Combining Injectable Plasma Scaffold with Mesenchymal Stem/Stromal Cells for Repairing Infarct Cavity after Ischemic Stroke

Abstract

Stroke survivors are typically left with structural brain damage and associated functional impairment in the chronic phase of injury, for which few therapeutic options exist. We reported previously that transplantation of human embryonic stem cell (hESC)-derived neural stem cells together with Matrigel scaffolding into the brains of rats after focal ischemia reduced infarct volume and improved neurobehavioral performance. Matrigel is a gelatinous protein mixture extracted from mouse sarcoma cells, thus would not be approved for use as a scaffold clinically. In this study, we generated a gel-like scaffold from plasma that was controlled by changing the concentration of CaCl₂. In vitro study confirmed that 10-20 mM CaCl₂ and 10-40% plasma did not affect the viability and proliferation of human and rat bone marrow mesenchymal stem/stromal cells (BMSCs) and neural stem cells (NSCs). We transplanted plasma scaffold in combination of BMSCs into the cystic cavity after focal cerebral ischemia, and found that the atrophy volume was dramatically reduced and motor function was significantly improved in the group transplanted with scaffold/BMSCs compared with the groups treated with vehicle, scaffold or BMSCs only. Our data suggest that plasma-derived scaffold in combination of BMSCs is feasible for tissue engineering approach for the stroke treatment.

Keywords: aging; aging-related diseases; diagnosis; frailty; regulation; senescence.

Figure 1.

The effect of CaCl₂, fibrinogen and thrombin on the formation of plasma gel in vitro. A) The time course of development of gelatinous clot of plasma in the presence of different concentrations of CaCl₂. B) The effect of different concentrations of fibrinogen on the formation of gelatinous clot of plasma. C) The effect of different concentrations of thrombin with 20 mM CaCl₂ on the formation of gelatinous clot of plasma. D) The comparison of different combinations of coagulation factors on formation of gelatinous clot of plasma. The experiments were repeated three times.

Figure 2.

The effect of different concentrations of CaCl₂ and plasma on cell survival and death in vitro. A) The human BMSCs (hBMSCs) were treated with different concentrations of CaCl₂ for 1, 2 and 3 days and CCK-8 proliferation assay was performed to determine the survival rate of hBMSCs. B) Human embryonic stem cells-derived NSCs were incubated with CaCl₂ at different concentration as indicated and the proliferation rate of NSCs was determined by CCK-8 proliferation assay. C) Quantitation of calcein-AM fluorescent staining of rat BMSCs (rBMSCs) treated with different concentrations of CaCl₂ for 1, 2 and 3 days. D) The HT-22 cells were treated with different concentrations of rat plasma and 10% FBS for 24 hrs and the survival rate of HT-22 cells was determined by MTT assay. E) The representative images of rBMSCs cultured in gelatinous plasma clot (30% rat plasma and 10uM CaCl₂). Left panel: bright view of rBMSCs; middle panel: 2D view of GFP-positive rBMSCs; right panel: 3D view of GFP-positive rBMSCs. The experiments were repeated three times.

Figure 3.

BMSCs/plasma-derived scaffold transplantation reduced damaged volume of young adult rats after MCAO. A) Representative images of lesion areas in H&E-stained stained coronal brain sections from scaffold and BMSCs/scaffold-treated rats at 6 weeks after dMCAO. B) Quantitative analysis of damaged volume in vehicle (control), scaffold, BMSCs and BMSCs/scaffold-treated young rats 4 weeks after transplantation.

Figure 4.

BMSCs/plasma-derived scaffold transplantation improves long-term recovery after experimental stroke in young rats. BMSCs, scaffold, vehicle (control) or BMSCs/scaffold was implanted into brain cavity 3 weeks after MCAO and neurological behavioral tests including Bederson's test (A), Cylinder Test (B), EBST (C), and limb placing test (D), were performed at 1-4 weeks after transplantation. Values presented as mean ± SEM. *P < 0.05, compared with vehicle-treated group.

Figure 5.

BMSCs/plasma-derived scaffold transplantation improves long-term recovery after experimental stroke in young rats. BMSCs, scaffold, vehicle (control) or BMSCs/scaffold was implanted into brain cavity 3 weeks after MCAO and staggered ladder test was performed at 1-4 weeks after transplantation. Values presented as mean ± SEM. *P < 0.05, compared with vehicle-treated group