The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury.

Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) have shown potential in facilitating recovery from spinal cord injury (SCI) through communicating with microglia/macrophages (MG/MΦ). We here focused on chemokines as a candidate for the communication. Selected MG/MΦ-related chemokines were determined gene expression after SCI and further focused CCL2/CCR2 and CCL5/CCR5 to estimate role of the chemokines by hMSCs. Male C57/BL6 mice were subjected to spinal cord transection. Gene expression was assayed in the spinal cords following SCI for selected MG/MΦ-related chemokines and their receptors. hMSCs (5×105 cells) were then transplanted into parenchyma of the spinal cord, and the expressions of the Ccl2/Ccr2 and Ccl5/Ccr5 axes, inflammation, MG/MΦ-polarization, and axonal regeneration were evaluated to measure the influence of the hMSCs. Finally, mouse CCL5 was injected into the spinal cords. Acute increases in gene expression after SCI were observed for most chemokines, including Ccl2; chronic increases were observed for Ccl5. CCL2+-cells merged with NeuN+-neurons. CCR2+ immunoreactivity was principally observed in Ly-6G+/iNOS+-granulocytes on postoperative day (pod) 1, and CCL5+ and CCR5+ immunoreactivity overlapped with NeuN+-neurons and F4/80+-MG/MΦ on pod 14. The hMSC transplantation enhanced Ccl2 and Ccl5 and improved locomotor activity. The hMSC implantation did not alter the number of Ly-6G+/CCR2+ but decreased Il1, Elane, and Mpo on pod 3. Conversely, hMSC transplantation increased expression of Zc3h12a (encodes MCP-1-induced protein) on pod 14. Moreover, hMSC increased the Aif1, and two alternatively activated macrophage (AAM)-related genes, Arg1 and Chil3 (Ym1), as well as axonal regenerative markers, Dpysl2 and Gap43. Gene expression indicative of AAM polarization and axonal regeneration were partially recovered by CCL5 injection. These results suggest that hMSC implantation increases Ccl2 and Ccl5, improves locomotor activity, enhances MG/MΦ polarization to AAM, and increases the gene expression of axonal regenerative markers. These functions of hMSCs might be partially mediated by the CCL2/CCR2 and CCL5/CCR5 axes

Intrapancreatic injection of human bone marrow-derived mesenchymal stem/stromal cells alleviates hyperglycemia and modulates the macrophage state in streptozotocin-induced type 1 diabetic mice

<div><p>Type 1 diabetes mellitus is a progressive disease caused by the destruction of pancreatic β-cells, resulting in insulin dependency and hyperglycemia. While transplanted bone marrow-derived mesenchymal stem/stromal cells (BMMSCs) have been explored as an alternative therapeutic approach for diseases, the choice of delivery route may be a critical factor determining their sustainability. This study evaluated the effects of intrapancreatic and intravenous injection of human BMMSCs (hBMMSCs) in streptozotocin (STZ)-induced type 1 diabetic mouse model. C57/BL6 mice were intraperitoneally injected with 115 mg/kg STZ on day 0. hBMMSCs (1 × 10⁶ cells) or vehicle were injected into the pancreas or jugular vein on day 7. Intrapancreatic, but not intravenous, hBMMSC injection significantly reduced blood glucose levels on day 28 compared with vehicle injection by the same route. This glucose-lowering effect was not induced by intrapancreatic injection of human fibroblasts as the xenograft control. Intrapancreatically injected fluorescence-labeled hBMMSCs were observed in the intra- and extra-lobular spaces of the pancreas, and intravenously injected cells were in the lung region, although the number of cells mostly decreased within 2 weeks of injection. For hBMMSCs injected twice into the pancreatic region on days 7 and 28, the injected mice had further reduced blood glucose to borderline diabetic levels on day 56. Animals injected with hBMMSCs twice exhibited increases in the plasma insulin level, number and size of islets, insulin-positive proportion of the total pancreas area, and intensity of insulin staining compared with vehicle-injected animals. We found a decrease of Iba1-positive cells in islets and an increase of CD206-positive cells in both the endocrine and exocrine pancreas. The hBMMSC injection also reduced the number of CD40-positive cells merged with glucagon immunoreactions in the islets. These results suggest that intrapancreatic injection may be a better delivery route of hBMMSCs for the treatment of type 1 diabetes mellitus.</p></div

hBMMSC distribution following intrapancreatic or intravenous injection.

<p>Representative time-course images of diabetic mice injected with NIR815-labeled hBMMSCs into the jugular vein or pancreatic region (A) and semi-quantification graphs of the signal intensities in the right supraphrenic chest (B) and left lateral abdomen under the subphrenic chest (C) (n = 8 per group). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 (Student’s t-test). (D) The injected PKH26-labeled hBMMSCs (red and arrows) were located adjacent to the pancreas on day 14 (7 days after transplantation). Immunostaining of amylase, an exocrine pancreatic marker, is shown in green. Scale bars = 500 μm.</p

Effects of hBMMSCs on plasma insulin and pancreatic morphology on day 56.

<p>Plasma insulin levels (A), pancreas weight (B), and islet histology with H.E. staining and insulin immunostaining (C) in mice intrapancreatically injected twice with hBMMSCs or the vehicle were determined on day 56. Simultaneously, these parameters were determined in STZ-untreated control (untreated) mice on day 7 after STZ treatment (STZ day 7 or STZ). Using histological sections, the islet area (D), insulin positive (+) area (E), and islet number (F) were determined. Sample numbers are indicated in each bar. Data are expressed as the mean ± SEM and compared between vehicle- and hBMMSC-injected groups on day 56 by the Student’s t-test. Scale bars in (C) = 100 μm.</p

Isotype controls used for immunohistochemistry.

<p>Isotype controls used for immunohistochemistry.</p

Intrapancreatic hBMMSC injection modulates the macrophage activation state.

<p>Representative images of Iba1 and CD206 immunostaining in the islets (A) and exocrine pancreas (B) of STZ-untreated control mice (untreated) and STZ-treated mice on day 28 after intrapancreatic injection of hBMMSCs or the vehicle. Scale bars = 100 μm. Semi-quantitative analysis of islets (C and D) and the exocrine pancreas (E and F) for Iba1 (C and E) and CD206 (D and F) in untreated (n = 3), vehicle-treated (n = 6), and hBMMSC-treated (n = 6) mice. Data are expressed as the mean ± SEM (Tukey post-hoc test). (G) Iba1 immunoreactions (green) were merged with F4/80 (red) immunoreactions in the islets as indicated by insulin immunoreactions (pink) in vehicle-injected mice. (H) Iba1 immunoreactions (green) were merged with CD206 immunoreactions (red) in the adjacent area of the islets (insulin immunoreactions; pink) in hBMMSC-injected mice. (I) Iba1 immunoreactions (green) were merged with MHC class II (red) in the islets and adjacent in vehicle-injected mice. DAPI staining (blue). Scale bars = 25 μm.</p

Primary and secondary antibodies used for immunohistochemistry.

<p>Primary and secondary antibodies used for immunohistochemistry.</p

Intrapancreatic hBMMSC injection reduces CD40 immunoreactivity in islets.

<p>Representative images (A) and semi-quantification of CD40 immunostaining in the islets (B) and exocrine pancreases (C) of STZ-untreated control mice (n = 3; untreated) and mice intrapancreatically injected with the vehicle or hBMMSCs (n = 6 per group). Scale bars = 50 μm. Data are expressed as the mean ± SEM. (Tukey post-hoc test). (D) CD40 immunoreactions (green) were observed inside of the islet (insulin immunostaining; yellow), indicated by a dashed circle, and were not merged with Iba1 immunoreactions (red) in vehicle-injected mice. (E) CD40 immunoreactions (green and arrow) mainly localized to the marginal area of the larger islet and were not merged with MHC class II (red and arrowhead) or insulin (yellow) in hBMMSC-injected mice. (F) CD40 immunoreactions (green) in vehicle-injected mice were found in the islet and were merged with glucagon (red) immunoreactions, indicated by arrows, but not insulin (pink) immunoreactions. DAPI staining (blue). Scale bars = 25 μm.</p

Effects of hBMMSC injection on blood glucose and body weight of STZ-induced diabetic mice.

<p>Mice were injected intravenously (A, C) or intrapancreatically (B, D) with hBMMSCs or the vehicle on day 7 after STZ treatment, and their blood glucose (A, B) and body weight (C, D) were monitored until day 28. Mice were intrapancreatically injected with hBMMSCs, HDFs, or the vehicle on day 7 after STZ treatment, and their blood glucose concentration (E) and body weight (F) were monitored until day 28. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 (vs vehicle injection); #p < 0.05 (vs HDF injection) (Tukey post-hoc test).</p