The Effects And Mechanisms Of Hvpoxia And Hvpoxia-mimicking Agents On Mesenchvmal Stem Cell Mobilization

Chapter1:The effects and mechanisms of hypoxia on mesenchymal stem cell mobilizationWe took continuous hypoxia as mobilizing stimulus and established a MSC-mobilizing model. Using CFU-F assay, we investigated the kinetics of hypoxia-induced mobilization. The results showed that MSCs could be mobilized into peripheral blood (PB) of rats exposed to short-term hypoxia. The CFU-F frequency was significantly increased in the PB of2-day hypoxic rats (5.80±0.58/3×106vs.1.40±0.24CFU-Fs per3×106, P<0.05). The mobilization efficiency increased in a time-dependent manner (2-14days). No significant differences in bone marrow (BM) CFU-Fs were seen among the groups (P>0.05). To identify the hypoxia mobilized PB cells, immunophenotye and in vitro trilineage differentiation potential were detected. The fibroblast-like cells derived from mobilized PB were similar to that from BM on morphology. They were positive for expression of CD90, CD29and CD44, and absent for expression of hematopoietic cell surface marker CD34and CD45. The mobilized PB derived cells could differentiate into adipocytes, osteoblasts, and chondrocytes. Thus hypoxia-mobilized PB-derived adherent cells were demonstrated to be bona fide MSCs.Using this model, we studied the related mechanisms further. The results showed that, during hypoxic exposure, hypoxia-inducible factor1α (HIF-1α) was upregulated and expressed continuously in BM. To clarify the role of HIF-la in hypoxia-induced MSC mobilization, we observed the effects of YC-1, a widely used HIF-1α inhibitor, on the mobilizing effect of hypoxia. HIF-1α expression was markedly inhibited by injection of YC-1, and HIF-1α blockade resulted in a notable inhibition of MSC mobilization. These results suggest that HIF-1α is essential for the induction of MSC mobilization by hypoxia.Furthermore, we investigated the potential role of HIF-1α target genes, vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α). VEGF was elevated from days2-7of hypoxia and may increase BM vascular permeability. Induced by VEGF, BM sinusoid vessels were increased in7and14-day hypoxic rats, possibly further facilitating the egress of MSCs. In addition, migration capacity of MSCs was remarkably enhanced under hypoxic conditions in response to SDF-1α, which was increased in PB of rats during MSC mobilization.These results suggest that HIF-1α plays a pivotal role in hypoxia-induced MSC mobilization, possibly acting via its downstream genes VEGF and SDF-1α. These data provide a novel insight into the mechanisms responsible for MSC mobilization and may help in the development of clinically useful therapeutic agents.Chapter2:The effects and mechanisms of hypoxia-mimicking agents on mesenchymal stem cell mobilizationHIF-1α plays a pivotal role in hypoxia-induced MSC mobilization. Chemical hypoxia-mimicking agents including cobalt chloride (CoCl2) and deferoxamine (DFX) are able to inhibit the degradation and stabilize the expression of HIF-la under normoxic condition. In this section, we investigated the effects of CoCl2and DFX on MSC mobilization and their related mechanisms. Firstly, we confirmed that CoCl2could up-regulate the expression of HIF-la of MSCs in vitro. To detect whether CoCl2may induce MSC mobilization, CFU-F and flow cytometry assay were performed. The results showed that the frequency of CFU-Fs in PB increased significantly in rats treated with CoCl210mg/kg/d for7days (2.37±0.19vs.1.27±0.08CFU-Fs/ml, P<0.05). In addition, the percentage of PB CD45-CD90+cells was enhanced in CoCl27d group. Intriguingly, the number of CFU-Fs and percentage of CD45-CD90+cells in BM were also increased following CoCl2treatment. These results suggested that hypoxia-mimicking agent CoCl2can induce MSC mobilization. The protein level of HIF-1α was increased in BM by CoCl2treatment. The concentrations of VEGF were higher in CoCl2treated rats in comparison to vehicle treated controls.Since CoCl2treatment increased the number of BM MSCs, we speculated that combination of the hematopoietic mobilizing agents G-CSF or CXCR4antagonist AMD3100might improve the mobilization efficiency of MSCs. We found that administration of G-CSF or AMD3100alone had no obvious effects on the mobilization of MSCs. No additive effect was observed by combination of CoCl2and G-CSF. Interestingly, CoCl2pretreatment followed by acute administration of AMD3100synergistically enhanced circulating number of MSCs (3.83±0.32vs.2.37±0.19CFU-Fs/ml, P<0.05). An additional increase of percentage and total mumber of PB CD45-CD90+cells was also detected. What's more, the number of BM CFU-Fs was decreased when AMD3100administered acutely to rats pretreated with CoCl2as compared to rats treated with CoCl2alone (13.8±1.07vs.18.4±1.50, P<0.05). These experiments definitively indicated that CoCl2pretreatment followed by administration of AMD3100increased the mobilization efficiency of MSCs.We also investigated the MSC mobilizing effect of another hypoxia-mimicking agent DFX. The data showed that DFX administrating enhanced the percent and number of circulating CD45-CD105+cells.Chapter3:The characteristics of MSCs derived from mobilized peripheral bloodIn this chapter, we investigated the characteristics of MSCs derived from mobilized PB (PB-MSCs) and compared the PB-MSCs and BM derived MSCs (BM-MSCs) for their proliferation character, immunophenotype, multi-lineage differentiation potential, migration capacity and expression of angiogenic cytokines.The growth curve of PB-MSCs and BM-MSCs both exhibited a typical "S shaped curve". Both of their proliferation possessed lag phase, exponential phase and stationary phase. But PB-MSCs appeared to grow at a relatively slow speed. Flow cytometric analysis of the cell cycle showed the proportion of PB-MSCs in S phase was significantly lower than BM-MSCs. These data suggested that the proliferation potential of BM-MSCs was greater than PB-MSCs.Cell surface markers were examined by flow cytometry assay. MSCs from both sources were negative for the hematopoietic lineage markers CD34and CD45, and positive for CD44,CD29and CD90. There was no significant difference between PB-MSCs and BM-MSCs.Adipogenic and osteogenic differentiation were assessed by special stains. The expressions of osteogenesis-specific genes Runx2and Bglap2, and adipogenesis-related genes Pparg2and Lpl were analyzed at0,3, and7days after induction by real-time RT-PCR. The mRNA levels of Runx2and Bglap2in PB-MSCs were higher at each stage of osteogenic induction. In addition, the mineralization of PB-MSCs increased more remarkablely than BM-MSCs. Compared with BM-MSCs, the expression of Pparg2and Lpl was decreased and the oil red O-positive area was smaller in PB-MSCs. These results revealed that PB-MSCs possessed a stronger osteogenic differentiation potential but lower capacity for adipogenic differentiation. Confirmed by the formation of sphere-like pellets and toluidine blue stain, PB-MSCs exhibited comparable chondrogenic differentiation potential with BM-MSCs. PB-MSCs and BM-MSCs could both differentiate into neuro-like cells induced by β-mercaptoethanol.The number of transmigrated PB-MSCs were fewer than BM-MSCs (46.0±4.74vs.70.4±4.51/200×, P<0.05), suggesting a lower migration ability of PB-MSCs. ELISA analysis identified that VEGF was secreted at a higher level in PB-MSCs compared with BM-MSCs (6935.6±617.2vs.2068.7±243.2ng/ml, P<0.05). However, IGF and IL-6were secreted at lower levels in PB-MSCs.In summary:(1) MSCs could be mobilized into PB by short-term hypoxia and the mobilization efficiency increased in a time-dependent manner (2-14days).(2) HIF-la is essential for hypoxia induced MSC mobilization.(3) HIF-1α plays a pivotal role in hypoxia-induced MSC mobilization, possibly acting via its downstream genes VEGF and SDF-la.(4) CoCl2was able to stabilize the expression of HIF-la and up-regulate the VEGF level in bone marrow of rats.(5) CoCl210mg/kg/d for7days induced MSCs mobilization into PB and increased the number of BM MSCs.(6) CoCl2pretreatment followed by administration of AMD3100increased the mobilization efficiency of MSCs.(7) DFX administration enhanced the percent and number of circulating CD45-CD105+cells.(8) MSCs derived from mobilized PB had the potential of proliferation, multi-lineage differentiation, migration and angiogenic cytokines secretion, but exhibited some differences in characteristics with BM-MSCs.