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Abstract

Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, β-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.

Keywords

Substantia nigra Striatum Graft 6-Hydroxydopamine Neuroprotection Cytokines Rat

Introduction

In Parkinson's disease (PD), the ability to control voluntary movements is gradually lost, as a consequence of the progressive degeneration of melanin-containing, dopaminergic neurons of the substantia nigra pars compacta (SNpc). This process causes dopaminergic denervation of the striatum, which, in turn, triggers profound modifications in the functional organization of the basal ganglia circuitry, leading to the onset of the typical triad of PD motor symptoms (bradykinesia, tremor, rigidity) (26,39,40).

Although four decades have passed since its introduction into the clinical practice, pharmacological replacement of deficient neurotransmitter dopamine with its direct precursor, L-3,4-dihydroxyphenylalanine (l-DOPA), is still the gold-standard therapy in PD. However, long-term treatment with l-DOPA is associated with considerable side effects, extremely discomforting for the patient, which include “on–off” fluctuations, freezing episodes, lack of responsiveness (“wearing off”), and, above all, abnormal, uncontrollable movements, known as dyskinesias (44). More importantly, the use of l-DOPA aims at relieving PD motor symptoms by replacing the deficient neurotransmitter, without modifying the course of the disease, which continues to progress, synchronous with progression of SNpc cell loss.

After initial experiences with neural tissue transplantation (7), researchers of the PD field have begun to focus on the regenerative potential of stem cells, to develop novel therapeutic strategies that may modify the course of the disease. Encouraging, but also controversial, results have been reported with embryonic or neural stem cells, over the years. Embryonic stem cells proved able to differentiate into dopaminergic neurons, after transplantation into the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA) (8,17) or of monkeys intoxicated with MPTP (53), and to improve—although partially—motor abnormalities associated with the nigrostriatal lesion. More controversial results have been obtained with neural stem cells, derived from adult or fetal brain, which tend to differentiate into astroglial cells rather than into dopamine neurons (51). In general, with these approaches, the degree of dopamine cell survival following transplantation is small and the magnitude of behavioral benefit is modest. If one considers that the potential use of these cells for PD treatment is also hampered by ethical concerns and limited tissue availability, alternative sources of pluripotent stem cells may be necessary.

Adult, bone-marrow-derived mesenchymal stem cells (MSCs) have recently emerged, among others, as an attractive alternative to heterologous embryonic or neural precursor cells (22). Their possible autologous derivation avoids the immunological or ethical concerns related to other sources (32); another considerable advantage is that MSCs seem devoid of oncogenic potential (18). Human MSCs (hMSCs) can be easily obtained through a safe procedure (bone marrow aspirate) and expanded in vitro, where they maintain stable cellular features, as well as the ability to migrate towards lesioned tissues (3,59); hMSCs have differentiative multilineage capacity, which also includes the ability to differentiate toward the dopaminergic phenotype, at least in vitro (27). Moreover, hMSCs possess immunoregulatory properties, which are exerted through the release of soluble factors (14,31,32). Therefore, these cells may have the capacity of modulating the inflammatory response associated with the neurodegenerative process that underlies PD (25,42), thereby providing neuroprotection.

In this study, we transplanted naive hMSCs into the striatum of rats that had previously received an intrastriatal injection of 6-OHDA. Striatal injection of 6-OHDA causes early lesion of dopaminergic terminals, followed by delayed, partial loss of dopaminergic cell bodies in the SNpc, thereby mimicking the progressive nature of nigrostriatal degeneration of PD (10,20,34,50)). The aim was to determine whether this procedure is capable of counteracting the gradual SNpc cell loss, and associated behavioral alterations, caused by the toxin.

Materials and Methods

In Vitro Analysis

hMSC Preparation.

Commercial hMSCs (Cambrex, Walkersville, MD, USA) were grown, following the manufacturer's instructions, until confluence. Multiple batches (n = 6) were used to exclude nonspecific effects related to a single cell culture. Vitality maintenance was tested before any experimental procedure by TUNEL analysis. To exclude possible chromosome aberrations, cultures were expanded in vitro for no more than three passages and subjected to analysis (using Quinacrine coloration standard analysis and fluorescent in situ hybridization), as previously described (41). All treatment conditions were replicated twice, using at least three independent cell cultures. Conditioned media (CMs), obtained from cell cultures during the log phase of growth (gCMs) or at confluence (cCMs), were centrifuged briefly at 1,000 x g for 15 min, and chilled rapidly in liquid nitrogen. CMs were utilized as specified in the protocols below.

hMSC Characterization.

Adipogenic and osteogenic differentiation properties, typical of hMSCs, were verified by culturing cells in the appropriate differentiating media (Cambrex) and successive specific staining protocols (12). Expression of specific markers (neural, glial, mesenchimal, proliferative, hematopoietic, mesodermal, undifferentiative, mitochondrial, monocytic/macrophagic) was analyzed in cultured hMSCs, with different techniques, as described below.

RT-PCR.

RT-PCR was performed on three independent hMSC samples. Total RNA was extracted with TRIzol® reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions, and retro-transcribed as previously reported (11). Amplification steps and number of cycles were defined for each primer set, as shown in Table 1, where experimental conditions are summarized.

Table 1.

RT-PCR Primers and Experimental Conditions

Gene	Primer Sequences	T _a	No. Cycles	Conditions
β-Tubulin III (315 bp)	F: 5′-AGATGTACGAAGACGACGAGGAG-3′ R: 5′-GTATCCCCGAAAATATAAACACAAA-3′	58°C	35	30-30-30
BDNF (369 bp)	F: 5′-CCTCCTCTTCTCTTTCTGCTG-3′ R: 5′-AATTCTCTTTTTGCTATCCAT-3′	54°C	40	45-30-90
GAP-43 (497 bp)	F: 5′-TTTCCCACCCAGTAGCCCTCTTTC-3′ R: 5′-TGATGCTGCCACAGAGCAGG-3′	58°C	40	30-30-30
GDNF (334 bp)	F: 5′- CACCAGATAAACAAATGGCAGTGC-3′ R: 5′-CGACAGGTCATCATCAAAGGCG-3′	62°C	40	60-45-60
GFAP (203 bp)	F: 5′-GCATTCGAGCCAGGGAGAG-3′ R: 5′-CAGTCTAGCCCACTCCTTC-3′	63°C	40	30-60-120
IL-6 (295 bp)	F: 5′-AAATTCGGTACATCCTCGAC-3′ R: 5′-CAGGAACTGGATCAGGACTT-3′	54°C	35	30-30-30
IL-8 (265 bp)	F: 5′-CTTGGCAGCCTTCTTGATTT-3′ R: 5′-CTCAGCCTTCTTCAAAAACT-3′	54°C	35	30-30-30
MAP-2 (428 bp)	F: 5′-CCATTTGCAACAGGAAGACAC-3′ R: 5′-CAGCTCAAATGCTTTGCAACTAT-3′	58°C	40	30-30-30
Nestin (209 bp)	F: 5′-CAGCTGGCGCACCTCAAGATG-3′ R: 5′- AGGGAAGTTGGGCTCAGGACTGG-3′	68°C	35	30-30-30
NGF (189 bp)	F: 5′-CCAAGGGAGCAGCTTTCTATCCTGG-3′ R: 5′-GGCAGTGTCAAGGGAATGCTGAAGT-3′	60°C	40	45-30-90
NSE (324 bp)	F: 5′-ATGTGATCACGCCTGGCTAATA-3′ R: 5′-GAACCTAGGGTTGGGGAGAGAT-3′	58°C	35	30-30-30
NT-3 (302 bp)	F: 5′-ACGCGGATAAGAGTCACC-3′ R: 5′-CCACCGCCAGCCCACGAGTTT-3′	60°C	35	30-30-30
p75 (200 bp)	F: 5′-GTGGGACAGAGTCTGGGTGT-3′ R: 5′-AAGGAGGGGAGGTGATAGGA-3′	65°C	35	30-45-30
TNF-α (330 bp)	F: 5′-AAGCCTGTAGCCCATGTTGT-3′ R: 5′-CAGATAGATGGGCTCATACC-3′	56°C	40	30-30-30
TNF-β (288 bp)	F: 5′-AAACCTGCTGCTCACCTCATT-3′ R: 5′-TGGATACACCATCTTCTGGG-3′	58°C	35	30-30-30

The number in parentheses after the gene name indicates the length of the amplified RT-PCR fragment, expressed as base pair (bp). T_a is temperature of annealing. Conditions indicated are denaturation–annealing–extension time (seconds). BDNF, brain-derived neurotrophic factor; GAP-43, growth-associated protein 43; GDNF, glial cell line-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; IL, interleukin; MAP-2, microtubule-associated protein-2; NGF, nerve growth factor; NSE, neuron-specific enolase; NT-3, neurotrophin-3; p75, neurotrophin receptor; TNF, tumor necrosis factor.

Immunocytochemical Analysis.

Expression of hMSC-specific markers was detected by phosphatase-antialkaline phosphatase technique (16), while the analysis of neural antigen expression was performed using fluorescent immunocytochemistry analysis, as described previously (11). Primary antibodies against hematopoietic, mesenchymal, mesodermal, mitochondrial, monocytic/macrophagic, neurotrophic, neural, nuclear, proliferative, and undifferentiative markers were used; secondary Cy2 and Cy3 fluorescent antibodies (Jackson Immuno Research, Suffolk, UK), in 0.1 M PBS containing 10% NGS, were used for double labeling in combination with a nuclear counterstaining bisbenzimide-Hoechst33258 (1 ng/ml). Finally, samples were rinsed and embedded with mounting medium Fluorsave®.

Western Blot.

Western immunoblotting for neuroglial proteins was carried out by homogeneizing hMSCs in 100 μl of lysis buffer (150 mM NaCl, 20 mM TRIS, 1% Triton X-100, pH 8) containing protease inhibitor (Complete Mini; Roche, Hoffmann-La Roche Ltd, Basel, Switzerland). Samples (50 μg) were separated on SDS-Page, blotted, and incubated with antibodies against tyrosine hydroxylase (TH), dopamine transporter (DAT), glial fibrillary acidic protein (GFAP) (Chemicon International Inc., Temecula, CA, USA), mature (Tuj-1) (Covance, Princeton, NJ, USA), and immature (nestin) neuronal proteins (Becton Dickinson, San Jose, CA, USA). A chemiluminescent system (ECL; Amersham Bioscience, Arlington, IL, USA) was used for protein detection.

Enzyme-Linked Immunosorbent Assay (ELISA).

Levels of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were measured in cell lysates and CMs (both cCMs and gCMs), using enzyme immunoassay kits (Emax ImmunoAssay System; Promega Corporation, Madison, WI, USA). Protein extracts (20 μg), diluted in 100 μl of 0.1 M PBS, or 100 μl of supernatants were plated in triplicate and incubated overnight, according to the manufacturer's instructions. In addition, an acidic treatment was performed on each sample before assaying.

Assay detection limits of GDNF, BDNF, and NT-3 assays were 15.6, 7.8, and 4.12 pg/ml, respectively.

TUNEL Assay.

Apoptotic nuclei were detected using the In Situ Cell Death Detection Kit, TMR-red (Roche, Basel, Switzerland) on cell cultures and hMSC-transplanted tissues, following the manufacturer's indications.

In Vivo Experiments

Male Sprague-Dawley rats (Charles River, Calco, LC, Italy), weighing 200 g at the beginning of the experiment, were used. Animals were housed two per cages at 20–22°C on a 12-h light/dark cycle, with food and water available ad libitum. All animal care and use were in accordance with the European Convention for Animal Care and Use of Laboratory Animals, and were approved by the Veterinarian Department of the Italian Ministry of Health.

Nigrostriatal Lesion.

Animals were anaesthetized with 50 mg/kg of sodium thiopental (50 mg/kg; Hospira, Lake Forest, IL, USA) and arbitrarily divided into two groups. Rats were then placed in a stereotaxic frame (Stoelting, Wood Dale, IL, USA); the 6-OHDA group (n = 17) received 3 μl of a 6-OHDA solution (20 μg in saline solution containing 0.02% ascorbic acid; Sigma-Aldrich, St. Louis, MO, USA) into the right striatum (1.0 mm anterior, 3.0 mm lateral, and 5.0 mm ventral, with respect to bregma and dura) (47) as described previously (10); the sham group (n = 18) received an equal volume of saline, at the same stereotaxic coordinates.

Intrastriatal Grafts.

Five days later, each group was further divided into two subgroups (n = 8–9/group). Animals received other two injections into the right striatum (8 μl/rat), rostrally (0.2 mm anterior, 3.0 mm lateral, and 5.0 mm ventral) and caudally (1.8 mm anterior, 3.0 mm lateral, and 5.0 mm ventral) to the lesion site: transplanted animals received 180,000 (90,000 × 2) hMSCs, suspended in PBS, while control animals received two injections of PBS. The following four experimental groups were obtained: 1) 6-OHDA/hMSCs: animals lesioned with 6-OHDA that received two intrastriatal injections of 90,000 hMSCs each (180,000 cells); 2) 6-OHDA/vehicle: animals lesioned with 6-OHDA that received two intrastriatal injections of PBS; 3) sham/hMSCs: unlesioned animals that received two intrastriatal injections of 90,000 hMSCs each (180,000 cells); 4) sham/vehicle: unlesioned animals that received two intrastriatal injections of PBS.

All intracerebral infusions were performed at 0.5 μ/min, using a Hamilton 10-μl syringe with a 26-gauge needle; to avoid reflux along the inoculation track, the needle was left in place for 10 min before being retracted. All animals received daily immunosuppressive treatment [10 mg/kg cyclosporine A (CsA), IP; Sandoz, Holzkirchen, Germany]; cyclosporine A administration was initiated 2 days after the intrastriatal injection of 6-OHDA or vehicle and maintained until animals were sacrificed.

Turning Behavior.

Rotational response to apomorphine, a classic functional measure of unilateral dopaminergic denervation was assessed 2 days before sacrifice by an observer unaware of group distribution, as described previously (10). Briefly, animals were injected with apomorphine (0.5 mg/kg in saline containing 0.2% ascorbic acid) and immediately placed into a circular cage, where they were tethered to an automated rotometer (Basile, Comerio, VA, Italy). The number of complete turns performed by the animals was recorded for 45 min starting 1 min after injection of the agonist. The rotational response was calculated by subtracting the total number of ipsilateral rotations to the total number of contralateral turns, and results were expressed as the number of total net contralateral rotations.

Cell Tracking.

To track the grafted cells, the day before transplantation, hMSCs were labeled overnight with fluorescent dye bisbenzimide-Hoechst33258 (1 ng/ml; Molecular Probes-Invitrogen). Hoechst dyes have been repeatedly used for this purpose (35,43); however, because potential occurrence of postgrafting apoptototic cell death and redistribution of the dye to surrounding host cells has been reported for high concentrations of Hoechst33242, we used Hoechst33258, which does not affect cell survival or function (61).

Immunohistochemical Staining of Striatal and Nigral Sections

Animals were sacrificed 28 days after 6-OHDA injection by transcardiac perfusion, under deep anesthesia, with ice-cold PBS followed by 4% paraformaldehyde (PFA) (in 0.1 M PBS). Brains were immediately removed, postfixed for 4 h at 4°C in the same solution, immersed in a 30% sucrose/0.1 M PBS solution (all reagents from Sigma-Aldrich) overnight at 4°C, then rapidly frozen in 2-methylbutane and stored at −80°C. Serial coronal sections (20 μm) containing the striatum and SNpc were cut with a cryostat (Leica CM 1850 UV; Leica Microsystems Ltd, Heidelberg, Germany) and mounted on polylysine-coated slides. For the analysis of lesion of dopaminergic neurons, TH immunohistochemistry was performed, as described previously (10).

For immunofluorescent stainings, brain slices were incubated overnight at 4°C with the specific antibodies diluted in 0.1 M PBS containing 0.1% Triton X-100 and 3% normal goat serum (NGS, all reagents Sigma-Aldrich). To unambiguously identify the grafted cells, specific anti-human antibodies—against human mitochondria and nuclear mitotic apparatus protein 1 (NuMA)—were used. Detection was performed using fluorescent secondary antibodies diluted in 0.1 M PBS containing 0.1% Triton X-100 and 3% NGS. Samples were washed with PBS/10% NGS and mounted with Fluorsave® (Calbiochem, La Jolla, CA, USA). For NuMA staining, an additional boiling step in Tris-HCl, 50 mM, pH 8, for 15 min was required to retrieve specific epitope (48).

Image Analysis

All analyses were performed by an investigator unaware of the experimental design.

Fluorescent samples were viewed under a Leica DMIRE2 microscope and images were captured with a CCD camera directly connected to the analytical system (Leica Microsystems Ltd). Cytokine autoradiograms were scanned and semiquantitative densitometric analysis performed, in triplicate, using a dedicated software (Quantity One-BioRad, Richmond, CA, USA).

Image analysis of TH immunohistochemistry and grafted Hoechst-positive hMSCs was performed using an AxioSkop 2 microscope connected to a computerized image analysis system (AxioCam MR5; Zeiss, Oberkochen, Germany) equipped with a dedicated software (AxioVision Rel 4.2; Zeiss).

The lesion of striatal dopaminergic terminals was evaluated by evaluating optical densities of TH-immunoreactive fibers at three rostrocaudal levels (AP 2.0, 0.8, and −0.4 mm with respect to bregma), considering three sections per level. Briefly, a picture of each section was captured at 1.25x magnification, maintaining the same parameters across all images. Readings were corrected for nonspecific background density, as measured from an area devoid of specific TH staining. Optical densities were analyzed in the striata of both hemispheres and averaged. Results were expressed as the ratio of the value in the lesioned striatum with respect to the intact side.

TH-positive cells in the SNpc of both hemispheres were counted, in every fourth section, throughout the entire nucleus. The anatomical levels considered, in the antero-posterior (AP) extension, fell within −5.20 and −5.80 mm, with respect to bregma (47). Results were expressed as the percentage of TH-positive cells in the lesioned SNpc with respect to the contralateral, intact side. In the absence of a stereological count, such approach was chosen to avoid methodological biases due to interindividual differences and has been previously used to assess the extent of 6-OHDA-induced lesion in the SNpc (9,10,30).

Statistical Analysis

All values are expressed as mean ± SEM. Comparisons between groups were made using the Student t-test (paired) or one-way analysis of variance (ANOVA, nonparametric) followed by a Tukey's HSD or Dunnet post hoc test using a dedicated statistical software (Prism 3 software, GraphPad Software, San Diego, CA, USA). Minimum level of statistical significance was set at p < 0.05.

Results

Neuroglial Potential of hMSCs In Vitro

Cultured hMSCs displayed typical spindle-shape morphology, without significant apoptotic features, and a 24–48-h doubling rate. Normal karyotype state and adipogenic and osteogenic differentiation properties, typical of hMSCs after induction, were confirmed by specific stainings (data not shown).

Cultured hMSCs expressed stem and neural cell messengers (Fig. 1A), as well as selected cytokine mRNAs (Fig. 1B). Moreover, specific human mesenchymal antigens (such as CD90) and markers of both undifferentiated and committed neural cells were present (Fig. 2A and B). In particular, nestin fibrillary net was clearly visible in the cytoplasm (Fig. 2B); cultured hMSCs did not express astroglial (GFAP) or neuronal markers, such as dopamine transporter (DAT) or TH (Fig. 2C).

Figure 1.

Neuroglial characterization of hMSCs in vitro. (A) RT-PCR expression of undifferentiated (nestin) and differentiated neural genes: growth cone-associated protein 43 (GAP-43), neuron-specific enolase (NSE), β-tubulin III, microtubule-associated protein-2 (MAP-2), glial fibrillary acidic protein (GFAP), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF). Lane M: molecular weight markers; lane 1: hMSCs; lane 2: positive control; lane 3: negative control. (B) RT-PCR expression of NGF and its receptor, p75, and hematopoietic markers (interleukin-6 and -8); no expression was detected for TNF-α or TNF-β. Lane M: molecular weight marker; lane 1: hMSCs; lane 2: positive control; lane 3: negative control.

Figure 2.

Further characterization of hMSC neuroglial potential and stromal origin in vitro. (A, B) Fluorescent double-labeling of hMSCs: proliferative state and typical mesenchymal phenotype are indicated by Ki67 and CD90 labeling, respectively (A); (B) colocalization of Ki67 (arrowhead) with undifferentiated neural marker nestin (arrow). (C) Western immunoblotting for neuroglial proteins in hMSCs under normal culture conditions. The presence of mature (Tuj-1) and immature (nestin) neuronal proteins was clearly detectable; no specific glial (GFAP) or dopaminergic [tyrosine hydroxylase (TH); dopamine transporter (DAT)] phenotypes were detected. M: molecular weight markers; lane 1: positive control, lane 2: hMSCs; β-actin antibody (35 kDa) was used as internal control. Scale bar: 50 μm.

Neurotrophin levels were determined in lysates and CMs from confluent or proliferative cells upon 7 days in vitro (Table 2). BDNF was present in confluent hMSC lysates, while GDNF could only be detected in CMs from cells in the proliferative phase; moreover, although NT-3 mRNA was detected, no appreciable levels of NT-3 were detected, either in CMs or cell lysates (Table 2).

Table 2.

Neurotrophin Levels in hMSCs Lysates and Conditioned Medium (CM)

	Confluent hMSCs		Proliferative hMSCs
	Lysate	CM	Lysate	CM
BDNF	16.7 ± 2.1	n.d.	n.d.	n.d.
GDNF	n.d.	n.d.	n.d.	110.5 ± 43.8
NT-3	n.d.	n.d.	n.d.	n.d.

Values are expressed in pg/ml (mean ± SD). n.d.: results below detection limit. BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; NT-3, neurotrophin-3.

Effects of hMSC Grafting in 6-OHDA-Lesioned Animals

Hoechst33528-labeled hMSCs were grafted into the ipsilateral striatum of rats, 5 days after the intrastriatal injection of 6-OHDA. To avoid rejection of the xenotransplant, all animals received a daily injection of CsA throughout the experimental period. Preliminary studies, performed in lesioned animals, showed that the immunosuppressive treatment per se had no influence either on the 6-OHDA-induced nigrostriatal neurodegeneration and associated inflammatory responses, or on hMSC viability (data not shown).

Survival and Phenotypic Changes in Transplanted hMSCs.

Grafted hMSCs survived and integrated in the striatum (Fig. 3); cells remained gathered around the site of injection, without evident migration. Morphological analysis of the striatum showed that transplantation per se did not damage the surrounding tissue.

Figure 3.

Fluorescent immunostainings of Hoechst-positive hMSCs transplanted into the striatum of a 6-OHDA-lesioned rat. (A) Nuclear mitotic apparatus protein 1 (NuMA) and (B) human mitochondria (hMito) immunostainings confirm the human origin of grafted cells. (C) TUNEL staining shows an extremely low rate of apoptotic cell death within transplanted hMSCs. (D) Staining for Ki67 (proliferation marker) shows absence of cellular overgrowth, because very few hMSCs are actively proliferating and, thereby, positive for Ki67. (E) Sparse immunoreactivity for human-specific, astroglial marker GFAP is present within grafted cells. (F) Magnification of encircled cells shown in (E). Scale bars: 10 μm (A, B, C, D, F) and 100 μm (E).

Human origin of Hoechst-labeled cells was confirmed using specific antibodies against NuMA and human mitochondria antigens; unambiguous staining was confined to Hoechst-positive cells, excluding any host cell picking of fluorescent dye or fusion, both for NuMA (Fig. 3A) and human mitochondria (Fig. 3B). An extremely low rate of apoptotic cell death was present within transplanted cells, as shown by the negligible degree of TUNEL and Hoechst colocalization (Fig. 3C). Analogously, negligible immunoreactivity for proliferation marker Ki67 was detected in transplanted hMSCs (Fig. 3D), indicating substantial absence of cellular overgrowth.

Immunohistochemical analysis of transplanted cells evidenced a maturation process toward a neuroglial phenotype. The presence of human specific GFAP-positive hMSCs was observed in transplanted lesioned animals (Fig. 3E and F); conversely, the expression of nestin—a marker of undifferentiated neural cells detected in cultured hMSCs before transplantation—was no longer detectable. In sham-operated animals, grafted hMSCs integrated and survived without detectable suffering features, but showed no shift toward the neuroglial phenotype.

Microtubule-associated protein-2 (MAP-2), TH, or DAT expression were not observed in grafted hMSCs, indicating that naive hMSCs did not undergo maturation toward dopaminergic neuronal phenotype after grafting.

Nigrostriatal Lesion and GDNF Immunoreactivity.

The intrastriatal injection of 6-OHDA induced massive loss of dopaminergic terminals (Fig. 4A) and subsequent loss of TH-positive cells in the ipsilateral SNpc (Fig. 4B). The extent of the neurotoxin-induced nigrostriatal damage was significantly reduced in animals transplanted with hMSCs. Indeed, grafting of hMSCs almost doubled both the density of surviving terminals in the striatum (Fig. 4A and C) and the number of dopaminergic cell bodies in the SNpc, in the injected hemisphere (Fig. 4B and D) compared to lesioned animals that received vehicle (6-OHDA/vehicle group). Transplantation of hMSCs in sham (intact) animals per se did not modify the density of dopaminergic terminals in the striatum or the number of TH-positive cells in the SNpc.

Figure 4.

Neuroprotective effect of transplanted hMSCs. Immunohistochemical staining for TH in the striatum (A) and SNpc (B) of a control rat (sham/vehicle), of a rat lesioned with 6-OHDA that received vehicle (6-OHDA/vehicle), and of a rat lesioned with 6-OHDA and transplanted with hMSCs (6-OHDA/hMSCs). Note how the area deprived of TH signal in the striatum (mirroring the lesion of dopaminergic terminals caused by 6-OHDA) is smaller in the transplanted compared to the nontransplanted rat; the same animal shows a clear increase in the number of surviving, TH-positive cell bodies in the SNpc. Scale bars: 500 μm (A) and 200 μm (B). (C) Density of TH-positive fibers in the striatum. Values (mean ± SEM) are expressed as the difference (%) between injected and noninjected striata. In animals that received a sham lesion of the nigrostriatal tract, striatal injection of vehicle (white columns) or hMSCs (black columns) did not modify TH immunoreactivity; in lesioned animals (6-OHDA), hMSC transplant prompted increased survival of TH-positive terminals. (D) Analysis of TH-positive cells in the SNpc. Values are expressed as the difference (%) in the number of nigral cells between injected and noninjected hemispheres. Also in this case, striatal injection of vehicle (white columns) or hMSCs (black columns) did not modify the number of dopaminergic neurons, while increased survival of TH-positive, SNpc neurons was observed in lesioned animals that received the hMSC graft. *p < 0.001 versus sham/vehicle; §p < 0.01 versus 6-OHDA/vehicle group.

We also carried out an immunofluorescent, double staining for GDNF and TH in SNpc sections (Fig. 5). In sham-operated (unlesioned) animals, GDNF was highly expressed in SNpc neurons and, mostly, colocalized with TH; a dense network of GDNF-positive fibers was also clearly visible (Fig. 5A). Dramatic decrease of GDNF immunoreactivity was found in the SNpc of 6-OHDA-lesioned animals that did not receive the hMSC graft (Fig. 5B). Conversely, considerable GDNF immunoreactivity was detected in 6-OHDA-lesioned animals transplanted with hMSCs, which showed a substantial degree of GDNF/TH colocalization and, above all, considerable preservation of GDNF-positive neuronal arborization (Fig. 5C).

Figure 5.

GDNF and TH expression in the substantia nigra pars compacta. Double immunofluorescent staining for glial cell line-derived neurotrophic factor (GDNF) and tyrosine hydroxylase (TH) in the substantia nigra pars compacta of a (A) sham-lesioned rat, (B) 6-OHDA-lesioned rat that received vehicle into the striatum, (C) 6-OHDA lesioned rat that received the striatal hMSC graft. Note the GDNF-positive network of fibers visible in the 6-OHDA/hMSCs animal as opposed to the virtual absence of GDNF signal in the 6-OHDA/vehicle animal. Scale bars: 100 μm (larger pictures) and 40 μm (insets).

Turning Behavior.

Lesioned rats that received the intrastriatal injections of vehicle (6-OHDA/vehicle) exhibited consistent circling behavior in response to systemic administration of apomorphine, while no rotations were observed in unlesioned, nontransplanted (sham/vehicle) animals (Fig. 6). Rotational response to apomorphine was significantly reduced in lesioned animals that received the striatal graft (6-OHDA/hMSCs) (Fig. 6). No turning behavior was detected in unlesioned, transplanted (sham/hMSCs) animals.

Figure 6.

Apomorphine-induced contralateral rotations. Results (mean ± SEM) represent the net number of contralateral turns performed, in 45 min, by unlesioned (sham) or lesioned (6-OHDA) animals that received a striatal injection of vehicle (white columns) or hMSCs (black columns). Rotational behavior was absent in lesioned animals, while lesioned, transplanted animals showed reduced number of contralateral turns with respect to lesioned animals treated with vehicle. *p < 0.001 versus sham/vehicle group; §p < 0.05 versus 6-OHDA/vehicle.

Discussion

In this study, we investigated the effects of hMSC transplantation into the striatum of rats previously lesioned with 6-OHDA. The aim was to evaluate whether this procedure can counteract the progressive nigrostriatal degeneration caused by intrastriatal injection of the toxin (10,50); an additional aim was to investigate whether phenotypic changes occurred in hMSCs, after grafting.

In Vitro Characterization of hMSCs

In addition to typical mesenchymal antigens, before transplantation, cultured hMSCs showed markers of immature neuronal proteins, such as nestin and Tuj-1; in particular, nestin was abundant in the hMSC cytoplasm. No glial or mature neuronal markers were detected. This observation confirms recent findings, suggesting that bone marrow-derived mesenchymal cells can express specific neural proteins before any differentiation (19,55). A clear feature shown by our naive cells was the ability to release neurotrophic factors, such as BDNF and GDNF, thereby confirming the immunomodulatory and neurotrophic potential recently postulated for these cells (32,49).

Survival and Phenotypic Changes in Transplanted hMSCs

Transplanted hMSCs promptly integrated and survived in the lesioned striatum, as shown by the extremely low rate of apoptotic cell death detected, which confirmed the homing potential of these cells (24).

The most prominent change observed in our grafted cells was a phenotypic shift towards the glial lineage. Transplanted hMSCs expressed the human-specific, astrocytic marker GFAP, which was absent in cultured cells before grafting; this was associated, in some cases, with a stellate morphology and presence of ramified processes, reminiscent of astroglial cells. On the other hand, hMSCs lost the remarkable positivity for nestin, an immature neural cell marker, that had been detected in vitro, before transplantation. A possibility that one has to exclude, in this case, is the occurrence of cell fusion between endogenous and human grafted cells; expression of human-specific GFAP was mostly localized within the inner region of the implant, while only few GFAP-positive cells were found at the periphery of the graft. Because this is the area where contact with the host tissue may favor the fusion process, we tend to exclude this possibility.

Interestingly, hMSCs transplanted in sham-lesioned animals integrated and survived to a similar extent as those transplanted into 6-OHDA-lesioned animals. However, in unlesioned animals, although cytoplasmic nestin positivity was lost, transplanted hMSCs never acquired GFAP immunopositivity. Therefore, it is likely that the phenotypic shift detected in grafted hMSCs was driven by the cell stress signals released within the degenerating area. This is in keeping with previous data obtained in a rodent model of Huntington's disease based on the intrastriatal injection of neurotoxin quinolinic acid, which showed that engraftment of transplanted neural progenitors and their shift toward the astroglial phenotype is favored by the striatal lesion (2,58).

In our experimental setting, transplanted hMSCs did not show TH or DAT immunopositivity, thereby indicating that our cells did not adopt the dopaminergic phenotype after grafting. This was not surprising, because no differentiation into dopaminergic neurons has been found, for example, in fetal-derived neural stem cells grafted in 6-OHDA-lesioned rats (60) while only limited dopaminergic fate acquisition has been previously shown for naive (37) or transfected stromal cells (28). In fact, use of specific differentiation protocols or transfection procedures with vectors containing a TH promoter are required to induce, in vitro, differentiation of hMSC into dopamine-producing neurons (5,23,27,45,57) before transplantation. An exception seems to be represented by the findings of Park et al. (46), who injected, through the tail vein, hMSCs into rats bearing a nigral lesion induced by systemic administration of proteasome inhibitor MG-132. In this case, in addition to a neuroprotective effect against the dopaminergic cell loss in the SNpc, the authors reported TH immunopositivity in about 36% of hMSCs tracked in this area. Experimental conditions, however, were quite different, with respect to ours. In particular, the longer survival time following hMSC administration (8 weeks instead of 4) and, more importantly, the completely different hMSC administration protocol used by Park et al. (repeated IV injections vs. single intrastriatal grafting) make the comparison with our findings quite difficult.

Effects of hMSC Graft on Nigrostriatal Lesion and GDNF Immunoreactivity

Transplantation of hMSCs was associated with marked reduction of 6-OHDA-induced neurodegeneration, as reflected by the increase in both density of striatal dopaminergic terminals and number of TH-positive nigral cell bodies in lesioned animals that received the hMSC graft. This finding confirms recent data, showing that transplantation of MSCs—of either human or rodent origin—into the striatum of rats lesioned with 6-OHDA exerts protective and/or regenerative effects on nigrostriatal neurons (13,36,49). Our results, in particular, are in keeping with those reported by Bouchez et al. (13). In this case, unlike in previous studies where the hMSC graft was simultaneous to 6-OHDA injection (36,49), authors transplanted hMSCs after inducing the nigrostriatal lesion, as we did. In addition, total amount of grafted hMSCs (180,000) and total dose of intrastriatal 6-OHDA (20 μg) where the same as in our study. As mentioned above, similar results, although in a different experimental context, have been reported by Park et al. (46). In this case, the authors administered hMSCs, IV, to rats treated, several weeks before, with a proteasome inhibitor. Proteasomal inhibition was associated with a substantial loss of TH-positive (dopaminergic) neurons in the SNpc, which was markedly reduced in rats infused with hMSCs.

In line with previous results (6,8,13,52,60), striatal graft also induced significant behavioral effects. Turning behavior in response to systemic apomorphine, a functional measure of unilateral nigrostriatal lesion, was dramatically reduced in lesioned animals transplanted with hMSCs, thereby indicating that the increased neuronal survival promoted by the graft translated into reduction of lesion-induced motor stereotypies. It can be concluded that transplantation of hMSCs counteracts the progressive degeneration of the nigrostriatal pathway caused by specific neurotoxins, and associated motor abnormalities, even when the neurodegenerative process has already been set in motion and has reached a medium/advanced stage. This supports the protective potential of hMSCs against neurodegeneration (56).

Our in vivo experimental setting did not allow us to dissect out the exact mechanisms underlying the protective effect played by hMSCs. However, the immunohistochemical study of GDNF expression in surviving nigral neurons provided us with an interesting clue. In intact (sham-operated) animals, GDNF was highly expressed in neurons of the SNpc, where a dense network of GDNF-positive fibers was also present. GDNF immunoreactivity decreased dramatically in the SNpc of 6-OHDA-lesioned animals that did not receive the hMSC graft; the reduction of GDNF signal was less evident in lesioned animals, which showed, in particular, substantial preservation of the GDNF-positive neuronal arborization. Recent findings have shown that dopaminergic nigral neurons do not synthetize GDNF (4). The presence of the neurotrophin in the SNpc, therefore, is dependent upon the uptake of GDNF by dopaminergic terminals, which internalize and transport to the SNpc the neurotrophin released within the striatum (33,54). Our hypothesis is that hMSCs grafted in the striatum released GDNF and, consequently, enhanced trafficking of the neurotrophin, promoting its retrograde transportation, from striatal terminals to SNpc cell bodies. The neurotrophic effect of GDNF on the nigrostriatal system has been repeatedly reported in experimental models of PD (21). Previous studies have also indicated that the therapeutic effect of hMSCs may depend on the release of soluble factors, thereby proposing immunomodulatory/anti-inflammatory properties for these cells (15,29,32).

MSC-Induced Neuroprotection: Relevance to PD Therapy

The initial experiences with neural tissue transplantation in PD patients (7) have prompted extensive investigation of the regenerative potential of stem cells, with the objective of generating transplantable, stem cell-derived dopaminergic neurons to replace those that are lost in the neurodegenerative process. Encouraging results have been obtained in animal models of PD with this approach, particularly when using embryonic stem cells. However, there is no evidence that these cells, once transplanted, can restore the complex neural network that is altered in PD, for example, by reinnervating the striatum or restoring the physiological release of dopamine at the striatal level (38). As an alternative approach, transplanted stem cells may exert protective actions through the release of trophic factors, thereby promoting survival of nigrostriatal neurons. In this perspective, hMSCs may represent a valid tool to promote neuroprotection in the damaged nigrostriatal pathway. Our in vitro study has clearly shown that hMSCs are capable of releasing neurotrophic factors, including GDNF; such capacity was likely reinforced by the postgrafting differentiation toward the astroglial phenotype. This is further supported by recent data, showing that hMSCs that are differentiated, before grafting, into astroglial-like cells synthesizing neurotrophins (including GDNF), induce amelioration of the behavioral disturbances caused by 6-OHDA, once transplanted into the lesioned rats (1).

In conclusion, our data show that, in vitro, hMSCs synthesize and secrete cytokines and, more importantly, neurotrophins. Transplantation of hMSCs into the striatum of 6-OHDA-lesioned rats, carried out after the intrastriatal injection of the toxin, effectively counteracted neurodegeneration of the nigrostriatal pathway and related motor stereotypies. This was associated with acquisition of GFAP positivity by grafted hMSCs, pointing to potential differentiation toward an astroglial phenotype, and increased GDNF expression in the SNpc of transplanted animals. Altogether, our data confirm the potential of undifferentiated hMSCs in promoting neuroprotective mechanisms in PD.

Footnotes

Acknowledgments

We dedicate this work to the loving memory of Prof. Davide Soligo. We wish to thank Dr. D. Giardino and the Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Milan, Italy for the karyotipic analysis and the helpful technical support. This work was supported by grants issued by the Italian Ministry of Health (ex art. 56, 2004), Fondazione Cariplo and by a Francesco Caleffi donation.

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Transplantation of Undifferentiated Human Mesenchymal Stem Cells Protects against 6-Hydroxydopamine Neurotoxicity in the Rat

Abstract

Keywords

Introduction

Materials and Methods

In Vitro Analysis

hMSC Preparation.

hMSC Characterization.

RT-PCR.

Immunocytochemical Analysis.

Western Blot.

Enzyme-Linked Immunosorbent Assay (ELISA).

TUNEL Assay.

In Vivo Experiments

Nigrostriatal Lesion.

Intrastriatal Grafts.

Turning Behavior.

Cell Tracking.

Immunohistochemical Staining of Striatal and Nigral Sections

Image Analysis

Statistical Analysis

Results

Neuroglial Potential of hMSCs In Vitro

Effects of hMSC Grafting in 6-OHDA-Lesioned Animals

Survival and Phenotypic Changes in Transplanted hMSCs.

Nigrostriatal Lesion and GDNF Immunoreactivity.

Turning Behavior.

Discussion

In Vitro Characterization of hMSCs

Survival and Phenotypic Changes in Transplanted hMSCs

Effects of hMSC Graft on Nigrostriatal Lesion and GDNF Immunoreactivity

MSC-Induced Neuroprotection: Relevance to PD Therapy

Footnotes

Acknowledgments

References