Calcitonin Gene-related Peptide Directs Transplanted MSCs Homing To Sites Of Spinal Cord Injury

Objectives: Human umbilical cord mesenchymal stem cells(HUMSCs) is one of the promising candidates to treat many diseases including spinal cord injury(SCI). However, the key chemokine guiding cells located into the epicenter of SCI still remains unknown. Here, we examined the function of Calcitonin gene-related peptide(CGRP), which increased and released after rat SCI, on the homing of HUMSCs to the injury site.Methods: Chemotactic migration of HUMSCs to CGRP ranging from 10-9 to 10-6 mol/L is assessed by Boyden chamber and Dunn chamber assay in vitro(treatment of medium with same volume PBS as control). CCK-8, Annexin V-FITC apoptosis assay and immunocytochemistry are performed to visualize effects of CGRP on cell proliferation, survival and differentiation. By western blot, PI3K/Akt and MAPK signaling pathways are analyzed in CGRP-induced HUMSCs migration. After T9 transection or contusion, CGRP in spinal cord tissues has been tested with ELISA(0~14d). HUMSCs are transplanted through lumbar puncture at 3rd after injury(transection and contusion) with administration of 0.1mol/L PBS, 10-6 mol/L CGRP 8-37 and 10-6 mol/L CGRP in transection injury sites. Relative distribution of HUMSCs is quantitated by immunohistochemistry 7 days after injection.Results: HUMSCs exhibited different chemotactic responses to various concentrations of CGRP, of which 10-6 mol/L CGRP was the most attractive(1.81 ± 0.13 times increase). Through time-lapse video analysis we found that elevating speed, chemotactic index(CMI) or forward migration index(FMI) contributed to the trans-filter migration. CGRP had no effects on HUMSCs proliferation and survival, but could increase MAP2-, MBP- and Nestin-positive cells. The promotions of Akt and p38 MAPK phosphorylation were involved in chemotaxis migration of HUMSCs to CGRP. Abolishment of these two pathways led to a decrease in trans-filter cells. In vivo, increased CGRP in the site of injury was detected followed by T9 injury and throughout the observation period. HUMSCs implanted in transetion epicenter were much more than which in contusion site(158.1 ± 8.6 vs. 19.38 ± 3.7 cells/section). With continuous injection of PBS, CGRP 8-37 and CGRP at sites of transection, 158.1 ± 8.6, 88.07 ± 6.1 and 205.3 ± 9.2(cells/section) homing HUMSCs were found in T9 respectively. CGRP 8-37 decreased HUMSCs homing to the injury site(P < 0.01)。Conclusions: With binding to its receptors, CGRP activates Akt and p38 MAPK signaling pathways and induces HUMSCs chemotactic migration. After spinal cord injury, the increase of CGRP guides transplanted HUMSCs homing to the epicenter. CGRP can be used as a model molecule of SCI to simulate and analyze chemotactic migration of stem cells for transplantation therapy.