Mesenchymal stem cells (MSCs) constitute a population of multipotential cells giving rise to adipocytes, osteoblasts and chondrocytes. Combining with their engraftment promoting capacity and immunosuppressive property, MSCs may be therapeutically useful for haematopoietic stem cell transplantation. There is growing evidence that these cells can, under the right circumstances, enter peripheral circulation. Previous study revealed that MSCs are mobilized into peripheral blood (PB) by 3 weeks of chronic hypoxia, but the mobilization effect of short-term hypoxia and the underlying mechanisms are currently unknown. In this study, we used rat model to determine whether short-term hypoxia can mobilize MSCs into PB and investigated the related factors which may regulate the mobilization process.
Rats were housed in a hypoxic chamber (FiO2=10%) for 1, 2, 3, 5, 7 and 14 days, respectively, while control ones were housed in normoxic environment for equal periods. To quantify the number of MSCs and evaluate mobilization efficiency, PB and bone barrow (BM) samples of each group were collected and colony-forming unit fibroblast (CFU-F) assays were performed. Mobilized PB derived MSCs were identified by immunophenotype and trilineage differentiation. Since BM is the main reservoir and typical microenvironment of MSCs, we investigated the response of BM environment exposed to hypoxia which may potentially facilitate MSCs mobilization. Hypoxia-inducible factor 1α (HIF-1α) expression of BM cells was detected by Western blot; vascular endothelial growth factor (VEGF) in BM was qualified by ELISA and Immunohistochemistry (IHC). To evaluate the change of BM sinusoid vessels (BMSVs), VEGFR3 was stained by IHC and positive vessels were counted. The levels of stromal cell-derived factor-1α (SDF-1α) and VEGF in PB were tested by ELISA. Moreover, we compared migration capacity of MSCs in hypoxic condition (PO2=1% or 8%) with normal condition (PO2=21%) in vitro using Transwell assay.
We found that MSCs were mobilized into PB by exposing to short-term hypoxia (2d) and the CFU-F frequency was 5.80±0.58 vs. 1.40±0.24 CFU-Fs per 3×106 cells (p<0.05, n=5). From 2d to 14d of hypoxic exposure, the number of CFU-Fs mobilized in PB of hypoxic group was gradually increased in a time dependent manner. However, no significant differences were observed in bone marrow CFU-Fs among varies groups (P>0.05). Mobilized PB derived adherent cells were positive for CD90, CD29 and CD44, but negative for CD34 and CD45 and they could differentiate into adipocyte, osteoblast, and chondrocyte, which indicated that mobilized PB-derived cells are bona fide MSCs. What's more, we showed here that during hypoxic exposure, HIF-1α was stabilized and expressed continuously in BM of rats which is a main niche of MSCs. Stabilization and up-regulation of HIF-1α suggested that BM is hypoxia-sensitive and during hypoxic exposure it became a lower oxygen environment (PO2<1%). Previous studies have proved that VEGF and SDF-1α are directly regulated by the transcription factor HIF-1α. Our results showed that, induced by HIF-1α, VEGF was elevated from 2d to 7d in the BM of hypoxic rats which may increase BM vascular permeability and induce vasodilatation; VEGFR3(+) BMSVs increased in 7d and 14d hypoxic BM which may further facilitate the egress of MSCs. SDF-1α in PB increased from 2d to 14d, especially 7d of hypoxia (1976.7±148.1 vs. 663.6±56.7pg/ml, P<0.01). In addition, exposure of MSCs to low oxygen (8% PO2) significantly promoted their in vitro migration and a further increase was observed under lower oxygen condition (1% PO2). MSCs migrated more rapidly in response to SDF-1α exposed to hypoxia.
Taken together, we show here that MSCs can be mobilized into PB by short-term hypoxia and the mobilization efficacy increased in a time dependent manner. Our results suggest the mechanisms of hypoxia inducing MSCs mobilization relate to the lower oxygen milieu of BM and stabilization of HIF-1α may play a pivotal role in MSCs mobilization. Our data provide meaningful clues to clarify the mechanisms of MSCs mobilization and important evidence for further exploring the exact agents that of clinical use.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.