Simultaneous isolation of mesenchymal stem cells and endothelial progenitor cells derived from murine bone marrow.

Mesenchymal stem or stromal cells (MSCs) are identified as sources of pluripotent stem cells with varying degrees of plasticity. Endothelial progenitor cells (EPCs) originate from either bone marrow (BM) or peripheral blood and can mature into cells that line the lumen of blood vessels. MSC and EPC therapies exhibit promising results in a variety of diseases. The current study described the simultaneous isolation of EPCs and MSCs from murine BM using a straightforward approach. The method is based on differences in attachment time and trypsin sensitivity of MSCs and EPCs. The proposed method revealed characteristics of isolated cells. Isolated MSCs were positive for cell surface markers, cluster of differentiation (CD)29, CD44 and stem cell antigen-1 (Sca-1), and negative for hematopoietic surface markers, CD45 and CD11b. Isolated EPCs were positive for Sca-1 and vascular endothelial growth factor receptor 2 and CD133. The results indicate that the proposed method ensured simultaneous isolation of homogenous populations of MSCs and EPCs from murine BM.

VEGF secreted by mesenchymal stem cells mediates the differentiation of endothelial progenitor cells into endothelial cells via paracrine mechanisms.

Stem cell therapy is a promising treatment strategy for ischemic diseases. Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) adhere to each other in the bone marrow cavity and in in vitro cultures. We have previously demonstrated that the adhesion between MSCs and EPCs is critical for MSC self­renewal and their multi­differentiation into osteoblasts and chondrocytes. In the present study, the influence of the indirect communication between EPCs and MSCs on the endothelial differentiation potential of EPCs was investigated, and the molecular mechanisms underlying MSC­mediated EPC differentiation were explored. The effects of vascular endothelial growth factor (VEGF), which is secreted by MSCs, on EPC differentiation via paracrine mechanisms were examined via co­culturing MSCs and EPCs. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to detect the expression of genes and proteins of interest. The present results demonstrated that co­culturing EPCs with MSCs enhanced the expression of cluster of differentiation 31 and von Willebrand factor, which are specific markers of an endothelial phenotype, thus indicating that MSCs may influence the endothelial differentiation of EPCs in vitro. VEGF appeared to be critical to this process. These findings are important for the understanding of the biological interactions between MSCs and EPCs, and for the development of applications of stem cell­based therapy in the treatment of ischemic diseases.

A method to establish a mouse model of bone marrow microenvironment injury.

A normal bone marrow microenvironment plays a very important role in the normal functioning of hematopoietic stem cells. Once disturbed, this microenvironment can become favorable for the occurrence of blood disorders, cancers, and other diseases. Therefore, further studies on the bone marrow microenvironment should be performed to reveal regulatory and stem cell fate determination mechanisms and promote the development of bone marrow transplantation, tissue repair and regenerative medicine, and other fields. A small animal model for further research is also urgently needed. In this study, an electric shock device was designed to elicit a femur bone marrow microenvironment injury in mice. A wire was inserted into the distal femur but not into the proximal femur, and the bone marrow microenvironment was evidently damaged by application of 100 ± 10 V for 1.5 ± 0.5 min ; mortality, however, was low in the mice. Gross observation, hematoxylin and eosin staining, immunohistochemistry, bright-field microscopy, and micro-CT scanning were also conducted. A large number of new blood capillaries and sinusoids appeared in the injured distal femur after 2 weeks. The capillaries in the injured femur disappeared after 4 weeks, and mature blood vessels were scattered throughout the injured area. Red blood cells disappeared, and the cellular structure and trabecular bone were better than those observed 2 weeks previously. Thus, we developed a simply operated, accurate, reliable, and easily controlled small animal model as a good technical platform to examine angiogenesis and segmentation damage in the bone marrow microenvironment.

Mesenchymal stem cells promote endothelial progenitor cell proliferation by secreting insulin­like growth factor­1.

Bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) interact with each other. EPCs are able to promote the self­renewal of MSCs as niche cells in murine bone marrow, and MSCs are able to promote EPC proliferation in vitro in a co­culture system. It has previously been reported that MSCs can secrete insulin­like growth factor­1 (IGF­1), which serves critical functions in EPC proliferation. However, the mechanism underlying the IGF­1­mediated proliferation of EPCs remains unclear. The aim of the present study was to reveal the molecular mechanisms regulating this process. The effects of IGF­1, which is secreted by MSCs, on EPC proliferation via the PI3K/Akt signaling pathway were examined by MTT assay, reverse transcription­quantitative polymerase chain reaction and western blot analysis. The present study treated EPCs with various concentrations of IGF­1. The results demonstrated that IGF­1 significantly induced the proliferation of cultured EPCs. However, this effect was offset by treatment with the phosphatidylinositol 3­kinase (PI3K) inhibitor LY294002. These results indicated that the pro­proliferative effects of IGF­1 are mediated in response to the PI3K/protein kinase B signaling pathway.

[Effects of long time different negative pressures on osteogenic differentiation of rabbit bone mesenchymal stem cells].

Objective: To investigate the effects of long time different negative pressures on osteogenic diffe-rentiation of rabbit bone mesenchymal stem cells (BMSCs). Methods: The rabbit BMSCs were isolated and cultured by density gradient centrifugation. Flow cytometry was used to analyze expression of surface markers. The third passage cells cultured under condition of osteogenic induction and under different negative pressure of 0 mm Hg (control group), 75 mm Hg (low negative pressure group), and 150 mm Hg (high negative pressure group) (1 mm Hg=0.133 kPa), and the negative pressure time was 30 min/h. Cell growth was observed under phase contrast microscopy, and the growth curve was drawn; alkaline phosphatase (ALP) activity was detected by ELISA after induced for 3, 7, and 14 days. The mRNA and protein expressions of collagen type I (COL-I) and osteocalcin (OC) in BMSCs were analyzed by real-time fluorescence quantitative PCR and Western blot. Results: The cultured cells were identified as BMSCs by flow cytometry. The third passage BMSCs exhibited typical long shuttle and irregular shape. Cell proliferation was inhibited with the increase of negative pressure. After induced for 4 days, the cell number of high negative pressure group was significantly less than that in control group and low negative pressure group ( P<0.05), but there was no significant difference between the low negative pressure group and the control group ( P>0.05); at 5-7 days, the cell number showed significant difference between 3 groups ( P<0.05). The greater the negative pressure was, the greater the inhibition of cell proliferation was. There was no significant difference in ALP activity between groups at 3 days after induction ( P>0.05); the ALP activity showed significant difference ( P<0.05) between the high negative pressure group and the control group at 7 days after induction; and significant difference was found in the ALP activity between 3 groups at 14 days after induction ( P<0.05). The greater the negative pressure was, the higher the ALP activity was. Real-time fluorescence quantitative PCR and Western blot detection showed that the mRNA and protein expressions of COL-I and OC protein were significantly higher in low negative pressure group and high negative pressure group than control group ( P<0.05), and in the high negative pressure group than the low negative pressure group ( P<0.05). Conclusion: With the increase of the negative pressure, the osteogenic differentiation ability of BMSCs increases gradually, but the cell proliferation is inhibited.

E-cadherin-mediated contact of endothelial progenitor cells with mesenchymal stem cells through ß-catenin signaling.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) are attached to each other in the bone marrow (BM) cavity and in in vitro cultures, and this adhesion has important physiological significance. We demonstrated that cell proliferation could be promoted when MSCs were co-cultured with EPCs, which was beneficial to angiogenesis, tissue repair, and regeneration. The adhesion of MSCs and EPCs could promote the pluripotency of MSCs, particularly self-renewal and multi-differentiation to osteoblasts, chondrocytes, and adipocytes. This study focused on the mechanism of adhesion between EPCs and MSCs. The results showed that E-cadherin (E-cad) mediated the adhesion of MSCs and EPCs through the E-cad/beta-catenin signaling pathway. The E-cad of EPCs occupied a dominant position during this process, which activated and up-regulated the beta-catenin (ß-catenin) of MSCs to improve cohesion and exert their biological function.