Extracellular Vesicles Improve Post-Stroke Neuroregeneration and Prevent Postischemic Immunosuppression

Abstract

Although the initial concepts of stem cell therapy aimed at replacing lost tissue, more recent evidence has suggested that stem and progenitor cells alike promote postischemic neurological recovery by secreted factors that restore the injured brain's capacity to reshape. Specifically, extracellular vesicles (EVs) derived from stem cells such as exosomes have recently been suggested to mediate restorative stem cell effects. In order to define whether EVs indeed improve postischemic neurological impairment and brain remodeling, we systematically compared the effects of mesenchymal stem cell (MSC)-derived EVs (MSC-EVs) with MSCs that were i.v. delivered to mice on days 1, 3, and 5 (MSC-EVs) or on day 1 (MSCs) after focal cerebral ischemia in C57BL6 mice. For as long as 28 days after stroke, motor coordination deficits, histological brain injury, immune responses in the peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Improved neurological impairment and long-term neuroprotection associated with enhanced angioneurogenesis were noticed in stroke mice receiving EVs from two different bone marrow-derived MSC lineages. MSC-EV administration closely resembled responses to MSCs and persisted throughout the observation period. Although cerebral immune cell infiltration was not affected by MSC-EVs, postischemic immunosuppression (i.e., B-cell, natural killer cell, and T-cell lymphopenia) was attenuated in the peripheral blood at 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Because MSC-EVs have recently been shown to be apparently safe in humans, the present study provides clinically relevant evidence warranting rapid proof-of-concept studies in stroke patients.

Significance: Transplantation of mesenchymal stem cells (MSCs) offers an interesting adjuvant approach next to thrombolysis for treatment of ischemic stroke. However, MSCs are not integrated into residing neural networks but act indirectly, inducing neuroprotection and promoting neuroregeneration. Although the mechanisms by which MSCs act are still elusive, recent evidence has suggested that extracellular vesicles (EVs) might be responsible for MSC-induced effects under physiological and pathological conditions. The present study has demonstrated that EVs are not inferior to MSCs in a rodent stroke model. EVs induce long-term neuroprotection, promote neuroregeneration and neurological recovery, and modulate peripheral post-stroke immune responses. Also, because EVs are well-tolerated in humans, as previously reported, the administration of EVs under clinical settings might set the path for a novel and innovative therapeutic stroke concept without the putative side effects attached to stem cell transplantation.

Keywords: Adult stem cells; Angiogenesis; Cellular therapy; Clinical translation; Mesenchymal stem cells; Nervous system; Tissue regeneration.

©AlphaMed Press.

Figures

Figure 1.

MSC-EVs reduce postischemic neurological impairment. Motor coordination, evaluated using the rotarod test (A), tightrope test (B), and corner turn test (C) at 7, 14, and 28 days after ischemia (DPI). Mice (n = 12 per condition) were exposed to middle cerebral artery occlusion, followed by delivery of normal saline (control), cells of 2 independent bone marrow-derived MSC lines (MSC I and MSC II), or EVs harvested from supernatants of both MSC lines (MSC-EVs I and MSC-EVs II). MSCs were infused at 1 DPI and MSC-EVs at 1, 3, and 5 DPI. MSC-EV-treated mice showed very similar neurological performance to that of MSC-treated mice. Data are mean ± SD. ∗, p < .05 compared with control. Abbreviations: EVs, extracellular vesicles; MSC, mesenchymal stem cell; MSC I and MSC II, group I and II of bone marrow-derived MSC lines, respectively; MSC-EVs I and MSC-EVs II, supernatants of the two MSC lines.

Figure 2.

MSC-EVs induce postischemic long-term neuroprotection. Neuronal survival as examined by immunohistochemistry in the ischemic striatum at 28 days after ischemia in mice (n = 12 per condition) exposed to middle cerebral artery occlusion followed by i.v. delivery of normal saline (control), two different MSC lines (MSC I and MSC II), or their corresponding EVs (MSC-EVs I and MSC-EVs II). Representative microphotographs are also shown. Scale bars = 50 µm. Data are mean ± SD values. ∗, p < .05 compared with control. Abbreviations: EVs, extracellular vesicles; MSC, mesenchymal stem cell; MSC I and MSC II, group I and II of bone marrow-derived MSC lines, respectively; MSC-EVs I and MSC-EVs II, supernatants of the two MSC lines.

Figure 3.

MSC-EVs increase postischemic cell proliferation. Cell proliferation was assessed by BrdU incorporation analysis in the ischemic striatum at 28 days after ischemia in mice (n = 12 per condition) exposed to middle cerebral artery occlusion followed by i.v. delivery of normal saline (control), cells of two independent bone marrow-derived MSC lines (MSC I and MSC II), or EVs harvested from supernatants of both MSC lines (MSC-EVs I and MSC-EVs II). Representative microphotographs of BrdU+ cells counterstained with DAPI are also shown. Scale bars = 100 µm. Data are mean ± SD values. ∗, p < .05 compared with control. Abbreviations: BrdU, 5-bromo-2-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; EVs, extracellular vesicles; MSC, mesenchymal stem cell; MSC I and MSC II, group I and II of bone marrow-derived MSC lines, respectively; MSC-EVs I and MSC-EVs II, supernatants of the two MSC lines.

Figure 4.

MSC-EVs stimulate postischemic neurogenesis and angiogenesis. Double-immunohistochemistry for the proliferation marker BrdU and the immature neuronal marker Dcx (A), the proliferation marker BrdU and the mature neuronal marker NeuN (B), and the proliferation marker BrdU and the endothelial marker CD31 (C), evaluated in the ischemic striatum at 28 days after ischemia. Mice (n = 12 per condition) were exposed to middle cerebral artery occlusion followed by i.v. delivery of normal saline (control), cells of two independent bone marrow-derived MSCs lines (MSC I and MSC II), or EVs harvested from supernatants of both MSC lines (MSC-EVs I and MSC-EVs II). Representative microphotographs of double-labeled cells counterstained with DAPI are also shown. Scale bars = 20 µm. Data are mean ± SD values. ∗, p < .05 compared with control. Abbreviations: BrdU, 5-bromo-2-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; Dcx, doublecortin; EVs, extracellular vesicles; MSC, mesenchymal stem cell; MSC I and MSC II, group I and II of bone marrow-derived MSC lines, respectively; MSC-EVs I and MSC-EVs II, supernatants of the two MSC lines.

Figure 5.

MSC-EVs do not influence acute immune responses at 2 days after ischemia. Absolute counts and relative proportions of peripheral blood leukocytes and leukocyte subsets at 48 hours after ischemia or sham surgery (n = 8 per condition). Sham-operated mice underwent the same operation procedure as for ischemia, except for occlusion of the middle cerebral artery. Normal saline (control) or EVs harvested from supernatants of MSC line I (MSC-EVs I) were i.v. delivered to mice exposed to middle cerebral artery occlusion at 24 hours after ischemia; sham mice received saline injections. (A): Total white blood cell counts were analyzed using a veterinary hematology analyzer. The absolute cell numbers of neutrophils (B), dendritic cells (C), macrophages (D), monocytes (E), and monocyte subsets [B lymphocytes (G), NK cells (H), and T lymphocytes (I)] were determined by flow cytometry. The proportion of inflammatory (Ly6Chigh) and resident (Ly6Cneg) monocytes (E, right) and that of CD4 and CD8 T lymphocytes (I, right) was also calculated. Activation of myeloid cells (monocytes and dendritic cells) (F) and lymphocytes (J) was evaluated by MHCII and CD69 expression analysis, respectively. Specifications of leukocyte subset identification according to their specific antigen expression are listed in the supplemental online Table 1 and show in supplemental online Figure 2. Data are mean ± SD values. ∗, p < .05 compared with sham surgery. Abbreviations: EVs, extracellular vesicles; MHCII, major histocompatibility class II; MSC, mesenchymal stem cell; MSC I and MSC II, group I and II of bone marrow-derived MSC lines, respectively; MSC-EVs I and MSC-EVs II, supernatants of the two MSC lines; NK, natural killer.

Figure 6.

MSC-EVs reverse postischemic lymphopenia at 6 days after ischemia. Absolute counts and relative proportions of peripheral blood leukocytes and leukocyte subsets at 6 days after ischemia or after sham surgery (n = 12 per condition). Sham-operated mice underwent the same operation procedure as for ischemia except for occlusion of the middle cerebral artery. Normal saline (control) or EVs harvested from supernatants of MSC line I (MSC-EVs I) were i.v. delivered to mice exposed to middle cerebral artery occlusion (MCAO) at 1, 3, and 5 days after ischemia; sham mice received saline injections. (A): Total white blood cell counts were analyzed using a veterinary hematology analyzer. Absolute cell numbers of neutrophils (B), dendritic cells (C), macrophages (D), monocytes (E), B lymphocytes (G), NK cells (H), and T lymphocytes (I) were determined by flow cytometry. The proportion of inflammatory (Ly6Chigh) and resident (Ly6Cneg) monocytes (E, right) and of CD4 and CD8 T lymphocytes (I, right) was also calculated. Activation of myeloid cells (monocytes and dendritic cells) (F) and lymphocytes (J) was evaluated by MHCII and CD69 expression analysis, respectively. Specifications of leukocyte subset identification according to their specific antigen expression are listed in supplemental online Table 1 and show in in supplemental online Figure 2. Data are mean ± SD. ∗, p < .05 or ∗∗∗, p < .001 compared with sham surgery or MCAO. Abbreviations: EVs, extracellular vesicles; MHCII, major histocompatibility class II; MSC, mesenchymal stem cell; MSC I and MSC II, group I and II of bone marrow-derived MSC lines, respectively; MSC-EVs I and MSC-EVs II, supernatants of the two MSC lines; NK, natural killer.

Figure 7.

Cerebral immune cell infiltration is not modulated by MSC-EVs. (A): Density of leukocytes in the ischemic striatum at 2 and 6 days after ischemia (DPI) evaluated by CD45 immunohistochemistry (n = 8 per condition). Total number of CD45+ leukocytes (B), neutrophils (C), dendritic cells (D), macrophages (E), monocytes (F), and lymphocytes (H) in the ipsilateral and contralateral hemisphere at 6 DPI as determined by flow cytometry. Activation of myeloid subsets (G) and lymphocyte subsets (I) as determined by MHCII and CD69 expression analysis in the ipsilateral and contralateral hemisphere at 6 DPI using flow cytometry in mice exposed to middle cerebral artery occlusion, followed by i.v. delivery of normal saline (control) or EVs harvested from supernatants of MSC line I (MSC-EVs I) at 1, 3, and 5 DPI. In the proportion of monocyte (F) and T lymphocyte (H) subsets was also measured. Three independent flow cytometry experiments were performed, for each of which four brain hemispheres per group were pooled. Data are mean ± SD. ∗, p < .05 or ∗∗, p < .01 compared with the contralateral hemisphere. Abbreviations: contra, contralateral; EVs, extracellular vesicles; ispi, ipsilateral; MHCII, major histocompatibility class II; MSC, mesenchymal stem cell; MSC I and MSC II, group I and II of bone marrow-derived MSC lines, respectively; MSC-EVs I and MSC-EVs II, supernatants of the two MSC lines; NK, natural killer.