Sonic hedgehog enhances the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) may be promising seed cells for tissue regeneration because of their self-renewal and multi-differentiation potential. Shh (sonic hedgehog) is involved in the skeletal formation during embryo development and skeletal regeneration. However, how Shh regulates the biological characteristics of BM-MSCs (bone marrow-derived MSCs) is poorly understood. We have investigated the effect of rShh-N (recombinant N-terminal Shh) on the proliferation and osteogenic differentiation of rBM-MSCs (rat BM-MSCs) in vitro. rBM-MSCs were treated with rShh-N at concentrations up to 200 ng/ml. Proliferation and colony-forming ability of rBM-MSCs were increased in a dose-dependent manner. rShh-N increased the ratio of cells in S and G2/M phase, as well as the number of Ki-67+ cells. In addition, ALP (alkaline phosphatase) activity and matrix mineralization were enhanced by 200 ng/ml rShh-N. Real-time PCR showed that rShh-N (200 ng/ml) up-regulated the expression of genes encoding Cbfa-1 (core-binding factor α1), osteocalcin, ALP and collagen type I in rBM-MSCs. This information reveals some potential of rShh-N in the therapeutics of bone-related diseases.     

人干细胞生长因子-α基因的克隆、表达及其协同rhGM-CSF对人脐带间充质干细胞的增殖作用

为了研究hSCGF-α对人脐带间充质干细胞 (Human umbilical cord mesenchymal stem cells,hUCMSCs) 的作用,采用基因工程技术获得重组人干细胞生长因子-α (Recombinant human Stem Cell Growth Factor-α,rhSCGF-α)。针对SCGF基因的高GC含量,采用PCR两步法获得hSCGF-α基因,插入pET-28a(+) 载体质粒,构建重组质粒pET-28a-SCGF-α,转化大肠杆菌BL21(DE3) 获得表达菌株。低温20 ℃诱导24 h,目标重组蛋白人干细胞生长因子-α以可溶性表达为主。通过Ni2+-NTA柱纯化,获得目标重组蛋白,电泳谱带扫描分析蛋白纯度可达90％以上。以巨噬细胞/粒细胞(Granulocyte/macrophage,GM)集落形成实验鉴定重组蛋白的生物学活性,并协同重组人巨噬细胞/粒细胞集落刺激因子(Recombinant human GM-colony stimulating factor,rhGM-CSF) 研究其对人脐带间充质干细胞的影响。结果显示,纯化的重组蛋白rhSCGF-α具有生物学活性;hSCGF-α及rhGM-CSF对hUCMSCs均有刺激增殖活性,但协同作用效果最强。     

Effect of microgravity on proliferation and differentiation of embryonic stem cells in an automated culturing system during the TZ‐1 space mission

Objective

Despite a great number of studies analysing the effects of microgravity on stem cell proliferation and differentiation, few of them have focused on real‐time imaging estimates in space. Herein, we utilized the TZ‐1 cargo spacecraft, automatic cell culture equipment and live cell imaging techniques to examine the effects of real microgravity on the proliferation and differentiation of mouse embryonic stem cells (mESCs).

Materials and methods

Oct4‐GFP, Brachyury‐GFP mESC and Oct4‐GFP mESC‐derived EBs were used as experimental samples in the TZ‐1 spaceflight mission. These samples were seeded into chambers, cultured in an automatic cell culture device and were transported into space during the TZ‐1 mission. Over 15 days of spaceflight, bright field and fluorescent images of cell growth were taken in micrography, and the medium was changed every day. Real‐time image data were transferred to the ground for analysis.

Results

Space microgravity maintains stemness and long‐term survival of mESCs, promising 3D aggregate formation. Although microgravity did not significantly prevent the migration of EBs on the ECM substrate, it did prevent terminal differentiation of cells.

Conclusions

This study demonstrates that space microgravity might play a potential role in supporting 3D cell growth and maintenance of stemness in embryonic stem cells, while it may negatively affect terminal differentiation.

     

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O*2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+ cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O*2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34+/CD38- cell population, where the level of O*2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34+/CD38- cell population, and this intracellular pH decreased as early as 4 h post-irradiation, virtually simultaneous with the significant elevation of O*2- generation. These results suggest that the CD34+/CD38- stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O*2-, compare to more differentiated CD34+/CD38+ and CD34-/CD38+ cells and that its intracellular pH declines at an early phase in the apoptosis process.     

Protein profiling and angiogenic effect of hypoxia-cultured human umbilical cord blood-derived mesenchymal stem cells in hindlimb ischemia

The aim of the present study was to investigate protein profiles of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions, and evaluate oxygenation effects on angiogenesis in an ischemic hindlimb mouse model using a modified ischemic scoring system. Hypoxic conditions did not change the expression of phenotypic markers and increased adipogenesis and chondrogenesis. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β RII, and vascular endothelial growth factor (VEGF) were upregulated in the conditioned medium of hypoxic hUCB-MSCs, which are commonly related to angiogenesis and proliferation of biological processes by Gene Ontology. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, significant enrichment of the phosphorylation of abelson murine leukemia viral oncogene homolog 1 (ABL1) (Phospho-Tyr204) and B-cell lymphoma-extra large (BCL-XL) (Phospho-Thr47) as anti-apoptotic pathways was observed in hypoxic hUCB-MSCs. Furthermore, hypoxic conditions induced proliferation and migration, and reduced apoptosis of hUCB-MSCs in vitro. Based on the results of protein antibody array, we evaluated the angiogenic effects of injecting normoxic or hypoxic hUCB-MSCs (1 × 10⁶) into the ischemic hindlimb muscles of mice. Ischemic scores and capillary generation were significantly greater in the hypoxic hUCB-MSC injection group than in the normoxic hUCB-MSC group. Our findings demonstrate that culturing hUCB-MSCs in hypoxic conditions not only significantly enriches phosphorylation in the anti-apoptosis pathway and enhances the secretion of several angiogenic proteins from cells, but also alleviates ischemic injury of hindlimb of mice.     

Bone marrow mesenchymal stem cells modified by angiogenin-1 promotes tissue repair in mice with oxygen-induced retinopathy of prematurity by promoting retinal stem cell proliferation and differentiation

Retinopathy has become one of the major factors that lead to blindness worldwide. Although many clinical therapies are concerned about such disease, most of them focus on symptoms alleviation. In this study, we aim to investigate whether coculture retinal stem cells (RSCs) with bone marrow mesenchymal stem cells transfected with angiogenin-1 (Ang-1-BMSCs) affects the damaged retinal tissue of oxygen-induced retinopathy of prematurity (OIR-ROP) mice. After OIR-ROP mouse model establishment, Ang-1-BMSCs, RSCs, and OIR-ROP retinal tissues were cocultured in a a transwell chamber. RSCs proliferation and the expression of Ang-1, insulin-like growth factor-1 (IGF-1) in the supernatant of RSCs, as well as β-tubulin and protein kinase C (PKC) expression were evaluated. Finally, the repair of OIR-ROP mice retinal tissues was observed by injecting Ang-1-BMSCs + RSCs. In the OIR-ROP mouse model, RSCs cocultured with OIR-ROP retinal tissues could be induced to differentiate into cells expressing β-tubulin and PKC and promote the expression of Ang-1 and IGF-1. coculture of Ang-1-BMSCs further enhanced the proliferation and differentiation of RSCs by promoting the expression of Ang-1 and IGF-1. Coculture of RSCs + Ang-1-BMSCs induced differentiation of Ang-1-BMSCs through interaction among intercellular factors and restored the damaged retinal tissue of OIR-ROP mice. Collectively, our study provided evidence that coculture of Ang-1-BMSCs and RSCs could promote the proliferation and differentiation of RSCs and improve the treatment for the damaged retina tissue of OIR-ROP mice.     

Telomere length analysis of human mesenchymal stem cells by quantitative PCR

Human mesenchymal stem cells (hMSCs) have attracted much attention for tissue repair and wound healing because of their self-renewal capacity and multipotentiality. In order to mediate an effective therapy, substantial numbers of cells are required, which necessitates extensive sub-culturing and expansion of hMSCs. Throughout ex vivo expansion, the cells undergo telomere shortening, and critically short telomeres can trigger loss of cell viability. Telomeres are nucleoprotein structures that cap the ends of chromosomes, and serve to protect the DNA from the degradation which occurs due to the end-replication problem in all eukaryotes. As hMSCs have only a finite ability for self-renewal like most somatic cells, assaying for telomere length in hMSCs provides critical information on the replicative capacity of the cells, an important criterion in the selection of hMSCs for therapy. Telomere length is generally quantified by Southern blotting and fluorescence in situ hybridization, and more recently by PCR-based methods. Here we describe the quantification of hMSC telomere length by real-time PCR; our results demonstrate the effect of telomere shortening on the proliferation and clonogenicity of hMSCs. Thus, this assay constitutes a useful tool for the determination of relative telomere length in hMSCs.     

Immunoregulatory effect of mesenchymal stem cell via mitochondria signaling pathways in allergic asthma

Asthma is a complicated lung disease, which has increased morbidity and mortality rates in worldwide. There is an overlap between asthma pathophysiology and mitochondrial dysfunction and MSCs may have regulatory effect on mitochondrial dysfunction and treats asthma. Therefore, immune-modulatory effect of MSCs and mitochondrial signaling pathways in asthma was studied.After culturing of MSCs and producing asthma animal model, the mice were treated with MSCs via IV via IT. BALf's eosinophil Counting, The levels of IL-4, −5, −13, −25, –33, INF-γ, Cys-LT, LTB4, LTC4, mitochondria genes expression of COX-1, COX-2, ND1, Nrf2, Cytb were measured and lung histopathological study were done.BALf's eosinophils, the levels of IL-4, −5, −13, −25, –33, LTB4, LTC4, Cys-LT, the mitochondria genes expression (COX-1, COX-2, Cytb and ND-1), perivascular and peribronchial inflammation, mucus hyper-production and hyperplasia of the goblet cell in pathological study were significantly decreased in MSCs-treated asthma mice and reverse trend was found about Nrf-2 gene expression, IFN-γ level and ratio of the INF-γ/IL-4.MSC therapy can control inflammation, immune-inflammatory factors in asthma and mitochondrial related genes, and prevent asthma immune-pathology.