Induce Bone Marrow Mesenchymal Stem Cells Differentiation Research Articles

Bone Marrow Mesenchymal Stem Cells (BMSCs) Restrain the Malignant Behaviors of A549 Lung Cancer Cells Under Hypoxia via miR-145

Deriving from bone marrow, the bone marrow mesenchymal stem cells (BMSCs) possess multipolar chemotaxis, proliferation potential, along with the capability to differentiate into various types of cells. Moreover, the hypoxic stimulation can effectively induce BMSCs differentiation. This study intends to explore the impediment of BMSCs on malignant behaviors of lung cancer stem cells under hypoxia. A co-culture system of BMSCs with A549 cells was established and then assigned into normoxia group, hypoxia group (50, 100, and 200 nmol/L) followed by analysis of cell viability by CCK-8 assay and miR-145 expression by qRT-PCR. In addition, A549 cells were grouped into NC group, miR-145-mimics group, and miR-145-inhibitors group followed by analysis of cell invasion and levels of miR-145 and Oct4. Hypoxia group exhibited a reduced cell viability and higher miR-145 expression (146.01±21.23%) compared to normoxia group (P < 0.05). Transfection of miR-145-mimic significantly upregulated miR-145 and decreased cell invasion (7.49±1.43%) compared with miR-145-inhibitors group or NC group (P < 0.05). Meanwhile, Oct4 level in miR-145-mimics group (0.934±2.98) was significantly decreased (P < 0.05). In conclusion, under hypoxia condition, the co-culture with BMSCs can upregulated miR-145 level, effectively reduce the viability of lung cancer stem cells and restrain proliferation capability.

Protein Expression Profile on Differentiation of Bone Marrow Mesenchymal Stem Cells into Retinal Ganglion-Like Cells.

To explore possible approaches to differentiating rat bone marrow mesenchymal stem cells (BMSCs) into retinal ganglion-like cells and to demonstrate the dynamic changes in protein expression profiles of BMSCs throughout the differentiation. BMSCs were isolated from adult rats and cultured in medium conditioned by neonatal rat retinal cells to induce BMSC differentiation into retinal ganglion-like cells. Immunostaining for neurofilament, nestin, Map2, and Thy1.1 was used to follow the differentiation process. Two types of protein arrays were employed to profile the BMSCs, the differentiated retinal ganglion-like cells, and the primary retinal ganglion cells (RGCs) using the Biomarker Wizard System. After 7 days of culture in conditioned medium, cells showing a neural-cell-like modality appeared. The differentiated retinal ganglion-like cells showed that network-like connections were positive for nestin, neurofilament, Map2, and Thy1.1. In total, 16 marker proteins were highly expressed in both retinal ganglion-like cells and RGCs and no obvious expression was observed in BMSCs. Among them, nine proteins were expressed more highly in RGCs than in retinal ganglion-like cells. BMSCs can be induced to differentiate into retinal ganglion-like cells by neonatal rat retinal cells, and the induced cells show protein profiles resembling those of isolated RGCs.

Knockdown of Tcf3 enhances the wound healing effect of bone marrow mesenchymal stem cells in rats.

The aim of the present study was to explore the wound healing effect of Tcf3 in rat bone marrow mesenchymal stem cells (BMSCs) and their effects on wound healing. Epidermal growth factor (EGF) and fibroblast growth factors (FGFs) were used to induce BMSCs differentiation into epithelial-like cells. Western Blotting analysis and RT-qPCR were performed to assess the expression levels of Tcf3 and the markers of epithelial-like cells, such as Cytokeratin-18 (CK-18), CK-19 and P63. Cell counting kit-8 (CCK-8) and clone formation assay were carried out to detect cell viability. Immunohistochemistry and HE staining were used to assess the level of Tcf3 protein and skin repair degree, respectively. Rat wound healing model was built to evaluate the effects of BMSCs with altered expression of Tcf3 on wound healing. Results showed that EGF and FGFs stimulation increased the expression of CK-18, CK-19 and P63, improved BMSCs viability, but decreased the expression of Tcf3. Knockdown of Tcf3 in BMSCs increased CK-18, CK-19 and P63 expression and improved cell proliferation, as well as accelerated wound healing process. Moreover, inhibition of Wnt/β-catenin signaling weakened the effect of Tcf3 down-regulation on BMSCs proliferation enhancement. And inhibition of Notch1 signaling impeded the epithelial-like cell differentiation of BMSCs induced by Tcf3 down-regulation. Our study reveals that knockdown of Tcf3 enhances the wound healing process of BMSCs in rat, which provides new approach for accelerating skin regeneration.

GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes

This study aimed to investigate whether exosomes secreted by mouse GATA-4-expressing bone marrow mesenchymal stem cells (BMSCs) could induce BMSC differentiation into myocyte precursors, decrease cardiomyocyte apoptosis, and improve cardiac function following myocardial infarction (MI). BMSCs were transduced with a lentivirus carrying a doxycycline (DOX)-inducible GATA-4 or control lentivirus, and secreted exosomes from these BMSCs were collected and co-cultured with BMSCs or cardiomyocytes under hypoxic and serum free conditions. Furthermore, exosomes were injected into mice 48 h after MI. Cardiac function was evaluated by echocardiography at 48, 72, and 96 h after exosome treatment. Quantitative PCR showed that co-culture of BMSCs with GATA-4-BMSC exosomes increased cardiomyocyte-related marker expression. Co-culture of GATA-4-BMSC exosomes with cardiomyocytes in anoxic conditions decreased apoptosis as detected by flow cytometry. Injection of GATA-4-BMSC exosomes in mice 48 h after MI increased cardiac function over the next 96 h; increased cardiac blood vessel density and number of c-kit-positive cells and decreased apoptotic cardiomyocyte cells were also observed. Differential expression of candidate differentiation- and apoptosis-related miRNAs and proteins that may mediate these effects was also identified. Exosomes isolated from GATA-4-expressing BMSCs induce differentiation of BMSCs into cardiomyocyte-like cells, decrease anoxia-induced cardiomyocyte apoptosis, and improve myocardial function after infarction.

Induction of mesenchymal stem cell differentiation in the absence of soluble inducer for cutaneous wound regeneration by a chitin nanofiber-based hydrogel.

Transplantation of bone marrow mesenchymal stem cells (BMSCs) has been considered to be a promising strategy for wound healing. However, poor viability of engrafted BMSCs and limited capabilities of differentiation into the desired cell types in wounds often hinder its application. Few studies report the induction of BMSC differentiation into the skin regeneration-related cell types using natural biopolymer, e.g. chitin and its derivative. Here we utilized a chitin nanofiber (CNF) hydrogel as a directive cue to induce BMSC differentiation for enhancing cutaneous wound regeneration in the absence of cell-differentiating factors. First, a 'green' fabrication of CNF hydrogels encapsulating green fluorescence protein (GFP)-transfected rat BMSCs was performed via in-situ physical gelation without chemical cross-linking. Without soluble differentiation inducers, CNF hydrogels decreased the expression of BMSC transcription factors (Oct4 and Klf4) and concomitantly induced their differentiation into the angiogenic cells and fibroblasts, which are indispensable for wound regeneration. In vivo, rat full-thickness cutaneous wounds treated with BMSC hydrogel exhibited better viability of the cells than did local BMSC injection-treated wounds. Similar to that of the in vitro result, CNF hydrogels induced BMSCs to differentiate into beneficial cell types, resulting in accelerated wound repair characterized by granulation tissue formation. Our data suggest that three-dimensional CNF hydrogel may not only serve as a 'protection' to improve the viability of exogenous BMSCs, but also provide a functional scaffold capable of enhancing BMSC regenerative potential to promote wound healing. This may help to overcome the current limitations to stem cell therapy that are faced in the field of wound regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Plasma treatment in conjunction with EGM-2 medium increases endothelial and osteogenic marker expressions of bone marrow mesenchymal stem cells

For many tissue engineering applications, an important goal is to create functional tissues in vitro. In order to make such tissues viable, they have to be vascularized. Bone marrow mesenchymal stem cells (BMSCs) are a promising candidate for vascularization. Growth supplements are commonly used to induce BMSCs differentiation, and further improvements in differentiation conditions can be achieved by modifying the cells’ growth environment, such as pretreating the culture dish with gas plasma to modify the surface functional groups that the cells are seeded on. In this work, we compare the effects of different gas plasmas on the growth and differentiation of BMSCs. We treat the growth dish with different plasmas (CO2, N2, and O2) and induce BMSC differentiation on them with endothelial growth medium-2 (EGM-2). We find that EGM-2 by itself does not have strong effect on endothelial differentiation. However, we find that plasma treatment markedly increases the mRNA expression of endothelial cell markers. Interestingly, we also find that the upregulation of osteogenic markers. The data show that plasma treatment in conjunction with EGM-2 can enhance BMSCs differentiation into both endothelial-like cells and osteogenic-like cells. The effects of EGM-2 on BMSCs differentiation are influenced by the plasma-modified surface of the substrate. Our findings provide a method to enhance EGM-2-based cell differentiation, with consequences for tissue engineering and stem cell biology applications.

In vitro differentiation into endothelial-like cells from bone marrow mesenchymal stem calls in diabetic rats

Objective To investigate the methods for separation and culture of bone marrow mesenchymal stem cells (BMSCs) in vitro in diabetic rats and the capability of differentiation into endotheliallike cells.Methods Rat diabetic model (DM) was induced by injection of stretopzin (STZ) in SD rats.After 12 weeks of DM duration,BMSCs were separated from bone marrow with density gradient centrifugation,wall sticking screening and amplified in vitro.The surface markers of BMSCs were evaluated with fluorescence-activated cell sorter (FACS).BMSCs of the third passage were obtained and divided into two groups.In the induction group,the cells were induced to differentiate in M199 medium containing 20％ fatal bovine serun ( FBS),vascular endothelial growth factor ( VEGF,10 μg/L) and basic fibroblast growth factor (bFGF,2 μg/L).No cytokine was added in the blank control group.Two weeks later,the cell morphology was observed under the phase contrast microscopy.The expression levels of VEGFR-2 were detected by using immunohistochemistry.EPC-specific marker CD34 was evaluated with FACS,and release of nitrous oxide (NO) from culture cells was also detected by NO detection reagent.Weibel-Palade (W-P) bodies in the cytoplasm were observed under the transmission electron microscopy.Results Rat diabetic model in SD rats that were injected with STZ intraperitoneally was established.Cultured BMSCs at the third passage were positive for CD44 and CD90 [ (97.8 ± 0.9 ) ％ and (96.8 ± 1.4) ％,respectively ],but negative for CD11 b/c and CD34 [ ( 13.2 ± 0.6 ) ％ and ( 1.2 ± 0.5 ) ％,respectively ].On the 14 day in the induction group,the cell morphology observed under the phase contrast microscopy was not distinctly different.Differentiation antigens VEGFR2 and CD34 in the induction group were higher [ (97.1 ± 1.0) ％,(65.0 ± 3.9) ％ ] than in the blank control group [ (7.0 ± 1.0 ) ％,( 0.9 ± 0.3 ) ％ ] ( P ＜ 0.05 ).The content of nitrous oxide in the induction group was also obviously increased as compared with the blank control group [ (94.14 ± 3.25 ) vs.( 70.37 ± 2.1 0) μmol/L,P ＜ 0.05 ).W-P bodies in the cytoplasm of the cultured cells were not found in the two groups.Conclusion BMSCs in diabetic rats can be isolated by density gradient centrifugation and adherent culture.VEGF and bFGF can induce differentiation of BMSCs in diabetic rats into endothelial-like cells in vitro.It is suggested that BMSCs may be used as a new type of seed cells for the diabetic lower limb ischemia disease. Key words: Diabetic; Bone marrow mesenchymal cell; Induction differentiation; Endothelial-like cell