O-linkedβ-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells

Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.

Neurotrophic Effect of Bone Proliferation and Committed Mesencephalic Precursors Marrow Stromal Cells on Differentiation of Ventral

OBJECTIVE To explore the potential neurotrophic effect of bone marrow stromal cells （BMSCs） on cell proliferation and committed neuronal differentiation of ventral mesencephalic precursors （VMPs） in vitro. METHODS Ventral mesencephalic precursors from Ell inbred rat embryos and BMSCs from adult rats were cultured both separately and in co-culture. After a 7-day incubation in vitro, three conditioned culture media were obtained, termed VMP or common medium, BMSC medium, and BMSC±VMP medium. Ventral mesen- cephalic precursors cells were cultured in each of these media and the effects on proliferation and VMP differentiation were assessed. The relative yield of TH± cells was calculated and compared by immunocytochemical staining. RESULTS After a 7-day culture and induction of VMPs, the total cell counts were increased by （44.13±4.75）-fold （common）, （60.63±5.25）-fold （BMSC）, and （64.00±7.63）-fold （BMSC±VMP）. The proportions of TH＋ cells were （18.76±5.20）%, （23.49±4.10）%, and （28.08± 5.42）%, respectively, with statistically significant differences among the treatment groups. CONCLUSION BMSCs release factors that promote the proliferation of VMPs and facilitate the committed differentiation of VMPs into dopaminergic neurons.

Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.

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

BACKGROUND Collagen 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.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We 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.RESULTS Transforming growth factorβ1(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.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.

Adipose-derived stromal cells resemble bone marrow stromal cells in hepatocyte differentiation potential in vitro and in vivo

AIM To investigate whether mesenchymal stem cells(MSCs) from adipose-derived stromal cells(ADSCs) and bone marrow stromal cells(BMSCs) have similar hepatic differentiation potential.METHODS Mouse ADSCs and BMSCs were isolated and cultured. Their morphological and phenotypic characteristics, as well as their multiple differentiation capacity were compared. A new culture system was established to induce ADSCs and BMSCs into functional hepatocytes. Reverse transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to identify the induced hepatocytelike cells. CM-Dil-labeled ADSCs and BMSCs were then transplanted into a mouse model of CCl4-induced acute liver failure. fluorescence microscopy was used to track the transplanted MSCs. Liver function was tested by an automatic biochemistry analyzer, and liver tissue histology was observed by hematoxylin and eosin(HE) staining.RESULTS ADSCs and BMSCs shared a similar morphology and multiple differentiation capacity, as well as a similar phenotype(with expression of CD29 and CD90 and no expression of CD11 b or CD45). Morphologically, ADSCs and BMSCs became round and epithelioid following hepatic induction. These two cell types differentiated into hepatocyte-like cells with similar expression of albumin, cytokeratin 18, cytokeratin 19, alpha fetoprotein, and cytochrome P450. fluorescence microscopy revealed that both ADSCs and BMSCs were observed in the mouse liver at different time points. Compared to the control group, both the function of the injured livers and HE staining showed significant improvement in the ADSC-and BMSC-transplanted mice. There was no significant difference between the two MSC groups.CONCLUSION ADSCs share a similar hepatic differentiation capacity and therapeutic effect with BMSCs in an acute liver failure model. ADSCs may represent an ideal seed cell type for cell transplantation or a bio-artificial liver support system.

In vitro differentiation of adipose-derived stem cells and bone marrow-derived stromal stem cells into neuronal-like cells

Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.

Tissue Extracts From Infarcted Myocardium of Rats in Promoting the Differentiation of Bone Marrow Stromal Cells Into Cardiomyocyte-like Cells

Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells （BMSCs） into cardiomyocytes. Methods Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract （IMTE）, normal myocardial tissue extract （NMTE） and cultured neonatal myocardial lysate （NML）] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction （RT-PCR） detection, immunocytochemical analysis, and transmission electron microscopy. Results After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Conclusions Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.

Neuronal differentiation effects of vascular endothelial factor on bone marrow stromal cells

BACKGROUND：Studies have demonstrated that bone marrow stromal cells （BMSCs） undergo neuronal differentiation under certain in vitro conditions.However,very few inducers of BMSC differentiation have been used in clinical application.The effects of vascular endothelial growth factor （VEGF） on in vitro neuronal differentiation of BMSCs remain poorly understood.OBJECTIVE：To investigate the effect of VEGF on neuronal differentiation of BMSCs in vitro,and to determine the best VEGF concentration for experimental induction.DESIGN,TIME AND SETTING：In vitro comparative study was performed at the Central Laboratory and Laboratory of Male Reproductive Medicine,Shenzhen Hospital of Peking University from October 2008 to August 2009.MATERIALS：Recombinant human VEGF165 was purchased from Peprotech Asia,Rehovot,Israel.Neuron-specific enolase （NSE） was purchased from Beijing Biosynthesis Biotechnology,China.METHODS：BMSCs were harvested from adult Sprague Dawley rats.The passaged cells were pre-induced with 10 ng/mL basic fibroblast growth factor for 24 hours,followed by differentiation induction with 0,5,10,and 20 ng/mL VEGF,respectively.MAIN OUTCOME MEASURES：Morphological changes in BMSCs prior to and following VEGF induction.Expression of NSE following induction was determined by immunocytochemistry.RESULTS：Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20 ng/mL VEGF.However,these changes were not observed in the control group.At 10 days after induction,the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group （P〈 0.05）.The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction （P〈 0.05）.Moreover,the number of NSE-positive cells was greater at 10 days compared with at 3 days after induction （P〈 0.05）.CONCLUSION：Of the VEGF concentrations tested,10 ng/mL induced the greatest number of neuronal-like cells in vitro from BMSCs.

Effects of corneal stromal cell-and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM： To explore the effects of conditioned media on the proliferation of corneal endothelial cells （CECa） and to compare the efficiency of different conditioned media （CM）. METHODS： Rat CECs, corneal stromal cells （CSCs）, bone marrow -derived endothelial progenitor cells （BEPCs）, and bone marrow-derived mesenchymal stem cells （BMSCs） were isolated and cultured in vitra CM was collected from CSCs, BEPCs, and BMSCSo CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed.~ RESULTS： After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone- like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na＋/K＋-ATP, aquaporin 1 （AQP1）, and zonula occludens 1 （ZO-1）. Based on quantitative polymerase chain reaction （qPCR） analysis, Na ＋/K ＋-ATP expression in CSC-CM was notably upregulated by 1.3-fold （＋0.036） （P〈0.05, n=3）. The expression levels of ZO-1, neuron specific enolase （NSE）, Vimentin, paired homebox 6 （PAX6）, and procollagen type VII （COL8A1） were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation.~ CONCLUSION： CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. KEYWORDS： conditioned medium; corneal endothelial cell; corneal stromal cell; bone marrow-derived endothelial progenitor cell; proliferation

Synergistic effects of brain-derived neurotrophic factor and retinoic acid on inducing the differentiation of bone marrow stromal cells into neuron-like cells in adult rats in vitro

BACKGROUND： Under induction of retinoic acid （RA）, bone marrow stromal cells （BMSCs） can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor （BDNF） can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE： To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN： Randomized controlled animal study SETTING： Department of Neurology, Affiliated Hospital of Xuzhou Medical College MATERIALS： The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification： SYXK （su） 2002-0038]. Materials and reagents： low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein （GFAP） antibody, SABC kit, and diaminobenzidine （DAB） color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS： Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group （primary culture）, BDNF group （20 μg/L BDNF） and BDNF＋RA group （20 μg/L BDNF plus 20 μg/L RA）. On the 3^rd and the 7^th days after induction, BMSCs were stained immunocytochemically with nestin （sign of nerve stem cells）, neuron-specific enolase （NSE, sign of diagnosing neurons） and GFAP （diagnosing astrocyte）, and evaluated cellular property. MAIN OUTCOME MEASURES ： Induction and differentiation in vitro of BMSCs in 3 groups RESULTS： （1） Induction and differentiation of BMSCs： Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. （2） Results of immunocytochemical detection： Three days after induction, rate of positive cells in BDNF＋RA group was higher than that in BDNF group and control group [（86.15±4.58）%, （65.43±4.23）%, （4.18±1.09）%, P 〈 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF＋RA group than that in both groups at 3 days after induction [（31.12±3.18）%, （29.35±2.69）%, P 〈 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction （P 〈 0.01）; meanwhile, the amount in BDNF＋RA group was remarkably higher than that in BDNF group （P 〈 0.01）. CONCLUSION： Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.

Effects of neurotrophin-3 intervention on on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis

Objective:To study the effects of neurotrophin-3 (NT-3) intervention on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis. Methods: Bone marrow mesenchymal stem cells were cultured and divided into control group, 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group, they were treated with different doses of NT-3 for 24 h, and then osteoblast marker gene, cell proliferation gene and apoptosis gene expression were determined.Results: RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression in 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group were significantly higher than those in control group whereas Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression were significantly lower than those in control group, and the larger the dose of NT-3, the higher the RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression whereas the lower the Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression.Conclusion: NT-3 intervention in bone marrow mesenchymal stem cells can promote osteoblast differentiation and cell proliferation and inhibit apoptosis.

Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells

Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed colony-forming unit-fibroblasts, and could be expanded by supple- mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from 13-galactosidase transgenic mice were also transplanted into focal ischemic brain （right corpus striatum） of C57BL/6J mice. At 8 weeks, cells positive for LacZ and 13-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both 13-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells

Age-related osteoporosis is associated with the reduced capacity of bone marrow mesenchymal stem cells （BMSCs） to differentiate into osteoblasts instead of adipocytes. However, the molecular mechanisms that decide the fate of BMSCs remain unclear. In our study, microRNA-23a, and microRNA-23b （miR-23a/b） were found to be markedly downregulated in BMSCs of aged mice and humans. The overexpression of miR-23a/b in BMSCs promoted osteogenic differentiation, whereas the inhibition of miR-23a/b increased adipogenic differentiation. Transmembrane protein 64 （Tmem64）, which has expression levels inversely related to those of miR-23a/b in aged and young mice, was identified as a major target of miR-23a/b during BMSC differentiation. In conclusion, our study suggests that miR-23a/b has a critical role in the regulation of mesenchymal lineage differentiation through the suppression of Tmem64.

Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells

Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

Gene expression profiles associated with osteoblasts differentiated from bone marrow stromal cells

Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isolated and cultured,and differentiation was induced by dexamethasone,β-glycerol phosphate and vitamin C.Cellular mRNA was extracted and reverse transcribed into cDNA,thus related genes expression differences were detected by gene expression profile chip.Results:Calcifying nodules were visible in the induced cells.There were27.7%genes expressed differentially,three times more than the normal and induced cells,and some genes were related to transcription,translation,glycosylation modification.Extracellular matrix,signal molecules and metabolism were up—regulated.Conclusions:The gene chip technique can be used to detect the multi-gene different expression in the differentiationinduceed rat BMSCs,and these differentially expressed genes are necessary genes related to rat BMSCs proliferation and induction of osteoblastic differentiation.

Chondrogenic differentiation of rat bone marrow mesenchymal stem cells induced by puerarin and tetrandrine

Objective: This study aims to clarify the effect of the active components puerarin and tetrandrine on the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods: Using network pharmacology, protein targets of puerarin and tetrandrine were predicted, and a database of cartilage formation targets was established. The protein target information related to disease was then collected, and the drug-targeting network was constructed by analyzing the protein–protein interactions. Genes related to chondrogenesis induced by puerarin and tetrandrine and chondroblast differentiation signaling pathways were searched. Finally, potential drug-and disease-related genes,as well as proteins, were screened and verified using real-time RT-PCR and western blotting.Results: Network pharmacological studies have shown that puerarin and tetrandrine are involved in BMSCs cartilage differentiation. The experimental results showed that puerarin and tetrandrine could regulate the expression of cartilage differentiation-related genes and proteins. Puerarin increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9,as well as the gene expression of Col2 a1, Mmp13, Tgfb1, and Sox-9. Tetrandrine increased the protein expression of COL2 A1,COL10 A1, MMP13, and SOX-9, as well as the gene expression of Col10 a1, Tgfb1, Sox-9, and Acan. The combination of puerarin and tetrandrine increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9 and the gene expression of Col2 a1,Col10 a1, Sox-9, and Acan.Conclusions: Puerarin, tetrandrine, and their combination can promote the proliferation of BMSCs and induce their differentiation into chondrocytes, and they are thus expected to be inducers of chondrogenic differentiation. These results suggest that puerarin and tetrandrine have potential therapeutic effects on osteoarthritis.

Bone morphogenetic protein-7 induced bone marrow stromal cells differentiate into neuron-like cells

Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for intermediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.

Experimental study on the induction of bone marrow stromal cells differentiating into cardiomyocyte-like cells with cardiomyocytes in vitro

Objective：To investigate the feasibility of bone marrow stromal cells （BMSCs） differenti ating into cardiomyocyte like cells in heterogeneous cardiomyocytes microenvironment in vitro. Methods： Mouse GFP-BMSCs were isolated by centrifugation through a Ficoll step gradient and purified by plating culture and depletion of the non-adherent cells. Neonatal rat cardiomyocytes （CMs） were isolated by enzymatic dissociation from hearts of 1-to 2-day old Sprague-Dawley （SD） rats and differentially plated to remove fibroblasts. Mouse GFP-BMSCs were cocuhured with neonatal rat CMs through direct and indirect contact, respectively. Cardiomyogenic differentiation of BMSCs was evaluated by immunostaining with an- ti-a-actin monoclonal antibody and observing synchronous contraction with adjacent CMs by phase contrast microphotography. Results： On day 7 of cocuhure, GFP-BMSCs （CMs ： BMSCs：4 ： 1）attached to nonfluorescent contracting cells （rat-derived CMs） showed myotube-like formation and started to contract synchronously with adjacent cardiomyocytes. About 10% of the fluorescent GFP-BMSCs were cardiomyocyte-like cells as determined by cell morphology and positive actin staining. Conclusion;Direct cell to-cell interaction with CMs is crucial for cardiomyogenic differentiation of BMSCs in heterogeneous CMs microenvironment in vitro. This provides a novel inducing pathway for directional differentiation of cardiovascular tissue engineering seed cells.

The effect of neuropeptides on proliferation of rat bone marrow mesenchymal stem cells

Objective To investigate the effects and mechanism of calcitonin gene-related peptide(CGRP)and substance P (SP) on proliferation of rat bone marrow mesenchymal stem cells.Methods The rBMSCs were isolated using whole bone marrow

Bone Marrow Stromal Cells Express Neural Phenotypes in vitro and Migrate in Brain After Transplantation in vivo

Objective To investigate the differentiation of bone marrow stromal cells （BMSC） into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC （hBMSC） to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilamentl （NF1）, nestin and neuron-specific enolase （NSE） in rat BMSC （rBMSC）. Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NFI and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S 100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated, rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia milliorrhiza could induce hBMSC to differentiate into neuron-like cells, If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.