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First published online July 26, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2797 -2808
doi:10.1634/stemcells.2007-0212; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Specification of a Dopaminergic Phenotype from Adult Human Mesenchymal Stem Cells

Katarzyna A. Trzaskaa,c, Eldo V. Kuzhikandathilb, Pranela Rameshwarc

aGraduate School of Biomedical Sciences,
bDepartment of Pharmacology and Physiology,
cDepartment of Medicine Hematology/Oncology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey, USA

Key Words. Dopamine • Stem cells • Parkinson disease • Neural repair

Correspondence: Pranela Rameshwar, Ph.D., University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, MSB E-585, Newark, New Jersey 07103, USA. Telephone: (973) 972-0625; Fax: (973) 972-8854; e-mail: rameshwa{at}umdnj.edu

Received on March 24, 2007; accepted for publication on July 16, 2007.

First published online in STEM CELLS EXPRESS  July 26, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Dopamine (DA) neurons derived from stem cells are a valuable source for cell replacement therapy in Parkinson disease, to study the molecular mechanisms of DA neuron development, and for screening pharmaceutical compounds that target DA disorders. Compared with other stem cells, MSCs derived from the adult human bone marrow (BM) have significant advantages and greater potential for immediate clinical application. We report the identification of in vitro conditions for inducing adult human MSCs into DA cells. Using a cocktail that includes sonic hedgehog and fibroblast growth factors, human BM-derived MSCs were induced in vitro to become DA cells in 12 days. Based on tyrosine hydroxylase (TH) expression, the efficiency of induction was determined to be ~67%. The cells develop a neuronal morphology expressing the neuronal markers NeuN and β III tubulin, but not glial markers, glial fibrillary acidic protein and Olig2. As the cells acquire a postmitotic neuronal fate, they downregulate cell cycle activator proteins cyclin B, cyclin-dependent kinase 2, and proliferating cell nuclear antigen. Molecular characterization revealed the expression of DA-specific genes such as TH, Pitx3, Nurr1, DA transporter, and vesicular monoamine transporter 2. The induced MSCs also synthesize and secrete DA in a depolarization-independent manner. The latter observation is consistent with the low expression of voltage gated Na+ and Ca2+ channels in the induced MSCs and suggests that the cells are at an immature stage of development likely representing DA neuronal progenitors. Taken together, the results demonstrate the ability of adult human BM-derived MSCs to form DA cells in vitro.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Dopamine (DA) is one of the most studied neurotransmitters in the brain because of its essential role in motor control, cognition, reward, and addiction [1, 2]. DA dysfunction underlies several psychiatric and neurological disorders. Control of motor behavior is lost in Parkinson disease (PD) due to the selective degeneration of mesencephalic DA neurons in the substantia nigra [3–6]. This progressive loss of DA neurons results in devastating symptoms for which there is no cure. Pharmacological agents that increase DA can alleviate motor symptoms; however, patients develop severe side effects with long-term use. As a result, cell transplantation therapy has been investigated as an alternative treatment. Cell replacement therapy is likely to be successful for PD, since one cell type is affected in a distinct location of the brain.

There are many reports on the derivation of DA neurons from human embryonic stem cells [7–12]. However, ESCs can form teratomas and are likely to elicit immune rejection if used for transplantation. Neural stem cells (NSCs) maintain lineage specificity and have the ability to differentiate into any type of brain cell [13]. Although NSCs show therapeutic value for neural disorders, NSC-derived DA neurons have some drawbacks with respect to efficiency and lineage polarization [14]. This by no means suggests that NSCs and ESCs will not transition from bench to bedside, but additional studies are warranted to identify other stem cells that have the ability to generate DA neurons with high efficiency.

The least characterized stem cells, with respect to their ability to generate DA neurons, are the mesenchymal stem cells. Of the adult stem cells, MSCs appear to have the best potential for regenerative medicine [15–21]. In fact, MSCs are currently in clinical trials for a number of disorders, including graft-versus-host disease, heart failure, and multiple sclerosis [21]. It has also been reported that subpopulations of MSCs have an inherent ability to promote neural repair [18]. Additionally, their multilineage potential may be explained by their similarities with ESCs [22].

MSCs are easily obtained from bone marrow (BM) aspirates, can be expanded in vitro, and have low potential to transform into malignant cells [19]. MSCs can differentiate along the multiple lineages of mesodermal cells [19]. These stem cells have been shown to exhibit immune suppressive functions, making them ideal candidates for cell therapy across an allogenic barrier [23]. Although autologous transplantation would be ideal, allogenic MSCs might be best in the event that the neural disorder is caused by inherited genetic mutations, as is the case for several forms of familial PD.

Several studies have demonstrated the ability of MSCs to transdifferentiate into the functional cells of the nervous system [24–31]. Generation of DA-type cells from MSCs has also been reported using rodent models and various induction methods but at low efficiency [32–36]. In this project, our goal was to develop a high efficiency method to generate DA cells from human MSCs obtained from the adult BM. By means of in vitro manipulation with sonic hedgehog (SHH) and fibroblast growth factors (FGF8 and basic bFGF), we induced adult human BM-derived MSCs to DA neuronal cells with an ~67% efficiency. We demonstrate that these cells not only express DA neuron-specific markers but also secrete DA. Our results support the feasibility of using MSCs as a source of in vitro generated DA cells for treatment of DA disorders such as PD.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Culture of Human MSCs
The use of human BM aspirates followed a protocol approved by the Institutional Review Board of the University of Medicine and Dentistry of New Jersey (UMDNJ), Newark Campus, and informed consent was obtained from all subjects. MSCs were obtained from four donors ranging in age from 18 to 35. Unfractionated BM aspirates (3 ml) were diluted in 3 ml of Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) containing 10% fetal calf serum (FCS) (HyClone, Logan, UT, http://www.hyclone.com) and then transferred to tissue culture Falcon 3003 Petri dishes. Plates were incubated and, at day 3, mononuclear cells were isolated by Ficoll Hypaque (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) density gradient and then replaced in the culture plates. Fifty percent of the media were replaced with fresh media at weekly intervals until the adherent cells were ~80% confluent.

Flow Cytometry
Cells were detached with Accutase Dissociation Solution (MP Biomedicals, Irvine, CA, http://www.mpbio.com) and incubated with respective fluorochrome-conjugated antibodies for cell surface glycoproteins: CD44, CD31, CD34 (BD Biosciences, San Diego, http://www.bdbiosciences.com), and CD105 (Fitzgerald Industries, Concord, MA, http://www.fitzgerald-fii.com). These are commonly used for the positive and negative detection of MSCs. Primary antibodies Stro-1 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), patched (PTCH) (Abcam, Cambridge, U.K., http://www.abcam.com), smoothened (SMO) (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), tyrosine hydroxylase (TH), and β III tubulin (Chemicon, Temecula, CA, http://www.chemicon.com) were labeled with secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Primary antibodies were incubated for 30 minutes on ice, and secondary antibodies were incubated for 20 minutes on ice. For the detection of PTCH, SMO, TH, and β III tubulin, cells were fixed with 2% paraformaldehyde for 30 minutes prior to labeling. For TH and β III tubulin detection, cells were permeabilized at room temperature with 0.1% Triton X-100 for 15 minutes. Cells were immediately analyzed by BD FACScan and analyses were done using BD CellQuest software (BD Biosciences), and percent statistics were given.

Neuronal Induction of Human MSCs
MSCs were trypsinized and then subcultured in Falcon 3046 multiwell plates or on round glass coverslips. Each plate contained ~1.0 x 105 cells. The next day, DMEM containing 10% FCS (HyClone) was replaced with Neurobasal medium and B27 supplement (Invitrogen). The 50x (100%) B27 supplement was diluted to a final concentration of 0.25x (0.5%). The cells were induced only once with a cocktail of 250 ng/ml SHH, 100 ng/ml FGF8, and 50 ng/ml bFGF (R&D Systems), and cells were cultured at 37°C with 5% CO2. The medium was not replaced during the induction period. As a control, MSCs were also cultured with Neurobasal and B27 supplement alone, without the induction cocktail. Cells were cultured at the same conditions previously mentioned.

Reverse Transcription-Polymerase Chain Reaction
RNA was extracted from cultured cells using RNAqueous-4PCR (Ambion, Austin, TX, http://www.ambion.com). Positive control samples were obtained by gross dissection of normal human substantia nigra tissue from cadavers (National Disease Research Interchange [NDRI], Philadelphia, http://www.ndriresource.org). The use of normal human substantia nigra tissue was approved by the Institutional Review Board of UMDNJ, Newark Campus. Tissue was disrupted using a tissue homogenizer, and RNA was extracted using the RNAqueous-4PCR Kit. Synthesis of cDNA was performed using SuperScript III Reverse Transcriptase (Invitrogen) following manufacturer's instructions. Samples were treated with DNase (Ambion) to remove traces of genomic DNA contamination. Polymerase chain reaction (PCR) amplification was carried out using Platinum Taq Polymerase (Invitrogen). PCR reactions were normalized by amplifying same cDNA samples with primers for glyceraldehyde-3-phosphate dehydrogenase. Each reaction had the following cycling parameters: 95°C, 30 seconds; 45°C–62°C, 30 seconds to 1 minute; 72°C, 30 seconds for 40 cycles. Each reaction was preceded by a denaturation at 95°C for 2 minutes and an extension at 72°C for 10 minutes. Table 1 displays the primer probe sets used for the reverse transcription-PCR experiments. PCR reactions were separated by electrophoresis on a 1% agarose gel containing ethidium bromide, and band sizes were compared with a 1-kilobase plus DNA ladder (Invitrogen).


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  		Table 1. Polymerase chain reaction primer pairs

 
Growth Curve Analysis
MSCs were plated at 5 x 103 and allowed to adhere overnight. The next day, cells were induced with neural induction media. At 3-day intervals, uninduced and induced MSCs were trypsinized and counted using a hemacytometer.

Western Analysis
Whole cell protein extracts were obtained by resuspension of cell pellets in 30 µl of 1x Lysis Buffer (Promega, Madison, WI, http://www.promega.com) followed by freeze/thaw in dry ice/ethanol bath. Positive control samples were obtained from normal human substantia nigra tissue taken from cadavers (NDRI) for the detection of DA and brain specific markers. The human neuroblastoma cell line SH-SY5Y (American Type Culture Collection, Manassas, VA, http://www.atcc.org) was used as a positive control for cell cycling proteins. Protein extracts (100 µg) were electrophoresed on 4%–20% Tris/HCl precast gels (Bio-Rad, Hercules, CA, http://www.bio-rad.com) and transferred onto polyvinylidene difluoride membranes (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). Membranes were incubated with primary antibodies in 5% milk–phosphate-buffered saline (PBS) overnight at 4°C. Dilutions were as follows: cyclin B, 1/1,000; cyclin-dependent kinase 2 (Cdk2), 1/1,000; proliferating cell nuclear antigen (PCNA), 1/1,000 (BD Biosciences); Bcl2, 1/200 (Santa Cruz Biotechnology); TH, 1/500; Pitx3, 1/200; Nurr1, 1/200; Nestin, 1/500; NeuN, 1/500; Olig2, 1/500; glial fibrillary acidic protein (GFAP), 1/1,000 (Chemicon); Kv4.2, 1/500 (Abcam); pan NaV, 1/500; β-actin, 1/1,000 (Sigma). The next day, membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1/2,000) (Pierce, Rockford, IL, http://www.piercenet.com) for 2 hours at 4°C. Membranes were developed with chemiluminescence Western blot detection reagents (Pierce). Membranes were stripped with Restore Stripping Buffer (Pierce) for reprobing. Band densities were analyzed using UN-SCAN-IT gel software (Silk Scientific, Orem, UT, http://www.silkscientific.com).

Immunocytochemistry
Cells were fixed with 2% paraformaldehyde and subsequently permeabilized and blocked with 0.1% Triton X-100 and 1% bovine serum albumin in PBS. Cells were stained with primary antibodies overnight at 4°C. Dilutions were as follows: TH, NeuN, β III tubulin, 1/500; GFAP, 1:1,000 (Chemicon); DA, 1:5,000 (Abcam); synaptic vesicle protein 2 (SVP2), 1:100 (Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk); vesicular monoamine transporter 2 (VMAT2), 1:200 (Santa Cruz Biotechnology). Appropriate secondary antibodies anti-rabbit IgG-phycoerythrin, anti-goat IgG-fluorescein isothiocyanate (FITC) (Open Biosystems, Huntsville, AL, http://www.openbiosystems.com), and anti-mouse IgG-FITC (Jackson Immunoresearch Laboratories); nuclear stain 4,6-diamidino-2-phenylindole (DAPI) (300 nM); and cytoskeletal stain Texas Red phalloidin (F-actin) (6.6 µM) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) were used for detection and visualization.

Electrophysiology
Currents were measured by the whole-cell patch clamp technique. The cells were held at –70 mV and currents elicited by stepping from –100 to +70 mV in 10-mV steps. The external solution (SES) used for current measurements was, in mM, 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 Hepes pH7.4, and 10 glucose, and the pipette solution contained, in mM, 130 K-aspartate, 20 NaCl, 1 MgCl2, 10 Hepes, 10 glucose, 0.1 GTP, 5 Mg-ATP, and 1 EGTA. The current responses were normalized to the cell capacitance to account for variation in cell size. Currents were measured using an Axopatch 200B amplifier and sampled through a Digidata 1322A interface using the pClamp 8.0 software (Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com). Data files were imported into SigmaPlot (SPSS, Chicago, http://www.spss.com) for analysis and display.

Dopamine Enzyme-Linked Immunosorbent Assay
DA levels were quantitated using an enzyme-linked immunosorbent assay (ELISA) kit obtained from Rocky Mountain Diagnostics (Colorado Springs, CO, http://www.rmdiagnostics.com) according to the manufacturer's instructions. After differentiation, culture medium was replaced with a balanced salt solution (SES). The SES solution does not contain any ingredients that would react with the antibodies. For the Ca2+-free SES, Ca2+ was substituted with 4 mM magnesium and 1 mM EGTA. Cells were treated with elevated K+ solution (56 mM) to induce depolarization, 500 nM DA transporter (DAT) inhibitor GBR12909 (Sigma; D052), or 100 µM ATP (Sigma). Cells were treated for 5 minutes at 37°C, except for ATP treatment, which was for 1 minute. One-hundred-microliter samples were collected and immediately used for DA quantitation through ELISA.

Calcium Imaging
Intracellular calcium ([Ca2+]i) transients were studied in MSCs induced for 12 days. Cells on coverslips were loaded with 5 µM Fura2-AM (Molecular Probes) for 30 minutes at 37°C. After washing the cells twice with PBS to remove free fura-2, cells were transferred to a recording chamber containing balanced salt solution (SES) for imaging. To induce changes in intracellular Ca2+ levels, bath solution was replaced with SES containing elevated K+ (50 mM) or 100 µM ATP. Fura-2 fluorescence images were acquired by alternating the excitation wavelength between 340 and 380 nm and capturing the resultant emissions through a 510-nm dichroic mirror and a 520-nm long-pass filter using a cooled charge coupled device camera. Fluorescence images obtained by excitation at 340 and 380 nm were acquired at 3-second intervals, and the ratio of fluorescence elicited at 340 and 380 nm was plotted. Images were processed and analyzed using SigmaPlot.

Statistical Analysis
Data are expressed as the mean ± standard error. Statistical differences were calculated using analysis of variance. Pairwise comparisons were evaluated using the post hoc Holmes test; p values <.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Induction of Human MSCs to Neuronal Cells
MSCs were isolated and cultured from human BM aspirates as described [23]. To verify that the isolated population contained pure MSCs, we performed positive and negative characterization by fluorescence-activated cell sorting (FACS) analysis. Figure 1A shows a representative example of the cell surface antigens expressed on uninduced MSCs after four cell passages. Isolated MSCs were approximately 100% positive for two known MSC markers, cell surface glycoproteins CD44 and CD105 (SH2, endoglin) (Fig. 1A1, 1A2). A subpopulation of MSCs was positive for Stro-1 (61.4%), an antigen that is only expressed on a subset of MSCs and identifies stromal precursors [37] (Fig. 1A5). MSCs were negative for CD34 and CD31, indicating no contamination with endothelial cells from the BM (Fig. 1A3, 1A4). We also confirmed that MSCs express both patched (PTCH) and smoothened (SMO), the receptors for the key DA inductive factor SHH (Fig. 1A6). A majority of the MSC population (71%) was double positive for PTCH and SMO. Only 2% were single positive for PTCH, and 16% were single positive for SMO, whereas 11% were negative for both.


Figure 1
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  		Figure 1. Flow cytometry analysis and bright-field images of MSCs in culture. (A): Human MSCs were isolated from bone marrow (BM) aspirates and analyzed after four cell passages. For (A1–A5), red traces indicate positive signal and green traces indicate negative signal. Isolated MSCs are positive for the MSC markers CD44 (A1) and CD105 (endoglin, SH2) (A2). A subset of MSCs is positive for Stro-1 (A5). MSCs are negative for the primitive hematopoietic marker CD34 (A4) and for the endothelial marker CD31 (A3). MSCs are 71% double positive for both PTCH and SMO (upper right), 2% are single positive for PTCH (upper left), and 16% are single positive for SMO (lower right), whereas 11% are negative for both (lower left) (A6). (B): Bright-field images of human MSCs in culture. Morphological change was observed in human MSCs after induction with SHH, FGF8, and bFGF. Unstimulated, pure population of MSCs isolated from BM aspirates after four cell passages (B1). MSCs induced for 6 days (B2) and 12 days (B3). (C): Control images of MSCs cultured with Neurobasal medium and B27 only. MSCs in culture for 3 days (C1), 6 days (C2), and 12 (C3) days do not show a change in morphology. Abbreviations: bFGF, basic fibroblast growth factor; FGF8, fibroblast growth factor 8; PTCH, patched; SHH, sonic hedgehog; SMO, smoothened.

 
We hypothesized that since the majority of MSCs expressed both PTCH and SMO (Fig. 1A6), the cells would respond to SHH, the key factor in DA development [38]. MSCs were treated with a cocktail of differentiating factors that included SHH, FGF8, and bFGF as described in Materials and Methods. Untreated MSCs are very flat, symmetrical, and spindle-shaped (Fig. 1B1). Neuronal induction is not immediate, as cells stimulated for 6 days only slightly change their morphology; however, they do adopt a radial glial-like appearance (Fig. 1B2). By day 12, the cell bodies became refractive with long thin processes (Fig. 1B3). This morphology resembles that of native neurons. MSCs that did not transdifferentiate remained flat. To verify that the observed morphological changes were a result of the induction cocktail, we cultured MSCs in Neurobasal and B27 alone, omitting SHH, FGF8, and bFGF (Fig. 1C). Shown in Figure 1C are images of MSCs in culture with Neurobasal and B27 at 3 days (Fig. 1C1), 6 days (Fig. 1C2), and 12 days (Fig. 1C3). The cells survive in the medium; however, neuronal morphology is not observed, suggesting that the morphological changes in Figure 1B are due to the addition of SHH, FGF8, and bFGF to the culture medium.

In vivo, differentiated neurons stop proliferating and become postmitotic, downregulating the expression of several cell cycle related proteins. To determine whether the MSCs treated with induction cocktail showed these characteristics of neuronal differentiation, we compared the growth rate (Fig. 2A) and expression of several cell cycle-related proteins (Fig. 2B–2D) in treated and untreated MSCs. Although the growth rate of untreated MSCs was 2.0 ± 0.23, the growth rate of MSCs treated with the induction cocktail was significantly reduced by approximately 70% (Fig. 2A). Consistent with the decrease in growth rate, in MSCs treated with the induction cocktail, the cell cycle activator protein Cdk2 (involved in G1-S progression) is rapidly and significantly downregulated by day 3 of stimulation (Fig. 2C, 2E). Expression of cyclin B protein (involved in G2-M progression) is downregulated more gradually, with significant reduction observed as early as day 3 (Fig. 2C, 2D). The expression of PCNA, involved in DNA synthesis, is also rapidly downregulated in MSCs treated with the induction cocktail (Fig. 2C, 2F). The expression level of these cell cycle activators was also reduced in untreated MSCs, albeit on a different time scale (Fig. 2B). The reduction of these proteins in untreated MSCs was most likely due to inhibition of cell proliferation as a result of confluency, an observation that is supported by the growth curve in Figure 2A, which begins to plateau by day 9. Finally, to determine whether the reduction in cell number in treated MSCs is due to apoptosis-mediated cell death, we measured the levels of the antiapoptotic protein Bcl2 (Fig. 2C). Results in Figure 2C show that the levels of Bcl2 are not significantly different from day 0 to day 12. Taken together, these results strongly suggest that the MSCs treated with the induction cocktail decrease proliferation and become postmitotic.


Figure 2
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  		Figure 2. Analysis of proliferation and cell cycle in stimulated MSCs. (A): Compared with unstimulated MSCs, cells induced to transdifferentiate slow their proliferation. The data points were fitted to a three-parameter sigmoid equation. (B): Western analysis of unstimulated MSCs in culture for 3, 6, 9, 12, and 15 days. Positive control was obtained from the tumorigenic cell line SH-SY5Y. (C): Western analysis of cyclin B, Cdk2, PCNA, and Bcl2 in MSCs induced for 3, 6, 9, 12, and 15 days. Day 0 represents uninduced MSCs. β-Actin is the normalizing control. (D–F): Cumulative data showing the differences in normalized protein levels of cyclin B (D), Cdk2 (E), and PCNA (F) between uninduced MSCs (filled circle) and induced MSCs (inverted triangle). Abbreviations: Cdk2, cyclin-dependent kinase 2; PCNA, proliferating cell nuclear antigen.

 
Induced MSCs Express DA-Specific Genes
To determine whether the induction protocol was inducing MSCs into neuronal cells with DA characteristics, we tested for the expression of mRNA and protein of genes known to be associated with DA neuron differentiation (Fig. 3). DA neurons are classically identified by expression of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of DA, and DAT that reuptakes the DA released from DA neurons. Additionally, genes involved in DA neuron differentiation in vivo include, among others, En1, En2, Lmx1a, Wnt1, Pitx3, and Nurr1 [39]. As shown in Figure 3A, 12-day stimulated MSCs express significantly higher levels of TH, DAT, En1, En2, Wnt1, Lmx1a, Nurr1, and Pitx3 mRNA compared with untreated MSCs. Uninduced MSCs express low levels of En1 that is transiently downregulated on day 6 and robustly upregulated on day 12. Consistent with these results, the Western blot analyses in Figure 3B show that the expressions of TH, Pitx3, and Nurr1 proteins are significantly upregulated in MSCs treated for 12 days with the induction cocktail. In addition to the DA markers, the treated MSCs also express the neural stem cell marker nestin (Fig. 3B). The neuronal characteristics of the 12-day induced MSCs were confirmed by the expression of neuronal markers NeuN and voltage gated K+ (Kv 4.2) and Na+ (NaV) channels and by the absence of glial markers GFAP and Olig2 (Fig. 3B, 3C). Interestingly, the 6-day induced MSCs express low levels of GFAP, consistent with the ability of the induction cocktail to transdifferentiate the MSCs into a neuro-glial lineage.


Figure 3
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  		Figure 3. Stimulated MSCs express dopamine (DA)-specific markers. Representative ethidium bromide stained gel (A) and Western blots (B, C) showing the expression of mRNA (A) and proteins (B, C) associated with DA neuron development in MSCs induced for 0, 6, and 12 days with the induction cocktail. The mRNA levels of various DA neuron-related proteins were detected using semiquantitative reverse transcription-polymerase chain reaction (A). The photograph of the ethidium bromide stained gel was inverted for clarity. Positive control (+ control) RNA and protein lysates were obtained from adult human substantia nigra tissue. Abbreviations: DAT, dopamine transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; TH, tyrosine hydroxylase.

 
The results in Figure 3 show that protein lysates obtained from MSCs treated for 12 days robustly express the proteins associated with DA neurons. To support this evidence, we performed immunocytochemical analyses for TH and DAT (Fig. 4A). Shown in the top panel of Figure 4A, TH expression is noticeable by day 6, but it is significantly upregulated by day 12, indicated by the green and yellow fluorescence. Day 0 and day 3 MSCs only show the nuclear and cytoskeletal labels DAPI and F-actin. The bottom panel of Figure 4A shows similar expression with the DAT, which is also upregulated by day 12, with very minimal expression on day 6. As expected, unstimulated MSCs and 3-day stimulated MSCs did not show any expression of the DAT. To determine the percentage of 12-day treated MSCs expressing TH, we used immunocytochemistry and FACS analysis (Fig. 4B, 4C). Shown in the top panel of Figure 4B is the upregulation of NeuN and TH by day 12. Quantitative cell count analyses of three independent immunocytochemistry experiments indicated that >90% of the 12-day treated MSCs expressed NeuN, and 68.7% of these cells expressed TH. Of a total of 202 NeuN positive cells counted, 137 cells were positive for TH. In parallel, we counted the cells that expressed TH from the β III tubulin positive population, which is shown in the middle panel of Figure 4B. Out of 155 β III tubulin positive cells counted, 107 cells were positive for TH. MSCs stimulated for 12 days also expressed >90% β III tubulin, and from this population 69.0% expressed TH. In contrast, uninduced MSCs and those treated for 3 days, probed with TH, NeuN, and β III tubulin antibodies, were negative and only show the nuclear stain DAPI (Fig. 4B, top and middle panels). To confirm that the cell populations were neuronal, we probed the cells with both NeuN and GFAP (Fig. 4B, bottom panel). Slight expression of both markers is noticeable by day 6 of stimulation, but by day 12, the cells solely express NeuN and not GFAP. These results coincide with the Western analyses (Fig. 3B, 3C). Control samples labeled with just secondary antibody were also negative (data not shown). To confirm the results from the immunocytochemistry experiments, we performed a FACS analysis of the MSCs induced for 12 days. The results from these FACS experiments were consistent with the immunocytochemistry experiments, showing that 98.1% of treated MSCs expressed β III tubulin, and, of this population, 65.4% expressed TH (Fig. 4C).


Figure 4
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  		Figure 4. Induced MSCs synthesize DA. (A): Representative images of MSCs induced for 0, 3, 6, and 12 days. The top row displays expression of TH (fluorescein isothiocyanate [FITC], green) on days 0, 3, 6, and 12, indicated by the green and yellow fluorescence. The bottom row shows the expression of DAT (FITC, green) on days 0, 3, 6, and 12 identified by the green and yellow fluorescence. Day 0 and day 3 MSCs stained for TH and DAT only display the nuclear label DAPI (blue) and the cytoskeletal label F-actin (red). (B): MSCs induced for 0, 3, 6, and 12 days were double stained for neural specific markers NeuN and β III tubulin and for the glial marker GFAP. The top row displays merged images of double staining with NeuN (FITC, green) and TH (phycoerythrin [PE], red). The middle row shows β III tubulin (FITC, green) double staining with TH (PE, red). The bottom row shows double staining of GFAP (PE, red) and NeuN (FITC, green). Colocalization was identified by the yellow fluorescence in the merged images. (C): Fluorescence-activated cell sorting analysis of MSCs induced for 12 days revealed that the cells were 65.4% double positive for TH and β III tubulin (upper right), and the remaining 32.7% were just positive for β III tubulin (lower right). (D): Representative fluorescent images show immunostaining of 12-day induced MSCs probed with SVP2, VMAT2, and DA. The top row displays three representative merged images showing the colocalization (yellow) of SVP2 (FITC, green) and DA (PE, red). The bottom row shows the colocalization (yellow) of VMAT2 (FITC, green) and DA (PE, red). Noninduced MSCs only display the nuclear label DAPI (blue). Abbreviations: DA, dopamine; DAPI, 4,6-diamidino-2-phenylindole; DAT, dopamine transporter; GFAP, glial fibrillary acidic protein; SVP2, synaptic vesicle protein 2; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2.

 
To determine whether the neuronal cells formed from MSCs synthesize DA, we labeled 12-day induced MSCs with an antibody to DA (Fig. 4D). To verify whether this corresponded to DA stored in synaptic vesicles, induced MSCs were also immunostained for SVP2, a protein associated with secretory vesicles. Three representative images in the top panel of Figure 4D show that DA staining colocalized with SVP2 staining, as indicated by the yellow fluorescence in the merged images. To support these data, we determined whether the induced MSCs expressed VMAT2, which is involved in accumulating DA into synaptic vesicles. VMAT2 is also a known mesencephalic DA marker, and thus its presence further indicates the cells are dopaminergic. Presented in the bottom panel of Figure 4D are three merged images showing that DA colocalized with VMAT2. Noninduced MSCs only display the DAPI nuclear stain. Induced MSCs stained without primary antibody did not reveal any staining (data not shown). These results suggest that the induced MSCs synthesize DA that is packaged into synaptic vesicles by the VMAT2. Taken together, the results shown in Figures 3 and 4 strongly suggest that the MSCs treated with the induction cocktail acquire a DA neuronal fate by day 12.

Excitable Properties of Induced MSCs
To determine whether human MSC-derived DA cells have excitable properties of neuronal cells, we performed whole cell recording in the current clamp mode. Resting membrane potential of the cells ranged from –50 mV to –70 mV, but none of the cells (n = 20) exhibited spontaneous and evoked action potentials (data not shown). This was surprising, since our group has previously shown that the same MSCs, when induced with retinoic acid to form substance P expressing neurons, exhibited spontaneous and evoked action potentials [24, 25]. To further assess the neuronal properties of the MSC-derived DA cells, we performed whole cell recording in the voltage clamp mode using a step protocol from –100 mV to +70 mV. This protocol elicits robust inward Na+ and Ca2+ currents at the appropriate membrane potentials in a positive control neuroendocrine cell (Fig. 5A) but failed to elicit these currents in the 6-day induced MSC-derived DA cells (Fig. 5B; n = 20) and elicited very small Na+ and Ca2+ inward currents in some of the 12-day induced MSCs (Fig. 5C, triangles; 2 out of 20 cells). These results suggest that although MSC-derived DA cells express functional K+ channels, they express very low levels of voltage-gated Na+ and Ca2+ channels. These results are consistent with their inability to fire spontaneous or evoked action potentials. This suggests that SHH, FGF8, and bFGF induced MSCs yield DA cells that have not acquired the properties of fully differentiated DA neurons. In neurogenesis, neuronal precursors and immature neurons often exhibit such properties, where only K+ channel current is present [40–44].


Figure 5
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  		Figure 5. Excitable properties of induced MSCs. Representative whole cell recordings from control AtT-20 neuroendocrine excitable cells (A) and MSCs treated for 6 (B) or 12 days (C) with induction cocktail. The cells were held at –70 mV and currents elicited by stepping from –100 to +70 mV in 10-mV steps. The current-voltage plot for each cell type is given below their individual current traces. MSCs induced for 12 days (C) express robust outward K+ currents (filled circle) but very small inward Na+ and Ca2+ currents (filled upright triangle). Abbreviations: nA, nanoampere; pA, picoampere.

 
Induced MSCs Secrete DA
To determine whether induced MSCs release DA, we measured the extracellular levels of DA secreted by 12-day induced MSCs under various conditions (Fig. 6A). The DA levels were measured with an ELISA kit following a 5-minute treatment in various external solutions. We attempted longer incubation up to 15 minutes and found optimal release at 5 minutes. This was expected since release is immediate. We also incorporated samples of Neurobasal medium and B27 as a control, in which dopamine was not detected (data not shown). Figure 6A shows that MSCs induced for 12 days constitutively release DA into the extracellular medium. This level is significantly enhanced by 500 nM GBR12909, a highly selective inhibitor of the DA transporter, by preventing the reuptake of the released DA. Interestingly, and consistent with our electrophysiological recordings (Fig. 5), under depolarizing conditions induced by elevated extracellular K+ (56 mM), there was no further increase in the constitutive release of DA. This suggests that the constitutive release of DA by 12-day induced MSCs is not mediated by neuronal activity. Furthermore, the release of DA was not dependent on extracellular Ca2+, as treatment of cells with GBR12909 in Ca2+-free external solution did not block the DA release. This is entirely consistent with the lack of effect of depolarizing conditions on DA release. Recent studies have shown that, in vivo, the release of DA from immature mid brain neurons does not require neuronal activity [45]. Together, these results suggest that the DA cells generated from the MSCs using the current induction protocol are not fully differentiated mature neurons. Additional inductive factors are likely required to complete the maturation process.


Figure 6
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  		Figure 6. Induced MSCs secrete dopamine (DA). (A): Cumulative data for DA secreted by control (day 0) and induced MSCs (day 12). Cells were treated for 5 minutes with control ES, external solution with elevated K+ (56 mM), external solution with 500 nM GBR12909, and Ca2+-free external solution with 500 nM GBR12909. The experiment was repeated three independent times and mean ± SE plotted; *, statistically significant p < .001; analysis of variance (ANOVA) followed by post hoc Holmes test. (B): Representative Fura-2 Ca2+ imaging data show that intracellular Ca2+ levels are transiently elevated in 12-day induced MSCs treated with 100 µM ATP. The arrow indicates the time point of addition of ATP. (C): Induced MSCs treated for 1 minute with 100 µM ATP show significantly elevated release of DA into the extracellular medium. The experiment was repeated three independent times and mean ± SE plotted; *, #, statistically significant p < .05 and p < .0001, respectively; ANOVA followed by post hoc Holmes test. The levels of DA were measured using an enzyme-linked immunosorbent assay kit and quantitated using a standard curve. Abbreviation: ES, external solution.

 
To confirm the results of our electrophysiological experiments, we performed Ca2+ imaging experiments to monitor changes in intracellular Ca2+ upon depolarization. MSCs induced for 12 days were loaded with the ratiometric Ca2+ indicator dye Fura2-AM. The levels of intracellular Ca2+ were not changed by depolarizing the cells using the elevated K+ extracellular solution (56 mM K+) (data not shown); this was entirely consistent with our electrophysiological observations. As a control, we treated the cells with 100 µM ATP, which, via P2Y purinergic receptors, can increase the release of Ca2+ from intracellular stores. The data in Figure 6B clearly show that ATP robustly increases intracellular Ca2+ levels. Given a recent observation that ATP induces Ca2+ oscillations in human MSCs [46], we hypothesized that the constitutive DA release might be mediated by extracellular ATP-induced release of Ca2+ from intracellular stores. To test this hypothesis, we treated 12-day induced MSCs with 100 µM ATP and measured DA secreted into the extracellular medium. The results in Figure 6C suggest that extracellular ATP causes DA release from the 12-day induced MSCs.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In the present study, we provide novel evidence that DA cells are generated by in vitro stimulation of MSCs obtained from adult human BM. Five previous studies have reported the transdifferentiation of human MSCs to DA neuronal cells; however, most of these reported low efficiency of induction ranging from 12.7% to 41% [32–36]. Prior experience with fetal nigral transplants suggests that a large number of DA neurons are required for successful clinical transplantation [14]. In comparison with other reports that required the use of rodent neuronal conditioned medium [33], gene transfection [32], or chemical inducers [36, 47] to transdifferentiate MSCs, we used a simple induction cocktail containing SHH, FGF8, and bFGF to induce MSCs to TH-expressing cells with ~67% efficiency. Initial studies done by Jiang et al. [35] showed that mouse multipotent adult progenitor cells (subpopulation of MSCs) could generate 25% of cells with DA markers after 21 days of sequential induction with SHH, FGF8, and other neurotrophic factors and chemical reagents. The study done by Fu et al. [33] used similar induction means but in rodent conditioned medium and, more importantly, the MSCs were derived from the umbilical cord. Umbilical cord MSCs have been reported to have different properties than adult MSCs [48], which may account for the low efficiency (12.7%) acquired by Fu et al. [39]. Dezawa et al. [32] reported a 41% efficiency of TH-expressing cells; however, their induction included gene transfection with Notch domain and did not consist of combined SHH and FGF8, which are required for DA induction [49]. Another group showed a 35% efficiency of TH-expressing cells from rat MSCs stimulated with glial-derived neurotrophic factor (GDNF) and interleukin-1β in glial cell conditioned medium; however, they did not report on DA secretion or the excitability of these cells [34]. A recent paper using multiple chemical and trophic factor inducers generated TH-expressing cells from human marrow-isolated adult multilineage inducible cells [47]; however, it is not known whether these induced cells secrete DA. The use of rodent neuronal conditioned media, gene transfection, and chemical inducers to transdifferentiate human MSCs is problematic if the in vitro generated DA neuronal cells are to be eventually used clinically. With regard to transplantation, it is necessary to have a simple protocol that generates a high efficiency of DA cells. Also, it has yet to be determined whether DA progenitors or mature DA neurons will be most efficient for transplantation, regeneration, and survival. In this paper, we used a simple induction cocktail containing SHH, FGF8, and bFGF to induce MSCs to transdifferentiate to TH-expressing cells with ~67% efficiency. We also demonstrated that induced MSCs express several DA neuron-specific markers and constitutively secrete DA.

The in vivo ontogeny of DA neurons has been extensively studied, but many of the mechanisms and factors involved, particularly in humans, remains unclear. However, studies during the past several years have discovered many extracellular and transcription factors involved in the cell-specific determination of DA neurons [50]. The development of DA neurons in the embryo is dependent on the interaction of two growth factors—SHH and FGF8 [49]. Additionally, basic fibroblast growth factor (bFGF, FGF-2) promotes neurogenesis and enhances differentiation and survival of DA neurons [51]. In particular, SHH is a potent morphogen that acts at multiple levels during vertebrate neural development [52–54]. In addition to specifying cell identity, both neuronal and oligodendrocyte fates, it governs proliferation, survival, and axonal guidance [52–54]. SHH is very versatile, coordinating both proliferation and differentiation, but it is diversified by interaction with other signaling pathways [52–54]. Hence, it is the synergistic action of both the SHH and FGF8 signaling cascades that promotes DA specification [49]. SHH binds receptors that include patched, patched 2, and smoothened, and FGF8 binds the FGFR family of receptors. The expression of these receptors on stem cells is therefore likely to be a key requirement to initiate the induction of DA fate. We determined that approximately 89% of our MSCs expressed one or more SHH receptor subtypes (Fig. 1A6). Coincidentally, the percentage of MSCs expressing both SMO and PTCH receptor subtypes (70%) corresponded well with the percentage of TH expressing cells generated (67%) following the 12-day induction. This raises the possibility that the final induction efficiency of DA cells might be dependent on the number of MSCs that express SHH receptors in the initial MSC population isolated from the BM. Thus, we propose that clonal isolation of MSCs expressing SHH receptors might be useful for therapeutic applications that require high efficiency generation of DA neurons.

Several studies have reported links between neural development and the cell cycle [55–59]. During cell fate determination, cell cycle activators, cyclins and cdks, are inhibited or downregulated as neuronal genes are activated [55–59]. In addition, the cells may be switching from symmetric, proliferative divisions to asymmetric divisions in which neural cells are generated [55–59]. Hence, we were interested in determining whether induced MSCs slowed their proliferation and downregulated cell cycle activators as they differentiated. Indeed, cell cycle activators Cdk2 (G1-S progression) and cyclin B (G2-M progression) are downregulated by day 3 and by days 6–9, respectively (Fig. 2). PCNA, a marker of proliferating cells, is downregulated by days 3–6 (Fig. 2). Growth inhibition of induced MSCs was also seen in the cell count analysis as compared with unstimulated MSCs (Fig. 2A). Although the growth rate of induced MSCs was significantly reduced, the cell population continued to increase over the course of the experiment. This suggests that some of the cells continue to divide even after treatment with the induction cocktail. We speculate that these dividing cells did not differentiate and could represent the subpopulation of MSCs (11% of the initial population) that did not express any of the receptor subtypes for SHH.

Several factors and pathways are involved in DA neuron development in vivo [39, 50]. In this study, we determined that the expression levels of many of these DA development-related genes are upregulated following treatment of MSCs with the induction cocktail (Figs. 3, 4). In addition, we showed that 12-day induced cells did not express glial markers GFAP and Olig2 but expressed the neuron specific markers NeuN, β III tubulin, and voltage-gated K+ and Na+ channels (Figs. 3, 4). Furthermore, 12-day induced MSCs express TH, DAT, and VMAT2, which are specific markers for mesencephalic DA neurons (Figs. 3, 4). However, our functional studies using electrophysiological methods revealed that the induced cells did not exhibit neuronal activity (Fig. 5). Although robust outward K+ currents were present in all cells tested, only a small fraction of the tested cells (2 out of 20 cells) showed weak inward Na+ and Ca2+ currents (Fig. 5), consistent with their inability to fire spontaneous or evoked action potentials. This suggests that the 12-day induced MSCs are not fully differentiated mature neurons and may require additional inducers such as retinoic acid [24, 25], brain-derived neurotrophic factor (BDNF), and nerve growth factor [22]. In neurogenesis, cells express K+ currents before Na+ currents are detectable [40, 41]. Immature neurons reportedly have very simple electrical properties, only exhibiting a delayed rectifier K+ current [40–44].

Despite the lack of neuronal activity, the 12-day induced MSCs synthesized and secreted DA (Figs. 4, 6) constitutively. The amount of constitutively released DA in the extracellular medium was increased by a selective inhibitor for DAT (GBR12909), suggesting that, 12 days after induction, the cells express functional DAT (Fig. 6A). As expected from the lack of neuronal activity, the constitutive release was not increased by elevated K+-induced depolarization and not prevented by the removal of free Ca2+ from the extracellular solution (Fig. 6A). The ability of extracellular ATP to enhance DA release from 12-day induced MSCs (Fig. 6C) suggests that, in the absence of electrical activity, the immature MSC-derived DA cells may use a novel receptor-mediated mechanism to release DA. ATP-induced release of Ca2+ from intracellular stores might mediate DA release in the 12-day-induced MSCs. A recent study has demonstrated that ATP released in an autocrine manner acts via purinergic receptors to induce Ca2+ oscillations in human MSCs [46]. In light of the fact that the cells express DAT and lack electrical activity, it is possible that the cells could release DA through the reversal of DAT [60, 61]. Ca2+-independent release of DA does occur in vivo as a result of shuttling DAT, as reported for amphetamine-induced release of DA [62]. However, our data show that the addition of a highly specific DAT inhibitor (GBR12909) increases the level of DA (Fig. 6A). As reported previously, if DA release was occurring through DAT, release of DA would be abolished by addition of the inhibitor; thus, we would not have seen an increase in DA [60, 63]. Nonetheless, we cannot exclude the reversal of DAT in releasing DA in MSC-derived DA cells because the mechanisms of DA release/uptake have yet to be identified. Our observations are in agreement with previous reports that, during neurogenesis, neurotransmitters are present very early, before the cells show electrical activity [64–66]. Immature neuronal cells utilize paracrine communication crucial for neuronal development before functional voltage-dependent Na+ and Ca2+ channels are present [40, 41, 64]. In addition, a recent study demonstrated that the release of DA in young, immature DA neurons is independent of neuronal activity [45].

Although we have demonstrated that the induced MSCs have polarized toward a DA phenotype, they are at an immature stage of differentiation due to their lack of neuronal excitability. In future studies, we will determine the microenvironmental influences of other factors important in DA neuron development and survival. Differentiation into excitable cells may require additional agents or cell-to-cell contact with mature neurons or astrocytes. The use of neurotrophins such as BDNF and GDNF is also warranted, since they have been previously shown to contribute to the maturation of the DA phenotype [67, 68]. Our protocol efficiently generates >90% neuronal cells, with a population of ~67% expressing TH (Fig. 4B, 4C). Although the remaining 20%–30% of cells lack TH, they are neuronal, as indicated by the cell count and FACS analysis (Fig. 4B, 4C). These cell types will be investigated further when determining other inductive factors for full functionality. We speculate that these cells could be either serotonergic neurons or GABAergic interneurons, since SHH is involved in the development of these cells [52–54]. Alternatively, the cells may be at different stages of development, as is often the case in neuronal development, where a population of cells contains mature neurons, progenitors, and neural stem cells. The cells may be a population at early stages of neural development in which TH is not expressed yet. Furthermore, in vivo transplantation studies will be necessary to evaluate the ability of MSC-derived DA cells to rescue motor deficits in animal models of PD. The expansion of DA neurons from human adult stem cells is important for developing cell transplantation therapies for PD. Our results encourage additional study of human MSCs derived from the adult BM for use in potential cell therapy for PD. Human MSCs are ideally suited for use in cell transplantation therapy for PD since they circumvent the ethical issues associated with embryonic stem cells and are easily isolated, expanded, and manipulated in culture. We envisage that the stem cell-derived DA cells can be used as an in vitro model to study the human DA system and to screen pharmacological compounds. In addition, in light of the recent reports of differences in human and rodent DA neuron development [69], the human MSCs described in this paper might provide a useful in vitro model to clarify the molecular ontogeny of the human DA system.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. Martha Nowycky and Nicola Pierobon for advice and technical assistance on Ca2+ imaging and analysis. We also thank Dana Stein for flow cytometry assistance. This work was supported by the F.M. Kirby Foundation. K.A.T. was supported by the Benigno Fellowship for research in neural regeneration and repair, and the work is in partial fulfillment of a Ph.D. thesis.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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