TY - JOUR
AU - Taguchi, Akihiko
AB - Abstract Transplantation of neural stem cells (NSCs) has been proposed as a therapy for a range of neurological disorders. To realize the potential of this approach, it is essential to control survival, proliferation, migration, and differentiation of NSCs after transplantation. NSCs are regulated in vivo, at least in part, by their specialized microenvironment or “niche.” In the adult central nervous system, neurogenic regions, such as the subventricular and subgranular zones, include NSCs residing in a vascular niche with endothelial cells. Although there is accumulating evidence that endothelial cells promote proliferation of NSCs in vitro, there is no description of their impact on transplanted NSCs. In this study, we grafted cortex-derived stroke-induced neural stem/progenitor cells, obtained from adult mice, onto poststroke cortex in the presence or absence of endothelial cells, and compared survival, proliferation, and neuronal differentiation of the neural precursors in vivo. Cotransplantation of endothelial cells and neural stem/progenitor cells increased survival and proliferation of ischemia-induced neural stem/progenitor cells and also accelerated neuronal differentiation compared with transplantation of neural precursors alone. These data indicate that reconstitution of elements in the vascular niche enhances transplantation of adult neural progenitor cells. Disclosure of potential conflicts of interest is found at the end of this article. Vascular niche, Endothelial cells, Neural stem cell, Ischemia, Transplantation Introduction Acute ischemic stroke caused by occlusion of a cerebral artery leads to sudden interruption of blood flow to the brain, resulting in the loss of neurons, astrocytes, and oligodendrocytes. Despite medical advances, stroke is still a leading cause of death and disability worldwide. A potential therapeutic strategy aiding recovery after stroke is cell transplantation, using neural stem cells (NSCs) derived from embryos, fetuses, or even adult brains to restore critical neuronal elements lost as a consequence of the ischemic episode. Although it has been reported that embryonic [1–3] or fetal neural stem/progenitor cells [4–6] transplanted after stroke may survive, differentiate into neurons, and promote recovery of neuronal functions, serious problems must be overcome before this approach can be used. Two such problems include the tumorigenic properties of the transplanted cells [1] and ethical issues related to the source of neural stem/progenitor cells. Because of these considerations, it is logical to consider other tissue sources, such as harvesting NSCs from adult brain tissue. In the adult mammalian brain, it is well known that neural stem/progenitor cells are present in conventional neurogenic regions, such as the subventricular zone (SVZ) of the lateral ventricle [7] and the subgranular zone (SGZ) within the dentate gyrus of the hippocampus [8]. Previous studies have examined transplantation of adult NSCs from the SVZ [9–11] and hippocampus [12] into the stroke-damaged brain. However, after transplantation of these NSCs, only a small proportion (1∼3%) of grafted cells survived [12]. Of the latter population, virtually none of the surviving cells [9, 11, 12] differentiated into mature neurons. In addition, the number of surviving cells after transplantation of adult neural stem/progenitor cells into the poststroke rat brain was considerably reduced compared with observations in rats engrafted with the same number of fetal NSCs [9]. These observations suggest the possibility that some essential factor(s) is missing that might allow successful adult NSC-based therapy. In the embryo, NSCs and their niches exist for only relatively brief periods as the brain develops. In contrast, in the adult brain, NSCs and their niches are maintained in restricted regions, such as SVZ [13, 14] and SGZ [15], throughout life. Adult neural stem/progenitor cells reside in a vascular niche, and the vasculature is regarded as a key element, especially in the adult SVZ [16]. Endothelial cells are thought to contribute importantly to this vascular microenvironment [15, 17]. In support of this viewpoint, coculture experiments have shown that endothelial cells increased proliferation of neural stem/progenitor cells derived from the adult SVZ [18, 19]. Accumulating evidence indicates that neural stem/progenitor cells are present in many parts of the adult brain, including cortex [20–22], subcortical white matter [23], and spinal cord [24, 25]; i.e., outside of conventional neurogenic zones including SVZ [7] and SGZ [8]. Recently, we also found that neural stem/progenitor cells developed in the poststroke area of the cortex in the adult murine brain (ischemia-induced neural stem/progenitor cells). These neural precursors have the capacity to differentiate into electrophysiologically competent neurons, astrocytes, and myelin-producing oligodendrocytes and can take part in cortical repair after stroke [22]. In this study, we transplanted these cortex-derived, ischemia-induced adult neural stem/progenitor cells into stroke-damaged cortex in the presence or absence of endothelial cells. Our goal was to study whether a vascular factor (i.e., the presence of endothelial cells) might contribute to graft survival, followed by differentiation into neurons and functional recovery. Materials and Methods All procedures were carried out under the auspices of the Animal Care Committee of Hyogo College of Medicine and National Cardiovascular Center and in accordance with the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science. Quantitative analyses were conducted by investigators who were blinded to the experimental protocol and identity of samples under study. Induction of Focal Cerebral Ischemia Male, 6-week-old CB-17/Icr-+/+Jcl mice (CB-17 mice) and CB-17/Icr-scid/scid Jcl mice (SCID mice; Clea Japan Inc., Tokyo, Japan, http://www.clea-japan.com) were used in these studies. Permanent focal cerebral ischemia was produced by ligation and disconnection of the distal portion of the left middle cerebral artery (MCA), as described [22, 26, 27]. In brief, the left MCA was isolated, electrocauterized, and disconnected just distal to crossing the olfactory tract (distal M1 portion) under halothane anesthesia. Infarct area using this protocol for induction of cerebral ischemia and mice of this strain is highly reproducible and limited to the ipsilateral cerebral cortex [22, 26, 27]. Preparation of Adult Ischemia-Induced Neural Stem/progenitor Cells Cortex-derived ischemia-induced adult neural stem/progenitor cells were obtained from poststroke CB-17 mice as described [22]. Briefly, tissue from stroke-affected cortex was mechanically dissociated by passage through 18- and 23-gauge needles to prepare a single-cell suspension. The resulting cell suspensions were incubated in medium promoting formation of neurosphere-like clusters [28]. Cells were incubated in tissue culture flasks (21 cm2) with Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) containing epidermal growth factor (EGF; 20 ng/ml; Peprotech, Rocky Hill, NJ, http://www.peprotech.com) and fibroblast growth factor-basic (FGF-2; 20 ng/ml; Peprotech). On day 7 after incubation, neurosphere-like cell clusters (primary spheres) had formed and were dissociated into single-cell suspensions and reseeded at a density of 1 × 105 cells/well into poly-D-lysine-coated 6-well plates (Nunc, Rochester, NY, http://www.nuncbrand.com) in DMEM/F12 medium including growth factors. Labeling Ischemia-Induced Adult Neural Stem/Progenitor Cells and Endothelial Cells Red fluorescent protein (RFP)-expressing lentiviral vector was used to label stroke-induced adult neural stem/progenitor cells, as described previously [29, 30]. In brief, primary neurospheres cultured for 4 days were transfected with lentivirus carrying RFP (7.4 × 104 TU/μl). Free virus was removed from the medium 2 days after infection, and 24 hours later (on day 7 in culture), expanded RFP-positive cell clusters were harvested, triturated by mechanical dissociation into single cells (a strong expression of RFP protein was detectable in the majority [∼90%] of cells), and used for cell transplantation. To label bovine pulmonary microvascular endothelial cells (CSC-2BM3-C75; Cell Systems, Kirkland, WA, http://www.cell-systems.com), green fluorescent protein (GFP)-expressing lentiviral vector (5 μl; 5.2 × 105 TU/μl) [29] was incubated in 9.6-cm2 tissue culture flasks. Two days after transfection, cells showing green fluorescence were selected by fluorescent-activated cell sorter. Coculture of Ischemia-Induced Neural Stem/Progenitor Cells with Feeder Cells To study the effect of a vascular niche/microenvironment in vitro, neural stem/progenitor cells were cocultured with feeder cells using transwell plates (Corning, Corning, NY, http://www.corning.com/lifesciences) according to a previously described method [18] with minor modifications. In brief, 1 day before coincubation, endothelial cells were plated onto transwell membrane inserts (24 mm) with 0.4-μm pore size at 5 × 105 cells/transwell in endothelial cell growth medium. Membranes were rinsed and placed above cultured neural/progenitor cells in DMEM/F12 medium including growth factors with fetal bovine serum (5%; FBS), the latter essential for the survival of endothelial cells. As a control, neural progenitor cells were replaced with WI26VA4 fibroblasts (ECACC-89101301; Health Protection Agency Culture Collections, Salisbury, Wiltshire, U.K., http://www.hpacultures.org.uk/collections/ecacc.jsp) or astrocytes (ACBRI-371; Cell Systems) and were plated in transwells at the same density and coincubated under the same conditions as endothelial cells. To study the role of cell-cell contact, neural stem/progenitor cells (1 × 105 cells/well) were directly plated onto wells that had already been seeded with GFP-positive endothelial cells (5 × 104 cells/well) in DMEM/F12 medium with EGF, FGF, and FBS (5%). Cell Transplantation into Poststroke Mice To suppress rejection of grafted cells, immunodeficient (SCID) mice were used as the recipients of cell transplantation. On day 7 after stroke, mice were anesthetized and tethered to a stereotactic apparatus, and the cranium was exposed through a midline skin incision. A burr hole was made using a small dental drill, and a suspension of RFP-positive neural stem/progenitor cells (0.2 μl; 5 × 104 cells/μl) was injected stereotactically at a depth of 500 μm from the dural surface at 3 mm lateral and 1.0 mm dorsal from the bregma. Where indicated, injection of neural progenitors was followed by transplantation of GFP-positive endothelial cells (0.2 μl; 2.5 × 105 cells/μl) at the same location. Immunohistochemical Analysis of In Vitro Samples Injury-induced neural stem/progenitor cells were fixed in paraformaldehyde (4%), cut on a cryostat (8-μm sections), and stained with antibody to nestin (Chemicon, Temecula, CA, http://www.chemicon.com). Differentiated neural stem/progenitor cells were fixed and stained with Tuj-1 (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), GFAP (Chemicon), and O4 (Chemicon), and counterstained using 4′6-diamidino-2-phenylindole (DAPI; Kirkegaard & Perry Laboratories, Gaithersburg, MD), as described previously [22]. In coculture experiments, neural stem/progenitor cells were stained using antibody to nestin followed by counterstaining with DAPI on day 4 after coincubation. To study cell proliferation, bromodeoxyuridine (BrdU, 10 μg/ml; Sigma, St. Louis, http://www.sigmaaldrich.com) was added 15 hours before removal of feeder cells, followed by staining with BrdU antibody (Abcam, Cambridge, U.K., http://www.abcam.com) after pretreatment with HCl (2.0 M) for 30 minutes at 37°C. To assess cell viability, neural stem/progenitor cells were stained with antibody to caspase-3 (Chemicon) at day 4 after incubation. After coculture for 4 days, transwells were removed, and cocultured NSCs were incubated for up to additional 14 days in Neurobasal Medium (Invitrogen) with B-27 supplement (Invitrogen), FGF-2, and all-trans retinoic acid (0.2 μM; Sigma) to induce differentiation. Staining was performed with antibodies to Tuj-1, GFAP, or O4. Anti-nestin or GFAP antibody was labeled with green fluorescence (Alexa Fluor 488) and anti-Tuj-1, O4, BrdU, caspase-3, or nestin antibody was labeled with red fluorescence (Alexa Fluor 555). The number of nestin-, BrdU-, caspase-3-, Tuj-1-, GFAP-, or O4-positive cells and the total number of DAPI-expressing cells were counted in 20 randomly chosen microscopic fields (magnification, ×200; LSM510; Carl Zeiss, Jena, Germany, http://www.zeiss.com) from samples of each experimental group. Immunohistochemical Analysis of the In Vivo Sample On days 5 and 28 after cell transplantation, mice were deeply anesthetized with sodium pentobarbital and perfused transcardially with 4% paraformaldehyde. Brains were removed, and coronal sections (20 μm) were stained with antibody to BrdU or NeuN (Chemicon). For immunofluorescence, anti-BrdU or NeuN antibody was labeled with blue fluorescence (Alexa Fluor 350). Samples were examined using confocal laser-scanning microscopy. For quantification of neural progenitor cells in vivo, BrdU (50 mg/kg; Sigma) was administered subcutaneously daily after transplantation. In mice injected with endothelial cells, the distance from closest transplanted endothelial cell (GFP-positive cell) was evaluated in each RFP/BrdU double-positive cell using fluorescence microscopy (Keyence, Osaka, Japan, http://www.keyence.com). Semiquantitative Analysis of Grafted Cells and Colabeling To estimate the number of cells in the transplant, serial coronal sections (20 μm) through the entire transplant were collected, and the sections containing grafted cells were evaluated. For each section, the number of grafted cells (RFP- or GFP-positive cells) or RFP/NeuN double-positive cells was counted. Behavioral Analysis To assess cortical function, mice were subjected to behavioral testing using a modification of the open field task [31] on day 28 after transplantation as described [26, 27]. In this behavioral paradigm, animals were allowed to search freely in a square acrylic box (30 × 30 cm) for 20 minutes. A light source on the ceiling of the enclosure was on during the first 10 minutes (light period) and was turned off during a subsequent 10-minute period (dark period). On the X- and Y-banks of the open field, two infrared beams were mounted 2 cm above the floor, spaced at 10-cm intervals, forming a flip-flop circuit between them. The total number of beam crossings by the animal was counted and scored as traveling behavior (locomotion). Statistical Analysis Results are reported as the mean ± SD. Statistical comparisons among groups were determined using one-way analysis of variance (ANOVA). Where indicated, individual comparisons were performed using Student's t-test. Significance was assumed when group differences showed p < .05. Results Endothelial Cells Promote Proliferation of Ischemia-Induced Adult Neural Stem/Progenitor Cells In Vitro Stroke-induced adult neural stem/progenitor cells were prepared (Fig. 1A) as described [22]. Expression of a neural stem/progenitor cell marker (nestin; Fig. 1B) and, after differentiation, expression of mature neural cell markers, Tuj-1 (Fig. 1C; red), GFAP (Fig. 1C; green), and O4 (Fig. 1D; red), were confirmed immunohistochemically (nuclei were counterstained with DAPI; Fig. 1B–1D; blue). 1 Open in new tabDownload slide ECs expand the ischemia-induced adult neural stem/progenitor cells in vitro. (A–D): Formation of neurosphere-like cell clusters was observed with cells harvested from the stroke-affected area of cortex (A). Immunohistochemistry showed an expression for nestin (B), Tuj-1/GFAP (C), and O4 (D). (E): Cells forming neurospheres from poststroke cortex (ischemia-induced adult neural stem/progenitor cells) were dissociated and attached in the bottom of culture dish (green, nestin). (F, G): Adhered cells were cocultured with feeder cells including ECs, ACs, or FBs for 4 days, and ECs markedly stimulated the proliferation of injury-induced adult neural stem/progenitor cells. (H): Significantly increased number of nestin-positive cells was observed in the neural stem/progenitor cells treated with ECs and ACs. There was a significant increase of the number of neural stem/progenitor cells treated with ECs compared with that observed in the treatment by ACs. (I): Population of bromodeoxyuridine-positive cells to 4′6-diamidino-2-phenylindole-positive cells showed a significant increase in endothelial-treated group. (J): There was no significant difference of the portion of caspase-3-positive cells among all groups. n = 5 for each experimental group. *, p < .05 versus control (H, I). #, p < .05 versus AC (H). Scale bar: 100 (A, E, G) and 50 μm (B, C). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: AC, astrocyte; EC, endothelial cell; FB, fibroblast. 1 Open in new tabDownload slide ECs expand the ischemia-induced adult neural stem/progenitor cells in vitro. (A–D): Formation of neurosphere-like cell clusters was observed with cells harvested from the stroke-affected area of cortex (A). Immunohistochemistry showed an expression for nestin (B), Tuj-1/GFAP (C), and O4 (D). (E): Cells forming neurospheres from poststroke cortex (ischemia-induced adult neural stem/progenitor cells) were dissociated and attached in the bottom of culture dish (green, nestin). (F, G): Adhered cells were cocultured with feeder cells including ECs, ACs, or FBs for 4 days, and ECs markedly stimulated the proliferation of injury-induced adult neural stem/progenitor cells. (H): Significantly increased number of nestin-positive cells was observed in the neural stem/progenitor cells treated with ECs and ACs. There was a significant increase of the number of neural stem/progenitor cells treated with ECs compared with that observed in the treatment by ACs. (I): Population of bromodeoxyuridine-positive cells to 4′6-diamidino-2-phenylindole-positive cells showed a significant increase in endothelial-treated group. (J): There was no significant difference of the portion of caspase-3-positive cells among all groups. n = 5 for each experimental group. *, p < .05 versus control (H, I). #, p < .05 versus AC (H). Scale bar: 100 (A, E, G) and 50 μm (B, C). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: AC, astrocyte; EC, endothelial cell; FB, fibroblast. To study the effect of endothelial cells on injury-induced neural stem/progenitor cells, neurospheres were dissociated into single cells and 1 × 105 cells/well were incubated in 6-well plates. Immunohistochemical analyses showed that almost all cells adherent to the dish expressed nestin (98 ± 3%; Fig. 1E). Five hours after plating, transwell inserts with either adherent endothelial cells, astrocytes, or fibroblasts were placed in the well (Fig. 1F). Then, cell proliferation of injury-induced neural stem/progenitor cells was quantified with anti-nestin and BrdU antibodies on day 4 after coculture (Fig. 1G–1I). Although coincubation with fibroblasts did not promote proliferation of injury-induced neural stem/progenitor cells, coincubation with endothelial cells and astrocytes increased the number of nestin-positive cells (Fig. 1G; nestin [green], nuclei [blue], 1H). These observations support previous findings indicating that endothelial cells [18, 19] and astrocytes [20, 32] have a positive impact on the proliferation of neural stem/progenitor cells in vitro. Only neural precursor cells cocultured with endothelial cells showed a significant increase in the population of BrdU-positive cells compared with controls (Fig. 1G; BrdU [red], nuclei [blue], Fig. 1I). To exclude the possible influence of cell death caused by culture conditions, the population of caspase-3-positive cells was quantified and found to be comparable among the groups (Fig. 1J). These observations indicate that an increase in nestin-positive cells occurs in the presence of endothelial cells and astrocytes and is not likely caused by a reduction of cell death, but rather, to enhanced proliferation of the neural stem/progenitor cells (especially in the case of coculture with endothelial cells). Endothelial Cells Promote Neuronal Differentiation of Injury-Induced Adult Neural Stem/Progenitor Cells In Vitro Next, we studied the character of the cell population generated by injury-induced neural stem/progenitor cells after coculture with endothelial cells, astrocytes, or fibroblasts. Cells were stained with markers for mature neural cells (Fig. 2A; Tuj-1 [red], GFAP [green], O4 [red], nuclei [blue]), and expression of each marker was quantified (Fig. 2B, 2C). ANOVA showed a significant increase in Tuj-1, GFAP, and O4-positive cells in the group coincubated with endothelial cells and astrocytes compared with controls (Fig. 2B). Further analysis showed that coincubation with endothelial cells enriched the population for Tuj-1-positive neurons (Fig. 2C). The effect of endothelial cells on neural stem/progenitor cell proliferation (Fig. 2D) and generation of Tuj-1-, GFAP-, and O4-positive cells (Fig. 2E) appeared to be dose dependent. 2 Open in new tabDownload slide ECs accelerate neuronal differentiation from ischemia-induced adult neural stem/progenitor cells in vitro. (A): After the removal of feeder cells, adhered cells differentiated into mature neural cells expressing Tuj-1, GFAP, or O4. (B): The significantly increased number of mature neural cells (Tuj-1-, GFAP-, or O4-positive cells) was confirmed in EC- and AC-treated groups. (C): The cell population expressing Tuj-1 was significantly increased in the endothelial group. (D): Cell expansion of the neural stem/progenitor cells was induced by ECs of various densities (5 × 104, 1 × 105, 5 × 105 or 1 × 106 cells/well). (E): Induction of Tuj-1-, GFAP-, and O4-positive cells was also accelerated with several densities of ECs (5 × 104, 1 × 105, 5 × 105 or 1 × 106 cells/well). n = 5 for each experimental group. *, p < .05 versus control (B–E). Scale bar: 100 μm (A). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: AC, astrocyte; EC, endothelial cell; FB, fibroblast. 2 Open in new tabDownload slide ECs accelerate neuronal differentiation from ischemia-induced adult neural stem/progenitor cells in vitro. (A): After the removal of feeder cells, adhered cells differentiated into mature neural cells expressing Tuj-1, GFAP, or O4. (B): The significantly increased number of mature neural cells (Tuj-1-, GFAP-, or O4-positive cells) was confirmed in EC- and AC-treated groups. (C): The cell population expressing Tuj-1 was significantly increased in the endothelial group. (D): Cell expansion of the neural stem/progenitor cells was induced by ECs of various densities (5 × 104, 1 × 105, 5 × 105 or 1 × 106 cells/well). (E): Induction of Tuj-1-, GFAP-, and O4-positive cells was also accelerated with several densities of ECs (5 × 104, 1 × 105, 5 × 105 or 1 × 106 cells/well). n = 5 for each experimental group. *, p < .05 versus control (B–E). Scale bar: 100 μm (A). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: AC, astrocyte; EC, endothelial cell; FB, fibroblast. Direct Contact of Endothelial Cells and Stroke-Induced Adult Neural Stem/Progenitor Cells In Vitro Enhances Proliferation To examine the effect of direct contact of endothelial cells with neural stem/progenitor cells, the latter were directly plated onto GFP-positive endothelial cells (Fig. 3A). The effect of indirect contact of these two cell populations was assessed using the same number of neural stem/progenitor cells and GFP-positive endothelial cells incubated separately using transwells (Fig. 3B; in the latter case, one cell population was on the apical aspect of the membrane [endothelial cell] and the other was on the bottom of the transwell [neural stem/progenitor cell] and 0.4-μm slits in the membrane allowed passage of macromolecules but not intact cells). On day 4 after incubation, cells at the bottom of plates (the original neural stem/progenitor population) were stained with antibody to nestin and DAPI (Fig. 3C–3F, direct cell contact, indirect cell contact; nestin [red], GFP [green], nuclei [blue]). Quantitative analysis showed a significant increase in nestin-positive cells in the group with direct contact between endothelial and neural stem/progenitor cells compared with the indirect contact group (Fig. 3G). 3 Open in new tabDownload slide Direct contact of ECs stimulates the proliferation of ischemia-induced adult neural stem/progenitor cells in vitro. (A, B): GFP-positive ECs (5 × 104 cells/well) were directly (A) or indirectly (B) coincubated with ischemia-induced adult neural stem/progenitor cells (1 × 105 cells/well). (C–F): On day 4 after incubation, direct contact of ECs induced further increase of nestin-positive cells (C, D) compared with a group coincubated with ECs under indirect contact using transwell membrane inserts (E, F). (G): By quantitative analysis, it was confirmed that, after direct incubation by ECs, further proliferation of injury-induced neural/progenitor cells was induced compared with that observed with indirect coincubation [EC(Indirect)]. D and F show higher magnification of insets in C and E marked by white squares, respectively. n = 5 for each experimental group. *, p < .05 versus EC(Indirect) (G). Scale bar: 200 (C) and 100 μm (D). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: DAPI, 4′6-diamidino-2-phenylindole; EC, endothelial cell; GFP, green fluorescent protein. 3 Open in new tabDownload slide Direct contact of ECs stimulates the proliferation of ischemia-induced adult neural stem/progenitor cells in vitro. (A, B): GFP-positive ECs (5 × 104 cells/well) were directly (A) or indirectly (B) coincubated with ischemia-induced adult neural stem/progenitor cells (1 × 105 cells/well). (C–F): On day 4 after incubation, direct contact of ECs induced further increase of nestin-positive cells (C, D) compared with a group coincubated with ECs under indirect contact using transwell membrane inserts (E, F). (G): By quantitative analysis, it was confirmed that, after direct incubation by ECs, further proliferation of injury-induced neural/progenitor cells was induced compared with that observed with indirect coincubation [EC(Indirect)]. D and F show higher magnification of insets in C and E marked by white squares, respectively. n = 5 for each experimental group. *, p < .05 versus EC(Indirect) (G). Scale bar: 200 (C) and 100 μm (D). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: DAPI, 4′6-diamidino-2-phenylindole; EC, endothelial cell; GFP, green fluorescent protein. Cotransplantation of Endothelial Cells Promotes the Survival and Proliferation of Transplanted Stroke-Induced Adult Neural Stem/Progenitor Cells In Vivo To study the effect of endothelial cells on the survival of transplanted neural stem/progenitor cells in vivo, mice on day 7 after stroke received neural stem/progenitor cells and endothelial cells administered into the poststroke area (Fig. 4A). On day 5 after cell transplantation, multiple transplanted neural stem/progenitor cells were observed in proximity to transplanted endothelial cells (Fig. 4B, 4C; transplanted neural stem/progenitor cells [RFP, red], transplanted endothelial cells [GFP, green], nuclei [blue]). In contrast, fewer RFP-positive cells were observed in mice who received neural stem/progenitor cells without endothelial cells (Fig. 4D, 4E; transplanted neural stem/progenitor cells [RFP, red], nuclei [blue]). This impression was confirmed by quantitative analysis, because the number of RFP-positive neural stem/progenitor cells was significantly higher in the CO transplanted group (endothelial cells + neural stem/progenitor cells) compared with the group receiving only neural stem/progenitor cells (Fig. 4F). 4 Open in new tabDownload slide ECs stimulate the survival and proliferation of transplanted injury-induced adult neural stem/progenitor cells in vivo. (A–E): On day 7 after stroke, GFP-positive ECs were transplanted at the site of poststroke area. On day 5 after treatment, plenty of RFP-positive neural stem/progenitor cells were observed around GFP-positive ECs (NSC+EC group) (B, C), whereas a fewer RFP-positive cells were observed in mice only receiving ischemia-induced neural stem/progenitor cells (NSC group) (D, E). (F): A significantly increased number of survived cells was observed in mice with ECs (NSC+EC group) compared with mice without ECs (NSC group). (G–I): BrdU was administered, and it was found that RFP/BrdU double-positive cells were increased in endothelial-injected groups (NSC+EC group; arrowheads) (H) compared with groups without ECs (NSC group; arrow; RFP [red], BrdU [blue], GFP [green]) (I). (J): By semiquantitative analysis, it was confirmed that population of RFP/BrdU double-positive cells to total RFP-positive cells was significantly increased in mice after grafting ECs (NSC+EC group) relative to that observed in mice without grafting ECs (NSC group). (K): In mice receiving ECs (NSC+EC group), it was a tendency that the cell population of RFP/BrdU double-positive cells was increased nearby GFP-positive ECs. n = 5 for each experimental group. *, p < .05 versus the NSC group (F, J) and versus the group remote from GFP-positive cells (401∼600 μm) (K). Scale bar: 1 mm (B), 200 μm (C), and 50 μm (H). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: BrdU, bromodeoxyuridine; DAPI, 4′6-diamidino-2-phenylindole; EC, endothelial cell; GFP, green fluorescent protein; RFP, red fluorescent protein. 4 Open in new tabDownload slide ECs stimulate the survival and proliferation of transplanted injury-induced adult neural stem/progenitor cells in vivo. (A–E): On day 7 after stroke, GFP-positive ECs were transplanted at the site of poststroke area. On day 5 after treatment, plenty of RFP-positive neural stem/progenitor cells were observed around GFP-positive ECs (NSC+EC group) (B, C), whereas a fewer RFP-positive cells were observed in mice only receiving ischemia-induced neural stem/progenitor cells (NSC group) (D, E). (F): A significantly increased number of survived cells was observed in mice with ECs (NSC+EC group) compared with mice without ECs (NSC group). (G–I): BrdU was administered, and it was found that RFP/BrdU double-positive cells were increased in endothelial-injected groups (NSC+EC group; arrowheads) (H) compared with groups without ECs (NSC group; arrow; RFP [red], BrdU [blue], GFP [green]) (I). (J): By semiquantitative analysis, it was confirmed that population of RFP/BrdU double-positive cells to total RFP-positive cells was significantly increased in mice after grafting ECs (NSC+EC group) relative to that observed in mice without grafting ECs (NSC group). (K): In mice receiving ECs (NSC+EC group), it was a tendency that the cell population of RFP/BrdU double-positive cells was increased nearby GFP-positive ECs. n = 5 for each experimental group. *, p < .05 versus the NSC group (F, J) and versus the group remote from GFP-positive cells (401∼600 μm) (K). Scale bar: 1 mm (B), 200 μm (C), and 50 μm (H). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: BrdU, bromodeoxyuridine; DAPI, 4′6-diamidino-2-phenylindole; EC, endothelial cell; GFP, green fluorescent protein; RFP, red fluorescent protein. To study the effect of endothelial cells on the proliferation of transplanted neural stem/progenitor cells in vivo, BrdU was administered to mice after cell transplantation (Fig. 4G–4I). In mice receiving endothelial cells, many transplanted RFP-positive neural stem/progenitor cells showed uptake/incorporation of BrdU (Fig. 4H, transplanted neural stem/progenitor cells [RFP, red], transplanted endothelial cells [GFP, green], BrdU [blue]). In contrast, significantly fewer BrdU-positive cells were observed in mice that received neural stem/progenitor cells alone (Fig. 4I, transplanted neural stem/progenitor cells [RFP, red], BrdU [blue]). The ratio of BrdU- and RFP-positive cells to RFP-positive cells was quantified and confirmed that the proportion of proliferating neural stem/progenitor cells was significantly increased in the presence of endothelial cells (Fig. 4J). Furthermore, RFP-positive neural stem/progenitor cells located closer to GFP-positive endothelial cells showed a significantly higher ratio of BrdU staining (Fig. 4K). Cotransplantation of Endothelial Cells Promotes Neuronal Differentiation of Transplanted Stroke-Induced Adult Neural Stem/Progenitor Cells In Vivo To study the longer-term effect of endothelial cells on differentiation of transplanted injury-induced neural stem/progenitor cells in vivo, the fate of RFP-positive cells was investigated at later times. On day 28 after cell transplantation (Fig. 5A), many transplanted RFP-positive injury-induced neural stem/progenitor cells were observed in mice CO transplanted with GFP-positive endothelial cells (Fig. 5B, lower magnification; Fig. 5C, higher magnification; RFP [red], GFP [green], nuclei [blue]) compared with mice receiving only neural stem/progenitor cells (Fig. 5D, lower magnification; Fig. 5E, higher magnification; RFP [red], nuclei [blue]). It is notable that many RFP-expressing cells migrated away from the graft core and were observed in the peri stroke area, including corpus callosum and striatum, in mice CO transplanted with endothelial cells (Fig. 5B, 5C). In contrast, mice receiving only injury-induced neural stem/progenitor cells showed fewer RFP-positive cells, and migration of the latter cell population to the peri stroke area was less evident (Fig. 5D, 5E). Quantitative analysis showed that the total number of RFP-positive cells was significantly higher in mice with CO transplanted with GFP-positive endothelial cells and neural stem/progenitor cells (Fig. 5F). 5 Open in new tabDownload slide ECs accelerate neuron production from grafted injury-induced neural stem/progenitor cells in vivo. (A–E): On day 28 after transplantation, many RFP-positive cells were distributed around the poststroke area in mice after grafting ECs (NSC+EC group) (B, C), whereas it was less observed in mice without ECs (NSC group) (D, E). (F): Survived RFP-positive cells were significantly increased in mice with ECs (NSC+EC group) compared with mice without ECs (NSC group). (G–I): At the same time, an increased number of RFP/NeuN double-positive cells was observed in endothelial-treated mice (NSC+EC group; arrowheads) (H) compared with mice without ECs (NSC group; arrow) (I). (J, K): Semiquantitative analysis showed the number of NeuN/RFP double-positive cells (J) or population of NeuN/RFP double-positive cells to total RFP-positive cells (K) was significantly increased in endothelial-treated mice (NSC+EC group) compared with mice without ECs (NSC group). n = 5 for each experimental group. *, p < .05 versus the NSC group (F, J, K). Scale bar: 1 mm (B), 200 μm (C), and 50 μm (H). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: DAPI, 4′6-diamidino-2-phenylindole; EC, endothelial cell; GFP, green fluorescent protein; RFP, red fluorescent protein. 5 Open in new tabDownload slide ECs accelerate neuron production from grafted injury-induced neural stem/progenitor cells in vivo. (A–E): On day 28 after transplantation, many RFP-positive cells were distributed around the poststroke area in mice after grafting ECs (NSC+EC group) (B, C), whereas it was less observed in mice without ECs (NSC group) (D, E). (F): Survived RFP-positive cells were significantly increased in mice with ECs (NSC+EC group) compared with mice without ECs (NSC group). (G–I): At the same time, an increased number of RFP/NeuN double-positive cells was observed in endothelial-treated mice (NSC+EC group; arrowheads) (H) compared with mice without ECs (NSC group; arrow) (I). (J, K): Semiquantitative analysis showed the number of NeuN/RFP double-positive cells (J) or population of NeuN/RFP double-positive cells to total RFP-positive cells (K) was significantly increased in endothelial-treated mice (NSC+EC group) compared with mice without ECs (NSC group). n = 5 for each experimental group. *, p < .05 versus the NSC group (F, J, K). Scale bar: 1 mm (B), 200 μm (C), and 50 μm (H). Results shown are representative of five repetitions of the experimental protocol. Abbreviations: DAPI, 4′6-diamidino-2-phenylindole; EC, endothelial cell; GFP, green fluorescent protein; RFP, red fluorescent protein. Next, we studied differentiation of transplanted neural stem/progenitor cells into neurons in the ischemic area (Fig. 5G–5I). On day 28 after cell transplantation, RFP/NeuN double-positive cells were observed at the corpus callosum and striatum in mice CO transplanted with endothelial cells and neural stem/progenitor cells (Fig. 5H; RFP [red], GFP [green], NeuN [blue]). In contrast, only a few RFP/NeuN double-positive cells were observed in mice receiving only neural stem/progenitor cells (Fig. 5I; RFP [red], NeuN [blue]). Quantitative analysis confirmed a significant increase in the number of RFP/NeuN double-positive cells in CO transplantation experiments (Fig. 5J). The ratio of NeuN-positive cells in the overall population of RFP-positive cells was also evaluated; there was a higher level of neuronal differentiation in the brains of mice subjected to CO transplantation versus those receiving only neural stem/progenitor cells (Fig. 5K). Cotransplantation of Endothelial Cells Promotes Cortical Functional Recovery Finally, functional recovery of mice subjected to stroke and treated with neural stem/progenitor cells, in the presence/absence of endothelial cells, was studied by behavioral testing. Dysfunction of the cortex is closely linked to disinhibition of behavior in the presence of light [26]. Thus, locomotion under light and dark conditions was investigated at 28 days after transplantation. Compared with mice receiving NSCs alone, mice treated with NSCs and endothelial cells showed improved cortical function (i.e., reduction of locomotion) during the light phase (Fig. 6A). In contrast, there was no significant difference in the locomotion between the two groups during the dark phase (Fig. 6B). Although there was a trend of improvement in the response to darkness (the ratio of dark phase to light phase) observed in mice receiving endothelial cells and neural precursors compared with animals treated with neural precursors alone, there was no significant difference between the two groups (Fig. 6C). 6 Open in new tabDownload slide ECs promote cortical functional recovery after stroke. SCID mice were subjected to stroke followed by transplantation of neural stem/progenitor cells alone (NSC) or in the presence of ECs (NSC+EC) as described in the text. (A–C): Behavioral analysis was performed on day 28 after transplantation. Suppression of locomotion during the light phase (A) was observed in mice receiving ECs (NSC+EC group), although no significant difference was observed under dark conditions (B) between the groups. There was a trend, although this did not reach statistical significance, toward improvement in response to dark conditions in the NSC+EC group compared with NSC alone (C). n = 7 for each experimental group. *, p < .05 versus the NSC group (A). Abbreviation: EC, endothelial cell. 6 Open in new tabDownload slide ECs promote cortical functional recovery after stroke. SCID mice were subjected to stroke followed by transplantation of neural stem/progenitor cells alone (NSC) or in the presence of ECs (NSC+EC) as described in the text. (A–C): Behavioral analysis was performed on day 28 after transplantation. Suppression of locomotion during the light phase (A) was observed in mice receiving ECs (NSC+EC group), although no significant difference was observed under dark conditions (B) between the groups. There was a trend, although this did not reach statistical significance, toward improvement in response to dark conditions in the NSC+EC group compared with NSC alone (C). n = 7 for each experimental group. *, p < .05 versus the NSC group (A). Abbreviation: EC, endothelial cell. Discussion Our findings showed, for the first time, that CO transplantation of endothelial cells with cortex-derived, injury-induced adult neural stem/progenitor cells promotes survival, proliferation, and neuronal differentiation in the ischemic brain. Although the niches for the cortical neural precursors in the adult brain remain unclear, recent studies have shown the close association of intermediate progenitors and vasculature during cerebral cortical development in the embryo [33, 34]. These observations support our current results that endothelial cells are likely to be an important element of a niche in the cerebral cortex for injury-induced adult neural progenitor cells. In view of the residence of NSCs in a vascular niche, we anticipated the possibility that coincubation of endothelial cells with neural stem/progenitor cells would enhance proliferation of the latter and lead to generation of an increased number of neuronal-like cells in vitro. In cell culture, the presence of endothelial cells seemed to induce a shift in cell differentiation favoring neuronal precursors, consistent with previous results [18, 35]. The latter affect of endothelial cells in coculture of neural stem/progenitor cells was more marked than that observed with astrocytes; fibroblasts were without apparent effect. Furthermore, maximizing the impact of endothelial cells on neural precursor/stem cells seemed to be dose dependent and optimal under conditions in which endothelial cell-neural stem/progenitor cell contact could occur. Thus, although the effect of endothelial cells might involve soluble mitogenic factors, such as vascular endothelial growth factor, FGF-2, insulin-like growth factor-1, platelet-derived growth factor, brain-derived neurotrophic factor, or unidentified mediators [17, 19, 36–43], there seems to be a role for direct contact with potential passage of other types of regulatory signals. The latter observation is consistent with a previous finding that coculture of NSCs with endothelial cells induced a proliferative response for the NSCs compared with that observed in NSC monocultures [44]. These results are also supportive of previous work on adult SVZ-derived NSCs [13, 16]. In this study, transplantation of RFP-positive neural stem/progenitor cells alone resulted in survival of only ∼2% of the grafted cells 28 days later in the ischemic brain. Although there is a paucity of experimental data regarding transplantation of adult NSCs in models of stroke, a previous study also found that, 1 month after grafting adult SVZ-derived NSCs, only a small fraction of grafted cells (1-3%) survived in the postischemic rat brain [12]. In contrast, CO transplantation of neural stem/precursor cells in the presence of endothelial cells resulted in markedly enhanced survival of RFP-positive cells on days 5 (4.5-fold) and 28 (6.2-fold) after grafting. Furthermore, a larger fraction of RFP-labeled cells proliferated in mice CO transplanted with endothelial cells, especially when the latter were proximal to engrafted GFP-positive endothelial cells. These findings indicate that a key component of the vascular niche, endothelial cells, has an important role in survival, proliferation, and differentiation (see below) of neural stem/progenitor cells transplanted into ischemic brain. In view of the importance of endothelial cells in determining the fate of grafted neural stem/progenitor cells, it was relevant to understand the survival of transplanted endothelium in vivo. We used immunocompromised (SCID) mice to mitigate immunologic rejection after grafting. However, on day 28 after transplantation, there were fewer GFP-positive endothelial cells (382 ± 85 GFP-positive cells) observed than on day 5 after transplantation (13,781 ± 1,917 GFP-positive cells). These findings are consistent with previous observations that a range of grafted cells gradually decreases over time [4, 45], even in the presence of immunosuppressive agents [2] or in immunocompromised mice (NOD SCID) [46]. Thus, there are likely to be multiple factors impacting on the survival of transplanted cells, some of which are probably related to the ischemic environment. In this context, our preliminary data have shown that more GFP-positive endothelial cells could be observed on day 28 after injection if they were grafted onto the nonischemic cortex of sham-operated mice (892 ± 85 GFP-positive cells; data not shown). A further level of complexity related to the interaction of endothelial and neural stem/progenitor cells is the apparent property of the neural cells to promote angiogenesis [19] and prevent endothelial cell death [47] under ischemic conditions. Previous studies using adult SVZ-derived neural stem/progenitor cells described lack [10] or rare [11] differentiation to neurons (MAP2-positive cells) after transplantation into poststroke animals. Similarly, grafting adult neural stem/progenitor cells from SVZ [9] or hippocampus [12] into postischemic animals resulted in only a small fraction of the cells, 5% or 3-9%, respectively, which survived and could differentiate into NeuN-positive neurons. Our transplantation findings of neural stem/progenitor cells in the absence of endothelial cells are similar; only 1.1% of the RFP-positive cell population (neural stem/precursor cells) differentiated into NeuN-positive neurons. In contrast, CO transplantation of endothelial cells and RFP-positive adult neural stem/progenitor cells resulted in enhanced cell survival, proliferation, migration (outside the poststroke area including the corpus callosum and striatum), and differentiation. For example, assessment of the migrating transplanted cell population showed a strong increase in the number of RFP/NeuN double-positive cells and the ratio of RFP/NeuN double-positive cells in the total population of RFP-positive cells of about 18- and 3.2-fold, respectively. These findings underscore the positive effect of endothelial cells on multiple properties of transplanted adult neural stem/precursor cells. Based on our observations thus far, we favor the concept that endothelial enhancement of the survival of neural stem/progenitor cells might be a critical factor enhancing subsequent differentiation into neurons. The precise mechanism by which endothelial cells exert their effect(s) on neural stem/precursor cells remains to be elucidated, and it would be more complex in vivo compared with in vitro because there are multiple factors that affected the fate of transplanted cells. However, the role for a vascular niche/endothelial cells in stroke-induced neurogenesis in vivo [48] is clear. These observations support a range of other findings, including a close relationship between angiogenesis and neurogenesis in poststroke adult mice [26]. We hoped that our current in vitro study would give us a clue to detect endothelial-induced key molecules responsible for the enhanced survival, proliferation, and neuronal differentiation of transplanted NSCs in the future. Although CO transplantation of endothelial cells with neural stem/progenitor cells enhanced neuronal differentiation in vivo, only a small population of cells expressing neuronal markers was produced. Of the surviving RFP-positive cells, only about 3.4% differentiated into NeuN-positive neurons in vivo. Most RFP-positive cells differentiated into GFAP-positive astrocytes (∼60%), consistent with previous observations with transplanted adult NSCs in animal models of stroke [9, 10]. Because of the differences in differentiation of our neural stem/progenitor cell pool, in the presence of endothelial cells, to neurons and astrocytes in vitro versus in vivo (i.e., greater differentiation towards neurons in vitro than in vivo), there is clearly a role for additional environmental factors (beyond those produced by the endothelial cells). For example, a variety of chemical mediators/cytokines is produced/activated at the site of brain injury, and several of these, such as interleukin-6, ciliary neurotrophic factor, and bone morphogenetic proteins, are known to promote differentiation of NSCs toward the astrocytic phenotype [49, 50]. In this study, we found that CO transplantation of neural stem/progenitor and endothelial cells in the poststroke period resulted in mild improvement of cortical function compared with transplantation of neural precursors alone. However, our current CO transplantation study does not allow us to discern whether such behavioral recovery is attributable to endothelial-induced neurogenesis from transplanted neural stem/progenitor cells. It is certainly possible that grafted endothelial cells also promoted proliferation and neuronal differentiation of endogenous ischemia-induced neural stem/progenitor cells, thereby resulting in/contributing to improved behavioral function. Furthermore, grafted endothelial cells may decrease cell death of proximal endogenous neurons, which also may lead the functional recovery. Our findings indicate the importance of factors in the local environment for neurogenesis. This is a well-accepted concept, because NSCs from non-neurogenic regions, such as the spinal cord, can generate neurons after transplantation into a conventional neurogenic zone (i.e., SGZ) [51]. On the other hand, diverse regions of the central nervous system have the capacity to exert a negative influence on neurogenesis [52]. In the context of our study, these results raise questions concerning the contribution of endothelial cells to the properties and potential of neural stem/progenitor cells. For example, through an understanding of the specific mechanisms by which endothelial cells exert their effects, could this information be exploited to optimize the particular type of endothelial cell or even to replace the endothelial cell with a cell engineered to express particular combination of mediators? Furthermore, one would want to understand which factors in the ischemic microenvironment impact on viability of the transplanted cells (both endothelial and neural precursor/stem cells)? Insight into these issues might provide insight into whether there is a particular time after the ischemic insult when survival of the transplanted cells would be optimal. It might also provide insight into what type of neural precursor/stem cell might best survive in the poststroke brain. In terms of clinical application, potentially, a stroke patient might have surgically administered endogenous NSCs during the immediate poststroke period. Of course, there are many additional issues and questions to be addressed. However, the current observations provide a concrete starting point by showing that the presence of endothelial cells enhances successful transplantation of exogenous ischemia-induced adult neural stem/progenitor cells into ischemic brain. The potential of this approach, presumably after multiple refinements, to enhance neurogenesis in the ischemic human brain is exciting. Conclusions We showed that CO transplantation of endothelial cells with stroke-induced neural stem/progenitor cells promotes the survival and proliferation of the neural precursors and promotes neuronal differentiation. Our observations provide the basis for a novel therapeutic strategy in stroke using adult NSCs. Acknowledgements This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (21700363), and Hyogo Science and Technology Association. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: N.N.: conception and design, collection and assembly of data, final approval of manuscript; T.N.: conception and design, collection and assembly of data, manuscript writing, final approval of manuscript; S.K.: provision of study material; A.N.-D. and O.S.: collection and assembly of data; M.T. and H.Y.: data analysis and interpretation; D.M.S., manuscript writing; T.M.: data analysis and interpretation, final approval of manuscript; A.T.: manuscript writing, final approval of manuscript. N.N. and T.N. contributed equally to this article. First published online in Stem Cells Express June 25, 2009. Telephone: 81-798-45-6822; Fax: 81-798-45-6823 Copyright © 2009 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Endothelial Cells Support Survival, Proliferation, and Neuronal Differentiation of Transplanted Adult Ischemia-Induced Neural Stem/Progenitor Cells After Cerebral Infarction
JF - Stem Cells
DO - 10.1002/stem.161
DA - 2009-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/endothelial-cells-support-survival-proliferation-and-neuronal-JHLMNwzs47
SP - 2185
EP - 2195
VL - 27
IS - 9
DP - DeepDyve
ER -