Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF-1α signalling.

Mesenchymal stem cells (MSCs) have drawn great attention because of their therapeutic potential. It has been suggested that intra-venous infused MSCs could migrate the site of injury to help repair the damaged tissue. However, the mechanism for MSC migration is still not clear so far. In this study, we reported that hypoxia increased chemotaxis migration of MSCs. At 4 and 6 hours after culturing in hypoxic (1% oxygen) conditions, the number of migrated MSCs was significantly increased. Meanwhile, hypoxia also increased the expression of HIF-1α and SDF-1. Using small interference RNA, we knocked down the expression of HIF-1α in MSCs to study the role of HIF-1α in hypoxia induced migration. Our data indicated that knocking down the expression of HIF-1α not only abolished the migration of MSCs, but also reduced the expression of SDF-1. Combining the results of migration assay and expression at RNA and protein level, we demonstrated a novel mechanism that controls the increase of MSCs migration. This mechanism involved HIF-1α mediated SDF-1 expression. These findings provide new insight into the role of HIF-1α in the hypoxia induced MSC migration and can be a benefit for the development of MSC-based therapeutics for wound healing.


| INTRODUCTION
The bone marrow (BM) stroma does not just contain a heterogeneous population of structural cells, including endothelial cells, fibroblasts, adipocytes, osteogenic cells, but also harbours the niche of stem cells like haematopoietic stem cells and mesenchymal stem cells. 1 Mesenchymal stem cells have the potential to protect, repair and possibly regenerate the damaged tissue upon arriving the injury site. Bone marrow-derived mesenchymal stem cells (MSCs) are a promising cell source for such cellular therapy because of their ability for self-renewal, multipotency and immunosuppressive properties. 2 Bone marrow MSCs are commonly thought to reside in hypoxic niches in the bone marrow, important for maintaining their undifferentiated state. 3 This suggests that oxygen tension plays an important role in stem cell regulation and indeed oxygen tension was recently found to affect MSC differentiation. 4 However, it is not clear if the migration of MSCs is also influenced by oxygen tension.
Mesenchymal stem cells are known as suitable cells that secrete several anti-inflammatory, angiogenic and antifibrotic factors and induce immunomodulation without significant activation of the immune response. Thus, MSCs induce regeneration in the surrounding tissues and cells, which is an important beneficial effect in cell therapy. 5 The systemic administration of stem cells is generally preferred to local injection due to being less invasive. 6 One critical barrier to effective MSC therapy is their insufficient homing capability to tissues of interest, especially when MSCs are infused through the vascular route. 7 Many factors contribute to the inefficient migration of these cells, among which low surface receptors' level is of high importance. 8 Some studies have highlighted that chemokine/-chemokine receptor interactions influence stem cell recruitment to the desired target, but to the best of our knowledge none of them have investigated whether hypoxia improves the migration of MSCs.
Wound healing is a complex process requiring cell migration, inflammation, angiogenesis, granulation tissue formation, re-epithelialization and extracellular matrix (ECM) remodelling. 9 MSCs have an active role through this process, and therapeutic application of MSCs has been shown to enhance and improve wound-healing outcomes. 10 Understanding the mechanism of MSCs migration upon chemotaxis stimuli would benefit the development of novel methods that can be used to increase MSC delivery and efficacy for treating wound healing.
In this study, we conducted investigations on the chemotaxis migration of MSCs under hypoxic condition. We also demonstrated the possible mechanism of the MSCs migration at molecular level.

| Isolation of bone marrow mesenchymal stem cells
Male inbred BALB/C mice, C57BL/6J mice (8 weeks old) were purchased from XXX. All experiments were performed after the approval by our local ethical committee at XXXX. The MSCs were isolated and cultured using standard protocols. Bone marrow cells from C57BL/6 mice were harvested by flushing the femurs and tibias. Then cells were seeded in DMEM with high glucose (Gibco, Grand Island, NY, USA), supplemented with 10% FBS (Lonza, Allendale, NJ, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco; Thermo Fisher Scientific, Inc, Waltham, MA, USA). The medium was slowly changed after 3 days to remove non-adherent cells.
Meantime, the adherent marrow cells were subcultured until obtaining purify population of mesenchymal stem cells with spindle-shaped morphology. 11

| RNA interference and transient infection
For the design of effective siRNA HIF-1α target sequences, a siRNA design tool was applied siRNA design tool (eurofins, USA) and siRNA target sequences were obtained according to published criteria. 14,15 For synthesis of siRNA HIF-1α via in vitro transcription, the Silencer ™ siRNA Construction Kit (Ambion, Austin, TX, USA) was used with modifications. 16   Biosciences, Grand Island, NY, USA). The primers used in the PCR are described in Table 2.

| Western blotting
The cells were washed with ice-cold PBS, lysed in RIPA buffer with protease inhibitors (Thermo Fisher Scientific, Rockford, IL, USA) on a rotation wheel for 1 hour at 4°C. After centrifugation at 10 000 g for 10 minutes, the supernatant was collected as protein extract.

| Statistical analysis
All data are expressed as the mean ± SEM from at least three independent experiments. The differences between two groups were analysed with the two-tailed unpaired Student's t test, and differences between multiple groups were analysed with one-way ANOVA followed by Tukey's test, using Prism GraphPad (La Jolla, CA, USA).
A value of P < 0.05 was considered statistically significant. with BM-MSCs in 21% oxygen ( Figure 1C).

| Hypoxia increases MSC migration by enhancing SDF-1/CXCR4 signalling
To validate the role of hypoxia in MSC chemotaxis migration, we performed transwell migration assays of MSCs at the presence of SDF-1, under normoxia (21% oxygen) and hypoxia (1% oxygen).
As shown in Figure

| Small interference RNA successfully knockdown the expression of HIF-1α
In investigate the role of HIF-1α in hypoxia-induced chemotaxis migration, we designed three pairs of small inferences RNA to knock down the expression of HIF-1α. Fluorescent images indicated the transfection efficacy of siRNA ( Figure 3A). siRNA-3 had a similar transfection efficiency as negative control. Results of qPCR indicated that siRNA-3 could reduce the expression of HIF-1α to the lowest level ( Figure 3B). In the following experiments, only siRNA-3 was used for knocking down of HIF-1α.

| Knocking down HIF-1α abolished hypoxia induced chemotaxis migration in MSCs
It has been reported that Hif-1α controlled chemotaxis towards the chemokine SDF-1 by regulating expression of its receptor CXCR4. 18 To demonstrate the function of Hif-1α in the chemotaxis migration of MSCs, we knock down the expression of Hif-1α by small interference RNA and examined the migration of MSCs by transwell device.
Methylene blue staining revealed the cells that pass through the membrane. Less cells were observed in HIF-1α knock down group, comparing to negative control group, in both normoxia and hypoxia, at 4 and 6 hours ( Figure 4A). Quantification of the cell numbers showed that differences in cell numbers in all conditions are statistically significant ( Figure 4B-E).

SDF-1/CXCR4 signalling axis in MSCs
As shown in Figure 5A, we cannot detect any expression of HIF-1α in normoxic condition by Western blot. SDF-1 expression was slightly reduced by HIF-1α knocking down, meanwhile, the expression of CXCR4 was shown at low level, and not changed too much. Under knocking down suppressed SDF-1 expression ( Figure 6B). CXCR4 expression seemed not be regulated by HIF-1α ( Figure 6C).

| DISCUSSION
It is previously reported that infusion of MSC overexpressing HIF-1α promotes myocardial healing in an experimental rat model of myocardial infarction. 19 As described in previous studies, signalling pathways related to several paracrine factors and interleukins are up-regulated in HIF-MSCs. 20 In this study, we reported that hypoxia increased chemotaxis migration of MSCs. Using small interference have not been fully elucidated. 24 HIF-1α is a pivotal transcription factor regulating the adaptive response to hypoxia 25 and numerous proteins interact directly with HIF-1 to enhance or reduce its function. 26 Our results further suggested that HIF-1α expression is necessary for hypoxia induced MSC migration. Previous work has shown that cytokine SDF-1 and its receptor CXCR4 are able to regulate cell migration. 27 And it has been reported that increased SDF-1 expression is responsible for the enhanced migration of MSCs. 28

| CONCLUSION
Hypoxia culture condition significantly improves the migratory effects of MSCs. Furthermore, the molecular mechanism of this procedure has been suggested to be controlled through HIF-1α medi- and data analysis, as well as preparing this manuscript.

CONFLI CT OF INTEREST
All the authors confirm that they have no competing interests.

AUTHOR CONTRI BUTION
WX, RX and YW designed the experiments; WX, RX and YW performed the experiments; WX and RX analysed the data; WX and YW drafted the manuscript. All authors discussed the results and commented on the manuscript.