Failure of Transdifferentiation of Adult Hematopoietic Stem Cells into Neurons - 《stemcells》干细胞学杂志干细胞之家



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作者：Laurent Roybona, Zhi Mab, Fredrik Asztelyc, Anna Fosumb, Sten Eirik W. Jacobsenb, Patrik Brundina, Jia-Yi Lia作者单位：a Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund, Sweden;b Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden;c Department of Clinical Neuroscience, Sahlgre 1 P, ?; Q, |) P' ^7 Y, Y6 m# p& O

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      【摘要】
   Previous studies of bone marrow-derived stem cell transdifferentiation into neurons have not involved purified cell populations and determined their exact phenotype prior to differentiation. The present study investigates whether highly purified mouse adult hematopoietic stem cells (HSCs), characterized by lineage marker depletion and expression of the cell surface markers Sca1 and c-Kit (Lin¨C Sca1  c-Kit  ), can be stimulated to adopt a neuronal fate. When the HSCLSK cells were cultured in vitro in neuronal differentiation medium supplemented with retinoic acid, 50% of the cells expressed the neural progenitor marker nestin and no cells had become postmitotic. Electrophysiological recordings on neuron-like cells showed that these cells were incapable of generating action potentials. When the HSCLSK cells either were grown in vitro together with neural precursor cells or were transplanted into the striatum or cerebellum of wild-type mouse, they either differentiated into Iba1-immunopositive macrophage/microglia or died. In conclusion, we demonstrate that adult HSCLSK cells do not have the capacity to leave the hematopoietic lineage and differentiate into neurons.
      【关键词】 Adult hematopoietic stem cells Fluorescence-activated cell sorting Plasticity Neural stem cells Microglia Macrophage Transdifferentiation
   INTRODUCTION
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Adult bone marrow contains a heterogeneous mixture of cells that is predominantly composed of bone marrow stromal cells and hematopoietic stem cells (HSCs) .$ t. A) X& Y% b& P" M( O
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Recently, bone marrow cells have been suggested to have a greater plasticity than previously anticipated and have been reported to cross the barrier of their commitment and differentiate along alternate lineages. The evidence for this came from sex-mismatched bone marrow transplantations (male to female) in irradiated mice and leukemia patients. Several months to years after HSC transplantation, cells expressing neuronal markers and the Y chromosome were found in the recipient brain. This suggested that they were neurons derived from the bone marrow donor cells . If this becomes true, it would avoid the rejection and immunosuppression problems associated with other source of tissues for transplantation and circumvent ethical issues associated with the use of embryonic and fetal cells.
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In our study, we isolated HSCs that were Lin¨C, Sca1 , and c-Kit  cells (HSCLSK) from adult mouse bone marrow as enriched or "pure" HSCs (more than 99% of purity after fluorescence-activated cell sorting . We used different approaches in attempts to induce neuronal differentiation. When exposed in vitro to soluble factors known to induce neuronal differentiation of neural stem cells and embryonic stem cells, a low number of these HSCLSK cells displayed some features of immature neural-like cells. However, in in vivo or in vitro neural environments, they either died or gave rise to cells with a macrophage/microglial phenotype.1 `8 q: B5 X4 ^7 ~: b8 Y5 K

MATERIALS AND METHODS
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HSCLSK Stem Cell Purification
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In this study, transgenic mice expressing green fluorescent protein (GFP) under the chicken ß-actin promoter were used. GFP transgenic mouse-derived HSCLSK cells were sorted on a FACS Vantage Cell Sorter (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com), equipped with 488 nm argon and 633 nm He-Ne lasers, at the rate of 2,000 to 8,000 cells per second. In brief, lineage-depleted (Lin¨C) cells were isolated from 7- to 9-week-old GFP transgenic mice after incubation of bone marrow cells with a cocktail of purified lineage (Lin) antibodies: RA3¨C6B2 (B220), RB6¨C8C5 (Gr1), M1/70 (Mac-1), 53¨C6.7 (CD8), 53¨C7 (CD5), H129.19 (CD4), and Ter-119, all anti-mouse from BD PharMingen (San Diego, http://www.bdbiosciences.com/pharmingen). Cells were incubated at 250 x 106 cells per ml with sheep anti-rat immunoglobulin G (IgG) (Fc)-conjugated immunomagnetic beads (Dynal Biotech, Oslo, Norway, http://www.invitrogen.com) at a cell/bead ratio of 1:0.3 at 4¡ãC for 45 minutes. Magnetic beads were removed with a magnetic particle concentrator (MPC-6; Dynal Biotech), and unattached cells were exposed to the same amount of magnetic beads and processed as for the first separation. Lin¨C cells recovered from the supernatant were further purified based on the expression of HSC markers Sca1 and c-kit. In brief, Lin¨C cells were resuspended at 100¨C400 x 106 cells per ml and incubated for 15 minutes on ice with CyChrome-conjugated goat anti-rat antibody (Caltag Laboratories, Burlingame, CA, http://www.caltag.com) and subsequently stained with phycoerythrin (PE)-conjugated rat anti-mouse E13¨C161.7 antibody (Sca1) and allophycocyanin-conjugated rat anti-mouse 2B8 (c-kit). Cells were stained with 7-amino actinomycin (7-AAD; Sigma, St. Louis, http://www.sigmaaldrich.com) to exclude dead cells. LSK GFP  cells showed a reproducible purity of 96%¨C99% and a cell viability of more than 99% when re-analyzed on FACS Vantage Cell Sorter (Becton, Dickinson and Company). Cell viability was confirmed using trypan blue exclusion method.

HSCLSK Stem Cell Proliferation
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Undifferentiated HSCLSK GFP  cells were expanded during 7 days in T75 flasks at the density 10,000 cells per ml in serum-free medium (X-vivo 15; BioWhittaker Molecular Applications, Rockland, ME, http://www.cambrex.com) supplemented by a cocktail of cytokines . Recombinant rat stem cell factor (rrSCF), recombinant mouse interleukin-3 (rmIL-3), recombinant human flt3 ligand (rhFL), and recombinant human thrombopoietin (rhTPO) (all from R&D Systems, Inc., Minneapolis, http://www.rndsystems.com) were used at the following concentrations: 25 ng/ml (rrSCF, rhFL, and rhTPO) and 10 ng/ml (rmIL-3).
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HSCLSK Differentiation in Vitro
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Two to three days before cell sorting, mouse stromal cell line, PA6 cells, (passage 7; Cell Bank, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan, http://www.brc.riken.jp/lab/cell/english) were plated as feeder cells on four- or eight-well glass chamber slides or collagen-coated glass coverslips on 24-well plates. HSCs were plated at the concentration of 1,000 cells per ml on monolayer PA6 cells. HSCs were differentiated using different conditions: (a) differentiation medium was supplemented with retinoic acid (RA) (1 µM; Sigma) during 6 days, then brain-derived neurotrophic factor (BDNF) was added until the 12th day (20 ng/ml; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and (b) RA was substituted by sonic hedgehog (SHH) and fibroblast growth factor (FGF) 8 (both 100 ng/ml; Invitrogen), also for 6 days before the addition of BDNF. In some experiments, in addition to BDNF, the medium was supplemented by ascorbic acid. As control, HSCs were cultured only on PA6 feeder cells in growth/neurotrophic factor-free basic differentiation medium. Alternative methods reported elsewhere have also been performed . The cells were fixed with 4% paraformaldehyde (PFA) after 4, 7, 12, and 14 days for immunocytochemistry.4 a* g, p! E# d/ A
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Sphere-Derived HSCLSK and Hematoneurosphere Formation and Differentiation In Vitro
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HSCs were seeded either alone to generate sphere-derived HSCs or mixed with ventral mesencephalic or cortical neural progenitor cells from embryonic day 14.5 (E14.5) rats at a density of 200,000 cells per ml in uncoated T25 flasks containing 10 ml of defined serum-free medium: Dulbecco¡¯s modified Eagle¡¯s medium-Ham¡¯s F-12 at the ratio 3:1 (Gibco), 2% B27 (Gibco), penicillin/streptomycin (50 u/ml:50 µg/ml; Sigma) supplemented with epidermal growth factor (EGF; 20 ng/ml), FGF-2 (20 ng/ml) (both from R&D Systems, Inc.), and Heparin (5 µg/ml). After 7 days, the sphere-derived HSCs or hematoneurospheres (HNSs) were plated in poly-L-lysine (PLL) glass coverslips and differentiated by withdrawing the mitogens EGF and FGF-2 (both at the concentration of 20 ng/ml; R&D Systems, Inc.) and supplementing the medium with SHH and FGF8 (both 100 ng/ml; Invitrogen) or RA (1 µM; Sigma) or without any supplements. Cultures were fixed after 3, 7, and 14 days for immunocytochemistry.6 U( @; _/ O5 S
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5-Bromo-2'-deoxyuridine Immunocytochemistry and Cytosine ß-D-Arabinofuranoside Exposure  i: s0 E4 Z0 a1 W7 R6 v% O4 G
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To examine whether differentiated HSCs can acquire a postmitotic phenotype, cells grown on PA6 stromal cells with medium supplemented with either RA or SHH FGF8 were treated with 5-bromo-2'-deoxyuridine (BrdU; 50 µM; Dako, Glostrup, Denmark, http://www.dako.com) for 48 hours after 6, 9, and 12 days differentiation. The cells were fixed with 4% PFA, and BrdU immunocytochemistry was performed after denaturation in 1 mM HCl at 65¡ãC for 10 minutes. In parallel, cultures, grown in the same conditions were exposed during 72 hours to cytosine ß-D-arabinofuranoside (AraC) (15 µg/ml; Sigma). Cultures were analyzed under fluorescent microscopy every day from the 1st day of incubation.

HSCLSK Differentiation In Vivo: Transplantations4 P# I, W! Y0 @" _! b) {4 _

Two microliters of GFP HSCs (50,000 cells/µl) from male adult GFP transgenic mice were injected by stereotaxic surgery into the striatum tooth bar  = ¨C3.8 mm or cerebellum (TB = 0.0 mm, AP = ¨C 6 mm, Lat = ¡À 2.5 mm, VD = ¨C 3 mm) of wild-type female adult mice recipients from the same strain. Mismatched-sex transplantations were performed in order to allow us to detect possible cell-fusion in case neuronal differentiation would have occurred. Mice were killed and perfused with 4% PFA 2 weeks and 1, 2, and 10 months after transplantation. Coronal brain sections were cut (thickness, 30 µm) for immunohistochemistry.& k2 m7 j4 J5 z# ]; ]1 D
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Electrophysiological Activity Recording7 M5 m$ y. {/ k8 `5 j' d6 E8 X; F
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For electrophysiological recordings, the glass chamber slides were transferred to a recording chamber with a medium consisting of 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, 1 mM NaH2PO4, and 11 mM glucose, which were gassed with 95% O2 and 5% CO2. The temperature of the recording chamber was kept between 21¨C23¡ãC. HSCLSK GFP  cells were identified and recorded under video-assisted differential interference contrast with an upright Zeiss Axioscope microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Whole-cell voltage-clamp recordings were made using pipettes (4¨C6 M) containing 122.5 mM potassium gluconate, 17.5 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 8 mM NaCl, 2 mM MgATP, and 0.3 mM GTP (pH 7.2; osmolarity 295 mOsm). Membrane currents were amplified and filtered at 2.9 kHz and sampled at 10 kHz with an EPC-9 patch-clamp amplifier. Liquid junction potential was calculated to ¨C 5.8 mV. Application of kainate (50 µM) was made by bath application. The depolarizing ramp (¨C 110 to 110 mV) experiments were performed in the presence of 1 µM tetrodotoxin to block possible voltage-dependent sodium channels . The current-voltage plot was calculated as the current at a given voltage in the presence of kainate subtracted by the current at the same voltage before the application of kainate./ R- b7 H# ?* r+ T
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Immunocytochemistry and Immunohistochemistry
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Immunocytochemistry and immunohistochemistry were performed according to standard protocol using the following primary antibodies: nestin (intermediate filament, 1:500, mouse; Pharmingen), Vimentin (intermediate filament, 1:200, mouse; Sigma), Musashi (RNA binding protein, 1:200, rabbit; Chemicon, Temecula, CA, http://www.chemicon.com), ß-tubulin isotype III (1:500, mouse; Sigma), ß-tubulin isotype III (1:500, rabbit; Covance, Princeton, NJ, http://www.covance.com), microtubule-associated protein 2 (MAP2; 1:500, mouse; Sigma), neurofilament (NF)-68 (neurofilament, 1:500, mouse; Sigma), NF-200 (neurofilament, 1:500, rabbit; Sigma), NCAM (1:500, rabbit; Chemicon), polysialic acid (PSA)-NCAM, (1:500, mouse; Chemicon), doublecortin (DCX; microtubule associated protein, 1:500, Guinea pig; Chemicon), glial fibrillary acidic protein (GFAP; 1:1200, rabbit; Dako), Gal-C (galactocerebroside, 1:200, rabbit; Sigma), O4 (O-antigen sulfatide, 1:200, mouse; Chemicon), TH (tyrosine hydroxylase, 1:500, rabbit; Chemicon), GABA(a) receptor ß-2/3 (1:100, mouse; Upstate Biotechnology, Inc., Lake Placid, NY, http://www.upstatebiotech.com), GABA(a) receptor -1 (1:200, rabbit; Chemicon), CD45 (protein tyrosine phosphatase, 1:200, rat; NeoMarkers, Fremont, CA, http://www.neomarkers.com), GFP (1:1,200, chicken; Chemicon), Ki67 (nuclear antigen expressed in all proliferating cells during late G1, S, M, and G2 phases of the cell cycle, mouse, 1:200; NOVOCASTRA, Newcastle, U.K., http://www.novocastra.co.uk), and Iba1 (Calcium-binding protein, 1:500, rabbit; Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp). Appropriate secondary antibodies biotinylated anti-rat IgG (1:200; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), streptavidin ALEXA-647, ALEXA-488, and ALEXA-595 (1:200; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) were used for confocal and fluorescent microscopy imaging. In some experiments, cells were counterstained using Hoescht staining (5 µg/ml; Molecular Probes). For immunocytochemistry of freshly sorted cells, centrifugation of cells was performed at 500g for 10 minutes between each step of the immunocytochemistry.1 ^8 n2 ]0 [' ^& R! p2 Y$ r
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Quantification and Image Processing" @+ [& C! ^( U0 B
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The number of cells immunopositive for neuronal markers was assessed using stereological analysis. The coverslip surface area covered by the cells was delimited using x 10 objective, whereas the different types of cells were counted using x 40 objective, under fluorescence microscope (Leica DMRBE, Leica, Heerbrugg, Switzerland, http://www.leica.com). Hoechst 33,342 (Molecular Probes) and GFP expression allowed us to distinguish the HSCs from the cocultured PA6 feeder cells that have a characteristically big nucleus with several spots of chromatin that appear as bright blue dots. Specimen analyses were performed using a Leica fluorescent microscope (Leica DMRBE, software Openlab) and Leica confocal microscope (Leica software, equipped with a GreNe and a HeNe lasers with the following lines of excitation: 488, 594, and 647 nm). Samples were analyzed using x 10, x 20, and x 63 objectives. Images of positive stainings were adjusted to make optimal use of the dynamic range of detection. Background settings were adjusted from the examination of immunostainings performed on positive control specimens. Figures were composed in CANVAS9 software.

RESULTS
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Phenotypic Conversion of HSCLSK Cells into Neural-Like Cells In Vitro3 m7 P8 `1 O3 J2 x0 r# K+ V+ Y9 T

To assess whether HSCLSK cells could convert their fate to neural differentiation, we first isolated GFP  HSCLSK cells prior to inducing their differentiation. GFP  HSCLSK cells were selected by FACS according to their size and granularity (Fig. 1A), their lack of lineage markers (lin¨C), viability (negative for 7AAD) and GFP expression (Fig. 1B), and finally their immunopositivity for the cell surface markers Sca1 and c-Kit (Fig. 1C). Subsequently, for all conditions tested, these GFP  HSCLSK cells were either proliferated for 1 week or directly exposed to neurogenic factors., Y, k5 x8 P+ U7 i# ?

Figure 1. Isolation and purification of adult HSCLSK cells. (A¨CC): FACS of GFP adult HSCs according to their size and granulosity (, black frame). (D¨CF): FACS-sorted HSCLSK cells express GFP (green) and CD45 (pink) and are immunonegative for the neural, neuronal, and microglial markers nestin (D), ß-III-tubulin (E), and Iba1 (F), respectively (red). For triple staining, (D¨CF) were taken using confocal microscope (objective x63). Scale bars = 50 µm (D¨CF). Abbreviations: 7AAD, 7-amino actinomycin D; APC, allophycocyanin; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FSC, forward scatter; GFP, green fluorescent protein; HSC, hematopoietic stem cell; HSCLSK, hematopoietic stem cell that is Lin¨C, Sca1 , and c-Kit ; PE, phycoerythrin; SSC, side scatter.9 t9 a! }( y# f# C/ ^. e& v( y
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Prior to neural differentiation, expression of neural markers in the GFP  HSCLSK population was assessed. Freshly FACS-sorted GFP  HSCLSK cells were fixed in 4% PFA, and immunocytochemistry was performed. The cells were positive for GFP and CD45 (Fig. 1D¨C1F), a panhematopoietic marker, but were negative for the following neural markers: nestin (Fig. 1D), ß-III-tubulin (Fig. 1E), and the macrophage/microglia marker Iba1 (Fig. 1F). GFP  HSCLSK cells were also found immunonegative for the markers Musashi, MAP2, and neuronal nuclei (NeuN) (data not shown).

GFP  HSCLSK cells were then plated at 10,000 to 30,000 cells per ml on PLL/laminin (PLL/L)-coated eight-well glass chamber slides. The medium was supplemented with RA or a combination of SHH and FGF8 for 6 days. Regardless of their concentration, GFP  HSCLSK cells did not survive in this environment.
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GFP  HSCLSK cells were then cultured on mouse bone marrow stromal cell feeder (PA6 cells, passage 7; Cell Bank, RIKEN BioResource Center) that have been shown to maintain HSC survival in vitro . GFP  HSCLSK cells were plated on PA6 stromal cells at a concentration of 1,000 cells per ml. GFP  HSCLSK cells survived and adopted star-shaped morphologies over time. However, when the GFP  HSCLSK cells were maintained in basic differentiation medium without any supplementation, none of the GFP  HSCLSK cells became immunopositive for the neural markers nestin and ß-III-tubulin.
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We then applied several additional cell differentiation protocols in attempts to induce neural differentiation of HSCLSK cells. These were based on protocols shown to promote neural differentiation of either neural or embryonic stem cells. We first supplemented the differentiation medium with SHH and FGF8, inducers of differentiation of midbrain neural stem cells into dopaminergic neurons. The GFP  HSCLSK cells adopted a flat morphology and proliferated extensively to confluence within 6 days (Fig. 2A). During a period of 8 days, the cells had formed multiple layers and started to detach. At this time, after 6 days of differentiation, less than 30% of the GFP  HSCLSK cells were immunopositive for nestin, but all were negative for neuronal or glial markers. In an alternate condition, we supplemented the media with RA. The GFP  HSCLSK cells proliferated less and adopted, after 6 days, a neuronal cell-like morphology, extending long bipolar processes (Fig. 2B). To prolong the survival time of these neuronal-like cells that mainly started to retract their bipolar extensions after 6 days in vitro, BDNF was added to the medium at day 6. After 6, 9, and 12 days of culture in vitro, cultures were fixed and immunocytochemistry was performed with neural markers. Fifty percent of the cells were found to express nestin after 6 days (Fig. 2C), but none of them was positive for the early neural marker Musashi (data not shown). In addition, another early neural marker, vimentin, was also examined. All cells in the culture, differentiated GFP  HSCLSK and PA6 stromal cells, were found to be immunopositive (data not shown). However, vimentin is not considered a specific early neural marker, because it can also stain fibroblasts . When we examined a total of approximately 100,000 cells from more than 15 separate FACS experiments, we found less than 10 cells to be immunopositive for ß-III-tubulin (Fig. 2D¨C2F). These cells were found in cultures grown on PA6 stromal cells, in medium supplemented with RA only. Interestingly, after neural differentiation, GFP  HSCLSK cells were not immunopositive for the astrocytic marker GFAP, the oligodendrocytic marker O4, or neuronal markers such as MAP2, NF-68, NF-200, DCX, NCAM, NeuN, or the dopamine-neuron related enzyme TH. More importantly, cells that were immunopositive for nestin had not downregulated the expression of the panhematopoietic marker CD45 (Fig. 2G), showing that they had kept their hematopoietic identity.. s: ~( f/ z6 ?. V5 n0 [

Figure 2. HSCLSK cells cultured on PA6 stromal cells adopt a neural-like morphology when differentiated in vitro with RA. (A): GFP HSCLSK cells cultured on PA6 stromal cells with medium supplemented with SHH and FGF8 extensively proliferate and give rise to star-shape morphology cells. (B): Cells cultured on PA6 stromal cells in presence of RA adopt a bipolar neuronal precursors-like morphology. (C): GFP HSCLSK cells (green) express the neural marker nestin (red) after 6 days in vitro cultures on PA6 stromal cells with medium supplemented with RA. Both GFP HSCLSK neural-like cells (arrowhead) and non-neural-like cells (asterisk), cultured on PA6 stromal cells in presence of RA, express the panhematopoietic marker CD45 (blue) and nestin (red). (D¨CF): GFP HSCLSK cells (green) immunopositive for the neural marker nestin (red) after 6 days culture on PA6 stromal cells with RA. (G¨CI): GFP HSCLSK cells (green) immunopositive for the neuronal marker ß-III-tubulin (red) after 6 days culture on PA6 stromal cells with RA. Arrowheads show bipolar processes cell-immunopositive for ß-III-tubulin. For triple labeling, (C) was taken using a confocal microscope. Scale bars = 100 µm (A, B, G¨CI), 15 µm (C), and 50 µm (D¨CF). Abbreviations: FGF8, fibroblast growth factor 8; GFP, green fluorescent protein; HSCLSK, hematopoietic stem cell that is Lin¨C , Sca1 , and c-Kit ; RA, retinoic acid; SHH, sonic hedgehog.9 C+ E. M' d0 `
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Furthermore, we tried to induce neural differentiation with protocols that have been successfully used to differentiate multipotent adult progenitor cells from bone marrow into neuron-like cells . There was an intense proliferation of the HSCLSK cells during to the initial step in which knockout serum was supplemented to the medium. Under none of the aforementioned conditions did we obtain cells that expressed neural markers (data not shown)., P! F/ r/ S: p( U) K
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Electrophysiological Activity of In Vitro-Differentiated HSCLSK Cells
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After 7 days of culture in presence of RA and BDNF, GFP  cells showing neuron-like morphology with long processes were selected and electrophysiological properties were recorded (Fig. 3A). Whole-cell current and voltage-clamp recordings were performed on single cells, using a K  -based electrode solution. The average resting membrane potential was ¨C39.3 ¡À 4.2 mV (n = 18), and the average input resistance was found to be 0.35 ¡À 0.07 G. None of these cells showed an ability to generate action potentials in current-clamp mode (Fig. 3B). In voltage-clamp mode, recordings in epochs of up to 5 minutes (n = 9, cells held at ¨C 60 or 0 mV) revealed no spontaneous postsynaptic activity. It has been described that application of kainite, a nonselective AMPA/kainate agonist, to neural progenitor cells elicits currents, suggesting that they express functional glutamatergic ionotropic receptor channels . We therefore performed experiments to determine whether our cells express functional glutamatergic ionotropic receptors. However, the addition of the nonselective AMPA/kainate agonist kainate (50 µM) did not induce any currents in these cells (n = 12; Fig. 3C).

Figure 3. Electrophysiological activity of in vitro differentiated HC-SLSK cells. (A): A cell with an attached recording pipette (scale bar = 10 µm). (B): The cells showed no spikes during current injections in current clamp recordings. (C): Responses to a depolarizing ramp (¨C 110¨C110 mV) before, during kainate (50 µM) application, and wash-out. No differences were found between the responses obtained in the presence of kainate and before and after washout (control), respectively. The corresponding I-V curve is shown in the bottom panel.( R2 O+ q9 H2 w

No Cell Cycle Withdrawal of Differentiated HSCLSK Cells6 @' k4 A! I; l) {/ F
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One of the criteria required for the classification of a neuron is its ability to withdraw from the cell cycle and become postmitotic. To assess whether the neuronal-like cells cultured on PA6 stromal cells in presence of RA were postmitotic, differentiated GFP  HSCLSK cells at 6, 9, and 12 days were exposed to BrdU for 48 hours and then fixed for immunocytochemistry. Less than 1% of the GFP  HSCLSK cells with a bipolar neuronal-like morphology were immunonegative for BrdU at any time point. To verify this observation, some of the cultures grown according to the above-mentioned conditions (e.g., with RA and BDNF) were stained for the proliferation marker Ki67 at 6, 9, and 12 days of differentiation. Less than 1% of the neuron-like cells were found to be immunonegative for Ki67 (Fig. 4A¨C4D). Thus, less than 1% of the cells were found to be in a non-proliferative state according to both assays.
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Figure 4. High proliferation rate of differentiated HSCLSK cells. (A¨CD): GFP HSCLSK cells (C) grown on PA6 stromal cells in presence of RA were incubated for 48 hours with BrdU. Less than 1% of the cells were found BrdU-immunonegative (, arrowhead). A similar proportion of BrdU-labeled cells was found at either time points of differentiation. (B): Cells were counterstained with Hoescht (blue). Scale bars = 100 µm.

In an attempt to determine conclusively whether the non-BrdU cells obtained in our culture were postmitotic neuron-like cells, 6-day in vitro cultures grown on PA6 stromal cells with medium supplemented with either RA or SHH FGF8 were exposed during 72 hours to AraC, a selective inhibitor of DNA synthesis. After 72 hours, living cultures were observed under fluorescent microscope. In both conditions, no GFP cells could be detected. We therefore conclude that rather than being post-mitotic, differentiated cells that were negative for BrdU or positive for Ki67 had undergone slow division, because 72 hours of treatment with AraC had killed all cells in the culture.' d. c$ v" f& e" K2 p/ c

Sphere-Derived HSCs Do Not Differentiate into Neurons4 w& b/ ^7 k/ l/ M: }
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It has previously been shown that neural stem cells are able to generate neurospheres when cultured on medium supplemented with mitogens such as EGF and FGF2 . Previous techniques successfully used to generate neurospheres from cortical and ventral mesencephalic embryonic tissues (Fig. 5A) were applied to the GFP  HSCLSK cells. The GFP  HSCLSK cells were expanded for 1 week and then seeded in neurosphere formation medium supplemented with EGF and FGF2. Under these conditions, they died within a few days and did not give rise to any neurospheres-like aggregates. In contrast, when the GFP  HSCLSK cells were exposed to hematopoietic proliferation medium supplemented by cytokines for more than 10 days, we observed free-floating aggregates (Fig. 5B). Their size increased with time when continuously exposed to proliferation medium, but the size of the cells was not homogenous. When the medium was exchanged to differentiation medium supplemented with RA or SHH and FGF8, HSCLSK-derived spheres did not give rise to any neural cells.$ ]* l, p# i" o
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Figure 5. GFP HSCLSK-derived spheres and hematoneurospheres do not give rise to neural cells upon differentiation. (A): Embryonic day-14.5 rat ventral mesencephalic neural progenitors grown as neurospheres (passage 1) in presence of EGF and FGF2. (B): GFP HSCLSK cells were seeded in proliferation medium supplemented by cytokines. After 10 days, GFP HSCLSK-derived spheres were generated. (C): Seven-day proliferating hematoneuropsheres. HSCLSK cells can be visualized according to their GFP expression, among the neural cells in the neurosphere core. (D): GFP HSCLSK-derived spheres are immunonegative for the neural marker nestin compared with the neural cells derived from the ventral mesencephalic progenitors. (E¨CP): Seven-day differentiation of hematoneurospheres. (E¨CH): GFP HSCLSK cells (E) are immunonegative for the neural marker nestin (, pink). Phase-contrast images were taken in (A, C). For (C), phase-contrast and fluorescent images were merged. Confocal microscopy images were taken for (E¨CP). Scale bars = 200 µm (A), 75 µm (B¨CD), and 50 µm (E¨CP). Abbreviations: BrdU, 5-bromo-2-deoxyuridine; EGF, epidermal growth factor; FGF2, fibroblast growth factor 2; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; HSCLSK, hematopoietic stem cell that is Lin¨C, Sca1 , and c-Kit ; MAP2, microtubule associated protein 2; RA, retinoic acid.

In Vitro Neural Environment Does Not Induce Neuronal Differentiation of HSCLSK Cells1 U' q4 ?+ o& W/ y3 K8 l# B4 e) Y
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We then tested whether an embryonic neural environment provided by tissue-specific-generated neurospheres could induce neural differentiation of the HSCLSK cells. Neurospheres are composed of progenitor cells derived from a single stem cell when cells are seeded at low density. The neighboring cells interact with each other and influence the differentiation of adjacent cells. Chimeric neurospheres were generated by seeding a mixture of GFP  HSCLSK cells and dissociated rat neural progenitors derived from either cortex or ventral mesencephalon of E14.5 embryos. We arbitrarily named these "chimeric HNSs." We generated such HNSs in neurosphere formation medium during 7 days (Fig. 5C) and stimulated their differentiation for 3, 7, and 14 days. Prior to differentiation, none of the GFP  HSCLSK cells present in the HNS core was immunopositive for the early neural marker nestin (Fig. 5D). The HNSs were differentiated as described above: cultured with RA or SHH FGF8 for 6 days, followed by the addition of BDNF up to 14 days, on PLL/L-coated coverslips. Regardless of the tissue origin of the chimeric neurospheres (E14.5 cortex or ventral mesencephalon), the differentiation medium used, or the number of days of BDNF supplementation, GFP  cells did not express any of the following neural, neuronal, and glial markers: nestin (Fig. 5E¨C5H), ß-III-tubulin (mono- and polyclonal primary antibodies tested; Figure 5I shows immunocytochemistry result for the monoclonal antibody), MAP2 (mono- and polyclonal primary antibodies tested; Figure 5J shows immunochemistry result for the monoclonal antibody), GFAP (Fig. 5K), and O4 (Fig. 5L). However, GFP  cells were all immunopositive for the macrophage/microglial cell marker Iba1 (Fig. 5M¨C5P). Furthermore, all GFP  cells were immunopositive for the panhematopoietic marker CD45 (Fig. 5G¨C5L, 5O, and 5P). We also tested whether differentiated GFP  HSCLSK cells could express the following neural markers: NF-68, NF-200, TH, NCAM, and GalC (galactocerebroside). No single differentiated GFP  HSCLSK cell was found immunopositive for those markers (data not shown).
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Adult Brain Environment Does Not Induce Neuronal Differentiation of HSCLSK Cells
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To evaluate the impact of an adult neural in vivo environment on neural differentiation, 50,000 FACS-sorted nondifferentiated GFP  HSCLSK cells were transplanted into the striatum or cerebellum of wild-type adult mice from the same strain. Surviving cells were detectable after 3 weeks, as evidenced by GFP immunofluorescence. Only a few (Fig. 6D, 6F, and 6G) or no grafted cells (Fig. 6A, 6C) were detectable in the brain sections after 1 month. Immunohistochemical analysis revealed that none of the surviving transplanted cells was immunopositive for the neuronal markers NeuN (Fig. 6A¨C 6C) or calbindin (Fig. 6D¨C 6G).

Figure 6. Transplanted GFP HSCLSK cells do not differentiate into neural cells in the striatum and the cerebellum. (A¨CC): GFP HSCLSK cells were stereotaxically transplanted into the striatum of wild-type mice (A). After 1 month, dead cells or necrotic bodies were found at the transplantation site (, blue). Triple staining in (D¨CG) was taken using a confocal microscope. Scale bars = 200 µm (A, B, D, F), 100 µm (C), and 50 µm (G). Abbreviations: GFP, green fluorescent protein; HSCLSK, hematopoietic stem cell that is Lin¨C, Sca1 , and c-Kit ; NeuN, neuronal nuclei.) O; F" o5 H) v  M8 J; Q5 q
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FACS-Sorted Cells from the Lin¨C Sca1  c-Kit  Population Express Nestin and ß-III-Tubulin
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Cord blood cells have also been shown to express neural proteins . When FACS-sorted, GFP  HSCLSK cells were not immunopositive for any neural and neuronal markers (Fig. 1D¨C1F). In an attempt to align these data with previous observations, we decided to analyze whether other hematopoietic populations could be immunoreactive for such markers. Interestingly, we found that a fraction of more committed hematopoietic cells were positive for certain neuronal markers depending on their subpopulation. After FACS, we collected three other pools of cells: Lin¨C Sca1¨C c-Kit¨C, Lin¨C Sca1¨C c-Kit , and Lin¨C Sca1  c-Kit¨C. We found that few Lin¨C Sca1¨C c-Kit¨C and Lin¨C Sca1  c-Kit¨C cells expressed the neural marker nestin (
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Figure 7. Nonadult HSCLSK cells can express neural markers. (A¨CF): Immunocytochemistry for neural markers on fluorescence-activated cell sorting (FACS)-sorted hematopoietic cell populations Lin¨C Sca1¨C cKit  and Lin¨C Sca1  cKit¨C. (A, C): Among the FACS-sorted Lin¨C Sca1¨C cKit¨C and Lin¨C Sca1¨C cKit  hematopoietic cell populations, cells express the neural marker nestin (red). (B, D): Neither Lin¨C Sca1¨C cKit¨C cells nor Lin¨C Sca1¨C cKit  cells express the neuronal marker ß-III-tubulin (red). (E, F): Cells in the Lin¨C Sca1  cKit¨C population express the neural and neuronal markers nestin and ß-III-tubulin. Scale bars = 50 µm. Abbreviation: GFP, green fluorescent protein.

When these cell populations were subjected to the above conditions designed to promote neural differentiation, they never exhibited immunopositivity for more mature neural markers or adopted a more mature neuron-like morphology than what we found for the GFP  HSCLSK cells. Instead, they retained a nestin expression and a bipolar morphology.8 K. F, |+ a9 ^& M8 B$ Q' {. F

In conclusion, immediately after purification, Lin¨C Sca1¨C c-Kit¨C and Lin¨C Sca1¨C c-Kit  populations expressed the neural-like marker nestin, whereas the Lin¨C Sca1  c-Kit¨C population coexpressed nestin and ß-III-tubulin; however, upon differentiation, none of these three populations gave rise to neurons (data not shown).
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DISCUSSION- C* Y! n& H8 n% C" e, S
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Blood cells and, more precisely, bone marrow stromal cells, mesenchymal stem cells, or cord blood cells have previously been reported to differentiate into neural cell types when exposed to neurogenic factors . To our knowledge, no study has addressed whether a pure population of HSCs has the potential to differentiate into neural cells.

We demonstrate that HSCLSK-derived cells cannot adopt features of mature neurons under the conditions we studied. The HSCLSK cells could not be maintained alive in vitro without the support of PA6 stromal cells or supplementation with specific cytokines. On stromal cells, when the differentiation medium was supplemented with RA, the HSCLSK cells adopted a bipolar neuronal-like morphology. They exhibited processes that increased in length over time. There was a high expression of the early neural marker nestin, but not Musashi, and very rarely (not more than 10 cells out of 15 FACS experiments of 200,000 HSCLSK cells) did cells express the immature neuronal marker ß-III-tubulin. In addition, very few cells had a neuron-like morphology. The fact that all cells were vimentin-immunopositive, including the PA6 cells, shows us that the commonly used early neural markers Vimentin and nestin are not appropriate when assessing transdifferentiation toward a neuronal fate. Indeed, both vi-mentin and nestin can be found in endothelial cells and different organs outside the brain .

Our observations of the electrophysiological properties of the HSC-derived cells are hardly compatible with their being mature neurons. We realized the difficulties in estimating and interpreting the resting membrane potential with whole-cell recordings. Nevertheless, the estimated shallow resting membrane potential (¨C39 mV) in combination with a rather low input resistance , an inability to generate action potentials, and no electrophysiological evidence of functional AMPA/kainate receptors suggest that the cells failed to differentiate into neurons. Taken together, the electrophysiological properties and the high expression of the neural marker nestin and very low or almost nonexistent expression of ß-III-tubulin and other neuronal markers suggest that these cells are not neurons.

Interestingly, the nestin-positive cells did not stop dividing when differentiated with RA. Some of the differentiated cells were thought to have withdrawn from the cell cycle and apparently became postmitotic, but this population was very small (' S. Y: {1 _4 j7 q

Furthermore, protocols that have been used to successfully differentiate multipotent adult progenitors and mouse embryonic stem cells , differentiation protocols cannot be standardized, because their responses to the same soluble factors and coculture conditions differ.% d, V% ~2 J# k$ ?  J
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In Vitro and In Vivo Neural Environments Do Not Induce Differentiation of HSCLSK Cells Toward a Neural Fate: w# l# y5 V, ^. u: F- @$ j  P, I4 D  j
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Secreted soluble factors and cell-cell interaction participate in the maintenance of survival of cultured cells. To exploit the possible differentiating potential of these factors, we generated chimeric HNSs in vitro and transplanted GFP  HSCLSK cells into two different regions of the adult brain. The GFP  HSCLSK cells survived, and we could detect them in both ventral mes-encephalic and cortical HNS cores. When induced to differentiate, neural cells that composed the chimeric spheres gave rise to all neural cell types: astrocytes, oligodendrocytes, and neurons (Fig. 5). However, none of the GFP  cells, all CD45 , expressed any neural markers, not even nestin. In contrast, they were immunopositive for the macrophage/microglial marker Iba1. This indicates that the signal(s) given in vitro by the embryonic developing cells in neurospheres do not promote neural differentiation of the HSCLSK cells. When grafted into the striatum, the majority of implanted HSCLSK cells did not survive for a month. At earlier time points, they did not express any neural markers, such as NeuN or ß-III-tubulin. Only necrotic cell bodies were found at the transplantation site. In the cerebellum, only a few grafted cells were found to be alive. These cells were immunonegative for general neural markers as well as the specific Purkinje cell marker calbindin. The HSCLSK cells gave rise only to macrophage/microglial cells immunopositive for Iba1 after grafting into the brain. This is in agreement with published reports concerning stem cells obtained from cord blood, aorta gonad mesonephros-derived, and stromal cells . Possibly, HSCLSK cells are programmed to give rise to hematopoietic cells when they reside in the bone marrow "niche"; however, they differentiate into macrophage/microglia when they interact within a neural environment.
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Subpopulation of Hematopoietic Cells in the Bone Marrow Can Express Neural Proteins
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We observed that other subpopulations of hematopoietic cells can coexpress neural markers. It has been previously reported that hematopoietic progenitors, as well as primordial HSCs, can express neural markers .
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CONCLUSION) P" C/ |' h' G6 Q5 U3 ^

The high potential of plasticity of stem cells has raised the possibility that they could be used to treat several diseases. HSCs have successfully and routinely been transplanted into patients suffering from leukemia and other blood disorders. Inconsistent evidence on plasticity of HSCs crossing different germ layers raises questions of whether transdifferentiation actually exists . The present study shows that plasticity of highly purified HSCs toward neural differentiation is very rare if not nonexistent. Under the current experimental paradigms, considerable numbers of HSCs could adopt primitive neural-like phenotypes but did not differentiate into mature neurons. Taken together, the inability of HSCs to transdifferentiate into neurons under different in vitro and in vivo conditions does not support the use of HSCs as a source of donor cells for neural transplantation.( r6 F& c1 G. ?' [: l0 q
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DISCLOSURES( X9 y9 {4 B" T" S9 w
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS# C7 x& n+ ~" a

This work was supported by the National Institutes of Health (grant one R21 NS043717-01A1 P1), the Swedish Research Council, and the Swedish Parkinson Foundation. The Lund Stem Cell Center is supported by a Center of Excellence grant from the Swedish Foundation for Strategic Research. We thank Birgit Haraldsson, Britt Lindberg, Jens Nygren, and Lisa Nyborg for technical advice and assistance, our colleagues at the Neuronal Survival Unit for critical discussions of the data, and Emma Idman for critical reading of the manuscript.0 `' E- V: i# c2 a/ Q
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