Mixed culture of human iPSCs with rabbit corneal endothelial cells induces differentiation of iPSCs

AIM:To investigate the morphology and protein expression of human induced pluripotent stem cells(iPSCs) in the mixed-culture environment with rabbit corneal endothelial cells(CECs) and to provide the experimental basis and the mechanism of iPSC differentiation into CECs. METHODS:Primary rabbit CECs were isolated with trypsin and subcultured. The human iPSCs were cultured and amplified by a feeder-free method and their characteristics were evaluated by Western blotting. iPSCs labeled with quantum dots of appropriate concentration were used to establish mixed-culture model with rabbit CECs. The morphology of iPSCs was evaluated by atomic force microscopy(AFM) and inverted microscopy. The protein expression of CD31, CD34, CD133 and aquaporin 1(AQP1) in iPSCs was tested by the method of immunofluorescence. RESULTS:The rabbit CECs were hexagonal and showed a typical cobblestone appearance. iPSCs grew in the cloning form, and 3 pluripotent proteins Oct4, Nanog and Sox2 were expressed positively. 1/4 suspension of iPSCs labeled with 10 nmol/L quantum dots and 60% confluence of rabbit CECs made best mixed culture for each other. Under AFM and inverted microscope, the volume of iPSC became bigger and the nuclear-cytoplasm ratio was decreased after 7 days of mixed culture. Some granular protrusions of the membrane were observed and the surface roughness of the cell membrane increased. The protein expression of CD31, CD34, CD133 and AQP1 in iPSCs was negative, while AQP1 was detected after mixed culture for 2 weeks. CONCLUSION:iPSCs morphologically change to endothelial-like cells after mixed culture with rabbit CECs and express the marker protein AQP1 of CECs at the same time.     

Generation,identification and differentiation of primary ovarian insufficiency patient-derived iPSCs

AIM: To generate and identify primary ovarian insufficiency (POI) patient-derived induced pluripotent stem cells (iPSCs) and to explore their differentiation potential to primordial germ cells. METHODS: Plasmid pEB-C5 expressing reprogramming factors Oct4, Sox2, c-Myc, Klf4 and Lin28, and plasmid pEB-Tg expressing SV40 T antigen, were transfected into peripheral blood mononuclear cells (PBMCs) derived from POI patients at the same time. PBMCs were reprogrammed into iPSCs, and the pluripotency of the cells was identified. After supplementation of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), the mRNA and protein expression of primordial germ cell markers was detected by real-time PCR and Western blot, respectively. RESULTS: iPSCs derived from the PBMCs of POI patient differentiated into 3-germ layer cells and maintained pluripotency by the detection of alkaline phosphatase staining, immunofluorescence, embryoid body and teratoma formation. After addition of TGF-β1 and BMP4, the primordial germ cell markers, including stem cell growth factor receptor (c-Kit), developmental pluripotency-associated 3 (STELLA/DPPA3) and DEAD box polypeptide 4 (VASA/DDX4) were increased at mRNA level (P<0.05), and VASA/DDX4 was also up-regulated at protein level in induced group. CONCLUSION: PBMCs of POI patient are reprogrammed into integration-free iPSCs in vitro and maintain pluripotency. They differentiate into primordial germ cells by adding TGF-β1 and BMP4.     

Differentiation of mouse iPSCs into insulin-producing cells and experimental study on treating diabetes

AIM: To induce mouse induced pluripotent stem cells (iPSCs) to differentiate into insulin-producing cells (IPCs) by a new 3-step method, and to detect the efficiency and maturity for the treatment of diabetic mice. METHODS: We constructed iPSCs from mouse embryonic fibroblasts of male C57/C mouse by piggyBac transposon, then induced the iPSCs into IPCs by a 3-step method. The cell morphological change was traced by microscopy during the process of differentiation. The expression of mRNA and protein associated with islet β cell development was determined by real-time PCR and immunofluorescence staining. Flow cytometry was used to analysis the efficiency of differentiation. Insulin and C-peptide secretions of IPCs in response to glucose at high (25 mmol/L) or low (5.5 mmol/L) level were measured by ELISA. The IPCs were transplanted into the capsul of left kidney in the male C57/C diabetic mouse model. Blood glucose was continuously monitored for 28 day, serum insulin was tested by ELISA in different stages. The glucose tolerance test was performed on the 28th day, and the left kidney was excised. RESULTS: IPCs were obtained from mouse iPSCs by the 3-step method. The cells expressed the marker genes (Pdx1, Ngn3, Pax6 and Ins2) and proteins (Pdx1, Nkx6.1 and insulin) of β cells. The glucose stimulation induced the secretion of insulin and C-peptide. The efficiency of differentiation was 28% detected by flow cytometry. After transplantation of IPCs to the diabetic mice, the blood glucose was decreased to normal level on the 3rd day,and serum insulin level and the ability of regulating glucose were improved. IPCs were still alive after 28 d of transplantation by pathological observation. CONCLUSION: iPSCs is efficiently induced into IPCs by a 3-step method , and the induction time is shortened significantly. The hyperglycemia of diabetes mice is reversed after transplanting IPCs to same sex inbred strain mice.     

Generation of thalassemia-specific integration-free induced pluripotent stem cells and determination of their differentiation ability

AIM: To generate thalassemia-specific integration-free induced pluripotent stem cells(iPSC) and to detect their ability of differentiation into hematopoietic precursors.METHODS: The plasmids pEB-C5 and pEB-Tg were transfected into the fibroblast cells from hemoglobin Bart's hydrops fetalis's skin by the method of nuclear transfection to reprogramm the cells into iPSC. The ability of the iPSC to differentiate into 3-germ layer cells was determined. The iPSC were cocultured with mouse OP9 cells to differentiate into hematopoietic precursors and the hematopoietic precursor specific antigens were detected. RESULTS: The integration-free iPSC from hemoglobin Bart's hydrops fetalis's skin fibroblasts were successfully derived, and had the ability to differentiate into 3 germ layers. When cocultured with OP9 cells for 9 d, the positive rate of hematopoietic progenitor cell marker CD34 was 18.7%, and the CD34 and CD45 double positive rate was 12.2%. CONCLUSION: Hemoglobin Bart's hydrops fetalis's skin fibroblasts can be successfully induced into "integration-free" iPSC. This cell line has the ability to differentiate into 3 germ layers, and can be differentiated into hematopoietic precursors when cocultured with OP9 cells.     

Comparison of 2 methods for inducing iPSC to differentiate into neural stem cells

AIM: To select an efficient way of promoting induced pluripotent stem cells (iPSC) to differentiate into neural stem cells (NSC) by comparing 2 methods. METHODS: The culture system in method A contained SB431542 (5 mmol/L) and drosomophorin (5 mmol/L) with 100% initial cell density, while that in method B contained SB431542 (5 mmol/L) and drosomophorin (1 mmol/L) with 30%~50% initial cell density. For comparison and identification of the 2 methods, the growth state was observed under microscope, and the expression of Pax6, nestin, Sox1 and Sox2 was quantitatively detected by real-time PCR and flow cytometry. The related protein expression and the ability of spontaneous differentiation were determined by immunofluorescence analysis. RESULTS: The cells derived from method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density achieved the higher expression of Pax6, nestin, Sox1 and Sox2. The growth state was better and the cells differentiated into neurons and astrocytes normally. CONCLUSION: The method A was superior to method B, and we recommend the method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density as the method for differentiating NSC.     

Roles of reactive oxygen species in pluripotent stem cell functions

Pluripotent stem cells are characterized by the properties of self-renewal and the ability to differentiate into multiple cell types. Reactive oxygen species (ROS) are highly reactive metabolites. High levels of ROS are toxic and involved in stem cell senescence and apoptosis. However, regulation of ROS has an important role in maintaining “stemness” and differentiation of the stem cells. The role of ROS in the stem cells varies among different stem cell types. NADPH oxidase is one of the major sources of ROS in stem cells. Excessive amounts of ROS are produced in various pathophysiological states such as atherosclerosis, heart failure, hypertension, diabetes, and aging. Induced pluripotent stem cells have the potential to be used in modeling of ROS-associated diseases.Understanding the molecular mechanisms how ROS regulate the functions of stem cells will greatly enhance their translational applications. In this review, we summarize the recent progress regarding the roles of ROS in regulating the functions of embryonic and induced pluripotent stem cells.     

Research progress in reprogramming induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have been first induced from mouse fibroblasts since 2006, and the research on iPSCs has made great progress in the following years. iPS cell lines were established from different somatic cells through DNA, RNA, protein, and small molecule compounds and various methods of transduction, making the induction of iPSCs more secure and effective, and more attractive prospect of clinical application. In this review, different somatic cell reprogramming, different levels of reprogramming, different transduction pathways, and prospect of application are discussed.     

Generation of induced pluripotent stem cells and neural cells from urine-derived cells of Alzheimer disease patients

AIM: In this study, we aim to obtain the induced pluripotent stem cells (iPSCs) from the patients with sporadic Alzheimer disease (AD). METHODS: Three typical Alzheimer's patients were chosen, and the epithelial cells were isolated from their urine. We reprogrammed these cells into induced pluripotent stem cells by transfection of 4 factors (Oct4, Sox2, Klf4 and SV40LT) with the technique of electro-transfection. After getting these iPSCs, we continue to differentiate them into neural cells by a specific method—dual inhibition of Smad signaling. RESULTS: The primary cells from 3 AD patients were successfully reprogrammed to iPSCs, and these patients-derived iPSCs were differentiated into neural cells. There was no significant difference, during iPSCs reprogramming and neural differentiation, between cells from AD patients and normal people. CONCLUSION: The urine cells from AD patients were able to transfer to iPSCs, functional neurons and neurogliocytes.     

Effects of maxadilan on proliferation of human induced pluripotent stem cells

AIM: To investigate the promoting effect of maxadilan, which specifically activate the type I receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), on the proliferation of human induced pluripotent stem cells (IPSCs). METHODS: PACAP type I (PAC1) receptor in IPSCs was detected by RT-PCR and Western blotting. maxadilan at various concentrations was added to the medium of IPSCs as experimental groups. The medium in control group was without maxadilan treatment. The effect of maxadilan on theproliferation of IPSCs was measured with Cell Counting Kit-8 (CCK-8). The changes of cell cycle caused by maxadilan in IPSCs were analyzed by flow cytometry. The analysis of karyotype was carried out in IPSCs treated with maxadilan. Proteins and gene expression levels of both Nanog and OCT4 in IPSCs treated with maxadilan were detected by real-time quantitative polymerase chain reaction (real-time-qPCR) and immunofluorescence. The gene expression levels of Nestin and PAX6 in both IPSCs treated with maxadilan and cells of embryonic body, which was birthed from IPSCs with maxadilan treatment, were detected by real-time qPCR. The ability of IPSCs treated with maxadilan differentiating into 3 embryonic layers was evaluated by analyzing the component of embryo using RT-PCR. RESULTS: The PAC1 receptor in IPSCs was identified by RT-PCR and Western blotting. Viability of the IPSCs with 100 nmol/L maxadilan treatment was increased by 16% compared with control group. The differences with statistical significance were found in the cell viability between 100 nmol/L maxadilan treatment group and control group (P<0.05). The average values of proliferation index (PI) in IPSCs with 100 nmol/L maxadilan treatment for 3 h, 6 h and 9 h were 47.23%, 59.70% and 55.67%,respectively, while that in control group was 37.00%. The differences with statistical significance were found in PI between 100 nmol/L maxadilan treatment for 3 h group, 6 h group, 9 h group and control group (P<0.05). Normal karyotype and unchanged pluripotent state in IPSCs treated with maxadilan were observed. Compared with control group, the gene expression levels of Nestin and PAX6 were not significantly different in both IPSCs and the cells of embryonic body birthed from IPSCs with maxadilan treatment. The ability of differentiation into 3 embryonic layers in IPSCs treated with 100 nmol/L maxadilan was found. CONCLUSION: PAC1 receptor presents in IPSCs. Maxadilan promotes the proliferation of IPSCs but does not affect their pluripotent state and karyotype.     

Effects of iPSC-MSCs on mitochondria of PC12 cells injured by CoCl2

AIM: To investigate the effects of induced pluripotent stem cells-derived mesenchymal stem cells (iPSC-MSCs) on cobalt chloride (CoCl₂)-induced injuries of PC12 cells and its possible mechanism. METHODS: PC12 cells were exposed to CoCl₂ to set up a chemical-induced cellular injury model and were cocultured with iPSC-MSCs. The cell viability was tested by CCK-8 assay. The apoptosis was measured by flow cytometry using Annexin V/PI staining. The mitochondrial membrane potential (MMP) was analyzed by flow cytometry using JC-1 staining. Immunofluorescence was employed to observe mitochondrial transfer from iPSC-MSCs to PC12 cells. RESULTS: Apoptosis of PC12 cells was increased and MMP of PC12 cells was decreased after exposed to CoCl₂ at concentration of 400 μmol/L for 24 h. Coculture of PC12 cells with iPSC-MSCs reduced the apoptosis and recovered the MMP of the PC12 cells. Tunneling nanotubes were formed between iPSC-MSCs and PC12 cells, through which the iPSC-MSCs transferred the mitochondria to the PC12 cells. CONCLUSION: iPSC-MSCs protect PC12 cells from CoCl₂-induced injuries, which may be associated with the mitochondrial transfer from iPSC-MSCs to PC12 cells.     

Differentiation of neurons from mouse induced pluripotent stem cells in vitro

AIM: To study the induction method of mouse induced pluripotent stem cells (iPSCs) that differentiate into neurons in vitro. METHODS: Mouse iPSCs were cultured in non-adherent culture dishes for 2 d to form embryoid bodies (EBs). The EBs were cultured for consecutive 2 d in the presence of retinoic acid (RA), and then were plated in the serum-free medium for adherent culture. Seven days later, Pasteur pipette was used to detach the differentiated cells around adherent EBs into “fragment” cell colonies with the help of dissecting microscopes, and these “fragments” were transferred to culture dishes with neural stem cell medium. Another 7 days later, the cells were plated onto the culture dishes using differentiation medium containing fetal bovine serum (FBS) and RA. The morphological changes of the cells were observed under inverted microscope. The iPSCs markers Oct4, Sox2 and SSEA1, the neural stem cell (NSC) marker nestin, the neuronal marker microtubule-associated protein 2 (MAP-2), the astrocyte marker glial fibrillary acidic protein (GFAP) and oligodendrocyte marker myelin basic protein (MBP) were detected by immunofluorescence method. The mRNA expression of GFAP, nestin, β3-tubulin, MAP-2 and MBP was detected by RT-RCR. MAP-2 gene sequence was identified. The proportions of NSCs differentiated from iPSCs and neurons from NSCs were detected by flow cytometry. RESULTS: Mouse iPSCs strongly expressed Oct4, Sox2 and SSEA1, and formed spherical EBs by suspended culture. The EBs were induced by RA and serum-free medium in adherent culture for 2 d, and rosette structure was observed under the microscope. “Fragments” separated by Pasteur pipette from the rosette structure formed neurosphere-like colonies. After the colonies were cultured in adherent condition for 5 d to 7 d in the presence of RA and FBS, the typical neurite was observed under the microscope. The neurospheres expressed nestin and their differentiated derivatives expressed MAP-2, GFAP and MBP, respectively. RT-PCR analysis and gene sequencing showed that the neurons were induced successfully. The results of flow cytometry demonstrated that 63.93%±1.47% of iPSCs differentiated into NSCs and 21.4%±1.70% of NSCs differentiated into neurons. CONCLUSION: Mouse iPSCs proliferate stably and differentiate into neurons in vitro, which provide a reliable source for the treatment of spinal cord injury.     

Relationship between apoptosis and differentiation from ESCs into cardiomyocytes induced by TGF-β1 and co-culture with visceral endoderm like END-2 cells

AIM: To provide the reference for optimizing the seed cells for tissue engineering,the relationship between apoptosis and differentiation in the process of induction on embryonic stem cells(ESCs) was investigated.METHODS: Day 2-3 embryoid bodys (EBs) were derived from ESCs,and then tissue growth factor-β₁(TGF-β₁) was added,and co-cultured with visceral endoderm like END-2 cell conditioned medium.The induced cells were evaluated by using immunofluoresence and transmission electron micrography.Cell apoptosis was analyzed using flow cytometry.RESULTS: The total percentage of beating EBs treated with TGF-β₁ was 43%.All the beating cardiomyocytes derived from ESCs expressed cardiac-specific proteins for TnT,and were observed the cardiac-specific ultrastructure.Interestingly,the total percentage of beating EBs treated with TGF-β₁ combined with END-2 cell conditioned medium was 88% (P＜0.01),and the beating areas were bigger.After 3 days of induction with different conditions,the apoptotic levels were (5.58%±0.65% and 9.60%±0.75%,P＜0.05),respectively.CONCLUSION: combination of TGF-β₁ with co-cultured visceral endoderm like END-2 cell conditioned medium could get a higher induction efficiency on the differentiation of ESCs into the cardiomyocytes.The partly inducible effect mechanisms may induce EDCs differentiation toward a cardiac phenotype and enhance apoptosis in cells not committed to cardiac differentiation.     

Effect of VEGF on proliferation and differentiation of neural stem cells from the hippocampal gyrus of rat in vitro

AIM: To observe the effects of vascular endothelial growth factor (VEGF) on the proliferation and differentiation of neural stem cells (NSCs) of rats in vitro.METHODS: NSCs isolated from the hippocampal gyrus of SD rats were primary cultured and subcultured,and then divided into two groups: (1) the cells in VEGF group were treated with 150 μg/L VEGF in the culture system,and VEGF was removed at the 7 th day;(2) control group (without VEGF treatment).The cellular morphology of two groups was observed by contrast phase microscope.Nestin and NF-200 expressing cells were detected via immunofluorescence method.The percentages of the immunostaining positive cells in each group at the 7 th day and at the 11 th day were determined.RESULTS: At the 7 th day,the percentage of nestin positive cells in VEGF group was 52.19%±7.95%,vs 29.26%±4.12% in control group (P<0.01).The percentage of NF positive cells in VEGF group was 22.33%±4.13%,vs 38.62%±5.31% in control group (P<0.01).At the 3 th day after VEGF was removed,the percentage of NF positive cells in VEGF group was 43.10%±3.70%,vs 30.56%±4.16% in control group (P<0.01).CONCLUSION: VEGF stimulates the proliferation of neural stem cells and inhibits their differentiation.     

Lithium chloride promotes neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy

AIM: To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process. METHODS: MSCs were isolated and cultured in vitro. The cells were divided into LiCl group and control group. MSCs were treated with β-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons. The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associated protein 1 light chain 3 (LC3) were measured by immunofluorescence method and Western blot. An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells. The protein expression of NSE and MAP-2 were determined by Western blot. RESULTS: After induction, the expression of NSE and MAP-2 were increased. The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05). After induction, the number of LC3-positive dots and the expression of LC3-Ⅱ in LiCl group were greater than those in control group (P<0.05). The expression of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated-cells, and on the contrary, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA. CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy.     

Akebia saponin D promotes differentiation of bone marrow mesenchymal stem cells into osteoblasts through MAPK signaling pathways

AIM:To explore the role of Akebia saponin D(ASD) in the differentiation of rat bone marrow-derived mesenchymal stem cells(BMSCs) into osteoblasts. METHODS:The rat BMSCs were cultured using routine methods. The effects of ASD on the differentiation of MSCs into osteoblasts were observed. The p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 and extracellular signal-regulated kinase(ERK) inhibitor PD098059 were used to evaluate the mechanisms. The activity of alkaline phosphate(ALP) and content of osteocalcin(OC) were assayed during differentiation. The mRNA expression of osteoprotegerin(OPG) and receptor activator of nuclear factor-κB ligand(RANKL) was determined by real-time fluorescence quantitative PCR. The activity of p38 MAPK and ERK was measured by Western blotting. RESULTS:Six days after treatment with ASD, the mRNA expression of OPG significantly increased, while the mRNA level of RANKL significantly decreased in induced cells. ASD increased the activity of ALP and the content of OC. Moreover, ASD enhanced the activity of both p38 MAPK and ERK, which was inhibited by SB203580 and PD098059. SB203580 and PD098059 also inhibited the positive role of ASD in the differentiation of MSCs into osteoblasts. CONCLUSION:Akebia saponin D significantly enhances differentiation of rat BMSCs into osteoblasts in vitro, which may be mediated by the p38 MAPK and ERK signaling pathways.     

Estradiol promotes growth of decidua derived mesenchymal stem cells by inhibiting miR-16 via estrogen receptor α

AIM: To study the effect of estradiol (E₂) on the viability of mesenchymal stem cells (MSCs) derived from the decidua of the placenta by regulating the expression of microRNA-16 (miR-16). METHODS: The concentration of E₂ in the peripheral blood of normal pregnant women and the patients with severe preeclampsia (PE) was measured. The effects of E₂ at different concentrations on the viability of MSCs were analyzed. The effect of E₂ at different concentrations on the expression of miR-16 in the MSCs was detected, and which estrogen receptor (ER) mediated the regulatory effect of E₂ on miR-16 expression was determined. RESULTS: The concentration of E₂ in peripheral blood of the patients with severe PE was significantly decreased (P<0.01). After treatment with E₂ at 5, 10 and 100 nmol/L for 48 h, the viability of MSCs was increased (P<0.05). The expression level of miR-16 was down-regulated in the MSCs treated with E₂ at 5, 10 and 100 nmol/L for 12 h. After treatment with E₂ at 10 nmol/L for different time (0 h, 3 h, 6 h, 12 h and 24 h), the expression level of miR-16 in the MSCs showed a clear time-dependent downward trend. E₂ significantly promoted the viability of MSCs, and the cell viability was significantly reversed after miR-16 pretreatment. Pretreatment with estrogen receptor antagonists ICI 182780 and tamoxifen for 6 h attenuated the inhibitory effect of E₂ on miR-16 expression. Only ERα agonist propyl pyrazole triol significantly inhibited the expression of miR-16 in MSCs but ERβ agonist diarylpropionitrile did not. CONCLUSION: E₂ promotes the growth of decidua-derived MSCs by inhibiting miR-16 via ERα.     

Strontium ranelate promotes differentiation of bone marrow mesenchymal stem cells to osteoblasts by increasing expression of bone morphogenetic protein 2

AIM: To explore whether strontium ranelate (Sr) promotes differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts by increasing the expression of bone morphogenetic protein 2 (BMP-2). METHODS: Rat BMSCs were isolated, purified and cultured, then were induced to differentiate into osteoblasts. The cells were treated with different concentrations of Sr or noggin (an inhibitor of BMP-2) according to the experimental purposes. The activity of alkaline phosphatase (ALP) was measured by colorimetry. Mineralized nodules were measured by alizarin red staining. The expression of BMP-2 was detected by Western blotting. RESULTS: Treatment with Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d obviously increased the activity of ALP,and Sr at concentration of 3 mmol/L produced the maximum effect. Exposure of the cells to Sr at concentration of 3 mmol/L for 21 d significantly increased mineralized nodules. Exposure of the cells to Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d markedly increased the expression of BMP-2. Preconditioning with noggin at concentration of 100 μg/L for 2 h not only inhibited Sr-induced expression of BMP-2, but also antagonized the increase in the activity of ALP and mineralization induced by Sr in BMSCs. CONCLUSION: Up-regulation of the expression of BMP-2 may be one of the mechanisms by which Sr promotes differentiation of rat BMSCs to osteoblasts.