Augmented migration of mesenchymal stem cells correlates with the subsidiary CXCR4 variant

Figures & data

Figure 1. Differentiation potential and Immunophenotypic results of Ad-MSCs. Evaluation of Ad-MSCs for minimal criteria. (A and B) Adherent stem cells in stromal vascular fraction (SVF) derived from adipose tissue could form colonies after primary culture. The cells at passage 4 were induced with osteogenic medium for 21 d. In contrast to the control cells (C) the calcifications produced by differentiated cells in the extracellular matrix, were stained with Alizarin Red as evident for osteogenesis (D). Additionally, compared to control (E) osteogensis was confirmed by ALP activity of osteoblasts (F). Moreover unlike the control cells (G) adipogenic differentiation of Ad-MSCs was validated by Oil Red O staining of lipid droplets, which were produced by adipogenic induction medium (H). Photographs are representative of 3 independent experiments for each group. (I) Flow cytometry Results showed that Ad-MSCs are positive for CD90, CD44, CD73, and CD105 and negative for CD11b (macrophage marker), CD45 (haematopoietic stem cell marker) and CD34 (endothelial cell marker).

Figure 2. Pretreated Ad-MSCs show high expression level of both CXCR4 variants. The effects of different pretreatments on the expression of CXCR4 variants. (A) CXCR4 variants expression in haematopoietic (HL- 60) cell line used as a positive control. (B) In semi q-RT PCR analysis, CXCR4-A (lo) variant expression was appeared only after treatment of Ad-MSCs with DFX for 24h. All treatments (VPA, CoCl₂, and DFX) resulted in overexpression of CXCR4-B variant as compared to untreated control. The ACTB was used as an independent housekeeping-gene for both semi q-RT PCR and real-time RT-PCR. (C and D) Quantitative Real time PCR showed that expression of CXCR4-A (lo) variant was more significantly increased after pretreatment with DFX as compared to other treatments rather than other pretreatments and Untreated control. Interestingly expression of CXCR4-B variant had oscillating pattern among VPA, CoCl₂, and DFX pretreatments. * p < 0.05, ** p < 0.01 and *** p < 0.001 was considered significant as compared to untreated control. VPA: valproic acid, CoCl₂: cobalt-chloride, DFX: deferoxamine mesylate, ACTB: β actin.

Figure 3. Validation of the results of Real time PCR with Western blotting. As shown by Western blot all treatments resulted in overexpression of CXCR4 protein. ImageJ Software was used to quantify CXCR4 expression in comparision to untreated cells after normalization to ACTB. * p < 0.05 was considered significant as compared to untreated control. VPA: valproic acid, CoCl₂: cobalt-chloride, DFX: deferoxamine mesylate, ACTB: β actin.

Figure 4. Pretreated Ad-MSCs display significantly higher migration toward SDF-1α. In vitro migration assay of Ad-MSCs pretreated with various hypoxia-mimicking agents. (A-D) Migration of untreated and pretreated Ad-MSCs toward SDF-1α. After fixation, migrated cells were stained with DAPI. DFX-treatment (D) could induce migration activity of Ad-MSCs more than VPA (B) and CoCl₂(C) treated cells. Migration of untreated cells toward SDF-1α was considerably lower (A) than treated cells (B-D). To ensure that enhanced migration of treated cells is because of SDF-1α/CXCR4 axis, AMD3100, a CXCR4-blocking agent, was used to inhibit CXCR4 (E-H). (I) Migration of untreated Ad-MSCs toward FBS was used as a positive control. (J) In absence of FBS and SDF1-1α in lower chamber (as negative control), very few untreated Ad-MSCs migrated only by chance. (K) Quantified average number of migrated Ad-MSCs toward SDF-1α after pretreatments with various hypoxia-mimicking agents (i.e. VPA, CoCl₂, and DFX) and also inhibition by AMD3100. Significance of the data was examined in the level of p < 0.001 using the one-way ANOVA and expressed as mean ± SD. # denotes significant difference relative to untreated control, VPA and CoCl₂ pretreatment. * denotes significant difference relative to untreated control. VPA: valproic acid, CoCl₂: cobalt-chloride, DFX: deferoxamine mesylate, SDF-1α: stromal derived factor-1α.

Table 1. Description of the semi-quantitative RT-PCR primers.

Gene	Strand	Primer sequence	Product size (bp)
ACTB	Sense	5′- GCTCAGGAGGAGCAAT -3′	187
	Antisense	5′- GGCATCCACGAAACTAC -3′
CXCR4-A (Lo)	Sense	5′- GAGGAGTTAGCCAAGATGTGAC -3′	766
	Antisense	5′- GACGCCAACATAGACCACCT -3′
CXCR4-B	Sense	5′- TCTGGAGAACCAGCGGTTAC -3′	501
	Antisense	5′- GACGCCAACATAGACCACCT -3′

In order to quantify the level of the CXCR4 variants, a Real-time RT-PCR assay was established. Quantitative Real-time PCR analysis was performed using SYBR Green PCR Master Mix according to the manufacturer's recommendations (Takara). Each experiment was performed 2 times. The sequences of the applied primers are shown in Table 2.

Table 2. Description of the Real time-PCR primers.

Gene		Primer sequence	Product size (bp)
ACTB	Sense	5′- GCTCAGGAGGAGCAAT -3′	187
	Antisense	5′- GGCATCCACGAAACTAC -3′
CXCR4-A (Lo)	Sense	5′- GAGGAGTTAGCCAAGATGTGAC -3′	116
	Antisense	5′- CCCATAGTGACTTCATTATATCCTTC -3′
CXCR4-B	Sense	5′- GCAGCAGGTAGCAAAGTG -3′	116
	Antisense	5′- CTCGGTGTAGTTATCTGAAGTG -3′