Introduction

Huntington disease (HD) is an inherited disorder hallmarked by movement and cognitive anomalies (Ross et al. 2014). It is genetically caused by repetitive CAG expansions within the coding region of the huntingtin gene (HTT). During the course of HD, striatum is initially disrupted, but over time, other brain regions including cerebral cortex and basal ganglia are severely disturbed (Waldvogel et al. 2014).

Cell therapy approaches in neurodegenerative conditions have concentrated on the replenishment of lost/dying neurons with functional ones. Moreover, this therapeutical platform might stimulate the proliferation and differentiation of endogenous neural progenitors (Clelland et al. 2008).

Recently, mesenchymal stem cells (MSCs) have been demonstrated as a potential therapy for neurodegenerative diseases (Lee et al. 2010). Multipotent MSCs with multilineage capacity are found in a variety of tissues including bone marrow, adipose tissue, and Wharton’s jelly (Boroujeni et al. 2012; Boroujeni et al. 2017; Wang et al. 2004) which the latter is a gelatinous connective tissue from umbilical cord (Secco et al. 2008). Despite the great plasticity of MSCs for differentiation, these cellular paramedics can secrete a plethora of growth factors, chemokines, angiogenic and neurogenic molecules, and anti-apoptotic and anti-inflammatory compounds (Chao et al. 2008; Hsiao et al. 2011)

There are various reports suggesting that MSCs might hold promise for the therapeutic purposes in HD (Bantubungi et al. 2008; Dey et al. 2010; Olson et al. 2012; Rossignol et al. 2011; Sadan et al. 2012). In order to reproduce cellular and molecular alterations of HD phenotype, several animal models have been introduced, namely non-genetic and genetic models. We used 3-nitropropionic acid (3-NP) which perturbs mitochondrial function followed by striatal atrophy and behavioral disturbances (Pouladi et al. 2013; Ramaswamy et al. 2007).

In this study, we evaluated the in vitro and in vivo efficacy of human umbilical cord matrix stem cells (UCMSCs) and their paracrine effect against oxidative stress with a specific focus on HD.

Materials and Methods

Isolation and Culture of UCMSC

The umbilical cord was obtained from Taleghani Hospital in Tehran, Iran, upon consent of its donor. The cord was rinsed with PBS and isolated of amniotic membrane. Then, the jelly fraction of the cords was cut into pieces and cultured in DMEM/F12 medium supplemented with 10% FBS and antibiotics. Two weeks later, the isolated umbilical cord matrix stem cells were fed with the same medium. The cells were grown to 80% confluence and passaged by trypsinization. We used the fourth passage of UCMSC for characterization and differentiation steps. For collecting the conditioned media of UCMSC (UCMSC-CM) for treatment, UCMSC was cultured in serum free medium. After 48 h, medium was collected, filtered, and kept at − 20 °C for future uses.

Flow Cytometry

A number of 1 × 105 cultured UCMSCs were washed, fixed, and incubated for 15 min at 4 °C with a 1:9 dilution of normal goat serum in PBS. They were then stained with the following antibodies for 1 h: FITC-conjugated anti-CD105, FITC-conjugated anti-CD44 (Chemicon; USA), FITC-conjugated anti-CD45 (Ediscience; USA), and FITC-conjugated anti-HLA-DR (Dako, Denmark). The cells were washed with 2% FBS in PBS and analyzed using a FACSCalibur machine (Becton Dickenson, USA). The control population was stained with matched isotype antibodies (FITC-conjugated and PE-conjugated mouse IgG monoclonal isotype standards). At least 10,000 events were recorded for each sample and data were analyzed using WinMDI software (USA).

Adipogenic and Osteogenic Differentiation of UCMSC

For adipogenic differentiation, cell medium was supplemented with 50 μg/ml ascorbate-1 phosphate, 10−7 M dexamethasone, and 50 μg/ml indomethacin. For osteogenic differentiation, we added 50 μg/ml ascorbate phosphate, 10−8 M dexamethasone, and 10 mM β-glycerophosphate. The medium for each differentiation setting was changed every 3 days. Oil red and alizarin red (Sigma) were used, respectively, for detection of adipogenic and osteogenic differentiation.

PC12 Cell Culture and Treatment

PC12 cells were obtained from Institute of Pasture (Iran, Tehran). The cells were cultured in DMEM/F12 media supplemented with 5% fetal bovine serum (FBS), 10% horse serum and penicillin, and streptomycin (1%). Then, PC12 cells were treated with UCMSC-CM (4:1 ratio of UCMSC-CM to DMEM/F12 medium) and simultaneously exposed to H2O2 (150 μM) for 24 h.

Examination of PC12 Cell Morphology

PC12 cells were seeded in 6-well plates. For morphological analysis, random images were acquired from each well, taking 20 images per well. A minimum of 50 cells per treatment were quantified. After co-administration of UCMSC-CM and H2O2, neurite length was assessed. It was defined as the sum of lengths of all primary branches and their associated twigs. Data analysis was done by the Cell^A software.

MTT Assay

PC12 cells were cultured in 96-well plate. Following treatment with H2O2, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated for 4 h. Then, supernatant was removed and dark blue crystal of formazan was dissolved in dimethyl sulfoxide. Absorption of the suspension was read at 570 nm, and the measurements were reported as percentage of control.

Cell Viability Assay

Living and dead cells were distinguished using the EukolightTM Viability/Cytotoxicity assay (Molecular Probes). Culture medium was replaced with 2 mM calcein acetoxymethyl ester and 4 mM ethidium homodimer-1. Viable (green fluorescent by calcein) and non-viable (red fluorescent by ethidium) PC12 cells present in 10–20 random microscopic fields per condition per experiment were recorded.

3-NP Toxicity Model

In this study, 30 adult male rats (Sprague–Dawley, 200–220 g) were obtained from the Laboratory Animal Center of Shahid Beheshti University Of Medical Sciences, Tehran, Iran. The Ethics Committee of the University approved this animal experiment (IR SBMU.PHNS.REC.1395.7). Rats were housed at 22 °C, under 12-h light/12-h dark conditions with ad libitum access to food and water. Besides, after post-cell transplantation and post-electromyography, an analgesic (buprenorphine hydrochloride, 0.1 ml of 0.3 mg/ml) was subcutaneously injected. Thirty rats were randomly assigned into three groups: control group (n = 10), 3-NP + vehicle group (n = 10), and 3-NP + UCMSC (n = 10). In the 3-NP groups, all animals received intraperitoneal (i.p.) 3-NP injections daily for five consecutive days. Concentrations of 3-NP (per injection) were as follows: 30 mg/kg.

Stem Cell Transplantation

Bilateral UCMSC transplantation was performed 7 days after the beginning of 3-NP administration. The animals were assigned to one of the three experimental/transplant groups: control or intact group (n = 10), 3NP + vehicle (n = 10), UCMSC + 3-NP (250,000 cells; n = 10). The UCMSC was maintained alive in suspension using a 2-μl DMEM/F12 aliquot, stored on ice during the surgery procedure. After being anesthetized (i.p.) with xylazine (10 mg/kg)/ketamine (75 mg/kg), the animals were bilaterally transplanted with UCMSC in each striatum (250,000 cells) labeled with Hoechst 33,258 (5 μg/ml) and DiI using a 10-μl Hamilton microsyringe placed at the following coordinates, relative to bregma: + 0.5 mm AP; ± 2.6 mm ML; − 6 mm and − 5 mm DV. UCMSCs were transplanted in the part of the striatum (medio-posterior part) which was devoid of 3-NP-induced cell loss and disruption of extracellular matrix, in order to maximize graft survival. In 3-NP group, rats received media as vehicle. Rats were sacrificed at 30 days post-transplantation.

Rotarod Performance Test

A behavioral test was performed the day before the injection of 3-NP and at the 1st, 2nd, 3rd, and 4th week after the last injection of 3-NP. Rats were placed on the accelerating cylinder at speeds increasing from 4 to 40 rpm over a 5-min test session. The test was stopped if the animal fell off the rungs or gripped the device and spun around for two consecutive revolutions without attempting to run. The maximum time that each animal remained on the device was recorded. The data prior 3-NP injections were regarded as the baseline and the collected data over the course of 4 weeks was displayed as the percentage change from baseline. There were ten rats in each group and there were five runs per rat. The mean of five runs were calculated for each rat and then all these values were averaged to present a data point in the chart for each week.

Electromyography

Animals were placed under general anesthesia via intraperitoneal injection of ketamine hydrochloride (60 mg/kg) and xylazine (8 mg/k). After that, the right hind limb of animal was shaved and cleaned with a betadine solution. A 3-cm skin incision was made longitudinally on the posterior aspect of each thigh, from the greater trochanter to the knee. Then, dissection was performed between the gluteus maximus and biceps femoris muscles, and sciatic were exposed, along with the gastrocnemius muscle. With appearance of the sciatic nerve using forceps and cautiously in order to avoid damage to nerve, it is separated from the surrounding connective tissue which stimulation electrodes could able to pass under sciatic nerve. For electrical stimulation, two monopolar subdermal teflon needle electrodes were used, arranged in parallel at a fixed distance of 7 mm from each other. The recording electrodes had an insulating coating over leaving the distal uncoated. The sciatic nerve was then stimulated (1 A, 0.2 Hz frequency, 100 s long), and the compound muscle action potential was recorded in gastrocnemius muscle on the side ipsilateral to the stimulation. The compound muscle action potential parameters analyzed were amplitude and latency. Also, during stimulation and recording, ringer solution was used in order to prevent drying of the nerve.

Western Blot

For detection of neurotrophic factors of GDNF and VEGF, UCMSCs were lysed in lysis buffer. To determine the protein concentration in the samples, Bradford test was performed. The equal amount of protein was loaded on 12% SDS-page gel and then separated proteins were electrophoretically transferred onto PVDF. After incubating blotted membranes with blocking solution for 75 min, VEGF and GDNF antibodies were added and incubated for overnight at 4 °C. Blots were washed three times for 10 min each with PBS-Tween and thence horseradish peroxidase-conjugated anti-mouse Ig secondary antibody incubated with each membrane for 70 min and finally, immunoreactivity of polypeptides was detected using ECL solution.

Immunohistochemistry

Rats were deeply anesthetized by chloral hydrate and perfused transcardially using chilled saline followed by fixative consisting of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Then, brain was placed in formalin, prepared and placed on slides. The primary antibody was diluted with PBS containing 0.3% Triton X-100 and 1% bovine serum albumin (BSA). Sections were incubated in primary antibodies against GFAP (1:300) overnight in 4 °C. Sections were then incubated with the avidin–biotin complex substrate and treated with 0.05% 3, 3-diaminobenzidine tetra-hydrochloride and 0.03% hydrogen peroxide in 0.05 m Tris-buffer (pH 7.6). After immunohistochemical reaction, sections were mounted, counter-stained, and observed under a light microscope.

Estimation of the Striatum Volume

The rat’s brain tissue samples were fixed in 10% formalin for 1 week. Following tissue processing, serial coronal sections of 10-μm thickness were prepared and stained with cresyl violet (0.1%). To measure the total volume of striatum using the Cavalieri’s principle (Noorafshan et al. 2014), the following formula was used:

$$ V\ (striatum)=\Sigma P\times a/p\times t $$

Estimation of Total Length of Dendrite

The length of dendrite was estimated using the oriented cycloid. The striatum was cut into systematic uniform random sectioning. They were embedded in a paraffin block, sectioned (60 μm thickness) and stained with 1% silver nitrate. Mean dendritic length was calculated using the following formula, according to the previous study (Noorafshan et al. 2015).

$$ In=\frac{Total\ dendritic\ length}{Total\ number\ of\ neurons} $$

To measure the length, a vertical section was considered. A cycloid grid and a counting frame were superimposed on the live images of the striatum. Using a microscope (Nikon E-200) equipped with an objective lens connected to a computer, the dendrite length per neuron was assessed; two values were measured: (i) the number (Q–) of cell bodies of the neurons using the optical disector method and (ii) the total number of intersections (I) between the dendrite axes and the oriented cycloid. The following formula was used:

$$ {I}_n=2\times \frac{a}{l}\times \frac{1}{asf}\times {M}^{-1}\times \frac{\sum Q}{\sum I} $$

where “al” is the test area per cycloid test length, “asf” is the area associated with the cycloid grid divided by the area of the counting frame, and “M” is the final magnification.

Data Analysis

All data are represented as the mean ± SEM. Comparison between groups was made by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The statistical significances were achieved when P < 0.05.

Results

Cultured UCMSC Resemble Spindle-Shaped Fibroblasts and Express Mesenchymal Stem Cell Markers

Once UCMSC culture-expanded, they demonstrated fibroblast-like cell morphology (Fig. 1a). Flow cytometric analysis displayed that UCMSCs do not express hematopoietic markers CD45 and HLA-DR; however, they expressed mesenchymal stem cell markers CD44 and CD105 (Fig. 1b). To determine the ability of UCMSCs in synthesis of neurotrophic factors GDNF and VEGF, we detected these factors by Western blot. Our data showed that UCMSC could express GDNF and VEGF at the protein level (Fig. 1c).

Fig. 1
figure 1

Culture expansion and in vitro characterization of UCMSC. a Fibroblast-like morphology of UCMSC after 2 weeks of isolation. Magnification: 4×. b Flow cytometry analysis of UCMSC for the positive expression of MSC markers of CD44 and CD105, while negative for hematopoietic markers CD45 and HLA-DR. c Western blot analysis revealed the synthesis of neurotrophic factors of GDNF and VEGF by UCMSC

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UCMSC Differentiate to Osteoblasts and Adipocytes Under Specific Differentiation Media

The UCMSCs were subjected to an osteogenic medium for a period of 2 weeks that induced osteogenic lineage formation. This differentiation was illustrated by precipitation of minerals when the cells were stained with alizarin red (Fig. 2a). Moreover, the UCMSCs that were incubated in adipogenic medium staining with oil red revealed lipid-containing vacuoles within the cells (Fig. 2b). These observations indicated the potentiality of UCMSC to differentiate to both adipocytic and osteoblastic lineages.

Fig. 2
figure 2

Multilineage differentiation of UCMSC. a Osteogenic cells showing mineral deposits under osteogenic differentiation medium, stained with Alizarin red S. Magnification: 4×. b Adipogenic cells displaying lipid droplets under adipogenic differentiation medium, stained with Oil red O. Magnification: 10×

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UCMSC-CM Protect PC12 Cells Against H2O2-Induced Cell Death

To evaluate the effect of UCMSC-CM on PC12 cells, viability of cells treated with UCMSC-CM and H2O2 was measured by live and dead cell assay. As shown in Fig. 3a, b, the number of dead cells (red fluorescent) under oxidative stress was noticeably surged in group treated with H2O2. Based on our data, in UCMSC-CM + H2O2 group, the number of live cells grew significantly in comparison to cells incubated with H2O2 (1.6-fold). In addition, MTT assay results showed that cell viability, 24 h after presence of UCMSC-CM and H2O2, improved notably contrary to the cells incubated only with H2O2. In the group treated with UCMSC-CM and H2O2 concomitantly, these values remarkably augmented (Fig. 3c).

Fig. 3
figure 3

The neuroprotective effect of conditioned media of UCMSC (UCMSC-CM). a PC12 cells were exposed to superoxide-induced oxidative stress, incubated with hUCMSCs-CM for 24 h, then the number of live and dead cells (green and red fluorescents, respectively) were counted. Magnification: 10×. b The percentage of viable cells determined in each group using viability/cytotoxicity assay. c Viability of H2O2-treated PC12 cells in the presence of UCMSC-CM measured by MTT assay, indicating the neuroprotection of PC12 cells under oxidative stress by UCMSC-CM treatment. # exemplifies the difference between the H2O2-treated group and control, whereas * shows the difference between UCMSC-CM-treated H2O2 and H2O2 group (**, ##P < 0.01). The values are expressed as means (±SEM; n = 5)

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UCMSC-CM Precludes the Disruption of Neurite Outgrowth Induced by H2O2

In the next step, we evaluated neurite outgrowth in PC12 cells treated with UCMSC-CM and H2O2 simultaneously after 24 h (Fig. 4a, b). Accordingly, neurite length in the group that only received H2O2 showed a significant decline in comparison to the control (P < 0.001), whereas the cells in UCMSC-CM + H2O2 group restored their neurite outgrowth in contrast to H2O2-treated cells (P < 0.001).

Fig. 4
figure 4

Examination of PC12 neuritogenesis under oxidative stress. a, b Incubation of H2O2-treated PC12 cells with UCMSC-CM for 24 h significantly decreased neurite outgrowth perturbation. Magnification: 4×. # represents the difference between the H2O2-treated group and control, whereas * shows the difference between UCMSC-CM-treated H2O2 and H2O2 group (***, ###P < 0.001). The values are expressed as means (±SEM; n = 5)

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Transplantation of UCMSC in Rats Pre-treated with 3-NP Enhances Motor Coordination and Muscle Activity

To appraise whether the transplantation of UCMSC in rat brains improved the coordination of movement after injection of 3-NP, the rotarod test was performed. Motor coordination was significantly decreased in 3-NP receiving vehicle opposed to control group (P < 0.001). However, after transplantation of UCMSC, motor coordination of the UCMSC + 3-NP group was statistically higher than the 3-NP receiving vehicle group (P < 0.01). Following the injection of UCMSC, the coordination of movement improved significantly over the 4-week period (Fig. 5; Supplementary File 1).

Fig. 5
figure 5

Motor coordination in rats pre-treated with 3-NP. UCMSC + 3-NP group exhibited better rotarod performance compared to 3-NP group. # represents the difference between 3-NP group and control, but * points to the difference between UCMSC + 3-NP group and 3-NP group (**P < 0.01; ###P < 0.001). The values are expressed as means (±SEM; n = 5)

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To measure the efficacy of UCMSC transplantation on muscle activity, the EMG was performed (Fig. 6a, b). Latency showed an increase in 3-NP receiving vehicle group in comparison with control group. Following grafting of UCMSC, latency reduced in UCMSC + 3-NP compared with 3-NP group.

Fig. 6
figure 6

Muscle activity in rats pre-treated with 3-NP. a The sciatic nerve was stimulated and the muscle action potential was recorded in gastrocnemius muscle, and b latency was measured in control, 3-NP and UCMSC + 3-NP group. Transplanted UCMSC improved muscle activity in rats pre-treated with 3-NP. Latency showed a significant reduction in UCMSC + 3-NP group contrary to 3-NP group. # represents the difference between 3-NP group and control, but * points to the difference between UCMSC + 3-NP group and 3-NP group (*P < 0.05; ##P < 0.01). The values are expressed as means (±SEM; n = 5)

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Grafted UCMSC in 3-NP-Lesioned Rats Survive and Decrease Gliosis

Prior to cell implantation, striatal atrophy under 3-NP administration was investigated. According to Fig. 7 a, b, upon 3-NP injection, chromatolysis occurred in many striatal cells accompanied by a decline in the number of nissl bodies and condensed appearance of pyknotic nuclei. After the injection of UCMSC into striatum of rats, our results confirmed that these cells survived in the brain after 30 days (Fig. 8a; Supplementary Fig. 1). For detection of astrocytic migration (gliosis), immunohistochemistry against GFAP was done (Fig. 8b). Migration of astrocytes in 3-NP group was statistically higher than the control. In the group receiving UCMSC, number of astrocytes decreased compared with 3-NP group (Fig. 8c).

Fig. 7
figure 7

Striatal atrophy following 3-NP administration. a Nissl staining of striatal tissues in different groups disclosed that upon 3-NP injection, chromatolysis occurred in many cells along with a reduction in the number of nissl bodies. Magnification: 10×. b Hematoxylin staining of striatum also revealead noticeable alterations including condensed apperance of pyknotic nuclei in 3-NP group as displayed by arrows. Magnification: 20×

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Fig. 8
figure 8

Examination of striatal transplantation of UCMSC and gliosis in 3-NP-lesioned rats. a Hoechst/DiI-labeled UCMSC implanted in the striatum of 3-NP-lesioned rats survived after 30 days. Magnification: 40×. b, c Migration of astrocytes in 3-NP group climbed significantly in comparison to UCMSC + 3-NP group. GFAP was used for astrocyte detection. Magnification: 10×. # represents the difference between 3-NP group and control; however, * displays the difference between UCMSC + 3-NP group and 3-NP group (***, ###P < 0.001). The values are expressed as means (±SEM; n = 5)

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UCMSC Implantation Increases Striatal Volume as Well as Mean Dendritic Length of the Striatum in HD Rats

Our stereological analysis showed that grafted UCMSC reduced the striatal atrophy and, as a result, the striatal volume was significantly higher in UCMSC + 3-NP group contrary to 3-NP lesioned group (P < 0.05) (Fig. 9a). Moreover, silver staining of striatal medium spiny neuron (MSN) illustrated that the striatum of UCMSC + 3-NP group had higher dendritic length in contrast to 3-NP group (P < 0.01) (Fig. 9b, c).

Fig. 9
figure 9

Stereological analysis of striatal volume and dendritic length of the striatum. a The volume of striatum in UCMSC + 3-NP group showed an increase opposed to 3-NP group. b The silver staining of striatal medium spiny neuron (MSN) dendritic length in UCMSC + 3-NP group. Magnification: 40×. c The striatum of UCMSC + 3-NP group revealed higher dendritic length compared with 3-NP group. # demonstrates the difference between 3-NP group and control, but * displays the difference between UCMSC + 3-NP group and 3-NP group (*P < 0.05; **, ##P < 0.01; ###P < 0.001). The values are expressed as means (±SEM; n = 5)

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Discussion

In this study, we investigated the neuroprotective action of UCMSC and UCMSC-CM on 3-NP lesioned rat model of HD and PC12 cells, respectively. Our findings indicated that UCMSC through trophic support thwart H2O2-induced PC12 cell death and also they are capable of amelioration in both motor function impairment as well as striatal atrophy associated with HD.

Umbilical cord-derived matrix stem cells are considered as an invaluable cell reservoir with potential benefits in neurodegenerative disorders (reviewed in Boroujeni and Gardaneh 2017; Fan et al. 2011). In this study, following isolation and culture of UCMSC, we initially confirmed the expression of typical MSC markers CD44 and CD105 (Dominici et al. 2006), while negative for MHC class II cell surface marker HLA-DR (Jendro et al. 1991), which the latter entitles MSCs immune privileged (Machado et al. 2013). Similar to other MSC types, UCMSC exhibited their plasticity by differentiation into osteoblasts and adipocytes under specific induction media (Aliaghaei et al. 2016).

Next, we detected the synthesis of the two main tropic factors of GDNF and VEGF at the protein level in UCMSC. Previous reports have indicated that GDNF is able to confer neuroprotection in several models of HD (Kells et al. 2004; McBride et al. 2003; McBride et al. 2006; Pineda et al. 2007). Likewise, VEGF exerted its neuro-survival effects dose-dependently in both in vitro and in vivo HD models (Ellison et al. 2013). In our study, when PC12 cells were exposed to H2O2, the UCMSC-CM precluded oxidative stress-induced cell death. Moreover, UCMSCs-CM reversed the effects of hydrogen peroxide on neuritogenesis of PC12 cells. These demonstrations suggest that MSC secretome harbors a host of cytokines and growth factors, as reviewed in Kim et al. (2013); thus, the factors such as VEGF in the collected CM could promote neuroprotective activity by inhibition of apoptosis and the induction of neurogenesis (Sun et al. 2003; Volm et al. 1999). Besides, it was shown that combined action of GDNF and VEGF facilitated prolonged survival and protection of neuronal cells in amyotrophic lateral sclerosis (Krakora et al. 2013).

Bilateral striatal transplantation of UCMSC (at passage 4) in 3-NP lesioned rat models ameliorated motor coordination as well as muscle activity. However, this is in contrast with a report that using low passaged murine UCMSC in R6/2 mice of HD, no significant reduction in motor impairment was observed (Fink et al. 2013). This discrepancy might be partially explained by implementation of various experimental models of HD. We used 3-NP chemical model since (1) this toxin could quickly mimic HD symptoms, (2) it exerts its neurotoxicity by induction of oxidative stress in striatum, leading to excessive reactive oxygen species (ROS) production, thus suitably paralleling our in vitro and in vivo observations (Kumar et al. 2010; Túnez et al. 2010). Moreover, increased gliosis were identified in 3-NP group, which is in agreement with the early studies indicating an increase in gliosis in human dorsal striatum of HD patients (Myers et al. 1991). However, upon hUCMSC transplantation, the reduction of astrocytic migration was apparently noticed. According to Lin et al. (2011)) and Snyder et al. (2010)), administration of human bone marrow-derived mesenchymal stem cells in HD models of mice (N171-82Q, R6/2-J2, and quinolinic acid) led to the improvement of striatum volume after 30, 16, and 16 days post-grafting, respectively. Besides, implantation of UCMSC in 3-NP group boosted the dendritic total length pointing to reduced striatal atrophy. Furthermore, transplanted UCMSC survived in the striatum after 30 days despite the fact that we did not measure the survival rate of UCMSC post-grafting. Altogether, our results implied that UCMSCs provide an enriched platform by largely their paracrine factors, which downgrades the unfavorable effects of oxidative stress, accompanied by restoration of damaged tissues (Murphy et al. 2013).

It essentially appears that transplantation of suitable cells releasing neurotrophic factors is more efficacious rather merely administration of trophic factors, since in order to deliver such proteins to their appropriate targets in central nervous system (CNS), they should cross the blood-brain barrier. Due to the large size of these molecules as well as their chemical assembly, the ingress into CNS is indeed problematic following peripheral injection (Peterson and Nutt 2008). Consequently, the delivery route of these therapeutic agents needs to be fine-tuned.

In summary, cell replacement strategies aimed at neurodegenerative disorders such as HD are seen encouraging. We have shown that CM of UCMSC neuro-protected PC12 cells against oxidative stress and significantly enhanced cell viability and neurite outgrowth. Additionally, intrastriatally transplanted UCMSCs were able to improve degenerated striatum and recover the functional motor skills. However, there are still no standardized consistencies regarding the effective dose/times of cell injections, appropriate implantation routes, and proper time frame for post-graft analysis, led to confounding results (Kerkis et al. 2015); therefore, well-tailored approaches are highly demanded should we seek to translate stem cell research into clinic.