day 1,7,14, and 21 using two-step RT-qPCR for the following bone markers: Alkaline Phosphatase (ALP), Vascular Endothelial Growth Factor (VEGF), Osteocalcin (OCN), and RUNX2. Substrates were compared at each time point for gene expression and cell count. METHODS Discs, 15mm in diameter, of TS, PEEK, and T3 were used to culture murine bone marrow mesenchymal stem cells (BM-MSCs). Cells were also cultured on tissue culture polystyrene (TCPS). Substrates were seeded with BM-MSCs at a density of ∼56x103 cells/cm2. RESULTS T3 exhibited higher cell count than other discs over 14 days, though significantly less than TCPS at all time points. By day 14, TCPS, T3, PEEK, and TS cultures resulted in the cell counts of (349±12, 207±10, 134±7, and 103±83)×103 cells, respectively. Cell count on T3 discs was significantly higher than TS at D1 (58±3×103 and 30±9×103 respectively), TS and PEEK at D7 (167±8 x103, 47±45×103, and 122±22×103 respectively), and PEEK at D14 (207±10×103 and 134±7×103 respectively) (p CONCLUSIONS This study suggests that 3D-printed, porous, titanium implants may provide a more osteogenic environment than smooth titanium and PEEK, since cells cultured on 3D-printed titanium exhibited higher cell counts and enhanced bone marker production compared to both smooth titanium and PEEK over 21 days. Further research is required to assess the osseointegration potential of 3D-printed porous, titanium in vivo. FDA DEVICE/DRUG STATUS HEDRON (Globus Medical) (Approved for this indication)

54. Titanium plasma spray enhances ability of PEEK to express genes related to bone formation

BACKGROUND CONTEXT Polyether-ether-ketone (PEEK) is a commonly used material in orthopedic implant applications. PEEK is often preferred for its radiolucency and mechanical properties that more closely resemble bone than metallic materials. However, several studies have shown that bone does not directly bond to PEEK. Titanium coatings have been hypothesized to improve the ability of PEEK to bond to bone while maintaining radiolucency. PURPOSE In the present study, we sought to evaluate cell-substrate interactions of smooth titanium (TS), PEEK, and titanium plasma sprayed PEEK (PT) by measuring the capability of each substrate to encourage bone-relevant gene expression. STUDY DESIGN/SETTING In vitro cell culture study. PATIENT SAMPLE Not applicable. OUTCOME MEASURES Cell count was evaluated using a cell viability assay on day 1, 7, and 14 for all materials. Gene expression was analyzed at day 1,7, and 14 using two-step RT-qPCR for the following markers of bone formation: Alkaline Phosphatase (ALP), Vascular Endothelial Growth Factor (VEGF), Osteocalcin (OCN), and RUNX2. METHODS Discs, 15 mm in diameter, of TS, PEEK, and PT were used to culture murine bone marrow mesenchymal stem cells (BM-MSCs). Cells were also cultured on tissue culture polystyrene (TCPS). Substrates were seeded with BM-MSCs at a density of ∼56x103 cells/cm2. Substrates were compared at each time point for gene expression and cell viability. RESULTS PT substrates exhibited higher cell count than TS and PEEK over 7 days, though all substrates had significantly lower cell counts than TCPS at all time points. The PT cell count was significantly (p CONCLUSIONS Titanium plasma sprayed PEEK exhibited higher osteogenic gene expression and cell viability than both smooth titanium and PEEK over 14 days. Future research in an animal model is required to assess the osseointegration potential of titanium plasma-sprayed PEEK in vivo. FDA DEVICE/DRUG STATUS SUSTAIN (Globus Medical) (Approved for this indication)

Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging.We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p.Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3′,4′-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.

Green Tea Catechin (-)-Epigallocatechin-3-Gallate (EGCG) Facilitates Fracture Healing.

Green tea drinking can ameliorate postmenopausal osteoporosis by increasing the bone mineral density. (-)-Epigallocatechin-3-gallate (EGCG), the abundant and active compound of tea catechin, was proven to be able to reduce bone loss and ameliorate microarchitecture in female ovariectomized rats. EGCG can also enhance the osteogenic differentiation of murine bone marrow mesenchymal stem cells and inhibit the osteoclastogenesis in RAW264.7 cells by modulation of the receptor activator of nuclear factor-kB (RANK)/RANK ligand (RANKL)/osteoprotegrin (OPG) (RANK/RANKL/OPG) pathway. Our previous study also found that EGCG can promote bone defect healing in the distal femur partially via bone morphogenetic protein-2 (BMP-2). Considering the osteoinduction property of BMP-2, we hypothesized that EGCG could accelerate the bone healing process with an increased expression of BMP-2. In this manuscript, we studied whether the local use of EGCG can facilitate tibial fracture healing. Fifty-six 4-month-old rats were randomly assigned to two groups after being weight-matched: a control group with vehicle treatment (Ctrl) and a study group with 10 µmol/L, 40 µL, EGCG treatment (EGCG). Two days after the operation, the rats were treated daily with EGCG or vehicle by percutaneous local injection for 2 weeks. The application of EGCG enhanced callus formation by increasing the bone volume and subsequently improved the mechanical properties of the tibial bone, including the maximal load, break load, stiffness, and Young’s modulus. The results of the histology and BMP-2 immunohistochemistry staining showed that EGCG treatment accelerated the bone matrix formation and produced a stronger expression of BMP-2. Taken together, this study for the first time demonstrated that local treatment of EGCG can accelerate the fracture healing process at least partly via BMP-2.

Stereolithography-Based Additive Manufacturing of High-Performance Osteoinductive Calcium Phosphate Ceramics by a Digital Light-Processing System.

Digital light processing (DLP) is one of the additive manufacturing (AM) technologies suitable for preparation of high-performance ceramics. The present study provided an optimized formula to fabricate osteoinductive calcium phosphate (CaP) ceramics with high precision and controllable three-dimensional (3D) structure. Among the four surfactants, monoalcohol ethoxylate phosphate was the best one to modify the CaP powders for preparing the photocurable slurry with high solid loading and good spreading ability. By testing the photopolymerization property of the 60 wt % solid loading slurry, the appropriate processing parameters including the slice thickness (50 μm), exposure intensity (10.14 mW/cm2), and exposure time (8 s) were set to perform the 3D printing of the ceramic green body in the DLP system. After the debinding and sintering, the final CaP ceramics were acquired. The stereomicroscope and SEM observation confirmed the high precision of the ceramics. The average compressive strength of the ceramics with 64.5% porosity reached 9.03 MPa. On only soaking in simulated body fluid for 1 day, an even layer of apatite formed on the ceramic surface. The cell culture confirmed that the ceramics could allow the good attachment, growth, and proliferation of murine bone marrow mesenchymal stem cells. After implantation into the dorsal muscles of beagle dogs for 3 months, abundant blood vessels and obvious ectopic bone formation were observed clearly by the histological evaluation. Therefore, with good bioactivity and osteoinductivity as well as high precision and adjustable mechanical strength, the 3D printed CaP ceramics in the DLP system could have good potential in customized bone-repairing applications.

Inducing Macrophages M2 Polarization by Dexamethasone Laden Mesoporous Silica Nanoparticles from Titanium Implant Surface for Enhanced Osteogenesis

The study conveys an idea to enhance the osseointegration of titanium implant (Ti) through modulating macrophages M2 polarization. The ~ 100 nm spherical mesoporous silica nanoparticles (MSN) that compromised of ~ 4-nm-diameter nano-tunnels were synthesized by the conventional “sol–gel” method, into which the dexamethasone (DEX) was loaded (DEX@MSN). The DEX@MSN could consistently release DEX and showed favorable cytocompatibility in RAW264.7 cells. The arginase-1 expression, a specific marker for macrophages M2 polarization, was also enhanced by DEX@MSN treatment. Then, the Ti was pre-treated with anodization under 5 V to generate the titania nanotubes with ~ 30 nm diameter (NT-30) and the DEX@MSN was introduced onto NT-30 surface via electrophoretic deposition, with the aid of chitosan. After optimizing the deposition parameters, the supernatants of RAW264.7 from the decorated implant surface could significantly promote the osteogenic differentiation of murine primary bone marrow mesenchymal stem cells. These findings demonstrate that delivery of DEX from implant surface can modulate the macrophages M2 polarization and result in favorable osteogenesis.

In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo.

Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.

(-)-Epigallocatechin-3-Gallate (EGCG) Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate bone loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (−)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine bone marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate bone loss and improve bone microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing bone defect healing partially via bone morphogenetic protein 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and 10 µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

Adipogenic differentiation of murine bone marrow mesenchymal stem cells induced by visible light via photo- induced biomodulation.

Bone marrow mesenchymal stem cells (BM-MSCs) are undifferentiated cells that can proliferate and differentiate into specialized cells for tissue self-repair. Low-level laser (LLL) can induce biomodulatory effects such as cellular proliferation, differentiation, and migration. We investigated the biomodulatory effects of the photoactive compound chloroaluminum phthalocyanine nanoemulsion (AlClPc/NE) on the adipogenic differentiation of BM-MSCs, when combined with LLL (AlClPc/NE-LLL). The BM-MSCs used in this work were isolated from green fluorescent protein-positive (GFP+) C57BL6 mice. Cells were first treated with AlClPc/NE, a well-designed photoactive nano-drug and were then subjected to in vitro expansion, morphological and immunophenotypic characterization, and cellular cytotoxicity analysis. Subsequently, BM-MSCs were induced to differentiate into adipocytes by photo-induced biomodulation with AlClPc/NE-LLL. Our results showed that the isolated cell population was consistent with murine BM-MSCs. The cellular cytotoxicity analysis revealed that the optimal nanoemulsion dose to induce BM-MSC biomodulation was 5.0 μmol/L. Twenty-four hours following treatment with AlClPc/NE, BM-MSC were subjected to visible light irradiation of 20 mJ/cm2 at 670 nm. Six days after photo-induced biomodulation, cells maintained high GFP expression level, and expressed detectable mRNA levels of adipogenic genes (lipoprotein lipase and PPARγ); formation of lipid vacuoles was observed, and the cells did not show any tumorigenic potential in vivo. Our results indicated that photo-induced biomodulation via visible light using AlClPc/NE and LLL can induce adipogenic differentiation of murine BM-MSCs. Therefore, cell therapy with BM-MSCs and photo-induced biomodulation may contribute to the development of new therapeutic strategies that are faster and more effective than traditional methods to trigger MSC differentiation.

A Mutant p53-Dependent Embryonic Stem Cell Gene Signature Is Associated with Augmented Tumorigenesis of Stem Cells.

Mutations in the tumor suppressor p53 are the most frequent alterations in human cancer. These mutations include p53-inactivating mutations as well as oncogenic gain-of-function (GOF) mutations that endow p53 with capabilities to promote tumor progression. A primary challenge in cancer therapy is targeting stemness features and cancer stem cells (CSC) that account for tumor initiation, metastasis, and cancer relapse. Here we show that in vitro cultivation of tumors derived from mutant p53 murine bone marrow mesenchymal stem cells (MSC) gives rise to aggressive tumor lines (TL). These MSC-TLs exhibited CSC features as displayed by their augmented oncogenicity and high expression of CSC markers. Comparative analyses between MSC-TL with their parental mutant p53 MSC allowed for identification of the molecular events underlying their tumorigenic properties, including an embryonic stem cell (ESC) gene signature specifically expressed in MSC-TLs. Knockout of mutant p53 led to a reduction in tumor development and tumorigenic cell frequency, which was accompanied by reduced expression of CSC markers and the ESC MSC-TL signature. In human cancer, MSC-TL ESC signature-derived genes correlated with poor patient survival and were highly expressed in human tumors harboring p53 hotspot mutations. These data indicate that the ESC gene signature-derived genes may serve as new stemness-based prognostic biomarkers as well as novel cancer therapeutic targets.Significance: Mesenchymal cancer stem cell-like cell lines express a mutant p53-dependent embryonic stem cell gene signature, which can serve as a potential prognostic biomarker and therapeutic target in cancer. Cancer Res; 78(20); 5833-47. ©2018 AACR.

(−)-Epigallocatechin-3-gallate (EGCG) enhances healing of femoral bone defect

BackgroundPreviously, we found that (−)-epigallocatechin-3-gallate (EGCG) enhanced osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and eventually mineralization. We further found EGCG supplementation preserved bone mass and microarchitecture in female rats during estrogen deficiency in the proximal tibia and lumbar spine at least in part by increasing bone morphogenetic protein-2 (BMP2). BMP2 can enhance de novo bone formation. PurposeIn this study, we evaluate the effect of local EGCG application in de novo bone formation in bone defect healing. MethodsTwenty-four rats aged 4 months were weight-matched and randomly allocated to 2 groups: defect control with vehicle treatment (control) and defect with 10 µM EGCG treatment (EGCG). Daily vehicle and EGCG were applied locally by percutaneous local injection 2 days after defect creation for 2 weeks. Four weeks after treatment, animals were sacrificed for micro-computed tomography (μ-CT) and biomechanical analysis. ResultsLocal EGCG at femoral defect can enhance de novo bone formation by increasing bone volume and subsequently improve mechanical properties including max load, break point, stiffness, area under the max load curve, area under the break point curve and ultimate stress. ConclusionsLocal EGCG may enhance bone defect healing via at least partly by the de novo bone formation of BMP-2.

Effect of Growing Liver Biological Set on Various Cellular Cultures

Aim . To analyze the effect of growing liver biological set on viability and proliferative activity of various cellular cultures in vitro. Material and Methods. The biological combination is an extract from the growing liver obtained by using of original technique. We have assessed extract’s effect on the following cellular lines: hepatic carcinoma Huh7, L-fibroblasts of mice, murine bone marrow mesenchymal stem cells. Viability and proliferative activity were assessed by staining the cells with trypan blue and visual counting of cells under phase contrast microscopy. Result. Biological combination from neonatal piglet’s growing liver dose-dependently protects hepatocyte-like cells from deprivation of fetal serum and stimulates cellular growth in presence of serum. High concentrations of HRS do not lead to growth arrest of the Huh7 cells. At the same time, it is a cytostatic (or cytotoxic) for murine L- fibroblasts. Limited protective effect of the combination on the deprivation of serum when exposed to bone marrow stem cells was revealed. Conclusion. Our data show that the extract may be considered as an important regulator of reparative regeneration of liver with protective and/or stimulating effect on mesenchymal stem and hepatic-like cells and cytostatic effect on fibroblasts. So, further trials are necessary.

ID3013 Comparative characterization of murine Bone marrow mesenchymal stem cells cultured using two different supplements

Preclinical studies on mesenchymal stem cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. The mouse is the most widely used species for studying the characteristics of MSC. In recent years, conflicting data were reported regarding growth kinetics, surface marker profile, differentiation capacity, genetic instability or malignant transformation and so forth, that may be a result of a range of factors. One of the factors probably is the culture medium formulation. Here we have made a comparative characterization of bone marrow-derived mesenchymal stem cells (mBM-MSC), under the same experimental conditions, cultured using two common supplements, fetal bovine serum (FBS) and MesenCultTM Stimulatory Supplement (MSS). mBM-MSC isolated from the tibias of C57BL/6 mice were cultured and expanded in Dulbecco’s Modified Eagle’s Medium supplemented with either 15% FBS or 15% MSS. Clonogenic potential, population doubling time, immunophenotyping, differentiation immunosuppression potentials and chromosome analysis of early and late passage of mBM-MSC were assessed.
 The findings showed that the immunophenotype and differentiation potential of mBM-MSC were similar when cultured using these supplements irrespective of passages. Variations were seen in clonogenic, growth, proliferation rate and immunosuppression potential of the mBM-MSC. This study also revealed that prolonged culture will disrupt their genetic stability regardless of the supplements used. The genetically mutated mBM-MSC were also found to maintain their stemness characteristics and immunosuppression potential.
 In conclusion, culture medium formulation causes variations in the cultured MSC and may influence downstream investigation findings.