Transplantation of Human Brain-Derived Ischemia-Induced Multipotent Stem Cells Ameliorates Neurological Dysfunction in Mice After Stroke

Abstract

We recently demonstrated that injury/ischemia-induced multipotent stem cells (iSCs) develop within post-stroke human brains. Because iSCs are stem cells induced under pathological conditions, such as ischemic stroke, the use of human brain-derived iSCs (h-iSCs) may represent a novel therapy for stroke patients. We performed a preclinical study by transplanting h-iSCs transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion (MCAO). Compared with PBS-treated controls, h-iSC transplantation significantly improved neurological function. To identify the underlying mechanism, green fluorescent protein (GFP)-labeled h-iSCs were transplanted into post-stroke mouse brains. Immunohistochemistry revealed that GFP+ h-iSCs survived around the ischemic areas and some differentiated into mature neuronal cells. To determine the effect on endogenous neural stem/progenitor cells (NSPCs) by h-iSC transplantation, mCherry-labeled h-iSCs were administered to Nestin-GFP transgenic mice which were subjected to MCAO. As a result, many GFP+ NSPCs were observed around the injured sites compared with controls, indicating that mCherry+ h-iSCs activate GFP+ endogenous NSPCs. In support of these findings, coculture studies revealed that the presence of h-iSCs promotes the proliferation of endogenous NSPCs and increases neurogenesis. In addition, coculture experiments indicated neuronal network formation between h-iSC- and NSPC-derived neurons. These results suggest that h-iSCs exert positive effects on neural regeneration through not only neural replacement by grafted cells but also neurogenesis by activated endogenous NSPCs. Thus, h-iSCs have the potential to be a novel source of cell therapy for stroke patients.

Keywords: cell therapy; cell transplantation; ischemic stroke; multipotent stem cells; neural regeneration; neural stem cells; stroke patients.

Graphical Abstract

Figure 1.

h-iSCs are a unique stem cell population as evidenced by comparative gene expression profiling. (A) PCA for h-iSCs, h-ECs, h-ACs, h-NSPCs, and h-MSCs. (B) Distribution of h-iSC and h-MSC clusters from single-cell RNA-seq analysis. (C-F) Comparison of NES (C), CD44 (D), ENG (E), and ALCAM (F) gene expression levels between h-iSCs and h-MSCs by violin plot analysis. (G, H) Immunohistochemistry showing that most putative h-iSCs (G) express nestin (H) [nestin (H); DAPI (H)]. (I) Heat map analysis of neural lineage-related gene expression patterns for h-iSCs and h-NSPCs. (J) Formation of neurosphere-like h-iSC clusters in free-floating culture. (K-O) Cells within neurosphere-like clusters (K) differentiated into Tuj1⁺ [Tuj1 (L); DAPI (L)] and NF⁺ neurons [NF (M); DAPI (M)], MBP⁺ oligodendrocytes [MBP (N); DAPI (N)], and GFAP⁺ astrocytes [GFAP (O); DAPI (O)]. (P) Electron microscopy also revealed myelin-like formation in cell clusters (arrows). Scale bars: 100 µm (G, H), 20 µm (K), 50 µm (L-O), and 1 µm (P). Abbreviations: AC, astrocyte; ALCAM, activated leukocyte cell adhesion; DAPI, 4ʹ,6-diamidino-2-phenylindole; EC, endothelial cell; ENG, endoglin; EM, electron microscopy; GFAP, glial fibrillary acidic protein; iSC, injury/ischemia-induced multipotent stem cell; MBP, myelin basic protein; MSC, mesenchymal stem cell; NES, nestin; NF, neurofilament; NSPC, neural stem/progenitor cell; PCA, principal component analysis.

Figure 2.

Intracranial transplantation of h-iSCs following MCAO improves neurological function. (A) Performance metrics on multiple tests were compared among the 4 mouse treatment groups: (1) Sham surgery group (n = 13); (2) MCAO followed by PBS injection (M-PBS group, n = 12); (3) MCAO followed by low dose h-iSC injection (M-iSC (low) group, n = 13); (4) MCAO followed by high-dose h-iSC injection (M-iSC (high) group, n = 13). (B-G) Performance levels on wire hang (B), basket (C), open-field (D), hot plate (E), Y-maze (F), and passive avoidance-learning (G) tests among the 4 mouse treatment groups. *P < .05 between groups (B-G). #P < .05 between the conditioning and test trials within groups (G). [n = 13 for Sham group (B-G), n = 12 for M-PBS group (B-G), n = 13 for M-iSC (low) group (B-G), n = 13 for M-iSC (high) group (B-G)]. Abbreviations: iSC, injury/ischemia-induced multipotent stem cell; PBS, phosphate-buffered saline; MCAO, middle cerebral artery occlusion; PBS,: phosphate-buffered saline.

Figure 3.

Transplanted h-iSCs differentiate into mature neurons that may contribute to recovery of neurological function following MCAO. (A, B) GFP-labeled h-iSCs (A) were transplanted at 6 weeks following MCAO (B). (C) GFP⁺ h-iSCs were transplanted around the ischemic areas of CB-17 WT mice. (D-F) Immunohistochemical staining of brain sections 3 days after transplantation showing GFP⁺ h-iSCs at the injury site expressing nestin (E, F) [GFP (E, F); nestin (E, F); DAPI (E, F)]. (D, G-I) Immunohistochemical staining of brain sections 10 days following transplantation showing GFP⁺ h-iSCs (G) expressing neuronal markers Tuj1 (H, arrowhead) [GFP (H); Tuj1 (H); DAPI (H)] and MAP2 (I, arrowhead) [GFP (I); MAP2 (I); DAPI (I)]. (D, J) Immunohistochemical staining of brain sections 7 weeks following transplantation showing GFP⁺ h-iSCs expressing MAP2 (J, arrowhead) [GFP (J); MAP2 (J); DAPI (J)]. (K) Comparison of Y-maze performance among Sham, M-PBS, h-iSC GFP(+), and h-iSC GFP(−) groups. Scale bars: 100 µm (A, E), 20 µm (F, H, I), 50 µm (G), and 10 µm (J). *P < .05 between Sham and M-PBS groups (K). #P < .05 between M-PBS and h-iSC GFP(+) groups (K). [n = 24 for Sham group, n = 20 for M-PBS group, n = 13 for h-iSC GFP(+) group, n = 10 for h-iSC GFP(−) group]. Abbreviations: DAPI, ­4ʹ,6-diamidino-2-phenylindole; GFP, green fluorescent protein; iSC, injury/ischemia-induced multipotent stem cell; MAP2, microtubule-associated protein 2; MCAO, middle cerebral artery occlusion; WT, wild-type.

Figure 4.

Transplanted h-iSCs activate endogenous NSPCs at the injury site following MCAO. (A) Schematic representation of a reproducible stroke model using Nestin-GFP TG mice. (B, C) mCherry-labeled h-iSCs (B) were transplanted 6 weeks after MCAO (C). (D) mCherry⁺ h-iSCs were transplanted around the ischemic area of Nestin-GFP TG mice. (E-G) Immunohistochemical staining of brain sections 3 days following transplantation showing many GFP⁺ NSPCs around transplanted mCherry⁺ h-iSCs (F, G) [GFP (F, G); mCherry (F, G); DAPI (F, G)]. (H-J) Immunohistochemical staining of brain sections 3 days following PBS injection showing few GFP⁺ NSPCs at the injection site (I) but numerous GFP⁺ NSPCs in the SVZ (J) [GFP (I, J); DAPI (I, J)]. Scale bars: 100 µm (B, F), 50 µm (G), and 200 µm (I, J). Abbreviations: DAPI, 4ʹ,6-diamidino-2-phenylindole; F1, first filial hybrid; GFP, green fluorescent protein; iSC, injury/ischemia-induced multipotent stem cell; MCAO, middle cerebral artery occlusion; N, numbers of backcrossing generations; NSPC, neural stem/progenitor cell; PBS, phosphate-buffered saline; SVZ, subventricular zone; TG, transgenic.

Figure 5.

Coculture with h-iSCs promotes the proliferation of m-NSPCs. (A, B) Evaluation of m-NSPC proliferative potential in mono-culture (A) or in coculture with h-iSCs (B). (C-E) Immunocytochemical staining showing that the number of m-NSPC-derived nestin⁺ cells (GFP⁻/nestin⁺ cells) was significantly higher in the presence of GFP⁺ h-iSCs [h-iSC(+) group] compared to m-NSPC mono-culture (control) [h-iSC(−) group] [nestin (C, D); GFP (D); DAPI (C, D)]. (F-H) Immunocytochemical staining showing that the number of m-NSPC-derived Sox2⁺ cells (GFP⁻/Sox2⁺ cells) was significantly higher in the presence of GFP⁺ h-iSCs compared to m-NSPC mono-culture [Sox2 (F, G); GFP (G); DAPI (F, G)]. (I-K) Immunocytochemical staining showing that the ratio of m-NSPC-derived proliferative cells (GFP⁻/Ki67⁺ cells to GFP⁻/DAPI⁺ cells) was significantly higher in the presence of GFP⁺ h-iSCs compared to m-NSPC mono-culture [Ki67 (I, J); GFP (J); DAPI (I, J)]. Scale bars: 50 µm (C, D, F, G, I, J). *P < .05 between h-iSC(−) and h-iSC(+) groups (E, H, and K). [n = 3 (9 data points) for each group]. Abbreviations: DAPI, 4ʹ,6-diamidino-2-phenylindole; GFP, green fluorescent protein; iSC, injury/ischemia-induced multipotent stem cell; NSPC, neural stem/progenitor cell.

Figure 6.

Coculture with h-iSCs promotes the neuronal differentiation of m-NSPCs. (A, B) Evaluation of m-NSPC-derived neurosphere differentiation potential in mono-culture (A) and in coculture with h-iSCs (B). (C-E) Immunocytochemistry showing that the ratio of m-NSPC-derived neurons (GFP⁻/Tuj1⁺ cells to GFP⁻/DAPI⁺ cells) was significantly higher in the presence of GFP⁺ h-iSCs compared to m-NSPCs alone (control) [Tuj1 (C, D); GFP (D); DAPI (C, D)]. (F-H) Immunocytochemistry showing that the ratio of m-NSPC-derived oligodendrocytes (GFP⁻/MBP⁺ cells to GFP⁻/DAPI⁺ cells) did not differ significantly in the presence of GFP⁺ h-iSCs compared to control conditions [MBP (F, G); GFP (G); DAPI (F, G)]. (I-K) Immunocytochemistry showing that the ratio of m-NSPC-derived astrocytes (GFP⁻/GFAP⁺ cells to GFP⁻/DAPI⁺ cells) did not differ significantly in the presence of GFP⁺ h-iSCs compared to control conditions [GFAP (I, J); GFP (J); DAPI (I, J)]. Scale bars: 100 µm (C, D, F, G, I, J). *P < .05 between h-iSC(−) and h-iSC(+) groups (E, H, and K). [n = 3 (9 data points) for each group]. Abbreviations: DAPI, 4ʹ,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; iSC, injury/ischemia-induced multipotent stem cell; MBP, myelin basic protein; NSPC, neural stem/progenitor cell.

Figure 7.

h-iSCs can form neuronal circuits with m-NSPCs in coculture. (A) Evaluation of neuronal network formation between h-iSC- and m-NSPC-derived neurons under adherent culture conditions. (B-H) Immunocytochemistry showing that h-iSC-derived neurons (GFP⁺ cells) and m-NSPC-derived neurons (GFP⁻ cells) likely formed neuronal networks expressing MAP2 [GFP (B-D); MAP2 (B, C, E); DAPI (B-E), arrowheads (C-E)] and SYN [GFP (F, G); SYP (F, H); DAPI (F-H), arrowheads (F-H)]. (I-K) GFP⁺ h-iSC-derived cells (arrowheads, I, J) made contact with GFP⁻/PSD-95⁺ m-NSPC-derived postsynaptic neurons (arrows, I, K) [GFP (I, J); PSD-95 (I, K); DAPI (I-K)]. Scale bars: 100 µm (B, C) and 50 µm (F, I). Abbreviations: DAPI, 4ʹ,6-diamidino-2-phenylindole; GFP, green fluorescent protein; iSC, injury/ischemia-induced multipotent stem cell; NSPC, neural stem/progenitor cell; PSD-95, postsynaptic density-95; SYP, synaptophysin.0