Department of Psychology, Central Michigan University, Mount Pleasant, Michigan, USA
Program in Neuroscience, Central Michigan University, Mount Pleasant, Michigan, USA
College of Medicine, Central Michigan University, Mount Pleasant, Michigan, USA
Field Neurosciences Institute, Saginaw, Michigan, USA
Correspondence: Gary L. Dunbar, Ph.D., Department of Psychology, Program in Neuroscience, and College of Medicine, Central Michigan University, HP 2182, E. Campus Dr, Mount Pleasant, Michigan 48859, USA. Telephone: 1-989-774-3282; e-mail: firstname.lastname@example.org
Stem cells have gained significant interest as a potential treatment of neurodegenerative diseases, including Huntington's disease (HD). One source of these cells is adult neural stem cells (aNSCs), which differentiate easily into neuronal lineages. However, these cells are vulnerable to immune responses following transplantation. Another source is bone-marrow-derived mesenchymal stem cells (MSCs), which release neurotrophic factors and anti-inflammatory cytokines following transplantation, and are less vulnerable to rejection. The goal of this study was to compare the efficacy of transplants of MSCs, aNSCs, or cotransplants of MSCs and aNSCs for reducing deficits in a transgenic rat model of HD. HD rats received intrastriatal transplantations of 400,000 MSCs, aNSCs, or a combination of MSCs/aNSCs, while wild-type and HD controls were given vehicle. Rats were tested on the rotarod over the course of 20 weeks. The results indicated that transplants of: (a) aNSCs produced a strong immune response and conferred short-term behavioral benefits; (b) MSCs elicited a relatively weak immune response, and provided a longer term behavioral benefit; and (c) combined MSCs and aNSCs conferred long-term behavioral benefits and increased survival of the transplanted aNSCs. The finding that cotransplanting MSCs with aNSCs can prolong aNSC survival and provide greater behavioral sparing than when the transplants contains only aNSCs suggests that MSCs are capable of creating a more suitable microenvironment for aNSC survival. This cotransplantation strategy may be useful as a future therapeutic option for treating HD, especially if long-term survival of differentiated cells proves to be critically important for preserving lasting functional outcomes. Stem Cells2014;32:500–509
Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by progressive decline in motor and cognitive function with symptom onset usually occurring in the third to fifth decade of life. HD is caused by an expanded CAG trinucleotide repeat (>38) leading to the synthesis of an aberrant protein (mutant Huntingtin) with an expanded N-terminal polyglutamine tract . In adults with HD, motor dysfunction typically appears in midlife, but progresses to rigidity, bradykinesia and dystonia by the late stages of the disease. By the end stage, HD patients are usually bedridden, with very limited capacity for voluntary movement. The initial pathological basis of HD is selective degeneration of the medium spiny neurons within the striatum , although progression of the disease is associated with degeneration of additional brain regions, most prominently, the cerebral cortex .
Presently, there is neither a cure nor an effective treatment for this devastating disease. Cell replacement therapy, which involves transplanting new cells that either can replace lost neurons or maintain the function of the compromised cells , provides a promising new approach for treating neurodegenerative diseases, including HD. Patients with HD have received clinical benefits from implants of fetal/embryonic stem cells [5-7]. Inspection of brains of patients transplanted with fetal/embryonic striatal tissue, which contains a large number of neural precursor and neuronal progenitor cells that are capable of proliferation and differentiation into mature neurons, has revealed evidence of some, albeit incomplete, graft survival . However, graft rejection in an HD patient has been observed 14 months after grafting fetal neural cells , suggesting embryonic cell transplants are vulnerable to rejection .
Despite some encouraging results, the use of fetal/embryonic cell sources for therapeutic transplantation is still subject to logistical, immunological, and ethical limitations . To avoid some of these complications, use of adult, bone-marrow-derived stem cells has gained considerable interest. Cultured mesenchymal stem cells (MSCs) are characterized by plastic adherence, rapid proliferation, and multipotency (i.e., the capacity to differentiate into several cell phenotypes) . In addition to their natural propensity to differentiate into mesodermic lineages, MSCs may also be able to acquire phenotypes of neuro-ectodermic cells, and some reports have indicated that these cells can differentiate into neural or glial morphological phenotypes in culture, although the extent to which this occurs remains controversial [13, 14].
The degree to which neuronal differentiation occurs in transplanted MSCs is even more controversial. Some researchers have found no evidence that differentiation of transplanted MSCs into neurons occurs at all . While, other investigators [16, 17] suggest that neural differentiation from MSCs is possible, albeit a rare (approximately 1% or less of the total MSCs implanted) occurrence. In support of this latter finding, work in our lab has confirmed that only a very low percentage of MSCs can differentiate into neurons or glial cells following transplantation into the striata of: (a) intact rats ; (b) 3-nitropropionic acid-treated rats ; or (c) in rats given intrastriatal injections of quinolinic acid .
Perhaps a more important property of MSCs is that they appear to have considerable immunomodulatory capabilities. Evidence from both in vitro and in vivo studies has suggested that MSCs are intrinsically hypoimmunogenic, and do not stimulate an alloreactive response . In contrast, MSCs have been shown to exert suppressive effects on the proliferation and stimulation of T cells as well as suppressing mixed lymphocyte reactions . We have recently reported that transplantation of human or rat MSCs into the rat striatum decreases the general activity of the hyperimmunogenic dendritic cells, macrophages, and T lymphocytes, which translates into reducing the probability of MSC rejection after allotransplantation and xenotransplantation, even after 63 days .
Transplantation of MSCs into the striatum of rodent models of HD has been shown to reduce behavioral deficits  and provide neurotrophic support [see  for review]. Given that MSCs are readily available, easily accessed, can modulate the immune response following transplantation, and provide functional efficacy, they hold considerable promise as a source for an effective cell therapy. However, one of the major limitations of MSCs is their inability to differentiate into neuronal phenotypes, at least in therapeutically significant numbers.
An attractive alternative to the use of MSCs for transplantation therapy is using adult neural stem cells (aNSCs), which provides a source of adult stem cells that have a greater propensity to differentiate into neuronal phenotypes. It is well-established that the mature mammalian central nervous system contains multipotent stem cells, such as aNSCs, which can differentiate into a variety of specialized cells, such as astrocytes or oligodendrocytes [5, 23, 24]. These adult-derived stem cells, including progenitors derived from the rat hippocampus, are well-characterized and express relatively low levels of the major histocompatibility complex antigens . However, the survival and differentiation of transplanted aNSCs, which depend extensively on both the intrinsic properties of the grafted cells and the regional environmental cues in the host [26, 27], have not been thoroughly studied.
Neural stem cells, isolated from embryonic tissue, have been shown to differentiate into neurons , albeit with limited survivability , following transplantation but it is unknown whether aNSCs will produce a similar outcome following intrastriatal transplantation. In a hemiparkinsonian rat, adult hippocampal progenitor cells displayed a relatively high survival rate following transplantation of rat-derived neural progenitor cells . Studies using adult neural precursors, derived from the subventricular zone (SVZ) of the lateral ventricles, have indicated that these cells have the ability to differentiate into neuronal and glial phenotypes, both in vitro and in vivo . When transplanted into the adult rat striatum, these cells survived for 6 weeks post-transplantation, and were able to differentiate, albeit, into predominately astrocytic phenotypes . To date, the fate of transplanted aNSCs obtained from the SVZ remains to be determined.
The purpose of this study was to: (a) determine the survivability aNSCs obtained from the SVZ after cotransplantation with MSCs in the striatum of the HD 51 CAG rat model of HD ; (b) compare and characterize the long-term (5 months) fate of both MSCs and aNSCs that have been transplanted either separately or together (as cotransplants) in the HD rats; and (c) evaluate the functional recovery of the HD rats following transplantation of MSCs, aNSCs, or both (cotransplant). We hypothesized that the enriched microenvironment created by the MSCs would create a protective environment which would enhance the ability of aNSCs to survive and differentiate, despite being implanted into a degenerating striatum. It was also hypothesized that transplants of aNSCs may provide short-term functional recovery, but would probably undergo systemic rejection over time. Conversely, it was expected that transplants of MSCs would exert beneficial behavioral effects, but would be severely limited in their ability to differentiate into neuronal phenotypes. Finally, it was hypothesized that the cotransplantation of MSCs and aNSCs would confer a relatively long-term beneficial functional effect, with greater survival and differentiation of the aNSCs because of a more protective environment produced by the MSCs within the cotransplants.
Materials and Methods
All procedures mentioned were reviewed and accepted under the guidelines of the Institutional Animal Care and Use Committee of Central Michigan University.
Isolation and Cultures of MSCs
Bone marrow cells were aspirated from the femur and tibia of adult (8–12-week old) nonsyngenic, male, Sprague Dawley rats via a 25-gauge syringe. Following extraction, cells were suspended in 10 ml of Alpha Modified Eagle's Medium (αMEM: Sigma, St. Louis) with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad), 10% horse serum (HS; Invitrogen), 5 mg/ml streptomycin, and 5 UI/ml penicillin (Sigma). MSCs are selected via plastic adherence. After incubating for 24 hours at 37°C, half of the medium was changed (replaced with fresh medium) to remove the nonattached cells. When the MSCs reached 85% confluence, the culture medium was aspirated, and the cells were detached by adding 0.25% trypsin-EDTA solution (Sigma), for 5 minutes while incubating, after which the trypsin was deactivated by adding 2 ml of FBS. Detached cells were collected and centrifuged at 1,500 rpm for 7 minutes at 4°C. The supernatant was removed and the pellet was then resuspended, counted, and replated at a density of 8,000 cells per square centimeter in a new 75 cm2 flasks (Phenix, Candler) with fresh αMEM/ FBS(10%)/HS medium(10%). This “passage” was repeated four times, at which point the MSCs were ready for transplantation.
Isolation and Cultures of aNSCs
Floating “neurosphere” cultures, containing neural stem and progenitor cells, were propagated from plating cells extracted from the adult rat SVZ. Tissue (1 mm3) around the lateral ventricle (at +0.5 mm and +1 mm AP from bregma) was dissected and dissociated mechanically in a 0.25% EDTA/trypsin solution (Sigma). After 10 minutes, the trypsin was deactivated with 2 ml of FBS. The cell preparation was exposed to 10 mg/ml of DNase I (Sigma) for 10 minutes at 37°C and tritrated sequentially with fire-polished Pasteur pipettes. After the removal of aggregates by decantation, cells were collected following centrifugation and plated onto poly(L-ornithine)-coated dishes (50 mg/ml; Sigma) at a final density of 105 cells per square centimeter in a mixture consisting of Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (1:1, vol/vol; Sigma), 33 mM glucose (Sigma), 5 mM HEPES (pH 7.2), 5 mg/ml streptomycin, and 5 UI/ml penicillin (Sigma), supplemented with 10% FBS. Twelve hours after plating, the floating cells were placed in a 75 cm2 flask in defined medium composed of DMEM/Ham's F-12 (1:1, vol/vol), 33 mM glucose, 5 mM HEPES (pH 7.2; Sigma), 5 mg/ml streptomycin, and 5 UI/ml penicillin (Sigma), supplemented with B27 supplement (Gibco, Grand Island), 10 ng/ml epidermal growth factor (EGF; Invitrogen), and 100 ng/ml fibroblast growth factor-2 (Invitrogen). Neurospheres were cultured until they reached confluence. At this point, the medium containing the floating neurospheres was removed and centrifuged at 750 rpm for 10 minutes to isolate the neurospheres, which were then dissociated using 0.25% trypsin-EDTA solution, before being centrifuged. The pellet was resuspended, counted, and replated in a new flask. This passage process continued four times, until single cells that were derived from dissociated neurospheres were obtained for transplantation.
Stem Cell Immunocytochemistry
Before transplantation, samples of MSCs and aNSCs were characterized by immunocytochemistry. Briefly, MSCs were plated into six-well plates containing poly(L-ornithine)-coated glass coverslips (25 mm #1; Fisher Scientific, Pittsburgh) and cultured in standard bone-marrow medium. To induce differentiation of aNSCs, cells were cultured in six-well plates containing poly(L-lysine)-coated glass coverslips and cultured with medium consisting of DMEM/Ham's F-12 (1:1, vol/vol), 33 mM glucose, 5 mM HEPES (pH 7.2; Sigma), 5 mg/ml streptomycin, and 5 UI/ml penicillin (Sigma), supplemented with B27 supplement (Gibco). At 80% confluence, both MSCs and aNSCs were fixed with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS). After 1 hour blocking of nonspecific binding sites with 10% normal goat serum (Sigma), the cells on the coverslips were incubated in primary antibodies overnight at 4°C. Primary antibodies used included: CD45 (1/500; Abcam, Cambridge), CD90 (1/500; Abcam), Nestin (1/500; Chemicon, Billerica), and NeuN (1/500; Chemicon). Following incubation in the primary antibodies, the cells on the coverslips were rinsed and secondary antibodies were added for 1 hour at room temperature. Secondary antibodies included AlexaFluor488 and AlexaFluor594 (1/300; Invitrogen). Following incubation in secondary antibodies, the cells on the coverslips were rinsed and incubated in Hoechst 33258 (1/1,000; Thermo Scientific, Rochester) for 5 minutes at room temperature to visualize cell DNA and then mounted onto glass slides using Vectashield (VectorLabs, Burlingame).
Thirty-three 12-month-old, male, HD  and wild-type (WT) rats were housed at 22°C under 12 hour light/12 hour dark conditions with food and water provided ad libitum. Rats were separated into five groups: (a) WT (n = 7), which included sham-operated (Hanks Balanced Salt Solution, HBSS, Gibco; injection in the striatum) WT rats; (b) HD (n = 6), which included sham-operated HD rats; (c) MSCs (n = 7), which included HD rats transplanted with MSCs; (d) aNSCs (n = 5), which included HD rats transplanted with aNSCs; and (e) MSC/aNSC (n = 8), which included HD rats receiving cotransplants of both MSCs and aNSCs.
All rats were initially sedated with a mixture of isoflurane gas and O2. A 2% lidocaine gel was placed on the tips of the ear bar prior to placement. Anesthesia was maintained with isoflurane gas and O2 over the course of the transplant procedure. After incision over the scalp and retracting the skin, two burr holes (0.5 mm) were placed over the two hemispheres, directly over the neostriatum (coordinates, relative to bregma: +0.5 mm AP; ±2.6 mm ML; −6 mm and −5 mm DV, with tooth bar at −3.3 mm). Prior to the transplantation, the aNSC neurospheres were mechanically dissociated to obtain a single-cell suspension and both MSCs and aNSCs were counted on a hemocytometer. Using a 10 µl Hamilton microsyringe, animals were bilaterally transplanted with MSCs, aNSCs, or a combination both of these cells. Rat MSCs and aNSCs were transplanted at a density of 400,000 in each striatum (2 µl), suspended in HBSS. The MSCs/aNSCs group received bilateral injections of 200,000 of each cell type in each striatum. Prior to transplantation, MSCs were labeled with Hoechst 33258 (5 µg/ml, Sigma) and aNSCs labeled with PKH-26 (Sigma). The WT and HD groups received bilateral injections of 2 µl of HBSS (the vehicle). All cells and the vehicle were injected at a rate of 0.33 µl/minute over a period of 3 minutes. Following the first injection, the syringe was left in place for an additional 3 minutes and was raised 1 mm and given a second injection, after which it was left in place for another 3 minutes. Following the second injection, the syringe was withdrawn at a constant rate of 2 mm/minute. The burr hole was sealed with bone wax, and the wound was closed using sterile wound clips (9 mm). Rats were sacrificed at 20 weeks following transplantation.
Motor Coordination Test
Motor coordination was assessed using the rotarod (Ugo Basile, Collegeville, PA). The animals were handled for approximately 10 minutes daily for 1 week preceding the start of behavioral testing. The week prior to transplantation, the rats were trained to remain on a 5-cm diameter rod, rotating at 8 rpm for a period of 120 seconds. Training lasted for a total of 3 days, with five trials each day (with a 5-minute inter-trial-interval). Baseline measures were taken 1 day prior to stem cell transplantations and testing consisted of one session every 14 days, starting 14 days after transplantation, for a total of 10 testing sessions. All testing procedures were conducted by two trained experimenters who were blind to the group identity of the rats. The dependent measure for this task was the latency to fall onto a foam cushion located directly below the rotating rod.
Rats were deeply anesthetized at 20 weeks following cell transplantation (when the rats were 17 months old) with an overdose of sodium pentobarbitol (i.p.), and transcardially perfused with 0.1 M PBS, followed by fixation with 4% paraformaldehyde in 0.1 M PBS at pH 7.4. Brains were rapidly removed, immersed in the same fixative for 1 hour at 4°C, and transferred to 30% sucrose in 0.1 M PBS for cryopreservation. Serial coronal sections were cut on a cryostat (Leica, CM 3050, Buffalo Grove) at a thickness of 25 µm, and were mounted on positive charged microscope slides (Globe Scientific Inc., Paramus).
The histology and immunohistochemistry followed protocols from our previous study . Briefly, after blocking of nonspecific binding sites with 10% normal goat serum (Sigma) for 1 hour, free floating sections were incubated in primary antibodies overnight at 4°C. Primary antibodies used included: NeuN (neuronal nuclei; 1/500 dilution; Chemicon); Nestin (neural progenitor cells; 1/500; Chemicon); Dopamine- and cAMP-regulated phosphoprotein (Darpp-32; GABAergic medium spiny neurons; 1/1,000; Abcam); and glial fibrillary acidic protein (GFAP; astrocytes; 1/1,000; Abcam). Following incubation in the primary antibodies, the tissue was rinsed and secondary antibodies were added for 1 hour at room temperature. Secondary antibodies included AlexaFluor488 and AlexaFluor594 (1/300; Invitrogen). Following incubation in secondary antibodies, the tissue was rinsed, mounted onto glass slides, and coverslipped using Vectashield (VectorLabs).
Measure of Transplant Area and Cell Counts
For each rat, the transplant area was visualized in the striatum under fluorescent light at three different striatal levels using a Zeiss Axiovert 200 M microscope at ×20 magnification and measured using ImageJ software (National Institutes of Health, Bethesda, MD). The MSCs were detected using a 350 nm filter cube and aNSCs were detected using a 594 nm filter cube. The first section (0.5 mm anterior to bregma) contained the needle tract and the other two locations contained grafted cells (175 µm anterior to- and 175 µm posterior to the first section). Cell counts, using ImageJ, were performed on the same images used to assess the area of transplantation in the aNSC and MSC/aNSC groups. Briefly, all cells within the circumscribed transplanted areas were counted if: (a) the entire cell body was within the borders of the graft area; (b) they showed positive PKH-26 labeling within the cell body; (c) and the cell body was intact.
Based on previous behavioral results using this animal model, testing sessions were grouped into four testing stages (Baseline, 52 weeks; early-stage, 54–60 weeks; intermediate-stage, 62–66 weeks; and late-stage, 68–72 weeks) . Data from each stage of rotarod testing and for the area of transplants were analyzed using one-way analysis of variance (ANOVA). When appropriate, a Fisher's protected least significant difference (PLSD) post hoc test was performed. Data from cell counts were analyzed using an independent samples t test. The p value was set at .05 for all analyses.
Stem Cells Characterization
MSCs and aNSCs were cultured and characterized by immunocytochemistry in vitro, prior to transplantation. MSCs showed typical morphology (Fig. 1A), positive expression of CD90, and negative expression of CD45 at passage 4 (Fig. 1B). Adult NSCs formed spheroid-neurospheres (Fig. 1C) expressing Nestin and NeuN (Fig. 1D and 1E, respectively), after being cultured in neural differentiation medium.
Motor Coordination Results
One-way ANOVAs revealed no significant between-group differences in rotarod performance during the Baseline [F(4, 32) = 0.281, p > .05] or early-stage [F(4, 32) = 1.027, p > .05] of the disease. However, significant between-group differences were observed during the Intermediate-stage [F(4, 32) = 2.707, p = .05] and Late-stage [F(4, 32) = 3.605, p < .05]. PLSD post hoc analysis revealed significant differences between HD and WT rats for latency to fall off the rotarod, as well as significant differences between the WT and NSC groups during the Intermediate- and Late-stage of the disease. PLSD post hoc analysis also revealed significant differences between the MSC and HD group as well as significant between-group differences the MSC/aNSC and HD group during the Late-stage of the disease (Fig. 2).
Transplant Area and Cell Counts
In the striatum of rats receiving transplants of MSCs, cells were still present around the injection site (Fig. 3A). In rats that received aNSCs transplants, the transplanted cells were also observed at the injection site, but the graft area was smaller than observed in the MSC group (Fig. 3B). When the cells were cotransplanted in the striatum (200,000 MSCs and 200,000 aNSCs), the size of the graft area was similar to that of the MSC group (Fig. 3C, 3D), suggesting that the MSCs protected the aNSCs from dying or being rejected. A one-way ANOVA revealed significant differences between groups for the area of the transplanted cells [F(2, 38) = 8.369, p = .001] with PLSD post hoc analysis revealing significantly reduced area containing aNSCs compared to MSC and MSC/aNSC and aNSC (Fig. 3E). An independent samples t test revealed a significant difference between the aNSC group and the MSC/aNSC group for counts of aNSC-positive cells [t(7) = 6.08, p = .001] with greater survival observed in the cotransplanted aNSCs (Fig. 3F).
Stem Cell Transplant Characterization
Twenty weeks after the transplantation, analyses of the immunohistochemistry data revealed observable cell survival within the implantation site in all transplant groups (Fig. 4). However, MSCs did not coexpress neuronal markers, such as Nestin, NeuN, or DARPP-32 (Fig. 4A, 4C, 4D, respectively). Although astrocytes (GFAP) were found in the vicinity of the transplants (Fig. 4B), they were not colocalized with the MSCs, suggesting that these glial cells were generated de novo.
Adult NSCs expressed immature neuronal proteins (Nestin) and a marker for mature neurons (NeuN), suggesting that the transplanted aNSCs are capable of differentiation into neuronal phenotypes (Fig. 4E, 4G, respectively). However, the aNSCs did not express Darpp-32 (GABAergic medium spiny neurons), demonstrating that the cells did not differentiate into highly region-specific cell types (Fig. 4H). Like the MSC transplants, the aNSCs were not colocalized with GFAP, but the grafts showed a greater infiltration of GFAP-labeled cells (Fig. 4B). The area of labeled cells in the rats that were cotransplanted with MSCs and aNSCs was larger and expressed Nestin (Fig. 4I) and NeuN (Fig. 4K), but did not express Darpp-32 (Fig. 4L). The histology of the brains from cotransplanted rats also revealed a profile of astrocyte infiltration, similar to what was observed in the MSC group (i.e., localized astrocytes around the transplantation, without infiltration).
This study evaluated the potential of transplanted aNSCs (expanded from the SVZ), MSCs (from bone marrow), and a combination of both cell types, to provide neuroprotection and behavioral sparing in a HD rat model. These findings suggest that cotransplanting MSCs create a microenvironment more conducive to the survival of the aNSCs in the HD brain. Adult NSCs transplanted alone provided transient behavioral sparing, but this effect dissipated, probably due to a growing immune response and subsequent rejection by the host tissue. When the MSCs were transplanted alone, they provided longer lasting behavioral sparing, but these transplanted cells did not differentiate into neuronal phenotypes. When both cell types were transplanted together, the resulting behavioral sparing and neuroprotection were more robust.
This study demonstrates that the use of adult stem cells, such as MSCs or aNSCs, can retard the progression of motor deficits after transplantation in the striatum of HD rats. Transplanted aNSCs appear capable of reducing the deficits during the early-stage of HD in this model, which is similar to other studies performed in rat models of Parkinson's disease [29, 34]. In both of the previous studies, SVZ neuroprogenitors were able to attenuate the behavioral deficits as measured by amphetamine-induced rotations for 6 weeks after the transplantation. In a unilateral quinolinic-acid rat model of HD, Vazey et al.  demonstrated a reduction in motor function impairment (apomorphine-induced rotation and asymmetry score) for 8 weeks following transplantation of aNSCs . These findings were confirmed in our rat model of HD, with an aNSC-induced reduction of motor deficits in the rotarod task up to 4 weeks following transplantation. However, starting at 6 weeks after the transplantation, the HD rats receiving the aNSCs transplants displayed motoric deficits similar to the sham-transplanted tgHD rats, suggesting that there may be limits for the long-term therapeutic potential of aNSCs.
In contrast, rats given MSC transplants showed a longer term beneficial effect on the rotarod task, similar to what has been found in a 3-nitropropionic-acid-treated rats that were given these types of MSC transplants . Other reports show that MSCs can reduce stroke-induced motor deficits in rats  and in a rodent model of PD . However, these tests were only carried out for 4 or 6 weeks. MSCs have been extensively studied in a variety of diseases and are now being considered for clinical trials for myocardial infarction, stroke, meniscus injury, limb ischemia, graft-versus-host disease, autoimmune disorders, amyotrophic lateral sclerosis, and HD [see  for review]. In this study, the effects of the transplanted MSCs were probably mediated by either their intrinsic production of trophic factors  or by enhancing the production of these factors by the host tissue. Interestingly, in the cotransplantation paradigm, the same early beneficial effects that were observed in the aNSC rats were apparent, but, importantly, this ameliorative effect remained stable for the entire 20 weeks of testing.
Currently, the long-term survivability of aNSCs following transplantation into the rat brain is unknown. Previous research has reported that when transplanted into striatum of a rat model of PD, aNSCs can survive up to 4 weeks ; however, following transplantation into the striatum of a healthy rat, aNSCs have been shown to survive as long as 8 weeks [39, 40]. One of the most important factors which could alter the survival of the grafted aNSCs is the level of inflammatory responses of activated microglia and macrophages. When aNSCs were transplanted into the brains of inbred, nonimmunosuppressed rats, extensive inflammatory responses, and glia reaction were observed 4 weeks after transplantation around the injection site, suggesting that an anti-inflammation modulation may be needed for long-term survival . However, other investigators reported limited activation of microglia after 7 weeks post-transplantation in an intact rat brain , suggesting that long-term survivability of transplanted aNSCs is possible, at least in the normal rat brain.
However, in a degenerating environment, such as in the HD brain, neuroinflammation and proapoptotic bodies can create an environment detrimental to graft survival. In a toxic lesion model of HD, a strong increase of CD11b- and GFAP-positively labeled cells was observed near the transplanted aNSCs, but it is unclear if this reaction was due to the pathological environment or an immune response to the graft . In this study, it was shown that aNSCs can survive in a HD transgenic rat brain for up to 20 weeks, with inflammation observed in and around the transplantation site, as indicated by GFAP-positive cells. Transplantation of dead aNSCs, labeled prior to cell death with PKH-26, into the brain of a normal rat showed no positively labeled cells at 8 weeks following transplantation, confirming that the fluorescent cell linker was not transferred to the host (data not shown).
The survivability of MSCs following transplantation into the striatum of healthy rats has been reported to last for more than 63 days . MSCs have been widely studied for their immunomodulatory properties . The ability of MSCs to survive is attributed to their immunomodulatory properties, such as the release of anti-inflammatory cytokines and neurotrophic factors . In this study, transplanted Hoechst-labeled MSCs were detected up to 20 weeks following transplantation into HD rats. Similar to PKH-26 labeled aNSCs, transplantation of dead MSCs labeled with Hoechst into the brain of a normal rat showed no positively labeled cells at 8 weeks following transplantation, confirming that the fluorescent cell linker was not transferred to the host (data not shown).
We observed that when MSCs were cotransplanted with aNSCs the survival of the aNSC graft was enhanced compared to the aNSC alone group. Furthermore, when aNSCs were transplanted alone, the area of the transplanted tissue was significantly smaller than that of the MSC transplant group and the number of surviving cells decreased significantly. This is especially notable because the initial number of transplanted aNSCs in the aNSC group was twice that used in the cotransplantation group of rats. The area of the graft in the MSC/aNSC group was similar to the area of MSC group, suggesting that the environment created by the immunomodulatory effects of the surviving MSCs improved survival of transplanted aNSCs. It is important to note that, for all the transplant groups, labeled cells were observed in the vicinity of the transplant site, with no migration observed for the either cell type, an observation which is in line with previous research [19, 42, 43]. Further studies are planned to examine the optimal ratio of aNSCs to MSCs for future cotransplantation paradigms, so that the detrimental effects of a degenerating environment can be maximally counteracted.
Stem cells extracted and expanded from the SVZ of rats have been shown to possess the capability to differentiate into neuronal lineages in vitro , and in vivo following transplantation . Previous research has demonstrated that the fate of aNSC differentiation in vivo is highly dependent on the region of transplantation. It has been shown when aNSCs are transplanted into non-neurogenic regions, such as the striatum or cerebellum, the majority of the aNSCs differentiate into a glial lineage [30, 43].
In this study, aNSCs were able to differentiate into neuronal lineages in vitro as confirmed by positive labels for mature neurons, (NeuN) and astrocytes (GFAP). Similarly, when only aNSCs were transplanted into the striata of HD rats, these cells demonstrated colocalization with NeuN, but little colocalization with the GFAP, suggesting that aNSCs derived from the SVZ have the capability to differentiate into neuronal lineages.
Bone-marrow-derived MSCs have also been reported to display properties of neuronal differentiation in vitro when cultured in specific media [44, 45]. In this study, no neuronal or astrocyte differentiation was observed when only MSCs were transplanted in the HD rats. While NeuN- and GFAP-positive cells were located around the graft site, there was no colocalization with the transplanted cells.
When aNSCs were cotransplanted with MSCs, colocalization with NeuN was observed, similar to the aNSC group, suggesting that the MSCs did not interfere with the aNSCs differentiation capabilities. Conversely, no colocalization of MSCs with NeuN was observed, suggesting that the presence of aNSCs did not enhance or promote the ability of MSCs to differentiate into neuronal lineages. It is interesting to note that neither aNSCs nor MSCs colocalized with GFAP in any of the transplant groups, which does not support the contention of Chen et al. who suggested that when NSCs are transplanted into the brain, they favor differentiation into glial cells .
This study provides evidence that cotransplantation of adult-derived MSCs and aNSCs is a potential therapeutic strategy for treating HD. It appears that transplants of aNSCs can provide a short-term reduction in deficits on the rotarod task, while MSCs can provide a longer term beneficial effect. The precise mechanism in which each of these cells exert their effects is still unknown. However, there is mounting evidence that aNSCs can differentiate into neuronal lineages when transplanted in the striatum, with the potential to acquire region-specific neuronal phenotypes  and that these cells can integrate in the host brain without tumor formation . The short-term beneficial effect of aNSC transplants is unlikely due to the integration of the progenitor transplants and their differentiation in mature neurons in such a short period of time. Given the decrease of the graft size and reduced number of surviving cells observed at 20 weeks in the aNSC group, it appears that the host immune system is rejecting the cells (as suggested by the activation of the host astrocytes in and around the graft).
Similarly, the beneficial effects of MSC transplants are probably not due to neuronal differentiation, but rather by their immunomodulatory properties  and their capacity to enhance the production of brain-derived neurotrophic factor and VEGF [36, 37]. MSCs have shown to release anti-inflammatory cytokines which provide local immunomodulation . This property could explain why aNSCs have increased survival when cotransplanted with MSCs. The combination of the local protective environment and the trophic support is likely to enhance the long-term benefits of aNSCs. As such, this study demonstrates that when MSCs are cotransplanted with aNSCs, the aNSCs are able to survive longer, which may ultimately provide for better long-term behavioral outcomes.
Results from this study show that cotransplantation of MSCs and aNSCs into HD rats can slow the progressive motor deficits and increase survivability of transplanted aNSCs for up to 20 weeks post-transplantation when compared with HD rats given sham transplantation surgery. Collectively, the results of this study provide new evidence that transplantation of MSCs in the brain can provide a beneficial microenvironment, which appears to include a favorable local immunomodulation that can facilitate the survivability and efficacy of other type of cells that may otherwise be rejected when transplanted. As such, this study suggests that use of MSCs in a cotransplantation protocol may be a useful therapeutic strategy, especially when long-term survivability and differentiation are needed for bona fide cell replacement.
This work was supported by funds from the Field Neurosciences Institute, College of Humanities and Social and Behavioral Sciences, and John G. Kulhavi Professorship (to G.L.D.) and the College of Medicine at Central Michigan University (to J.R.). tgHD rats were obtained through a generous gift from Stephan von Hörsten.
R.J.: conception and design, data collection, assembly, analysis, and interpretation, and manuscript writing; F.K. and C.A.: collection, assembly, analysis, and interpretation of data, and manuscript writing; D.K., C.S., M.J., L.S., and D.N.: collection and assembly of data; L.L: manuscript writing; D.G.L.: analysis and interpretation of data, manuscript writing, and final approval of manuscript.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.