Functional Neuronal Differentiation of Bone Marrow-Derived Mesenchymal Stem Cell - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:57 |只看该作者 |倒序浏览 |打印

作者：Philippe Tropela, Nadine Plateta, Jean-Claude Platelb, Danile Nolc, Mireille Albrieuxb, Alim-Louis Benabida, Franois Bergera作者单位：aNeurosciences Prcliniques, INSERM U, Universit Joseph Fourier, CHU de Grenoble, Grenoble, France;bCanaux Ioniques et Signalisation, INSERM E, Universit Joseph Fourier, CEA-Grenoble, Grenoble, France;cImmunopathologie des Maladies Tumorales et Autoimmunes, INSERM U, Montpellier, France , W, h4 |9 N. X/ m
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      【摘要】
   Recent results have shown the ability of bone marrow cells to migrate in the brain and to acquire neuronal or glial characteristics. In vitro, bone marrow-derived MSCs can be induced by chemical compounds to express markers of these lineages. In an effort to set up a mouse model of such differentiation, we addressed the neuronal potentiality of mouse MSCs (mMSCs) that we recently purified. These cells expressed nestin, a specific marker of neural progenitors. Under differentiating conditions, mMSCs display a distinct neuronal shape and express neuronal markers NF-L (neurofilament-light, or neurofilament 70 kDa) and class III &szlig;-tubulin. Moreover, differentiated mMSCs acquire neuron-like functions characterized by a cytosolic calcium rise in response to various specific neuronal activators. Finally, we further demonstrated for the first time that clonal mMSCs and their progeny are competent to differentiate along the neuronal pathway, demonstrating that these bone marrow-derived stem cells share characteristics of widely multipotent stem cells unrestricted to mesenchymal differentiation pathways. ( ]  C! `4 |7 {2 k# r
      【关键词】 Adult bone marrow stem cells Transdifferentiation Pluripotent stem cells Neural differentiation Multipotential differentiation Mesenchymal stem cells& L# F. f; O7 a+ ]8 D! j$ l
   INTRODUCTION

Classic dogmas restrict stem cell differentiation potentialities to lineages that are specific to their tissue of origin. However, numerous recent publications reported evidence that bone marrow-derived cells do not strictly observe this restriction. Notably, they can give rise to brain cells such as neurons of various areas (hippocampus, cortex, cerebellum, olfactory bulb, etc.) after in vivo transplantation, in both mouse and human . This may suggest that such occurrence strongly depends on experimental conditions and on important unidentified factors. Alternatively, another hypothesis could be that hematopoietic stem cells (the presence of which is tested by grafting experiments) cannot differentiate along the neuronal pathway and thus this differentiation may involve another stem cell population housed in the bone marrow such as MSCs.  m0 J, [3 L1 P9 R
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MSCs derived from adult bone marrow were first described as able to differentiate along three main pathways: osteoblastic, adipocytic, and chondrocytic pathways  and MAPCs are not widely available. Therefore, the ability of mMSCs to acquire a neural phenotype in vitro has not been investigated to date. It would be of great interest to firmly establish the presence of neural progenitors in the mammalian bone marrow before investigating their in vivo fate in genetically or chemically induced mouse models of human pathologies.5 t4 |& [0 l' ^8 `- v
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The major aim of this work was to investigate the ability of mMSCs to differentiate in vitro toward functional neuronal cells in response to a treatment involving bFGF as a physiologic inducer. mMSCs were shown to express nestin, a marker for NSCs in embryo and adult. Neuronal differentiation was characterized by two parallel and complementary means: first, the expression of specific markers and, second, the acquirement of some neuron functions. This dichotomic approach was crucial to fully and unambiguously demonstrate the neuronal differentiation of mMSCs. After induction, the majority of mMSCs adopted a neuron-like morphology in parallel to the expression of neuronal markers NF-L (neurofilament-light, or neurofilament 70 kDa) and class III ß-tubulin (ß3-tub). This expression of neuronal markers is combined with a functional acquisition of neuronal properties such as an increase of cytosolic calcium concentration in response to various specific neuronal agonists. Our results firmly demonstrate that mMSCs can differentiate along the neuronal pathways toward a functional phenotype. Moreover, we also provide evidence that clonal mMSCs can differentiate along this pathway, demonstrating that these bone marrow-derived stem cells exhibit wide potentialities and are not restricted to differentiate into cell types of mesodermal origin.$ \( ]# M9 R* x
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MATERIALS AND METHODS9 ~6 N  ~( M! N, ^

Culture and Neuronal Differentiation of MSCs* |/ V6 o3 \! Q. s& W. v/ ]. y
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mMSCs were isolated and cultured as previously described . Cells were cultured between 10 and 20 passages. No significant differences in either renewal or differentiation abilities were observed between early and late passages. Cells were retrieved from subconfluent culture by trypsinization (trypsine/EDTA solution; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), counted, and plated to a density of 3,000 cells per cm2 on poly(lysine)-coated plates. Plates were previously coated overnight with a 10 µg/ml poly(lysine) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) solution in phosphate-buffered saline (PBS) (Cambrex Bio Science Verviers S.p.r.l., Verviers, Belgium, http://www.cambrex.com). Cells were subsequently cultured for 7 days in the amplification medium, Dulbecco's modified Eagle's medium-low glucose (Sigma-Aldrich), glutamine 2 mM (Cambrex Bio Science Verviers S.p.r.l.), penicillin/streptomycine (50 U/ml and 50 mg/ml, respectively; Cambrex Bio Science Verviers S.p.r.l.), and fetal calf serum 10% (Sigma-Aldrich) supplemented with bFGF 25 ng/ml (either from R&D Systems Inc., Minneapolis, http://www.rndsystems.com or Peprotech, Rocky Hill, NJ, http://www.peprotech.com). For immunofluorescence analysis of markers of expression, mMSCs were plated on poly(lysine)-coated glass or plastic culture slides in the same inductive conditions.6 w- q8 W% r& f9 B  e2 q% _

Isolation, Amplification, and Characterization of Monoclonal mMSCs
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A cell suspension was retrieved from subconfluent culture of the initial mMSC population by trypsinization and maintained in complete medium supplemented with HEPES 20 mM. Cells were isolated and distributed in a 96-well plate by means of microscopic examination and micromanipulation. Each well was then independently checked for the presence of a solitary cell. After amplification, a part of each cell population was frozen, and another part was tested for differentiation along mesodermal (osteoblastic, adipocytic, and chondrocytic ) and neuronal pathways (see above).. E: j8 [) T$ w8 z; c* b

RNA Extraction and Analysis  I8 e& ]; p9 |. l& c

Total RNA was extracted and analyzed as previously described . All primers were selected in different exons.

Immunofluorescence Analysis. H* Z6 O2 G+ K8 J4 ~( F+ |- k5 P
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Primary antibodies included mouse anti-nestin (dilution 1:250; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), mouse anti-ß3-tub (dilution 1:500; BAbCO, Richmond, CA,), and mouse anti-NF-L (dilution 1:500; DAKO, Glostrup, Denmark, http://www.dako.com). Donkey anti-mouse secondary antibodies coupled to cyanine 3 were provided by Jackson ImmunoResearch Laboratories (West Grove, PA, http://www.jacksonimmuno.com) (dilution 1:1,000). A nonspecific signal was evaluated as negligible in all experiments using anti-ß-galactosidase monoclonal antibody as a control (Promega, Madison, WI, http://www.promega.com). All antibodies were diluted in PBS supplemented with bovine serum albumin (BSA) 3% and donkey serum 2%. Untreated or bFGF-treated mMSCs were rinsed twice with PBS and then fixed 20 minutes in a paraformaldehyde 4% solution in PBS. Slides were treated with a blocking/permeabilizing solution consisting of PBS supplemented with BSA 3%, donkey serum 2%, and Triton X-100 0.3% for at least 20 minutes at room temperature. Slides were then sequentially incubated with primary antibodies overnight at 4¡ãC and with secondary antibodies for 1 hour at room temperature. Cells were finally incubated in a PBS solution containing 1 µg/ml 4,6-diamidino-2-phenylindole to stain nuclear DNA. Washed slides were then mounted with FluorSave (Calbiochem, San Diego, http://www.emdbiosciences.com) and analyzed using an epifluorescence microscope (Olympus, Tokyo, http://www.olympus-global.com).
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Calcium Imaging
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Cells were loaded with the calcium indicator dye Fluo-4 by bath application for 30 minutes at 37¡ãC in ACSF-HEPES (artificial cerebral spinal fluid with HEPES, which contained ." I. {# f6 f! J) x
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Statistical Analysis4 B! K2 V- M+ j: A6 Q( p/ ]2 V
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Data were then exported to Microsoft Excel (Microsoft, Redmond, WA, http://www.microsoft.com) or SigmaPlot (Systat Software Inc., San Jose, CA, http://www.systat.com) softwares for statistical analysis. All results are presented as mean ¡À SEM. Statistical tests were performed using either Student's t test or Mann-Whitney rank sum test as appropriate.& T" Z- I0 c/ ?, @

RESULTS
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mMSCs Acquire a Neuron-Like Shape in the Presence of bFGF and Poly(lysine)

We tested several culture conditions to induce neuronal differentiation of mMSCs. In our hands, previously published protocols using chemical inducers were not able to induce a neuron-like shape in mMSCs, possibly due to species differences (data not shown). We then investigated the efficiency of physiological inducers of neuronal differentiation. We first tested the efficiency of bFGF because of its well-known role in neuron differentiation and physiology. In preliminary experiments, bFGF was added to the culture medium of mMSCs spread on fibronectin-coated dishes but this gave poor results at the morphological level (data not shown), in contrast to the reported effect on mouse MAPCs . Because neuronal cells are classically cultured on poly(lysine)-coated dishes, we hypothesized that mMSC neuronal differentiation may be improved by this way. When plated on poly(lysine), mMSCs showed a smaller size than on fibronectin but their fibroblastoid shape was maintained (Fig. 1A). When bFGF was added to the medium, some short neurite-like extensions were visible after 2 days, easily recognizable after 4 days, and fully developed after 1 week (Fig. 1A). At this time, most cells presented with neuron-like morphology, including a small-cell body and long neurite-like extensions, sometimes a unique long extension and others shorter (Fig. 1B) or bipolar morphologies. From day 7, cells readopted a fibroblastoid shape, suggesting that such conditions could not support long-term cell differentiation.
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Figure 1. Mouse mesenchymal stem cells (mMSCs) can progressively acquire a neuron-like morphology. (A): mMSCs plated on poly(lysine) were treated with basic fibroblast growth factor. Shape change is illustrated by phase-contrast micrographs of various fields at days 0 (D0), 2 (D2), 4 (D4), and 7 (D7). (B): Cells progressively developed neurite-like extensions, with a maximum size at day 7. Some cells presented a unique long extension (arrows) from the small-cell body (arrowheads) as shown in this image of a May-Grunwald Giemsa stained field. Scale bars = 100 µm.: n8 \4 q$ T* F3 e; j1 M
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Treatment Increases the Number of Nestin-Positive mMSCs

Nestin is widely considered a specific marker of NSCs and progenitors  suggest that nestin expression is a common feature of mMSCs. Immunolabeling analysis further demonstrated that 50.3% ¡À 8.2% (mean ¡À SEM; n = 3) of untreated cells expressed the marker (Fig. 2B). bFGF treatment dramatically increased the number of nestin-positive cells to 94.5% ¡À 3.4% (mean ¡À SEM, three independent experiments; p  .02; Fig. 2C). This may be due to the growth of a neural progenitor subpopulation because our culture is heterogeneous in origin or to the commitment of multipotent mMSCs toward a neural fate. Alternatively, nestin could be expressed by all resting cells, but below the detection threshold in 50% of them, and then increased by bFGF treatment. Whatever the mechanism involved, the presence of nestin-positive cells in untreated mMSCs was a clue suggesting that those cells carried a true neuronal potentiality ready to be activated under appropriate conditions.

Figure 2. Mouse mesenchymal stem cells (mMSCs) expressed nestin before and after treatment. (A): Reverse transcription-polymerase chain reaction analysis of the expression of nestin in amplified subconfluent mMSCs (lane 1: culture on fibronectin in the amplification medium ) and treated mMSCs (lane 2: cells cultured for 1 week on poly-lysine in the amplification medium supplemented with basic fibroblast growth factor 25 ng/ml, as described in Materials and Methods). HPRT amplification is shown as a control of mRNA quality. Immunofluorescent analysis of nestin expression without (B) and after (C) treatment of mMSCs. Nestin-specific antibodies labeled a filamentous network in both cells, but with a stronger signal in treated cells, may be due to the concentration of filaments in thin extensions. Scale bars = 100 µm. Abbreviation: HPRT, hypoxanthine-guanine phosphoribosyltransferase.# i+ x5 q) U+ g- ]* K4 X
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Treatment Induces Expression of Other Neuronal Markers NF-L and ß3-Tub in mMSCs

Nevertheless, shape change and nestin expression may appear as coincidental and without links to a neural phenotype. We therefore attempted to correlate these results with the induction of neuronal marker expression. Neuronal cells are classically characterized by the expression of cytoskeletal proteins such as NF-L and ß3-tub, which are both early markers of this lineage. NF-L mRNA was below detection thresholds of our RT-PCR test in undifferentiated mMSCs but was easily amplified from treated cell samples (Fig. 3A). Immunofluorescence analysis showed that the rate of NF-L-positive cells increased from 12.4% ¡À 11.7% (mean ¡À SEM, n = 3; Fig. 3B) in the mMSC population to 88.5% ¡À 1.8% in treated culture (mean ¡À SEM, n = 4; p  .02; Fig. 3C). NF-L proteins were detected in dotted structures . Therefore, the treatment engages the cells in the neuronal pathway but does not support full differentiation. No expression of astrocytic and oligodendrocytic markers, respectively, glial fibrillary acidic protein (GFAP), and myelin basic protein, was detected using RT-PCR (N. Platet, unpublished data), supporting the idea that the treatment induced a coordinated genetic program and not a stochastic activation of irrelevant genes. Nevertheless, those results do not rule out the possibility of glial differentiation of mMSCs in other conditions.
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Figure 3. Dramatic induction of major neuronal markers in mouse mesenchymal stem cells (mMSCs) treated with basic fibroblast growth factor. (A): Reverse transcription-polymerase chain reaction analysis of the expression of NF-L in amplified (lane 1) and treated mMSCs (lane 2) showed a strong induction of the gene in cells after treatment. HPRT amplification is shown as a control of mRNA quality. (B¨CD): Immunofluorescent analysis of NF-L before (B) and after (C, D) treatment of mMSCs. NF-L-specific antibodies labeled punctate structures mainly in the perikaryon zone (C). (D): A higher magnification of bipolar cells presenting NF-L-positive dots distributed both in the cell body and in neurite-like extensions. (E, F): Immunofluorescent analysis of ß3-tub expression before (E) and after (F) treatment of mMSCs. ß3-tub was found widely distributed in the whole cell, without any preferential localization. Scale bars = 100 µm. Abbreviations: ß3-tub, class III ß-tubulin; HPRT, hypoxanthine-guanine phosphoribosyltransferase; NF-L, neurofilament-light, or neurofilament 70 kDa.* h% q* J+ v! [3 a7 J! c* |
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Treated Cells Can Respond to Classic Neuron Activators by Increasing Their Cytosolic Calcium/ ]% _: u; G, h1 A4 S$ a; }8 _

To confirm that expression of neuronal markers by mMSCs is the result of a specific program, we investigated the functionality of treated cells by imaging calcium signaling in response to various neuron activators: (a) glutamate, which is able to induce Ca2  rise through ionotropic or metabotropic receptors activation (reviewed in . Differentiated mMSCs responded to 100 µM glutamate (Fig. 4A) and 50 µM veratridine (Fig. 4B) as observed by a large and immediate calcium increase in 51.6% ¡À 12% (mean ¡À SEM; n = 6; p  .01) and 68.4% ¡À 11.6% of cells (mean ¡À SEM; n = 4; p  .03), respectively. These effects were blocked by 100 µM MK801 (specific N-methyl-D-aspartic acid receptor antagonist) and 1 µM tetrodotoxin (specific Na  channel antagonist), respectively, confirming the nature of activated receptors (J.-C. Platel, unpublished data). Under the same conditions, undifferentiated mMSCs did not respond to these activators regarding calcium movement. In differentiated mMSCs, dopamine induced a Ca2  response in 53.7% ¡À 10.4% of cells (mean ¡À SEM; n = 3; p  .05) within minutes after agonist addition (Fig. 4C). Response to dopamine was also punctually observed in control cells (approximately 30% responsive cells in one out of three experiments), possibly revealing spontaneous differentiation. These results suggest that dopamine acts as a neuromodulator in differentiated mMSCs. All these results demonstrate that treated mMSCs expressed functional neuronal receptors and voltage-dependent channels. Thus, bFGF-treated mMSCs clearly displayed a neuronal phenotype.

Figure 4. Classic neuron agonists induce a cytosolic calcium response in mouse mesenchymal stem cells treated with basic fibroblast growth factor. (A¨CC): Variation of the cytosolic calcium concentration is displayed in the bottom panel as a function of time for the cell(s) pinpointed (arrowheads) in the microphotographs above. Glutamate 100 µM (A) and veratridine 50 µM (B) induced an immediate increase in the cytosolic calcium concentration. Moment of addition is indicated by an arrow. (C): Imaging of calcium response to dopamine 100 µM. Dopamine was added at time = 0. Responses displayed by two characteristic cells (1 and 2, indicated by arrowheads) were shown as an example of dopamine effects. Classic response to this agonist is delayed in time and not synchronous between cells of a same field. Scale bars = 50 µm. Abbreviations: s, seconds; t, time.

Clonally Derived MSCs Can Differentiate Along the Neuronal Pathway9 Y, B( G+ s4 ~( d+ M
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To determine which mMSC subpopulation can differentiate along the neuronal pathway, we performed experiments on clonally derived cultures. We established 17 clonal populations and first tested their ability to differentiate along the three pathways that define MSCs: osteoblastic, adipocytic, and chondrocytic (Table 1). Three clones out of 17 were able to differentiate along all three pathways, demonstrating that they derived from clonal MSCs. Then, we analyzed the expression of neuronal markers in such populations with and without neurogenic induction (Table 1). Before differentiation, all clones were positive for nestin and negative for NF-L. A subset (9 of 17) was also positive for ß3-tub. After bFGF treatment, nestin and ß3-tub expression was maintained or increased in all clones in which they were already expressed. Treatment also induced de novo expression of NF-L in all clones (Table 1). All three MSC-derived clones (so-called A O C in Table 1) were found strongly positive for nestin and NF-L after neurogenic treatment, but only one was weakly positive for ß3-tub. This suggests that the treatment acted more efficiently on engaged progenitors than on stem cells. Nevertheless, these results clearly demonstrate for the first time that fully characterized clonally derived MSCs and their progeny can differentiate along the neuronal pathway.
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Table 1. Differentiation potentialities of clonally derived mesenchymal stem cells, |' _% k6 \9 x2 y' e/ p

DISCUSSION' E2 [# L9 \/ F* L5 O7 e5 l
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With the increasing amount of data about gene expression, few previously described differentiation markers remain. To demonstrate the neuronal differentiation of mMSCs, we adopted a "triangulation" strategy involving both marker and function analysis. If nestin is no longer a NSC marker (see above), NF-L and ß3-tub expression seems to be limited to neuronal cells. Many cell types express glutamate and dopamine receptors . Nevertheless, muscles cannot respond to glutamate, and only neurons express ß3-tub or NF-L. Altogether, our results clearly demonstrate that mMSCs can acquire a functional neuronal phenotype with early-stage differentiation characteristics.
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In the field of neuroscience, nestin has long been considered a specific marker of NSCs in the developing and adult brain. In contrast, publications reported nestin expression in testis , we have not been able to investigate nestin expression in either unamplified mMSCs or early passages. In the absence of any protocol to purify homogeneous MSC population from bone marrow, the demonstration of nestin expression by native MSCs remains an unsolved issue.: [% K  d9 M" z" a" y

bFGF has long been known as a key regulator of NSC proliferation and differentiation . In our hands, bFGF induced mMSCs to differentiate toward the neuronal pathway when they were plated onto poly(lysine)-coated culture dishes. To our knowledge, poly(lysine) receptor(s) has (have) not been identified but may participate in cell fate by modulating the bFGF signaling and/or by transducing a differentiating message on its own. It appears in our experience that both messages are necessary for efficiently inducing morphological changes. This may nicely illustrate synergies between growth and adhesive factors in regulation of cell fate but requires further experiments to specifically highlight the molecular mechanisms involved.
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The neuronal differentiation of rat or human MSCs was often successfully induced using chemical compounds such as BHA, IBMX, DMSO, or 2-mercaptoethanol. Whether such compounds may induce major perturbations of cell functions is a poorly investigated topic. One of these treatments, mixing ß-mercaptoethanol, DMSO, BHA, and other chemicals, clearly increased the number of dead cells in the culture  showed that an artifactual neuronal differentiation could be induced in MSCs by a wide variety of compounds as a stereotypical response to stress. The neuronal morphology previously observed was due to the cytoplasm shrinkage and not to the growth of neurites. In our case, mMSCs plated on poly(lysine) displayed a smaller body size than on fibronectin, and neurite-like structures were then clearly observed growing day after day only in presence of bFGF (Fig. 1). Moreover, in contrast to the results of Lu et al., we confirmed the de novo expression of NF-L by means of RT-PCR and the acquirement of neurons' functional properties by using calcium imaging. Conjunction of structural (expression of markers) and functional (ability to efficiently respond to neuroagonists) evidence allows us to exclude the idea of a nonspecific response but not to definitively rule out the "stress" hypothesis. In that hypothesis, our data actually suggest that such observations would be the expression of a very specific and regulated cell answer using neuron markers and functions in mesenchymal cells, which has not been suggested to date.. ^9 [% I2 C3 z
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Calcium imaging and immunostaining experiments showed that, in bFGF-treated culture, at least 50% of polyclonal mMSCs could differentiate toward the neuronal lineage. As the number of cells clearly increased with time, this phenomenon could result either (a) from the growth of a neurogenic population or (b) from the commitment to the neuronal lineage of individual mMSCs. To distinguish between these two hypotheses, we established and characterized the differentiation potentials of clonal mMSC populations. MSCs were first characterized at the clonal level by Pittenger and co-workers in 1999 .
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Despite the presence of functional nerve fibers . In this model, plasticity should be another distinctive property of stem cells with a controversial relationship with self-renewal and multipotentiality. Nevertheless, the conditions and molecular mechanisms of plasticity should be carefully studied in the future, leading to an improvement in adult stem cells manipulation.

CONCLUSION  D4 b+ q% x/ t
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Our results firmly establish the possibility of functional neuronal differentiation of mMSCs. This in vitro model will allow us to address molecular events inducing and controlling this differentiation. The firm establishment of a mouse model will further allow us to accumulate insights into in vivo neurogenicity of MSCs by transplantation into the brain. This new model with the disposability of hundreds of transgenic mouse strains opens up completely new fields for investigating MSC physiology and potentialities. From a clinical point of view, further investigations should also be initiated on physiological inducers of the neuronal differentiation of human MSCs. In this context, whether such a potentiality allows differentiation after in vivo transplantation in the brain will have to be clarified in the mouse model and others before the elaboration of any bone marrow-based cell therapies of nervous system.' x! W1 I  D# `" O/ I: O
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DISCLOSURES

The authors indicate no potential conflicts of interest.  Q; ^, S1 {3 o

ACKNOWLEDGMENTS
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We thank Dr. Michel Villaz for his crucial help for the completion of this work and Dr. Keyoumars Ashkan, Wesley J. Harrison, and Dr. Graham W. Neill for reading and corrections. This work was supported by Association Française contre les Myopathies. M.A. is currently affiliated with Dynamique des R¨¦seaux Neuronaux, INSERM U704, Universit¨¦ Joseph Fourier, Grenoble, France. J.C.P. is currently affiliated with the Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut, USA.& p: `4 _: k4 x6 J
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