Rat Bone Marrow Mesenchymal Stem Cells Research Articles

Cardiomyocyte-like cell differentiation by FGF-2 transfection and induction of rat bone marrow mesenchymal stem cells

Objective(s)To investigate and test the hypotheses that FGF-2 enhanced myocardial differentiation with rat bone marrow mesenchymal stem cells (BMSCs). Materials and methodsLentiviral vectors carrying the FGF-2 gene were transfected into rat BMSCs firstly. According to the different inducing agents, they were divided into the following four groups: group A (BMSCs blank control group), group B (FGF-2 induction group), group C (Lenti-FGF-2-GFP lentivirus transfection group), and the group D (Lenti-control-GFP lentiviral transfer). Then several kinds of experimental methods such as real-time PCR, immunocytochemical staining, immunofluorescence staining, Western blot, and transmission electron microscopy were used to elucidate the effects by which FGF-2 adjusts myocardial differentiation in rat BMSCs. ResultsThe results of real-time PCR showed that GATA-4 and Nkx2.5 were expressed in all groups of cells. Compared with the experimental control group, the expression of GATA-4 and Nkx2.5 genes was the strongest after induction of 2 weeks in each induction group, and gradually decreased after induction of 4 weeks. Among them, the relative expression levels of GATA-4 and Nkx2.5 genes in Lenti-FGF-2-GFP were highest at all time points. The expressions of cTnI, cTnT, Cx43, and Desmin were detected by immunocytochemical staining and immunofluorescence staining. After 4 weeks of induction, cTnI, cTnT, Cx43, and Desmin were positively expressed in the cytoplasm of cells. Statistical analysis showed that the integrated optical density (IOD) values of the markers in the Lenti-FGF-2-GFP were the strongest. Cx43 and cTnI were weakly positive or negative in the experimental control group. There was a significant difference in the positive expression of each marker in each induction group and the experimental control group. Western blot analysis showed that Tromyosin (Tm) and Desmin were expressed in the blank group, FGF-2 drug-induced group, Lenti-FGF-2-GFP, and empty virus control transfection group after 4 weeks of induction, among which FGF-2 lentivirus transfected. The expression levels of Tm and Desmin were the highest in the staining induction group. Statistical analysis showed that the positive expressions of Tm and Desmin in each experimental group were statistically significant. Transmission electron microscopy showed that the nucleus of the cells transfected and induced by FGF-2 was located at the center of the cells. Myofilaments, rough endoplasmic reticulum, and mitochondria, and ribosomes were seen in the cytoplasm. ConclusionThese results indicate that FGF-2 can transfect and induce differentiation of BMSCs into cardiomyocyte-like cells. Lentivirus-mediated FGF-2 transfection induces the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells better than FGF-2 direct induction.

Effect of Sirtuin1 (Sirt1) on Bone Marrow Mesenchymal Stem Cells (BMSCs) Osteogenesis/Adipogenesis via β-Catenin/The Transcription Factor T Cell Factor 1 (TCF1)/Runt-Related Transcription Factor 2 (RUNX2)

Bone marrow mesenchymal stem cells (BMSCs) have self-renewal potential. Sirt1 regulates cell differentiation and apoptosis. However, Sirt1’s effect on BMSCs osteogenic/adipogenic differentiation has not been fully elucidated. SD rats were randomly divided into Osteoporosis (OP) group and sham operation group. OP rat BMSCs were isolated and assigned into control group, NC group and Sirt1 siRNA group followed by analysis of Sirt1 level by Real-time PCR, cell proliferation by MTT assay, expression of OC, OPN and FABP4 level by real time PCR, and β-Catenin/TCF1/Runx2 protein expression by Western blot. In OP group, Sirt1 expression was significantly increased and BMSCs proliferation was decreased along with reduced OC and OPN mRNA expression, increased FABP4 expression and reduced β-Catenin/TCF1/Runx2 expression compared with sham operation group (P < 0.05). In Sirt1 siRNA group, Sirt1 expression was significantly reduced, BMSCs proliferation was increased, OC and OPN mRNA expression was increased, FABP4 expression was decreased, and β-Catenin/TCF1/Runx2 expression was increased compared to OP group (P < 0.05). Sirt1 is increased in osteoporosis. Down-regulating Sirt1 in osteoporotic BMSCs can regulate β-Catenin/TCF1/Runx2 signaling and promote BMSCs osteogenic differentiation and inhibit adipogenic differentiation.

RNF213 gene silencing upregulates transforming growth factor β1 expression in bone marrow-derived mesenchymal stem cells and is involved in the onset of Moyamoya disease.

Moyamoya disease (MMD) is a chronic and progressive cerebrovascular occlusion disease, the precise etiology of which is poorly understood. Ring finger protein 213 (RNF213) has been previously identified as a susceptibility gene that serves an important role in angiogenesis, where it has been shown to be closely associated with the onset of MMD. Patients with MMD exhibit increased expression levels of various pro-inflammatory molecules and angiogenic factors. Under certain conditions, bone marrow mesenchymal stem cells (BMSCs) have the ability to differentiate to form neuron-like and microglia-like cells. In the present study, a total of 40 MMD patients and 40 healthy individuals were enrolled. ELISA assays revealed that the expression of serum vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) were higher than that in healthy controls. Furthermore, rat BMSCs (rBMSCs) were isolated and cultured using the whole bone marrow adherence method, which were then phenotyped using flow cytometry. Osteogenic and adipogenic differentiation were determined by using Alizarin red and oil red O staining, respectively. RNF213 was knocked-down using a lentivirus-mediated short hairpin RNA system in passage three rBMSCs, and successful transfection of the RNF213 was confirmed by RT-qPCR and fluorescence imaging. The expression levels of VEGF and TGF-β1 in these rBMSCs were measured on days 7 and 14, respectively. The results demonstrated that RNF213 knockdown upregulated TGF-β1 at both protein and mRNA levels, but did not exert any effect on VEGF gene expression. In conclusion, these findings suggested that that RNF213 knockdown may contribute to aberrant TGF-β1 expression via a pathway that remains to be unidentified, indicating that quantitative changes in RNF213 gene expression may serve an important role in the pathogenesis of MMD.

Rapamycin's osteogenic effects on bone marrow mesenchymal stem cells through triggering autophagy

Background: Although autophagy is crucial for the self-renewal and differentiation of stem cells, it is yet unknown how it affects bone marrow mesenchymal stem cells (BMSCs). This work used rapamycin (RAPA), a traditional autophagy agonist with osteo-regulatory effects, to examine the relationship between autophagy and osteogenic differentiation. Methods: After osteo induction (0, 7, 14, and 21 d), real-time quantitative polymerase chain reaction, western blotting, and immune-fluorescence were used to examine the autophagy of rat BMSCs (RT-qPCR). Alizarin red staining, alkaline phosphatase tests, and RT-qPCR/Western blotting quantification of bone sialoprotein, type 1 collagen, alkaline phosphatase, osteopenia, and Runt-related transcription factor 2 mRNA and protein levels were also used to assess osteogenic differentiation.Results: During osteogenic differentiation, the BMSCs' baseline autophagy level steadily reduced due to changes in BECN1, the ratio of lapidated (LC3-II) to unelucidated (LC3-I) microtubule-associated protein 1 light chain 3, and the expression of the specific autophagic target p62. On the other hand, it grew when RAPA concentration increased. Additionally, whereas 5 nM RAPA inhibited osteogenesis on days 14 and 21, 2 nM RAPA increased BMSC osteogenic differentiation on days 7 and 14. The autophagy inhibitor 3-methyladenine may reduce RAPA's ability to increase osteogenesis in BMSCs.Conclusions: During osteogenic differentiation, the baseline autophagy level of the BMSC reduced with time. Yet, a suitable RAPA concentration encouraged BMSC osteogenic differentiation through activation of autophagy.

Expression of angiogenesis-related proteins in bone marrow mesenchymal stem cells induced by osteoprotegerin during osteogenic differentiation in rats

This study aimed to discuss the expression of angiogenesis-related proteins in bone marrow mesenchymal stem cells (BMSCs) induced by osteoprotegerin (OGP) during osteogenic differentiation in rats, and to analyze the effect of fracture healing inflammatory factor TNF-ɑ on the osteogenic differentiation of BMSCs of rats. BMSCs isolated and cultured from the third generation rats were taken as the research object. According to the addition amount of OGP, BMSCs were divided into control group, OGP (10−7 mol/L) group, OGP (10−8 mol/L) group, and OGP (10−9 mol/L) group. The cell growth and morphological characteristics of each group were observed by inverted phase contrast microscope, the cell proliferation rate was measured by MTT method, angiogenesis-related markers (platelet growth factor (VEGF), cingulate protein 5 (Fbln5), and angiogenin-like protein 4 (Angptl4)) were quantitatively detected by Western blot, and the effect of TNF-ɑ on osteogenic differentiation was detected by CCK. Compared with the control group, MTT results showed that the value-added rate of cells in the OGP (10−8 mol/L) group reached the maximum at 9 days (P < 0.05). The ALP activity in osteoblasts in the OGP (10−8 mol/L) group reached the maximum at 9 days (P < 0.01). The OGP (10−8 mol/L) group had the highest expression of vascular regeneration proteins (VEGF, Fbln5, and Angptl4) (P < 0.05). CCK analysis showed that the TNF-ɑ (1.0 ng/mL) group showed a significant increase in absorbance compared with the control group on 6 days (P < 0.05), and the OD value of the TNF-ɑ (10 ng/mL) group decreased at all time points (P < 0.05). Overall, 10−8 mol/L OGP can induce the proliferation and osteogenic differentiation of MSCs, and promote the expression of angiogenesis-related proteins (VEGF, Fbln5, and Angptl4) during osteogenic differentiation. Besides, 1.0 ng/mL of TNF-ɑ can also promote osteogenesis differentiation of BMSCs in the short term.

Retraction Note to: Effects of BDNF-Transfected BMSCs on Neural Functional Recovery and Synaptophysin Expression in Rats with Cerebral Infarction.

The purpose of this study was to investigate the effects of brain-derived neurotrophic factor (BDNF)-transfected bone marrow mesenchymal stem cells (BMSCs) on neural functional recovery and synaptophysin expression in rats with cerebral infarction (CI). A total of 120 healthy Sprague Dawley rats were randomly divided into sham group, control group, and model group. Craniotomy was conducted and neurological function defect scoring was used to verify the model. BDNF containing recombinant plasmid was transfected into rat BMSCs, which was verified by flow cytometry and Western Blot. After injection of the transfected BMSCs, neural functional recovery of the CI rats and synaptophysin expression were measured. After the CI rat model was established, magnetic resonance (MR) imaging, 2, 3, 5- triphenyl tetrazolium chloride (TTC) staining, and the neurological function defect scoring determined the success of the model. CD34 (-), CD45 (−), CD29 (+), and CD90 (+) cells detected showed that the obtained BMSCs have high purity. BDNF protein was highly expressed in the BMSCs successfully transfected with the recombinant plasmid. Balance beam walking score, rotating bar walking score, and screen test score were significantly lower, while synaptophysin expression was higher in the BDNF model group than those in the non-BDNF model group and sham group with time extension. BDNF can increase synaptic plasticity and neurogenesis and have a promotional role in neural functional recovery and synaptophysin expression in rats with CI. BDNF-transfected BMSCs may therefore have better treatment efficacy for CI clinically.

LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

BackgroundImproved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. MiR-127-5p has been reported to promote cartilage differentiation of rat bone marrow MSCs (rMSCs); however, the regulatory mechanisms underlying hypoxia-stimulated chondrogenic differentiation remain unknown.MethodsrMSCs were induced to undergo chondrogenic differentiation under normoxic or hypoxic conditions. Expression of lncRNA DNM3OS, miR-127-5p, and GREM2 was detected by quantitative real-time PCR. Proteoglycans were detected by Alcian blue staining. Western blot assays were performed to examine the relative levels of GREM2 and chondrogenic differentiation related proteins. Luciferase reporter assays were performed to assess the association among DNM3OS, miR-127-5p, and GREM2.ResultsMiR-127-5p levels were upregulated, while DNM3OS and GREM2 levels were downregulated in rMSCs induced to undergo chondrogenic differentiation, and those changes were attenuated by hypoxic conditions (1% O2). Further in vitro experiments revealed that downregulation of miR-127-5p reduced the production of proteoglycans and expression of chondrogenic differentiation markers (COL1A1, COL2A1, SOX9, and ACAN) and osteo/chondrogenic markers (BMP-2, p-SMAD1/2). MiR-127-5p overexpression produced the opposite results in rMSCs induced to undergo chondrogenic differentiation under hypoxic conditions. GREM2 was found to be a direct target of miR-127-5p, which was suppressed in rMSCs undergoing chondrogenic differentiation. Moreover, DNM3OS could directly bind to miR-127-5p and inhibit chondrogenic differentiation of rMSCs via regulating GREM2.ConclusionsOur study revealed a novel molecular pathway (DNM3OS/miR-127-5p/GREM2) that may be involved in hypoxic chondrogenic differentiation.

Member Domain 3 (LRIG3) Activates Hypoxia-Inducible Factor-1 α/Vascular Endothelial Growth Factor (HIF-1α/VEGF) Pathway to Inhibit the Growth of Bone Marrow Mesenchymal Stem Cells in Glioma

Member domain 3 (LRIG3) of the LRIG gene family is down-regulated in several cancers. However, its role in bone marrow mesenchymal stem cells (BMSCs) in gliomas and the related mechanisms is unknown. The qRT-PCR assessed LRIG3 mRNA level. Rat BMSCs were randomly assigned into glioma group (BMSCs cultured in glioma microenvironment); LRIG3 overexpression group; and si-LRIG3 inhibitor group followed by analysis of LRIG3 expression, cell proliferation, PCNA and Ki-67 apoptosis, TNF-α; and HIF-1α/VEGF mRNA level. LRIG3 mRNA expression was decreased in gliomas patients (P &lt; 0.05). BMSCs cultured in glioma microenvironment showed decreased LRIG3, increased cell proliferation, decreased PCNA, Ki-67 and TNF-α secretion as well as elevated HIF-1α and VEGF level (P &lt; 0.05). Transfection of LRIG3 siRNA further promoted the above changes. Conversely, LRIG3 plasmid transfection significantly promoted its expression in glioma BMSCs (P &lt; 0.05), inhibited cell proliferation, promoted PCNA, Ki-67, and TNF-α secretion, and increased HIF-1α and VEGF level (P &lt; 0.05). LRIG3 in rat BMSCs cultured in the glioma microenvironment is decreased. Down-regulation of LRIG3 inhibits TNF-α secretion by activating HIF-1α/VEGF pathway regulating BMSCs proliferation and apoptosis.

MicroRNA-210-3p Promotes Chondrogenic Differentiation and Inhibits Adipogenic Differentiation Correlated with HIF-3α Signalling in Bone Marrow Mesenchymal Stem Cells.

Cartilage injury of the knee joint is very common. Due to the limited self-healing ability of articular cartilage, osteoarthritis is very likely to occur if left untreated. Bone marrow mesenchymal stem cells (BMMSCs) are widely used in the study of cartilage injury due to their low immunity and good amplification ability, but they still have disadvantages, such as heterogeneous undifferentiated cells. MicroRNAs can regulate the chondrogenic differentiation ability of MSCs by inhibiting or promoting mRNA translation and degradation. In this research, we primarily investigated the effect of microRNA-210-3p (miR-210-3p) on chondrogenic and adipogenic differentiation of BMMSCs in vitro. Our results demonstrate that miR-210-3p promoted chondrogenic differentiation and inhibited adipogenic differentiation of rat BMMSCs, which was related to the HIF-3α signalling pathway. Additionally, miR-210-3p promotes mRNA and protein levels of the chondrogenic expression genes COLII and SOX9 and inhibits mRNA and protein levels of the adipogenic expression genes PPARγ and LPL. Thus, miR-210-3p combined with BMMSCs is a candidate for future clinical applications in cartilage regeneration and could represent a promising new therapeutic target for OA.

Effect of Liver Kinase B1 on Osteogenic/Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in High Glucose Environment

Bone marrow mesenchymal stem cells (BMSCs) present reduced proliferation under high glucose condition. Liver kinase B1 (LKB1) can maintain the homeostasis of hematopoietic stem cells. However, whether LKB1 regulates BMSCs osteogenic/adipogenic differentiation under high glucose is unclear. Rat BMSCs were isolated and separated into control group, high glucose group, and LKB1 group (BMSCs were transfected with pc-DNA 3.1-LKB1 plasmid under high glucose condition) followed by analysis of LKB1 expression by Real time PCR and Western blot, osteocalcin, type I collagen, RUNX2 and OPN mRNA level by real-time PCR, FABP4 and PPARγ2 level by western blot. In high glucose group, LKB1 expression was significantly decreased, with reduced expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA and elevated FABP4 and PPARγ2 level compared to control group (P &lt; 0.05). Transfection of LKB1 plasmid reduced LKB1 expression, upregulated osteocalcin, type I collagen, RUNX2 and OPN mRNA and downregulated FABP4 and PPARγ2. Compared with the high glucose group, there was a statistical difference (P &lt;0.05). High glucose can inhibit LKB1 expression and BMSCs osteogenic differentiation, and promote adipogenic differentiation. Upregulating LKB1 expression can promote BMSCs osteogenic differentiation.

Effects of Melatonin on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Inflammatory Environment by Regulating Mammalian Target of Rapamycin/Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling

Human bone marrow mesenchymal stem cells (BMSCs) differentiation into special cell types is affected by inflammation. Melatonin has various effects such as anti-oxidation and immune regulation. However, melatonin’s effect on BMSCs osteogenic differentiation during inflammation has not been elucidated. Rat BMSCs were isolated and assigned into control group, inflammation group (1 μg/ml lipopolysaccharide, LPS) and melatonin group (100 μM melatonin was added to LPSstimulated BMSCs cells) followed by analysis of BMSCs proliferation by MTT assay, Caspase 3 and ALP activity, expression of Runx2 and OP by Real time PCR, ROS content and SOD activity, TNF-α and IL-1β secretion by ELISA and mTOR/PI3K/AKT signaling protein level by Western blot. LPS action on BMSCs significantly inhibits BMSCs proliferation, promotes Caspase 3 activity, inhibits ALP activity, decreases Runx2 and OP expression and SOD activity, increases ROS content and TNF-α and IL-1β secretion as well as reduced mTOR and p-PI3K level (P &lt;0.05). Melatonin addition significantly reversed the above changes (P &lt;0.05). Melatonin can regulate oxidative stress, inhibit inflammation, and promote BMSCs proliferation and osteogenic differentiation in inflammatory environment by activating mTOR/PI3K/AKT signaling pathway.

The Role of Small Extracellular Vesicles Derived from Lipopolysaccharide-preconditioned Human Dental Pulp Stem Cells in Dental Pulp Regeneration

IntroductionRegenerative endodontics has created a desirable shift in the treatment paradigm despite current limitations of regenerative outcomes. Mesenchymal stem cells (MSCs) facilitate tissue regeneration and repair in a mild inflammatory environment. Small extracellular vesicles (sEVs) derived from MSCs play an imperative role in the paracrine modulation of regenerative responses modulated by MSCs. However, it remains unknown whether MSCs enhance dental pulp regeneration or whether this enhancement is mediated by sEVs in a mild inflammatory environment. The present study aimed to elucidate the effects of sEVs originated from lipopolysaccharide (LPS)-preconditioned human dental pulp stem cells (hDPSCs) on dental pulp regeneration. MethodsAll sEVs were isolated from hDPSCs cultured with or without LPS (ie, N-sEVs and L-sEVs, respectively). The effect of N-sEVs and L-sEVs on proliferation, migration, angiogenesis, and differentiation of rat bone marrow MSCs was identified in vitro. Moreover, N-sEVs or L-sEVs were implanted into rat pulpless root canal models, and the regenerated tissue in root canals was assessed via hematoxylin-eosin staining, Masson staining, and immunohistochemistry after 30 days of transplantation. ResultsBoth N-sEVs and L-sEVs could modulate BMSC proliferation, migration, angiogenesis, and differentiation. Both kinds of sEVs enhanced the structure of the regenerated tissue closer to that of a normal dental pulp in vivo. L-sEVs had a more significant effect than N-sEVs. ConclusionssEVs released by hDPSCs in a mild inflammatory microenvironment are capable of facilitating the regeneration of dental pulp through functional healing instead of scar healing, which has potential applications in regenerative endodontics.

MicroRNA-182 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Smad1.

The present study aimed to screen abnormally expressed microRNAs (miRs/miRNAs) in patients with postmenopausal osteoporosis (POP) and explore their mechanisms via functional verification. Bone marrow mesenchymal stem cells (BMSCs) were extracted from healthy controls and patients with POP. Differences in osteogenic differentiation and proliferation of human BMSCs were compared between the two groups using Cell Counting Kit-8 (CCK-8) assay and alizarin red staining. A rat model of POP was established. Compared with patients with POP, human BMSCs in healthy controls had significantly enhanced viability at 24, 36, 48 and 72 h. The results of alizarin red staining revealed that the deposition of calcium minerals in human BMSCs were significantly lower in patients with POP. Based on miRNA microarray and reverse transcription-quantitative polymerase chain reaction (PCR) results, the expression levels of miR-7010 and miR-467c decreased, while miR-132 and miR-182 expression increased in the human BMSCs of patients with POP. Alizarin red staining showed that miR-182 markedly suppressed the osteogenic differentiation of primary rat BMSCs in rats. Western blotting and immunofluorescence assay revealed that miR-182 inhibited the expression of osteogenesis markers runt-related transcription factor 2, osterix and actinin-associated LIM protein. The results of the luciferase reporter assay showed that Smad1 is the direct target of miR-182. In rat primary BMSCs, Smad1 overexpression abolished the inhibitory effect of miR-182 on osteogenesis, indicating that miR-182 inhibits osteogenic differentiation of primary rat BMSCs in rats by targeting Smad1. Finally, in vivo experimental results showed that the biomechanical characteristics of bone tissues in POP rats were significantly enhanced by miR-182 inhibition, while they were significantly weakened by miR-182 overexpression. MiR-182 inhibits osteogenic differentiation of rat BMSCs, thus aggravating POP in rats.

Moringa oleifera leaf extracts protect BMSC osteogenic induction following peroxidative damage by activating the PI3K/Akt/Foxo1 pathway

ObjectiveWe aimed to investigate the therapeutic effects of Moringa oleifera leaf extracts on osteogenic induction of rat bone marrow mesenchymal stem cells (BMSCs) following peroxidative damage and to explore the underlying mechanisms.MethodsConditioned medium was used to induce osteogenic differentiation of BMSCs, which were treated with H2O2, Moringa oleifera leaf extracts-containing serum, or the phosphatidyl inositol-3 kinase (PI3K) inhibitor wortmannin, alone or in combination. Cell viability was measured using the MTT assay. Cell cycle was assayed using flow cytometry. Expression levels of Akt, phosphorylated (p)Akt, Foxo1, and cleaved caspase-3 were analyzed using western blot analysis. The mRNA levels of osteogenesis-associated genes, including alkaline phosphatase (ALP), collagen І, osteopontin (OPN), and Runx2, were detected using qRT-PCR. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and ALP activity were detected using commercially available kits. Osteogenic differentiation capability was determined using alizarin red staining.ResultsDuring osteogenic induction of rat BMSCs, H2O2 reduced cell viability and proliferation, inhibited osteogenesis, increased ROS and MDA levels, and decreased SOD and GSH-PX activity. H2O2 significantly reduced pAkt and Foxo1 expression, and increased cleaved caspase-3 levels in BMSCs. Additional treatments with Moringa oleifera leaf extracts partially reversed the H2O2-induced changes. Wortmannin partially attenuated the effects of Moringa oleifera leaf extracts on protein expression of Foxo1, pAkt, and cleaved caspase-3, as well as mRNA levels of osteogenesis-associated genes.ConclusionMoringa oleifera leaf extracts ameliorate peroxidative damage and enhance osteogenic induction of rat BMSCs by activating the PI3K/Akt/Foxo1 pathway.

Bone Morphogenetic Protein-7 (BMP-7) Promotes Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs) In Vitro.

This study is aimed at investigating the effects of bone morphogenetic protein-7 (BMP-7) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells in vitro. The rat BMSCs were isolated and identified, which were divided into the control, empty, recombinant rhBMP-7 transfection, and Lv-BMP-7 transfection groups. BMSCs were induced under different conditions. CCK-8 assay was performed to detect cell proliferation. ALP was used to detect cell activity. Cellular morphology after induction was observed. Immunofluorescence was conducted to detect the expression and location of nerve cell markers. Quantitative real-time PCR and Western blot analysis were performed to detect the mRNA and protein expression levels, respectively. The rhBMP-7 and Lv-BMP-7 promoted the proliferation of BMSCs, accompanied with increased ALP activities. Morphological observations revealed that rhBMP-7 and Lv-BMP-7 induced BMSCs to differentiate into neuron-like cells. Immunofluorescence revealed that the rhBMP-7 and Lv-BMP-7 groups showed positive expression of MAP-2 and Nfh in BMSCs. MAP-2 was mainly distributed in the cell body and cellular protrusion, while Nfh was mainly distributed in the cytoplasm and cell protrusion. Positive mRNA and protein expressions of MAP-2 and Nfh were observed in the cells of the rhBMP-7 and Lv-BMP-7 groups, and the expression levels were significantly higher than the control and empty groups. Both exogenous BMP-7 (rhBMP-7) and endogenous BMP-7 (Lv-BMP-7) can induce BMSCs to differentiate into neuron-like cells highly expressing the neuronal markers MAP-2 and Nfh.

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold.

BACKGROUNDCollagen is one of the most commonly used natural biomaterials for tendon tissue engineering. One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment, proliferation, and tenogenic differentiation of cells. However, there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in a collagen sponge-based 3D culture system.AIMTo identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODSWe constructed a 3D culture system based on a type I collagen sponge scaffold. The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy. Primary BMSCs were isolated from Sprague-Dawley rats. Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay. The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot, respectively. The deposited collagen was assessed by Sirius Red staining.RESULTSTransforming growth factor β1 (TGF-β1) showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7 (GDF-7) and insulin-like growth factor 1 (IGF-1) in both the 2D and 3D cultures, and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment. In the 2D culture, the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1, IGF-1, or GDF-7 treatment. However, TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture. Strangely, we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1. Moreover, TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSIONCollagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.

Icariin Promotes the Osteogenesis of Bone Marrow Mesenchymal Stem Cells through Regulating Sclerostin and Activating the Wnt/β-Catenin Signaling Pathway.

Osteoporosis (OP) is a metabolic disease characterized by decreased bone mass and increased risk of fragility fractures, which significantly reduces the quality of life. Stem cell-based therapies, especially using bone marrow mesenchymal stem cells (BMSCs), are a promising strategy for treating OP. Nevertheless, the survival and differentiation rates of the transplanted BMSCs are low, which limits their therapeutic efficiency. Icariin (ICA) is a traditional Chinese medicine formulation that is prescribed for tonifying the kidneys. It also promotes the proliferation and osteogenic differentiation of BMSCs, although the specific mechanism remains unclear. Based on our previous research, we hypothesized that ICA promotes bone formation via the sclerostin/Wnt/β-catenin signaling pathway. We isolated rat BMSCs and transfected them with sclerostin gene (SOST) overexpressing or knockdown constructs and assessed osteogenic induction in the presence or absence of ICA. Sclerostin significantly inhibited BMSC proliferation and osteogenic differentiation, whereas the presence of ICA not only increased the number of viable BMSCs but also enhanced ALP activity and formation of calcium nodules during osteogenic induction. In addition, the osteogenic genes including Runx2, β-catenin, and c-myc as well as antioxidant factors (Prdx1, Cata, and Nqo1) were downregulated by sclerostin and restored by ICA treatment. Mechanistically, ICA exerted these effects by activating the Wnt/β-catenin pathway. In conclusion, ICA can promote the proliferation and osteogenic differentiation of BMSCs in situ and therefore may enhance the therapeutic efficiency of BMSC transplantation in OP.

Donor heart preservation with hypoxic-conditioned medium-derived from bone marrow mesenchymal stem cells improves cardiac function in a heart transplantation model

BackgroundIn heart transplantation, donor hearts inevitably suffer from ischemia/reperfusion (I/R) injury, which leads to primary graft dysfunction and affects patients’ survival rate. Bone marrow mesenchymal stem cells (BMSCs) have been reported to attenuate myocardial I/R injury via their paracrine effects, which can be enhanced by hypoxic preconditioning. We hypothesized that the donor heart preservation with hypoxic conditioned medium (CdM) derived from BMSCs would improve post-transplant graft function.MethodsNormoxic or hypoxic CdM were isolated from rat BMSCs cultured under normoxic (20% O2) or hypoxic (1% O2) condition. Donor hearts were explanted; stored in cardioplegic solution supplemented with either a medium (vehicle), normoxic CdM (N-CdM), or hypoxic CdM (H-CdM); and then heterotopically transplanted. Antibody arrays were performed to compare the differences between hypoxic and normoxic CdM.ResultsAfter heart transplantation, the donor heart preservation with normoxic CdM was associated with a shorter time to return of spontaneous contraction and left ventricular systolic diameter, lower histopathological scores, higher ejection fraction, and fractional shortening of the transplanted hearts. The cardioprotective effects may be associated with the inhibition of apoptosis and inflammation, as reflected by less TUNEL-positive cells and lower levels of plasma proinflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α) and cardiac troponin I in the N-CdM group compared with the vehicle group. These therapeutic effects can be further enhanced by hypoxic preconditioning. Antibody arrays revealed that nine proteins were significantly increased in hypoxic CdM compared with normoxic CdM. Furthermore, compared with vehicle and N-CdM groups, the protein levels of PI3K and p-Akt/Akt ratio in the transplanted hearts significantly increased in the H-CdM group. However, no significant difference was found in the phosphorylation of Smad2 and Smad3 for the donor hearts among the three groups.ConclusionsOur results indicate that the cardioplegic solution-enriched with hypoxic CdM can be a novel and promising preservation solution for donor hearts.

EMF frequency dependent differentiation of rat bone marrow mesenchymal stem cells to astrocyte cells

The numerous factors regulate the bone marrow mesenchymal stem cell (BMMSC) self-renewal and differentiation response. We aimed to analyze the influence of electromagnetic field (EMF) as an external inducing factor on rat BMMSC differentiation and proliferation to neuron and astrocyte cells. BMMSCs extracted from the rats femurs and tibias and incubated in a cell-cultured CO2 incubator. After the third passages, the plates selected randomly and then divided into seven groups (Sham exposed, three groups of square, and three groups of sinusoidal waveform EMF (25, 50, and 75 Hz, 400 μT, 1 h/day). The BMMSCs exposed to EMF at the middle of a Helmholtz coil for 7 days. The viable cell counting and proliferation performed by the MTT test and BMMSC differentiation into the neuron and the astrocyte cell was studied by immunocytochemistry staining.The results confirmed BMMSC viability and proliferation rate reduction in sinusoidal 25 Hz, square 50 Hz and sinusoidal 75 Hz EMF groups compare to sham. The maximum BMMSC differentiation to neuron was considered in sinusoidal 50 Hz and 75 Hz EMF groups. The increase of BMMSC differentiation to astrocyte cell was frequency dependent and the most differentiation was shown in square 75 Hz, and sinusoidal 75 Hz EMF groups. In conclusion, the results suggest that both square and sinusoidal EMF could affect BMMSC development and differentiation to neuron and astrocyte cells. Further studies for the consequence of EMF with wider flux density and frequency on BMMSC are recommended.

Synthesis of aligned porous polyethylene glycol/silk fibroin/hydroxyapatite scaffolds for osteoinduction in bone tissue engineering

BackgroundThe physical factors of the extracellular matrix have a profound influence on the differentiation behavior of mesenchymal stem cells. In this study, the effect of the biophysical microenvironment on rat bone marrow mesenchymal stem cell (BMSC) osteogenesis was studied both in vitro and in vivo.MethodsTo prepare cell culture scaffolds of varying stiffness, increasing amounts of hydroxyapatite (HAp) were mixed into a polyethylene glycol/silk fibroin (PEG/SF) solution. The amount of HAp ranged from 25 to 100 mg, which provided for different ratios between HAp and the PEG/SF composite. In vitro, the effect of stiffness on the osteogenic differentiation of rat BMSCs was studied. The outcome measures, which were verified in vivo, included the protein expression of runt-related transcription factor 2 and osteocalcin, alkaline phosphatase activity, and the mRNA expression of osteogenesis-related markers.ResultsIncreasing amounts of HAp resulted in an increased elastic modulus of the cell culture scaffolds. The PEG/SF/HAp fabricated with HAp (50 mg) significantly increased cell adhesion and viability (p < 0.05) as well as the expression of all the osteogenesis-related markers (p < 0.05).ConclusionsWe developed a novel cell culture scaffold and demonstrated that substrate stiffness influenced the osteogenic differentiation of rat BMSCs.