Induction of neural-like differentiation in human mesenchymal stem cellsderived from bone marrow, fat, spleen and thymus
Mauro Krampera a,⁎, Silvia Marconi b, Annalisa Pasini a, Mirco Galiè c, Gino Rigotti d,Federico Mosna a, Martina Tinelli a, Laura Lovato b, Elena Anghileri b, Angelo Andreini a,
Giovanni Pizzolo a, Andrea Sbarbati c, Bruno Bonetti b,⁎
a Departments of Clinical and Experimental Medicine, Section of Haematology, Italyb Departments of Neurological Sciences and Vision, Section of Neurology, Italy
c Departments of Morphological-Biomedical Sciences, Section of Anatomy, University of Verona, Italyd Departments of Plastic and Reconstructive Surgery, Azienda Ospedaliera of Verona, Italy
Received 19 June 2006; revised 23 August 2006; accepted 7 September 2006Available online 16 October 2006
Mesenchymal stem cells (MSCs) are non-hematopoieticprogenitor cells initially obtained from bonemarrow (BM) [1–4]with multilineage differentiation potential into tissues ofmesenchymal origin (including BM stromal cells, adipocytes,osteoblasts, chondrocytes, tenocytes and myocytes) as well as
⁎ Corresponding authors. M. Krampera is to be contacted at Sezione diEmatologia Policlinico ‘G.B. Rossi’ Policlinico P.le L.A. Scuro 10, 37134Verona, Italy, fax: +39 045 501807. B. Bonetti, Sezione di NeurologiaPoliclinico ‘G.B. Rossi' P.le L.A. Scuro 10, 37134 Verona, Italy, fax: +39 045585933.
tissues of both endodermal (hepatocytes) and ectodermal origin(neural cells) [5–7], thus allowing to speculate about theirpluripotency. In addition,MSCs have been obtained from tissuesother than BM, such as fat, umbilical cord blood or fetal tissues[8–12]. As there are no specific markers, human MSCs arerecognized on the basis of a complex immune phenotype,including the lack of hematopoietic stem cell markers (such asCD45 and CD34), as well as endothelial markers (such as CD31/PECAM-1), and the expression of a number of surfacemolecules, including CD105, CD73, CD106, CD44, CD90,CD29 and STRO-1 [1–4,13,14].
The increasing interest around MSC biology is due to theirpotential use for hematopoietic stem cell transplantation, tissueregeneration and immunosuppressive cell therapy [14,15]. In
fact, MSCs support the microenvironment and the regenerationof tissues of mesenchymal and non-mesenchymal origin andcan provide a target for gene therapy strategies [2,15–18].These lines of evidence have recently generated interest aboutthe potential use of MSCs for the treatment of degenerative andautoimmune diseases of the nervous system. In addition to theirimmune regulatory properties [14,19–21], MSCs have beensuggested to undergo neural differentiation under appropriatestimuli [6,7,22–24]. In this regard, a large body of evidence hasestablished the neural differentiation potential of MSCs derivedfrom BM. The treatment of BM-MSCs with different moleculesand growth factors induced very rapid morphological changesthat are typical of neural cells together with the expression ofneural markers, such as nestin, neurofilaments, MAP-2, orNeu-N [22–28]. However, it has been recently shown thatmorphological and phenotypic changes are not necessarilyassociated with definite electrophysiological properties ofneurons [29]. In fact, the acquisition of neuron-like excitabilityhas been observed after co-culture with mature neural cells ortransplantation into the brain [24,28]. Noteworthy, most studieshave been focused on the capacity of MSCs to differentiate intoneural cells of the central nervous system (CNS), while verylittle information is available regarding the possibility that adultstem cells may differentiate into Schwann cells (Sc), themyelin-forming cells of the peripheral nervous system (PNS).In this contest, a very recent study has suggested that Sc mayinduce neuronal differentiation of BM-MSCs [29].
The great majority of studies regarding the neural differ-entiation potential of MSCs have been focused on the capacityof adult BM-derived progenitors to differentiate into neuronsand/or glial cells of the CNS. A more limited number of studieshave explored the neural differentiation potential of adult MSCsderived from tissues other than BM; cells with morphologicaland biological features of neural cells have been obtained fromfat-derived MSCs [8,9,30–32], but the neural differentiationpotential of other adult tissues is still unknown.
Aim of our study was to evaluate and compare the neuraldifferentiation potential of adult human MSCs obtained fromdifferent tissues (BM, fat, thymus, and spleen) using previouslypublished protocols of chemical induction [22] with particularattention to the duration and selectivity of such process; inaddition, we assessed the effects of MSC-Sc co-cultures on theprocess of neural differentiation of MSCs of different origin.
Materials and methods
Cell cultures
Human MSCs were obtained from BM aspirates of healthy donors, fromlipoaspirates of abdominal fat, and from normal thymus and spleen samplesremoved during heart and abdominal surgery [8,9,13,14]; all tissues werecollected after informed consent. BM mononuclear cells were obtained withdensity gradient centrifugation (Lymphoprep, Nycomed Pharm, Oslo, Norway).Human spleen and thymus samples were fragmented with needles and filteredthrough cell strainers (BD Falcon™, Becton Dickinson, Milan, Italy) to removethe stromal capsule and obtain a homogenous cell suspension. The isolation ofstromal-vascular fraction derived from adipose tissue was carried out on 40 mlof lipoaspirates, which was extensively washed with sterile Hank's balanced saltsolution (HBSS). Extracellular matrix was digested at 37°C in HBSS with type I
collagenase and BSA (Sigma Immunochemicals, Milan, Italy). After incubation,digestion enzyme activity was neutralized with Dulbecco's modified Eaglemedium (DMEM) containing 10% fetal bovine serum (FBS) (all from Gibco,Milan, Italy) and centrifuged at 1200×g to obtain high-density pellet. This wasthen resuspended in 160 mM NH4Cl and incubated for 10 min to lysecontaminating red blood cells. The stromal fraction was collected bycentrifugation and filtered through a 70-μm nylon mesh to remove cell debris.Cells derived from the different tissues were then cultured in 25 cm2 flasks (BDFalcon™, Becton Dickinson) at a concentration of 30×106 nucleated cells inDMEM, with high glucose concentration, GLUTAMAX I™, 15% heat-inactivated adult bovine serum, penicillin and streptomycin (Gibco) at 37°C in a5% CO2 atmosphere. After 72 h, non-adherent cells were removed and thereaftermedium was changed twice a week. When 70–80% adherent cells wereconfluent, they were trypsinized, harvested and expanded in larger flasks[13,14]. A homogenous cell population was normally obtained after 3 to5 weeks of culture.
Schwann cells were obtained from benign schwannomas of acoustic nervethat have been shown to be phenotypically and functionally similar to non-neoplastic Sc and to express the distinctive features of normal Sc [33,34].Dissociated Sc were obtained according to a previously described protocol [33]and yielded 85–90% S-100 positive Sc cultures. Briefly, specimens were mincedand incubated in Earle'sMEMwith 15% fetal calf serum, hyaluronidase, dispase,collagenase overnight at 37 °C and then in trypsin and DNAse for 30 min. Cellswere further dissociated by gentle pipetting, washed and seeded in flasks coatedwith poly-L-lysine. For co-culture experiments, 2×103 cells/cm2 MSCs wereseeded on glass coverslips treated with poly-L-lysine and cultured for 24 h incomplete DMEM. In a set of experiments, MSCs were then exposed to the neuraldifferentiation protocol (see below). After washing with PBS, 1×104 Sc wereplated on MSCs at 1:2 ratio. Co-cultures were maintained in DMEMsupplemented with 10% FBS for 4–12 days and then fixed for immunocy-tochemistry. To assess the role of cell fusion among Sc and MSCs from differentorigin, Sc were irradiated (2800 cGy for 30 min) before the co-culture. In eachexperimental condition, all MSCs present on the coverslip were counted (about1×103) after chemical differentiation or co-culture with Sc (see below).
CFU-F assay
We used the fibroblast colony forming unit (CFU-F) assay to compare thefrequency of mesenchymal cell precursors inside the tissues of different origin,starting from the same number of cells in suspension, as previously described[35]. We seeded 5×104 cells/cm2 human BM, spleen, thymus and peripheralblood-derived mononuclear cells in triplicate in 75 cm2 flasks with the completemedium described above. After 72 h, non-adherent cells were removed andthereafter medium was changed every 3 days. Ten days later the number offibroblastic colonies with more than 50 cells was counted and expressed on theentire seeded cell population, as previously described [35]. Thus, we comparedthe number of CFU-F obtained from the different tissues.
MSC immunophenotyping
Human MSCs were recognized by immunophenotype using monoclonalantibodies (mAbs) specific for CD105 (endoglin), CD73, CD29, CD44, CD90,and CD106 (VCAM-1). In addition, the lack of hematopoietic (anti-CD45,-CD14, -CD11c, CD123 and -CD34 mAbs) and endothelial cell markers (anti-CD31 mAb) was assessed as previously described [7,8,13,14]. All mAbs werepurchased from Pharmingen/Becton Dickinson. For immunophenotypic analy-sis, MSCs were detached using trypsin/EDTA for 5 min, immediately washedwith phosphate-buffered saline to remove trypsin, and resuspended at 106 cells/ml. Cell suspension was incubated at +4°C for 10 min with 15% adult bovineserum, followed by incubation with the specific mAb at +4°C for 30 min. Atleast 10,000 events were analyzed by flow cytometry (FACScalibur, BectonDickinson) using Cell Quest software.
MSC differentiation assay
Multilineage differentiation potential was assessed by testing the ability ofhuman MSCs derived from BM, fat, spleen and thymus to differentiate into
adipocytes, osteoblasts, and chondrocytes, as previously described [1,13,14].Briefly, adipocyte differentiation was achieved after 3-week culture of MSCswith adipogenic medium, containing 10−6 M dexamethasone, 10 μg/ml insulinand 100 μg/ml 3-isobutyl-1-methylxantine (Sigma). Osteoblast differentiationwas achieved after 3-week culture with osteogenic medium containing 10−7 Mdexamethasone, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate(Sigma). Chondrocyte differentiation was achieved after 3-week culture withchondrogenic medium, containing 10−7 M dexamethasone, 10 ng/ml TGF–μ1,50 μg/ml ascorbic acid, 40 μg/ml proline (Sigma) and ITS+ Premix (BDBiosciences, Bedford, MA, USA). Oil-red-O, von Kossa and toluidine blue dyeswere employed to identify adipocytes, osteoblasts and chondrocytes,respectively.
To induce neural differentiation, MSCs were cultured according to amodified protocol from Woodbury et al. [22]. Briefly, basal medium was
Fig. 1. Neural differentiation medium produced identical morphological and phenotypBM stainedwith hematoxylin (A) showed similar morphology toMSCs derived from sImmuno-peroxidase for PMP-22 (C) and GFAP (D) on BM-MSCs reveals low levelsneural differentiation medium in terms of morphology (E–H) and phenotype (E1–H1)(H, H1); the majority of BM MSCs assumed an astrocytic/neuronal morphology (E–electron microscopy and intense immunoreactivity for PMP-22 (E1), GFAP (F1) and Nwithmulti-branched, irregular shape (H) and selective up-regulation of GalC (H1).MSand faint immunoreactivity for GFAP (J) 72 h after removal of neural induction meantibody on MSCs stimulated with neural differentiation medium. Scale bar: 10 μm.
replaced with DMEM and FBS supplemented with 5 ng/ml basic fibroblastgrowth factor (bFGF; Sigma) for 24 h; after this pre-induction, cells werewashed with PBS and induction medium including DMEM with N2supplement, butylated hydroxyanisole, KCl, valproic acid and forskolin (allfrom Sigma) was added for 2 to 16 h. Cells were either fixed forimmunocytochemistry or transferred to basal medium for the assessment oftheir phenotype and differentiation potential.
Light and electron microscopy
To detect changes of cell morphology, MSCs in basal conditions and aftertreatment with neural differentiation medium were either stained withhematoxylin (for light microscopy) or fixed in glutaraldehyde and evaluated
ic changes of MSCs obtained from different sources. Spindle-shapedMSCs frompleen (as in B at scanning electronmicroscopy), thymus or fat in basal conditions.of expression in basal conditions. (E–H) Dramatic changes after the exposure towere observed on MSCs from spleen (E, E1), thymus (F, F1), BM (G, G1) and fatF) with bi- and tripolar shape with primary and secondary processes at scanningeuN (G1). About 15–20% of MSCs displayed oligodendroglial-like morphologyCs fromBM (as well as from the other tissues) regained their basal morphology (I)dium. (K) Background immunostaining obtained with the omission of primary
MSC immunophenotype has been analyzed by flow cytometry in standardconditions (proliferating cells). Results are expressed as intensity of expression(−: negative; ±: weak expression, +: 1 log shift from negative control, ++: 2 logshift from negative control; +++ 3 log shift from negative control) and derivefrom five different experiments.
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with scanning electron microscopy; for this purpose, the tissues were post-fixedin 1% osmium tetroxide for 1 h, dehydrated in graded acetone, critical pointdried (CPD 030, Balzers, Vaduz, Liechtenstein), fixed to stubs with colloidalsilver, spattered with gold with a MED 010 coater and examined with scanningelectron microscope (DSM 950 Zeiss).
Immunocytochemistry
To evaluate the immune phenotype, MSCs derived from BM, fat, spleen andthymus, cultured without or with neural differentiation medium, were seededonto chambered slides and subjected to immunocytochemistry according tostandard protocols [33,34]. For this purpose, cells were incubated with specificantibodies directed against different phenotypic markers, including: CD105(expressed by MSCs, 1:500, Caltag Laboratories, Burlingame, CA); theneuronal markers NeuN (a marker of intermediate and mature neurons,1:1000; Chemicon, Inc., Temecula, CA), microtubule-associated protein 2(MAP2, a cytoskeletal protein required for neuronal development, 1:1000;Chemicon), PSA-NCAM (polysialic acid-neural cell adhesion molecule, 1:500;Chemicon); the neural precursor marker nestin (1:2000; Chemicon); theastrocytic marker GFAP (1:10,000; Dako); the oligodendroglial markerGalactocerebroside (GalC, 1:100; Chemicon); the Sc markers PMP22(1:5000; Chemicon) and S-100 (1:5000, Dako). After washing, appropriatebiotinylated secondary antibody and ABC amplification kit (Vector Labora-tories, Burlingame, CA) were added and the reaction visualized withdiaminobenzidine. In co-culture experiments, MSCs were labeled with thefluorescent marker PKH-67 (10−3 M; Sigma), according to manufacturer'sinstruction. Neural markers were assessed by double immunofluorescenceanalysis using appropriate biotinylated secondary antibodies and StreptavidineTexas Red (Vector). Experiments were performed in triplicate and thepercentage of positive cells by immunofluorescence was counted by twoindependent investigators (SM and BB). Images of immune-peroxidase wereobtained with Zeiss Axiophot microscope and AxioCam camera withAxioVision software; pictures of double immunofluorescence were obtainedwith Zeiss MC80 microscope.
Proliferation assay
To determine the mitotic activity of MSCs before, during and after inductionof neural differentiation, cells were exposed to BrdU 10 μM (Sigma) for 4 h,fixed with ethanol 70% at −20°C for 20 min, treated with HCl 2 N and then withNaBo 0.1 M, pH 9. After incubation with fluorescein-conjugated anti-BrdUmAb (Boehringer, 1:10), cells were observed at the fluorescence microscope.Rate of mitotic activity was calculated dividing the number of cells BrdU-positive in their nucleus by the total number of cells.
Statistical analysis
Statistical comparison of the results obtained with the different kinds ofMSCs treated with neural differentiation medium and/or co-cultured with humanSc was carried out according to the Student's T-test. Differences wereconsidered statistically significant when p<0.05.
Results
Biological features of BM-, fat-, spleen- and thymus-derivedMSCs
A homogeneous and proliferating adherent cell populationwas obtained from human BM, spleen and thymus, but not fromperipheral blood, after 3 to 5 weeks of culture. CFU-F assayshowed a higher frequency of fibroblastic colonies using BMmononuclear cells (58.1±3.2) rather than spleen (16.5±3.1)-and thymus (20.6±4.1)-derived cells, while no CFU-F wasobtained from PB MNCs (p<0.01, 3 experiments). The various
types of MSCs differed in replication rate: BM- and fat-derivedMSCs doubled within 1.5–1.8 days (mean 1.6±0.2), whereasMSCs from spleen in 3±0.5 days and those from thymus in 5±0.9 days.
MSCs derived from fat, thymus, and spleen showed in basalconditions the same morphology of BM MSCs, forming amonolayer of large and flat cells and assuming spindle-shapedmorphology at confluence (Figs. 1A, B). All kinds of MSCsshowed the same phenotype (Table 1), with expression ofCD105, CD73, CD29, CD44, CD90, CD106, absence ofhematopoietic and endothelial markers, and displayed the sameability to differentiate into adipocytes, osteoblasts and chon-drocytes, both qualitatively and quantitatively (Fig. 2). As far asmolecules of the neural lineage are concerned, all MSCs fromthe different tissues expressed in basal conditions low levels ofPMP22 (Fig. 1C), GFAP (Fig. 1D), and MAP2 (not shown) andwere negative for the other investigated markers (NeuN, PSA-NCAM, nestin, GFAP, S-100 and Gal-C).
Morphological and phenotypic changes of MSCs followingneural induction
The MSC populations used for differentiation experimentswere phenotypically homogeneous. After exposure to neuralinduction medium, all kinds of MSCs exhibited very rapidmorphological changes: most cells retracted their cytoplasm,forming spherical cell body, emitted cellular protrusions andcompletely stopped to proliferate, as compared to MSCs inbasal conditions (13.9±3.1% of MSCs incorporating BrdU, 3experiments). At the end of this process, we could identifythree morphologically distinct subsets of MSCs, regardless oftheir different origin (summarized in Fig. 3): 50–70% ofMSCs appeared as sharp, elongated bi- or tripolar cells withprimary and secondary processes (Figs. 1E–F), morphologi-cally reminiscent of neurons or astrocytes and showed intenseexpression of PMP-22 (Fig. 1E1), GFAP (Fig. 1F1), NeuN(Fig. 1G1), PSA-NCAM, nestin, S-100, MAP2 and Gal-C (not
Fig. 2. Multilineage differentiation of MSCs. Confluent MSCs from human BM, adipose tissue, spleen and thymus were cultured for 14 days in flasks with adipogenic,osteogenic or chondrogenic media. Then cells were stained with Oil-red-O, von Kossa, and toluidine blue methods, respectively, which confirmed their multilineagepotential that was qualitatively and quantitatively the same for all the MSCs of different origin. The figure shows a representative case of three different experiments.
Fig. 3. Effect of neural differentiation medium on MSC from different sources.After exposure to neural differentiation medium, the majority of MSCs fromBM, adipose tissue, spleen and thymus showed profound morphological andphenotypic changes. Cells were considered reminiscent of: astro-neuronalmorphology because of bi- or tripolar shape with up-regulation of all neuralmarkers; oligodendroglial morphology because of irregular shape and short,multi-branched processes with selective expression of GalC; or MSC-likebecause of unchanged morphology and absence of neural markers.
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shown); irrelevant mouse or rabbit IgG gave no staining (Fig.1K). The second population (about 15%, Fig. 3) was charac-terized by elements with irregular shape and short, multi-branched processes, morphologically similar to cells of theoligodendroglial lineage (Fig. 1H), and selectively expressedGal-C (Fig. 1H1). The last population (15–20%, Fig. 3) wassimilar to MSCs in basal conditions, i.e., remained undiffer-entiated and did not up-regulate any neural marker. Irrespectiveof the morphological changes, all MSCs retained CD105expression (data not shown). These events started after 1–2 hand reached their peak within 6–8 h; the only exception wasrepresented by fat-derived MSCs, in which the entire processlasted 2 h. With longer culture times (i.e., 12–16 h), signs of celltoxicity were observed in all MSCs, with retraction of cellprocesses, detachment from plastic and eventually apoptosis ofvirtually all MSCs.
The neural differentiation protocol induced a transientdifferentiation of all kinds of MSCs, as cells graduallyrecovered basal morphology and phenotype within 48–72 hof culture with basal medium (Figs. 1I, J). Proliferation assayat 72 h showed that 7.5±2.5% of MSCs incorporated BrdU(i.e., half the proliferation rate of MSCs in basal conditions);no relevant signs of cell death were observed. In addition,MSC multilineage differentiation potential into adipocytes,osteocytes and chondrocytes was still achievable (data notshown).
Co-cultures with Schwann cells induces long-lasting neuraldifferentiation of MSCs
To assess the possibility of inducing long-lasting and moreselective neural differentiation, MSCs derived from the different
Fig. 4. Time course of myelin protein expression on MSCs co-cultured withSchwann cells. PKH-67-labeled MSCs from spleen and adipose tissue were co-cultured with human Schwann cells for 4, 7 and 12 days and then evaluated forthe expression of PMP-22 by double immunofluorescence.
Fig. 5. Schwann cell differentiation of MSCs induced by co-culture with Schwann cwith phenotypic markers (A2–E2) (red) in basal conditions (A, B) and after co-culturenot PMP-22 (B2), as evidenced with merge images (A3, B3). After 7 days of co-culturand S-100 (D2), but not CNS phenotypic antigens as NeuN (E2). Scale bar: 10 μm.
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tissues were co-cultured with human Sc. PKH-67-labeled MSCswere either pre-cultured or not with neural differentiationmedium, and incubated with Sc. MSC morphology andphenotype were assessed by double immunofluorescence withphenotypic markers. A preliminary set of time course experi-ments to assess the kinetics of neural differentiation, carried outwith spleen- and fat-derived MSCs co-cultured with Sc for 4, 7and 12 days, showed maximal expression of myelin proteins byMSCs at day 7 (Fig. 4). On the basis of this result, MSCs fromBM, spleen, thymus and adipose tissue were co-cultured withSc for 7 days. In basal conditions, all kinds of MSCs werespindle-shaped, CD105-positive and expressed barely detect-able levels of PMP-22 and S-100 markers (Figs. 5A, B). Afterthe sole co-culture with Sc, a proportion of PKH-67-labeledMSCs acquired the morphological features of Sc with bipolar,elongated morphology and irregular shape; an elevatedproportion of PKH-67-labeled MSCs selectively expressed S-100 and PMP-22 myelin proteins (Figs. 5C, D), with no
ells. PKH-67-labeled (green) MSCs from adipose tissue (A1–E1) double stainedwith Schwann cells (C–E). When cultured alone, MSCs display CD105 (A2) bute with Schwann cells, MSCs up-regulate the Schwann cell markers PMP-22 (C2)
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detectable levels of CD105 and of any neuronal or oligoden-droglial molecules employed (Fig. 5E). The proportion of these“Sc-like” cells obtained without any neural pre-treatmentdepended on the MSC origin. Fat-derived MSCs showed thehighest percentage of cells (almost one third) acquiring thephenotype of Sc (PMP-22/S-100+) after 7 days of co-culture,while such proportion was significantly lower with MSCs fromthymus and, particularly, from spleen and BM (Fig. 6).Interestingly, pre-treatment with the neural differentiationmedium before co-culture significantly increased the percentageof PKH-67-labeled MSCs from spleen, BM and thymusexpressing S-100 and PMP-22 (Fig. 6) molecules, up to levelscomparable with those observed with fat-derived MSCs. Evenin this experimental condition, no expression of CNS markerswas observed by MSCs.
To investigate the mechanisms by which Sc induced theneural differentiation of MSCs, we tested the effect of solublefactors. Sc-conditioned medium induced no morphologicalchanges in MSCs, which failed to express any myelin protein(data not shown), thus suggesting the importance of cell–cellcontacts. Experiments with irradiated Sc were then performed toassess whether cell fusion between MSCs and proliferating Scoccurred. Irradiation almost abolished Sc proliferation rate, asassessed by BrdU assay. After treatment with neural differ-entiation medium and Sc co-culture, the proportion of MSCs(from either BM- or thymus-derived MSCs) expressing myelinproteins (29.8±3.2%) was not significantly different from thoseco-cultured with non-irradiated Sc (27.9±2.8%; p>0.5).
Discussion
Evidence of neural differentiation has been obtained byseveral groups using MSCs derived from BM, with theacquisition of morphological, phenotypic and functionalfeatures of neural cells [22–28]; more recently, the samefindings have been shown with MSCs from adipose tissue
Fig. 6. Neural differentiation potential of MSCs of different origin after co-culture with Schwann cells. Fat-derived MSCs showed the highest percentage ofcells (almost one third) acquiring the phenotype of Schwann cells after 7 days ofco-culture, while the proportion of differentiated MSCs from thymus and,particularly, from spleen and BM was significantly lower. Pre-treatment withneural induction medium (NIM+) before co-culture significantly increased thepercentage of BM-, spleen-, and thymus-derived MSCs expressing PMP-22molecules, up to levels comparable with those observed in MSCs from adiposetissue. *p<0.0001. **p<0.03. ***p<0.009.
[8,31]. We have applied the same conditioning mediumemployed with BM MSCs [22] to induce neural differentiationof MSCs obtained from spleen and thymus. Our purpose was toassess whether cells with neural features could be achieved fromtissues that differ from BM in terms of development andfunctions. We show here that spleen and thymus have areservoir of MSCs with the same differentiation properties intolineages of mesodermal origin of those in BM; in addition, theseMSCs derived from lymphoid tissues can differentiate into cellswith morphological features of ectodermic origin after co-cultures with Schwann cells.
The process of neural differentiation of MSCs obtained withchemical factors is still matter of open debate. In very recentstudies, the morphological and phenotypic changes observed inBM MSCs after such treatment have been claimed to be a mereconsequence of cytoskeletal alterations [36–38]. In fact, similarmorphological changes have been observed also in fibroblastsor after chemical disruption of cytoskeletal architecture; inaddition, Western blotting and gene expression analyses spokeagainst a true up-regulation of neural markers [37]. In thisregard, we also observed very rapid morphological changes andgeneralized expression of neural markers after exposure ofMSCs from different tissues to neural differentiation medium,without a clear correlation between morphology and phenotype(for example, the expression of myelin antigens on neuron-likecells). In addition, we showed that prolonged exposures to suchchemical factors resulted in massive cell death; alternatively,when MSCs subjected to neural differentiation mediumreturned to basal conditions, cells retained their multilineagedifferentiation potential, thus suggesting the reversibility ofsuch process. Taken together, these lines of evidence stronglyquestion about the occurrence of a full neural differentiation. Onthe other hand, a total lack of specificity of the process of MSCneural differentiation after chemical treatment seems unlikely, ifwe consider the distinct effect exerted by these chemical factorson a homogeneous cell population as MSCs. In fact, combiningphenotypic and morphological analyses, we identified three cellpopulations of MSCs with different responses to neuraldifferentiation medium: about 60% of cells showed the above-mentioned changes (cells with bi- or tripolar shapes withprimary and secondary processes, and up-regulation of allphenotypic markers), about 20% of MSCs was induced toassume an oligodendrocyte-like morphology with selectiveexpression of GalC and the remaining MSCs showed noapparent change. Thus, it is likely that such chemical factorsplayed as a potent stimulus inducing initial, though poorlyspecific, changes of neural differentiation upon MSCs, assuggested by a recent study on the neuronal differentiation ofBM MSCs [29]. As shown for the acquisition of neuron-likeexcitability [24,28], additional factors provided by co-culturewith mature neural cells or by transplantation into the brain arethen needed to complete the process of neural differentiation.
Differently from neural differentiation induced by chemicalfactors, co-cultures with human Sc induced important and long-lasting (up to 12 days) morphological and phenotypic changesin all kinds of MSCs, resembling the typical features of Scdifferentiation, thus indicating that cell plasticity is a common
feature of all kinds of MSCs. In fact, co-culture with Sc resultedin the selective expression by MSCs of the Sc-specific proteinsPMP-22 and S-100, but not of CNS glial or neuronal markers.The comparison of the biological changes of MSCs derivedfrom different tissues after co-culture with Sc showed that thepercentage of fat-derived MSCs undergoing Sc differentiationwas significantly greater than that achieved with MSCs from theother tissues. In these latter cases, pre-treatment with neuraldifferentiation medium increased MSC ability to differentiatetoward Sc phenotype, up to levels comparable with fat-derivedMSCs. These observations suggest that neural differentiationmedium may start the initial steps of neural differentiation [29]and that Sc co-culture may provide the additional, necessaryconditions to complete such process. Regarding the mechan-isms underlying Sc differentiation of MSCs, we showed that Sc-MSCs direct contact was necessary to induce MSC morpholo-gical and phenotypic changes, as demonstrated by the lack ofany effect by simply adding to MSCs the supernatant of Sccultures. In addition, Sc irradiation before co-culture did notproduce any change in the proportion of MSCs undergoingdifferentiation, thus ruling out a major role of cell fusion in theexpression of myelin proteins by MSCs.
The main new result is the evidence that adult lymphoidtissues such as spleen and thymus, both with embryologicalorigin and functions distinct from BM, contain mesenchymalcell precursors with multilineage stem cell potential, includingneural-like differentiation. These MSCs are more rare inlymphoid tissues than in BM and fat, as shown by clonogenicassay (CFU-F, which measures the frequency of mesenchymalcell precursors inside the tissues of different origin), but onceexpanded in vitro they have qualitatively and quantitatively thesame differentiation potential into fat, bone, cartilage, andneural-like cells than BM and fat-derived MSCs. It is unclearwhether all these MSC pools originate inside the differenttissues or derive from the colonization by circulating BM-MSCs, but their existence throws new light upon theregenerative potential of adult tissues. MSCs could be involvedin normal local reconstruction of tissue structures of differentorigin in case of injury, and might be useful for clinical purposesif expanded or mobilized.
It has been suggested that BM-derived MSCs may haveimportant implications for chronic peripheral neuropathies,where disability is largely related to the limited regenerativecapacity of Sc. In fact, Sc normally undergo de-differentiation,proliferation and re-differentiation in response to injury [39],however, such processes may eventually fail if the damagepersists, as it happens in chronic neuropathies [40]. In the adultnervous system, the limited regenerative capacity of glial andneuronal cells could be compensated by local progenitors and/orstem cells. In this regard, it has been already shown that neuralstem cells, once transplanted in vivo, may promote tissue repairin the CNS [41]. By contrast, little is known about the presenceand hence the therapeutic potential of neural stem cells in PNSdisorders. In this contest, MSCs could represent a validalternative to neural stem cells; in fact, BM MSCs have beenshown to ameliorate CNS diseases by inducing remyelination[42]. As co-culture procedure may re-create (at least partially)
the microenvironment of peripheral nerves, our results suggestthat MSCs, once penetrated in the PNS, could differentiate intoSc and have a regenerative effect. In line with this hypothesisand with our results in vitro, chemical trans-differentiation of ratBM-MSCs into Schwann-like cells has been recently achieved;in addition, these trans-differentiated MSCs were able toimprove nerve regeneration and directly take part in themyelinating process in vivo [43]. Based on this evidence andon the regulatory effect described in CNS autoimmune diseases[44], we hypothesized that MSCs could be used in chronic,autoimmune neuropathies also considering their immuneregulatory properties, which we are currently investigating toassess if they persist along with neural differentiation. Inaddition, the evidence that MSCs with neural-like differentia-tion potential normally reside in central (i.e., thymus) andperipheral (i.e., spleen) lymphoid tissues, may help to under-stand how naïve and memory autoimmune responses againstmyelin proteins can occur far from the PNS.
Acknowledgments
This work has been funded by MIUR (Italian Ministry ofUniversity and Scientific Research, ex 60%) and FondazioneCariverona 2005.
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