| Mesenchymal stem cells (MSCs) are widely distributed in a variety of tissues in the adult human body (e.g., bone marrow (BM), kidney, lung, and liver), as well as in the fetal environment (e.g., blood, liver, BM, and kidney). However, MSCs are a rare population in these tissues. Despite the fact that bone marrow mesenchymal stem cells (BMSCs) represents the main available source of MSCs, the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection, tumor or significant drop in cell numbers with age. Thus, the search for possible alternative MSCs sources remains to be validated. Here we tried to identify the cells with MSC-like potency in human placenta. We examined these cells for morphology, surface markers, and differentiation potential, and the feasibility of their transplantation in the intracerebral hemorrhage, as well as the possible mechanism of the therapy were investigated.Part1: Isolation and culture of Human Placenta-Derived Mesenchymal-Like Stem Cells (HPMSCs) and character study of the cellsSpecimens of human placenta (n=7) were obtained after caesarean section performed at 35 to 40 weeks of gestation, after digestion with 0.1%â…¥ collagenase the suspicious cells were obtained after cultured for about a month in vitro. Then, these cells were identified by immuno-fiuorescence staining and flow cytometric analysis (FACS) or immunocytochemistry assay; HPMSCs were cocultured with T lymphocytes with ~3H-TdR were mixed to analysis its immunogenicity.We found the adherent cells isolated from placenta contained a high number of MSC-like elements forming colonies of fibroblastoid cells. These cells contain no endothelium- orleukocyte-specific antigens but similar to bone mesemchymal stem cells, they expressed CD29, CD44, CD 105 (SH2), ASMA, collagen type I and III, but not CD1 lb, CD34, CD45, CD19, CD106 (VCAM-1), CD117,HLA-DR, vWF or MySM. FASC results also showed that HPMSCs did not express HLA-DR, CD40, CD40L, CD28, CD80 and CD86, but expressed PDL-1, the negative costimulator ; When PMSCs cocultured with T lymphocytes. The proliferation of T cells was not found. It suggested the low immunogenicity of the HPMSCs. They also showed adipogenic differentiation potentials in N2 medium.Part 2: HPMSCs can be induced to express neural antigensHPMSCs cells were induced towards neuronal-liked cells with butylated hydroxyanisole (BHA) and dimethylsulfoxide (DMSO) or Salviamiltiorrhiza or {3-dimersol (BME). Identified with immunocytochemistry and RT-PCR/Real-time PCR , these cells expressed NPCs marker-Nestin, and could be induced to neuron-like cells, which developed rounded cell bodies with multiple neuron-like extension as well as several neuronal proteins such as neuron specificenolased (NSE) , neurofilament (NF), microtuble associate protein-2(MAP-2) and astrocyte marker glial fibrillary acid protein(GFAP). Markers for oligodendrocytes Galactocerebroside (Gal-C) and Oligodendrocyte marker 04(04) could also be detected.Part 3: The experimental study of HPMSCs transplantation in theintracerebral hemorrhage in ratsNon-heparinized arterial blood of the rat was sterotaxically injected into its caudate peutamen unit to establish a model of intracerebral hemorrhage. Hoechst 33258 marked HPMSCs were injected into the model rats, and control sham rats were similarly injected with phosphate-buffered saline on the 4th to 7th days after intracerebral hemorrhage. Fluorescence microscope was used to observe the distribution of the marked cells in the brains of the rats. Elevated body-swing test (EBST) and Bederson 4 grade criterion were used to estimate the neural function of the rats.The transplantation experiment showed that HPMSCs could transcend the blood - brain barrier (BBB) and could migrate to the area around the lesion site. EBST and Bederson score suggestted that the rats in the experiment groups, i.e. HPMSCs were implanted into the intact site or the lesion site, or the common carotid artery all had better results than the control groups on the 5th and 14th days after transplantation; no neoplasia was found in the brain of the host 28 days after transplantation. This suggested the HPMSCs transplantation could accelerate the recover of the neurologic impairment.Part 4: HPMSCs secrete stromal cell derived factor-la and chemoattractneural progenitor cells in vitroHuman fetal derived neural precursor cells (HNPCs) were cultured in vitro and chemoattracted respectively by PMSCs and BMSCs culture supernatants. In addition to detecting the concentration of stromal cell derived factor-la (SDF-la) in the supernatant of PMSCs and BMSCs by ELISA and its receptor CXCR4 expression on the surface of NPCs by immuno-fluorescence staining and FACS, inhibition test was designed to confirm the role of chemoattraction was mediated by SDF-1 alpha and its receptor CXCR4.The influence of tumor necrosis factor-a (TNF-a) and gpl30 signal on the expression of CXCR4 on the HNPCs were analysed by immuno-fluorescence & FACS, and the migration of treated NPCs were measured by Boyden Chamber Assay .HPMSCs were confirmed by ELISA to secret high degree of SDF-la. Sugestted by Boyden Chamber Assay, HPMSCs were able to chemoattract the CXCR4 positive NPCs migration in vitro, and the monoclonal antibody of CXCR4 could neutralize this effect. It is speculated that TNF-a and gpl30 signal may up regulat the expression of CXCR4 on the NPCs, thus, enhanc the migration ability of NPCs.ConclusionHuman placenta derived mesenkymal-like stem cell has become a favorable alternative for stem cell transplantation, thanks to its rich resource, convenient availability, low immunogenicity, high plasticity, reliability and proliferation ability make it a goodresource of stem cell transplantation. When implanted into the intracerebral hemorrhage rats, HPMSCs could transcend the BBB, secreting SDF-la to chemoattract NPCs, which contributes to the improvement of the neurologic impairment. These exciting findings suggest that, placenta derived mesenchymal-Iike stem cells which can meet clinical needs, may be a new resource of MSCs .
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