Annexe I :
Enrichment and isolation of neural crest stem cells from adult bone marrow as a source of neurons.
Laudet E, Alix P, Neirinckx V, Shakhova O, Sommer L, Rogister B*, Wislet-Gendebien S*. Soumis.
Enrichment and isolation of neural crest stem cells from adult bone marrow as a source of neurons
Emerence Laudeta, Philippe Alixa, Virginie Neirinckxa, Olga Shakhovab, Lukas Sommerb, Bernard Rogister*a, c, d and Sabine Wislet-Gendebien*a
a GIGA Neurosciences, University of Liège, Liège, Belgium
b Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland
c Department of Neurology, CHU, University of Liège, Liège, Belgium
d Stem Cells and Regenerative Medicine, GIGA Development, University of Liège, Liège, Belgium
* Equally contributed
Recent studies have described presence of multipotent neural crest-derived cells in accessible locations, such as skin or bone marrow, and raised new hopes regarding their potential use for cell replacement therapies. However, specific markers have not been yet identified and isolation of those cells still remains impossible without any transgenic construct. Consequently, to consider neural crest stem cells (NCSC) from adult bone marrow as a potential source for cellular therapy protocol, development of strategies for specific isolation of NCSC was mandatory. In this study, two protocols have been developed: 1) an enrichment protocol using several membrane markers which increased proportion of NCSC in BMSC culture at around 60 % after 3 passages. 2) Sphere-forming protocol allowed us to obtain a pure population of NCSC after 4 passages. Furthermore, sphere formation at clonal density and subcloning experiment demonstrated the self-renewal capacity of NCSC and the recruitment of immature cells. Multipotency of those NCSC has then been addressed demonstrating their ability to differentiate into chondrocytes, melanocytes, osteocytes, glia and neurons. More interestingly, NCSC displayed a higher potential of immature neural differentiation in neurotrophins-containing medium, however only the contact with neuronal progenitors allowed them to reach a further mature/functional stage as they expressed voltage- gated Na+ and K+ channels.
Keyword: Neural crest, bone marrow, self-renew, multipotency, neurotrophins
Abbreviations: APA, Alkaline phosphatase activity; BMC, Bone marrow cells; BMSC, Bone marrow stromal cells; CGN, Cerebellar granule neurons; GFAP, Glial fibrillary acidic
protein; GFP, Green fluorescent protein; MSC, Mesenchymal stem cells; NC, Neural crest;
NCC, Neural crest cells; NCSC, Neural crest stem cells.
Although the adult brain contains small numbers of stem cells in restricted areas, the central nervous system exhibits limited capacity of regenerating lost tissue. Therefore, cell replacement therapies of damaged brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. The generation of neuronal cells from stem cells obtained from various adult mesenchymal tissues including bone marrow is of significant clinical interest. Indeed, stem cells could be isolated from the patient himself and, after minimal manipulations, those cells would be grafted in the compromised tissue. This idea has been even more reinforced, since several groups have identified neural crest stem cells (NCSC) in various accessible adult tissues such as skin or bone marrow, making them potential source of stem cells for therapies as those cells could be a source of neuronal precursors [1-4].
Neural crest cells (NCC) appear during neurulation. They delaminate from the dorsal part of the forming neural tube and undergo an epithelial-to-mesenchymal transition .
Afterwards, NCC migrate along characteristic pathways to differentiate into a wide variety of mature cells [6, 7]. Pathways of migration and multipotency of NCC have been largely described, especially in the avian model [8, 9]. Recently, the use of double transgenic mice allowed a finer lineage tracking of NCC in mammals. In this model, transgenic mice expressing CRE-recombinase under promoters of genes activated during neural crest (NC) formation, were mated to a Cre-reporter mouse, i.e. ROSA26-LacZ or -GFP/-EYFP [10, 11].
Promoters of genes that have been used in such transgenic mice were: Wingless/INT-related 1 (Wnt1) , tissue plasminogen activator (tPA) , myelin protein zero (P0)  and SRY- related HMG box (Sox10)  to track the final NCC fate and location. By this approach, every NCC can be permanently labeled and “followed” from embryonic stage to adulthood.
Wnt1-Cre construct has been used by several authors because of the transient expression of
the proto-oncogene Wnt1 in the dorsal neural primordium and in the migrating NCC .
Using this transgenic model, Sucov’s group followed the distribution of mammalian NCC in the pharyngeal and cardiac region , but also attested the contribution of cranial NCC during tooth and mandibular morphogenesis .
Regarding the potential interest of using bone marrow NCC in therapeutic protocols, a better characterization of those cells appears to be essential. However, we have to face several major issues like the fact that NCC constitute a minority cell population in this tissue and that there is no specific marker to identify them. Consequently, development of strategies for specific isolation or enrichment of NCC is mandatory.
In this study, we described approaches to enrich NCC in stromal cultures, first by using membrane markers with a yield of around 60 %, and second by a sphere-forming protocol to obtain a pure NCC culture. Self-renewal ability and multipotency of those sphere- derived NCC were also addressed and we observed that they were able to differentiate into chondrocytes, melanocytes, osteocytes, neurons and glia. Furthermore, by specifically addressing neuronal fate, we showed that neuronal differentiation efficiency of NCSC was largely increased in response to neurotrophins, whereas only co-culture with neuronal progenitors promoted formation of neurons expressing active voltage-gated channels.
1. Enrichment of adult bone marrow neural crest-derived cells based on membrane marker expression.
The presence of neural crest cells (NCC) in adult bone marrow was first confirmed using double transgenic mice (Wnt1-Cre/R26R-EYFP or Wnt1-Cre/R26R-LacZ mice). Bone marrow cells (BMC) were extracted from adult mice (Wnt1-Cre/R26R-EYFP) and directly analyzed by flow cytometry. In those conditions, 0.38% ± 0.04% (n = 3) of cells were EYFP- positive (Fig. 1A). Bone marrow stromal cells (BMSC) extracted from Wnt1-Cre/R26R-LacZ mice were cultured in MesenCult® Proliferation Kit medium usually used for mesenchymal stem cells (MSC) culture expansion. NCC were identified in those BMSC cultures by X-gal reaction (Fig. 1B). Despite the low proportion of NCC in vivo, proportion among BMSC was easily increased with passages, from 4.89% ± 0.96% of NCC at P2 to 18.31% ± 10.3% of NCC at P3 and 27.7% ± 5.8% of NCC at P4. (p = 0.0065 ; n = 4 ; repeated measures ANOVA followed by Newman-Keuls post-test; Fig. 1C) as it was already observed .
As NCC were not the only one stromal population in the bone marrow, one objective of this work was to identify specific markers to discriminate NCC and mesenchymal cells.
Recently, we isolated clones that were NC-derived (NC clones) and mesenchymal-derived (MSC clones) from Wnt1-Cre/R26R-LacZ mice. A Microarray comparison between both type of clones was performed and data were published on GEO dataset under GSE30419 reference number . Based on those Microarray data, we selected several membrane markers differentially expressed by NCSC and MSC (Fig.2.A). We validated those results by semi- quantitative RT-PCR and confirmed differential expression levels for plp1, l1cam, abca1, nrcam, s1pr3, cd141, cd106, cd56, erbb3, and p75NTR (Fig.2B). Even if numerous genes looked promising as selecting markers to discriminate NCSC and MSC, we failed to find efficient antibodies working in immunocytochemistry for most of those markers.
Transcriptomic results could only be confirmed at the proteic expression level for p75NTR, ErbB3 and NrCAM.
Ex vivo expression of p75NTR, ErbB3 and NrCAM markers were first assessed before starting any purification experiment. A smear of freshly aspirated-bone marrow cells (BMC) from Wnt1-Cre/R26R-LacZ confirmed the expression of p75NTR, ErbB3 and NrCAM by NCC (Fig. 3A). We then analyzed the potential use of those markers as selecting markers for NCC, using cell-sorting technique on low-passage (P2-P3) BMSC. NrCAM antibodies failed to be efficient in flow cytometry technique and cell sorting was thus only performed using p75NTR and ErbB3 labeling. Proportion of NCC in BMSC culture was significantly enriched from 15.32% ± 8.3% before cell-sorting to 52.81% ± 10.59% in p75NTR–positive fraction (p = 0.005 ; n = 4 ; repeated measures ANOVA ; Figure 3B-C). In the same way, the number of NCC was increased from 21.8% ± 2.42% in unsorted-cells to 58.08% ± 6.96% in ErbB3- positive fraction (p = 0.002 ; n = 5, repeated measures ANOVA ; Figure 3B-C). However, proportion of NCC was not significantly decreased in p75NTR- and ErbB3-negative fractions, suggesting that only a small proportion of NCC was recovered after cell sorting. Moreover, those results prevent us to use the negative populations as a pure population of BMSC depleted in NCC. Looking at those results, it appeared that differential level of expression of p75NTR, and ErbB3 would not be sufficient to perform a purification of NCC, as positive- sorted fraction still contained many MSC. Similar results were obtained by combining ErbB3 and p75NTR (data not shown).
2. Isolation of bone marrow neural crest-derived cells based on sphere-forming ability.
Using cell-sorting technique, we obtained a subsequent NCC enrichment, however we failed to strictly purify NCC from adult bone marrow. Consequently, instead of using classical characteristics (membrane markers) to isolate those cells, we decided to use a more unusual
characteristic as we exploited the ability of NCC to form spheres as an isolation protocol.
Indeed, it has been previously demonstrated that BMSC were able to grow as spheres when placed in culture conditions usually employed for neurosphere formation from neural stem cells [20, 21]. Therefore, proportion of NCC in spheres obtained from BMSC cultures from Wnt1-Cre/R26R-LacZ mouse was quantified. Floating spheres were obtained by resuspending BMSC at low density (1 x 104 cells / ml) in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) for 10 days. Interestingly, we observed that those spheres were only composed of -galactosidase-positive cells showing their NC origin (Fig.
4A). In those conditions, 0.3% ± 0.05 % of BMSC were able to form spheres (n = 4) with a sphere-diameter of 145.17 μm ± 35.63 μm (n = 4). Primary spheres were then dissociated and sub-cultured in the same culture conditions for 10 days to form new spheres. The efficiency of secondary sphere formation was significantly increased to 1.99% ± 0.53% of cells (p = 0.029, n = 3, paired t-test; Fig. 4B) while sphere diameter remained roughly identical (152.76 μm ± 17.72 μm; n = 3).
3. Self-renewal of adult bone marrow NCC obtained from sphere-forming protocol.
At this point of the study, we obtained a pure population of NCC isolated from medullar stromal cells. However, we have to confirm that the self-renewal and differentiating characteristics of those NCC remain unchanged, compared to what was already described in previous studies . We first decided to address the self-renewal ability and to rule out the possibility that those spheres would be due to cell aggregates. NCC were resuspended at single-cell dilution in neurosphere culture condition. In those conditions, 0.23 % ± 0.01% of cells were able to form clonal spheres after 12 days (n = 2; Fig. 4C). Furthermore, immunocytofluorescence characterization of those spheres was performed, showing expression of the NCC markers Sox10, ErbB3 and p75NTR. Likewise, those spheres were also
positive for the neural stem cell marker Nestin and for NrCAM (Fig. 4D), however, were negative for N-Cadherin, E-cadherin, CD133 and Sox2 (data not shown).
4. Multipotency of adult bone marrow NCC obtained by sphere-forming protocol.
We then decided to characterize the multipotency of sphere-derived NCC. Indeed, during development, neural crest gives rise to a wide range of mature cell types including among others, skeletal cells, myofibroblasts, melanocytes and nearly all the cells of the peripheral nervous system including enteric nervous system, sympathetic and parasympathetic ganglia, adrenal medulla, autonomic and sensory neurons and supporting glial cells .
Differentiation capacity of sphere-dissociated cells was assessed by plating and culturing them into various specific differentiating media. We observed that those NCC were able to differentiate into melanocytes that were identified by Trp2 staining (Fig. 5A) and a DOPA reaction (Fig. 5B). Osteocyte differentiation was assessed by Alizarin Red S staining (Fig.
5C) as well as by measurement of alkaline phosphatase activity (1.71 a.u. ± 0.68 a.u.; Fig.
5D). Chondrocytes formation was confirmed by toluidin blue staining (Fig. 5C). NCC were not able to generate any adipocytes or smooth muscle cells, similarly to previous results obtained with clonal populations .
We then characterized the neural differentiating abilities of sphere-derived NCC.
Three differentiating culture conditions were tested: 1) Serum-containing medium: NCC were cultured in a serum-containing medium. In those conditions, some NCC expressed the glial marker glial fibrillary acidic protein (GFAP) (3.54 % ± 0.84 % of cells; Figs. 6A and D) or the neuronal marker -III-tubulin (labelled by the TuJ1 antibody; 7.87 % ± 3.03 % of cells;
Figs. 6A and D). 2) Neurotrophin-containing medium: NCC were seeded in a medium containing nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). In those conditions, we also observed cells expressing GFAP (10.63
% ± 2.02 % of cells; Figs. 6B and D) or -III-tubulin (26.71 % ± 9.65 % of cells; Figs. 6B and D). 3) Co-culture with cerebellar granule neurons (CGN): direct contact with those immature neurons resulted in differentiation of NCC into GFAP-positive cells (2.61 % ± 1.22 % of cells; Figs. 6C and D) or -III-tubulin-positive (10.73 % ± 3.32 % of cells; Figs. 6C and D).
Comparison of differentiation efficiency of those three media revealed a higher potency of neurotrophin-containing medium to induce the neural fate of NCC. Indeed, this medium increased the proportion of GFAP-expressing NCC 4-fold (p = 0.00052) compared to co- culture condition and 3-fold compared to serum-containing medium (p = 0.0007, n = 3, repeated-measure ANOVA followed by Tukey-Kramer’s post hoc test; Fig. 6D). -III- tubulin-expressing NCC were also increased 2.5-fold compared to co-culture condition (p = 0.0076) and 3.4-fold compared to serum-containing medium (p = 0.0034, n = 3, repeated- measure ANOVA followed by Tukey-Kramer’s post hoc test; Fig. 6D). Nevertheless, if the number of -III-tubulin-positive cells was higher in the neurotrophin condition, no Map2 expression, a mature neuron marker, was detected in those conditions or even in serum condition, whereas few NCC were Map2-positive when cultured with CGN for 8 days (4.83
% ± 0.82 %; Fig. 6C).
5. Electrophysiological analysis of differentiated-NCC obtained from sphere-forming protocol.
As the culture of sphere-dissociated NCC with CGN was the only condition that allowed cells to express a mature neuron marker (i.e. Map2), we decided to characterize differentiated-neurons obtained in that condition. Electrophysiological analysis was performed on sphere-dissociated NCC cultured with GFP-positive CGN for 7-17 days. Using fluorescent microscope, we thus selected cells that were clearly GFP-negative and that exhibited a neuron-like shape (rounded cell body with extended processes). Differentiated-
NCC were recorded in whole cells patch-clamp mode. The presence of voltage-gate channels was first investigated by applying voltage steps from a holding potential of -50 mV to voltage-clamped cells. In those conditions, 36 cells were recorded and 5 differentiated-NCC revealed fast inward currents blocked by tetrodotoxin (TTX, 0.5 μM) whereas tetraethylamonium-sensitive (TEA, 20 mM) outward currents were recorded in 16 cells (Fig.
7). Those data confirmed that a percentage of differentiated NCC express voltage-gated Na+ and K+ channels. If injection of positive current pulses in current clamp mode failed to elicit any action potential, some spikelets (small amplitude depolarizations) could be observed, demonstrating apparition of a functional activity in those NCC-derived neurons (data not shown).
Although the field of stem cell research has evolved into promising therapies for brain repair, this field still faces many challenges. One main challenge is the origin of stem cells that should be used in such procedures . Recently, several studies characterized the presence of neural crest stem cells (NCSC) in adult tissues and their wide variety of cell fate, suggesting those cells as potential source for cellular therapies . However, several problems have to be resolved before considering those cells as a tool for therapy protocols.
One of them is the lack of specific protocol for NCSC isolation from those tissues. Indeed, as quantified here, the amount of NCC in adult bone marrow is very low and no specific markers have been already described to identify those cells.
In this study, we first described selective markers to identify and enrich NCC from BMSC culture, like p75NTR, ErbB3 and NrCAM. Cell sorting of NCC using p75NTR positivity has already been performed from explants of E10.5 trunk neural tubes , from post- migratory sciatic nerve and gut [26, 27], and from post-natal and adult gut . ErbB3 is known to play a key role in NCC neural fate restriction during development [29, 30] but so far, has not yet been described for NCC identification and/or selection. Likewise, NrCAM is involved in neuron-neuron cell adhesion during nervous system development , however, it is described for the first time, as expressed by NCC ex vivo. Unfortunately, we failed to efficiently sort cells using these membrane markers. We think that one reason of this failure, besides technical problems (no efficient antibodies available for FACS), is due to a basal p75NTR-expression level by MSC in adult bone marrow, making NCC isolation difficult even if cell size or roughness were taking into account.
Consequently, we changed our strategy to isolate and characterized NCC and used sphere-inducing culture condition to obtain pure NCC population. Purified sphere-derived
NCC were analyzed to share self-renewal and multipotent characteristics of the cells. Sphere formation was encouraging to suspect the presence of stem cells, as it has already done for NCC from other adult tissue [1, 32]. However, it is important to remember that generation of a sphere is not a conclusive evidence of stemness as both stem cells and progenitors are able to proliferate and form spheres . As previously demonstrated for neural stem cells (NSC), sphere subcloning led to an increasing number of spheres after passages and thus to a selection and an increasing number of more immature NCC. Self-renewal capacity of sphere- dissociated NCC was therefore confirmed by their capacity to form spheres at clonal dilution . The multipotency of bone marrow NCSC was also addressed and we demonstrated that sphere-dissociated cells were able to differentiate into melanocytes, osteocytes, chondrocytes, glia (GFAP-positive cells) and neuronal cells (Tuj1- and Map2-positive cells). At this point, we were consequently able to validate sphere-forming capacity of NCC as a purification protocol for adult bone marrow multipotent NCSC.
A deeper characterization of the neuronal differentiation ability allowed us to observe a highest proportion of neuronal differentiated cells when NCC were cultivated in presence of neurotrophins (i.e. NT-3, BDNF and NGF), confirming the importance of those factors for neural differentiation of NCC. Those results were not surprising as neurotrophins are known to be essential for proliferation, survival and differentiation of NCC during development .
Furthermore, it has been demonstrated that the transduction of BDNF or NT-3 genes into BMSC promoted their neural differentiation . However, presence of neurotrophins in the culture medium was not sufficient to allow a full maturation of NCC-derived neurons as no Map2ab-positive cells were detected. On the other hand, co-culture of NCC with CGN induced mature neurons formation as around 4.8 % of Map2-expressing cells were obtained.
Furthermore, a number of cells exhibited functional voltage-gated Na+- and K+-channels that led to the appearance of spikelets when recorded in current-clamp condition. Indeed,
expression of voltage-gated Na+ channels is a critical step for the generation of action potentials and could be use to assess neuronal maturation of differentiating cells . Similar results have been reported by Arthur and collaborators as they characterized NCSC from adult dental pulp and showed those cells functional voltage-gated channels without firing . It remains to be elucidated whether the incomplete maturation of NCSC-derived neurons was due to the inability of cells to fully maturate or to non-appropriate culture conditions.
In conclusion, our study described a straight forward protocol to isolate multipotent NCSC from adult bone marrow. Neuronal differentiation of those cells was encouraging to investigate efficacy and safety of NCSC transplantation in experimental animal models for neurological diseases to qualify those cells for clinical purposes.
MATERIALS AND METHODS 1. Animals
BMC were isolated and cultured from Wnt1-Cre/R26R-EYFP and Wnt1-Cre/R26R- LacZ double transgenic mice [10-12]. Transgenic green fluorescent protein (GFP) C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) were used for CGN cultures. Mice were bred and kept at University of Liege Central Animal Facility and euthanized in accordance with the rules set by the local animal ethics committee as well as the Swiss Academy of Medical Sciences.
2. Cell Culture Protocols
BMC were obtained from femoral and tibial bones of adult mice (8–10-weeks-old).
Bone marrow was extracted by aspiration and cells were resuspended into 5 ml of MesenCult® Proliferation Kit medium (StemCells Technologies, Grenoble, France). After 24 hours, non-adherent cells (hematopoietic cells) were removed as we conserved only adherent cells, which correspond to BMSC. At 80% confluence, BMSC were resuspended using trypsin-EDTA 0.05% (Invitrogen, Carlsbad, CA, USA) and then sub-cultured at 30 000 cells / cm², defining a passage (P).
Primary sphere-forming protocol was adapted from skin sphere culture protocol .
BMSC were seeded at 10 000 cells/ml in Dulbecco's Modified Eagle Medium (D-MEM)/F-12 supplemented with B27 (Invitrogen), 20ng/ml FGF2 (PeproTech, Rocky Hill, NJ, USA) and 10 ng/ml EGF (PeproTech) in a flask coated with poly(2-hydroxyethylmethacrylate) (Poly- Hema) (Sigma-Aldrich, St. Louis, MO, USA). Secondary spheres were obtained after dissociation of primary spheres in a solution containing 0.05 % trypsin-EDTA (Invitrogen)for 3–5 min at room temperature (RT). 400 μl of Ovomucoid solution(1 mg/ml Trypsin inhibitor [Sigma-Aldrich] and 10 mg DNase [Roche, Vilvoorde, Belgium] in 25 ml medium) were
added and spheres were dissociated mechanically. Cell suspension was centrifuged and resuspended for 10 days in the same condition initially described for primary spheres. For clonal analyses, P6-BMSC were seeded in 96-well plates at a dilution of 1.2 cell/well in DEM/F12 supplemented with B27, 20 ng/ml FGF2 and 10 ng/ml EGF. An inverted microscope was used to identify and mark single cell-containing wells and eliminate wells containing no or at least two cells. Single cell-containing wells were marked on the flask to follow the potential proliferation of those cells.
3. Differentiation protocols
Spheres were dissociated and plated on poly-ornithine/laminine-coated slides. Several protocols have been used to analyze differentiating abilities of bone marrow NCC into neural cells, smooth muscle cells, melanocytes, adipocytes, osteocytes and chondrocytes. For neural differentiation: 1) Serum-containing medium: Neural (and smooth muscle cell) differentiation has been monitored by placing dissociated spheres in DMEM/F-12 supplemented with 10%
fetal bovine serum (FBS) (Invitrogen) for 10 days. 2) Neurotrophin-containing medium:
Neural differentiation was observed after culture of dissociated cells in DMEM/F12 supplemented with B27 (Invitrogen), FBS 1%, 50 ng/ml BDNF (PeproTech), 50 ng/ml NGF (PeproTech) and 10 ng/ml NT-3 (PeproTech) for 2 weeks, as used by Li et al. . 3) Co- culture with cerebellar granule neurons (CGN): Neural differentiation was also tested by culturing cells for 7-17 days with GFP-expressing CGN, as previously described . CGN cultures were obtained from cerebella of 3-days-old mice which express GFP under control of the -actin promoter . For smooth muscle cell differentiation, cells were also incubated in DMEM/F12, B27, Chicken Embryo Extract 5% and 1 nM transforming growth factor (TGF- 1) (PeproTech) for 6 days. Melanocyte formation was obtained after 10 days in MEM containing FBS 10%, 50 ng/ml murine stem cell factor (mSCF) (PeproTech) and 100nM
endothelin-3 (Sigma-Aldrich). Cells fixed with paraformaldehyde (PFA) 4% were incubated for 5h at 37°C in phosphate-buffered saline (PBS) containing 3-(3,4-dihydroxyphenyl)-L- alanine (L-Dopa) 0.1% (Sigma-Aldrich). For osteogenic induction, cells were cultured in StemXVivoTM Osteogenic media (R&D Sytems, Minneapolis, MN, USA). Osteogenic differentiation was measured using p-nitrophenyl phosphate, a substrate for alkaline phosphatase (Sigma-Aldrich). Level of APA was detected by development of soluble yellow reaction product that may be read at 405 nm using Thermo Labsystems Multiskan Ascent 354 (Lab Recyclers, Gaithersburg, MD, USA). Cells maintained in Mesencult were used as negative control. APA was expressed in unit of absorbance (u.a.). Cells were also stained with an Alizarin Red S solution (Sigma-Aldrich) to detect presence of calcific deposition.
Adipogenic differentiation was induced by treatment with DMEM containing 0.5 mM 1- methyl-3-isobutylxanthine (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 0.01 mg/ml bovine insulin and 0.2 mM indomethacin (Sigma-Aldrich). Cells were cultured in this above-described adipocyte induction medium for 21 days. Differentiation was evaluated by accumulation of lipid vacuoles and an Oil Red O staining (Sigma-Aldrich) following fixation with PFA 4%. Chondrocyte formation was obtained after 1 month in DMEM containing 0.1 μM dexamethasone (Sigma-Aldrich), 1 mM sodium pyruvate (Invitrogen), 50 μg/ml ascorbic- 2-phosphate acid (Sigma-Aldrich), 6.25 μg/ml mM transferin (Sigma-Aldrich), 6.25 μg/ml bovine insulin, 6.25 μg/ml selenic acid (Sigma-Aldrich), 5.35 μg/ml linoleic acid (Sigma- Aldrich), 1.25 mg/ml BSA and 10 ng/ml TGF-3 (PeproTech). Pellets were fixed with PFA 4%, paraffin-embedded, cut in 5 m sections and stained with toluidine blue. In each case, medium was refreshed every 3 – 4 days.
4. Flow Cytometry
For the quantification of ex vivo bone marrow NCC content, cells aspirated from tibial bones of Wnt1-Cre/R26R-EYFP mice were filtered through a 40 μm cell strainer (BD Biosciences, San Jose, CA, USA) and resuspended in PBS with 2% FBS. NC-derived cells were detected using BD FACStar PLUS Flow Cytometer and BD CellQuest software (BD Biosciences). Bone marrow cells from ROSA26 Cre reporter mice were used as negative control. For the cell sorting, P2-P3 cells (previously extracted from Wnt1-Cre/R26R-lacZ mice) were filtered through a 40 μm cell strainer (BD Biosciences) and incubated for 30 min, on ice, with anti-ErbB3 (1:100 ; LifeSpan Biosciences, seatle, WA, USA) or anti-p75NTR (1:150; Millipore, Temecula, CA, USA) primary antibodies and then with FITC- or PE- conjugated secondary antibodies (1:250; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). ErbB3- and p75NTR-stained cells were analyzed and sorted on BD FACSAriaII using FACSDiva software (BD Biosciences).Data were finally treated using FlowJo software (Tree Star, Ashland, OR, USA). In each case, dead cells and doublets were excluded by gating on forward and side scatter.
5. Transcriptomic analysis
Microarray procedures have been previously described . For RT-PCR, total RNA was extracted from NCSC and MSC clones using a silica-gel column-based extraction (RNeasy Minikit, Qiagen) and quantified by a Nanodrop 3300 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Reverse transcription was performed using Moloney-murine leukemia virus (M-MLV) Reverse transcriptase (Promega, Madison, WI, USA), to reach a final concentration of 50 ng/μl of cDNA, and PCR reactions were then performed using Taq polymerase kit (Promega) on a PTC-200 thermocycler (MJ Research, St. Bruno, Canada).
Amplification steps and number of cycles were standardized for each set of primers (30”
[94°C] - 30” [Tm] - 30” [72°C]), except for melting temperatures that differ according to primer’s sequences, which are described in Table 1.
6. Electrophysiological recordings
Sphere-dissociated cells were cultivated for 5–20 days with CGNs, as previously described.
During recordings, cover slip was continuously perfused with artificial cerebrospinal fluid (ACSF; 137 mM NaCl, 5.4 mM KCl, 2 mMCaCl2, 22.2 mMD-glucose and HEPES; pH 7.4) at room temperature. ACSF and drug applications were performed using gravity and a BPS-8 valve control system (ALA Scientific, Westbury, NY, USA). Then, 1 μM tetrodotoxin (TTX;
Tocris Biosciences, Ellisville, MO, USA) and 20 mM tetraethylammonium (TEA; Sigma- Aldrich) were added to the ACSF to respectively block Na+ or K+ current. Neurons from clones were discriminated using an Axiovert microscope (Zeiss, Oberkochen, Germany).
Pipettes were pulled on a P-87 micropipette puller (Sutter Instruments, Novato, CA, USA) using borosilicate glass capillary tubing (2.0 mm OD 1.16 mm ID; Hilgenberg, Malsfeld, Germany). The resistance of the electrodes was 5–8 M when filled with the intracellular solution: 130 mM KCl, 2 mM CaCl2, 2 mM HEPES, 2.5 mM ATP-Mg, 2.5 mM ATP-Na2, 10mM EGTA and 11.1 mM D-glucose; pH 7.4. Non-fluorescent cells (NCC, as CGN were originated from GFP mice) were sealed at a gigaohm and placed in whole-cell configuration.
Membrane potentials and currents were recorded using an EPC9 amplifier (HEKA, Lambrecht/Pfalz, Germany) connected to Patchmaster software (HEKA). Liquid junction potentials were corrected. Only recordings in which the series resistance was lower than 30 M and remained stable (variations 20%) were used. No compensation of the series resistance was performed.
7. X-gal Staining
X-gal staining was performed on 2% PFA-fixed cells. Cells were incubated overnight in PBS supplemented with 20 mM Tris (pH 7,4), 2 mM MgCl2, NP-40 0.02%, Na- deoxycholate 0.01%, 5 mM K3Fe(CN)6 (Sigma-Aldrich), 5 mM K4Fe(CN)6 (Sigma-Aldrich), and 1mg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (Sigma–Aldrich) in dimethylsulfoxyde (DMSO). The reaction was stopped by PBS washes, and classical fluorescent immunostainings were then performed as described below.
Briefly, cells on coverslips were fixed with 2% PFA for 10 min at RT, followed by 3 washes of 10 minutes in PBS, normal donkey serum (NDS) 10% (Jackson ImmunoResearch Laboratories) in PBS (supplemented with Triton X-100 0.1% for intracellular antigen stainings) for 45 min. Cells were then incubated overnight at 4°C in NDS 1.5% blocking solution containing primary antibody: anti-Sox10 (1:200; ABR/Thermo Fisher Scientific, no more available), anti-p75NTR (1:200; Millipore), anti-ErbB3 (1:100 ; LifeSpan Biosciences), anti-Nestin (1:300; Novus Biological, Littleton, CO, USA), anti-GFAP (1:1000;
DakoCytomation, Glostrup, Denmark), anti--III-tubulin (1:1000; Covance, Princeton, NJ, USA), anti-Map2ab (1:500; Sigma-Aldrich), anti-SMA (1:400; Sigma-Aldrich). After 3 washes of 10 minutes in PBS, cells were incubated with FITC- or Rhodamine Red X- conjugated secondary antibody (1:500; Jackson ImmunoResearch Laboratories) for 1h at RT and finally counterstained with Vectashield HardSet Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Preparations were observed using a Nikon TE 2000-U epifluorescent microscope (Nikon, Amstelveen, The Netherlands) or an Olympus laser scanning confocal microscope (Olympus, Tokyo, Japan). The digitized images were adjusted for brightness and contrast, color-coded, and merged, when appropriate, using the NIH
program ImageJ or the Adobe Photoshop 6.0 program (Adobe Systems Incorporated, San Jose, CA).
9. Statistical Analysis
Data were analyzed statistically using Statistica 10 program (StatSoft, Inc., Tulsa, OK). Data are reported as mean ± standard deviation, with the number of experiments (n) between parentheses. Level of statistical significance was set at p < 0.05.
This work was supported by a grant from the Fonds National de la Recherche Scientifique (FNRS) of Belgium, by the Belgian League against Multiple Sclerosis associated with the Leon Fredericq Foundation, and by the Swiss National Science Foundation. The authors thank Patricia Ernst and Alice Marquet for providing technical assistance, Dr. Sandra Ormenese and Rafaat Stefan from the GIGA-Imaging and Flow Cytometry platform for cell sorting, Dr.
Hennuy and Christophe Poulet from GIGA-Management and transcriptomic platform, but also Pr. Albert and Dr. Donneau from “Informatique médicale et biostatistique” department for statistical advices.
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Figure 1. Identification of neural crest cells (NCC) in adult bone marrow. (A) Bone marrow was extracted from femoral bones of Wnt1-Cre/R26R-EYFP mice. Bone marrow NCC content was defined by flow cytometry. Approximately 0.38% ± 0.04% (n = 3 independant cultures) of cells were NCC. ROSA26-EYFP reporter mice were used as negative controls for EYFP-fluorescence. (B) NCC were also identified in stromal culture of bone marrow extracted from Wnt1-Cre/R26R-LacZ mice. X-gal reaction led to a blue staining of NCC. As passages increased, proportions of NCC were increased until 27% at P4 (p <
0.05). Scale bar = 30 μm.
Figure 2. Comparison of membrane marker expression in NC clones and MSC clones.
(A) Microarray analysis revealed some differences between NC and MSC clones in expression level of genes encoding various membrane markers. (B) Using semi-quantitative RT-PCR, those variations of expression between NC and MSC clones for selected genes were confirmed. a.u., arbitrary unit.
Figure 3. ErbB3, p75NTR and NrCAM expression by bone marrow NCC. (A) Aspirated- bone marrow smear from Wnt1-Cre/R26R-LacZ mice was realized and a X-gal staining confirm the presence of NCC, ex vivo. Immunocytofluorescence in smears reveals expression of p75NTR, ErbB3 and NrCAM by X-gal-positive cells. (B-C) Gates targeted for the sorting of p75NTR- and ErbB3-positve cells from a P2-BMSC culture. Percentage of NCC were significantly increased in p75NTR- and ErbB3-FACS-enriched populations comparing to their basal level before sorting (US, unsorted) (n = 4 and 5 independent cultures; ** p < 0.01, ***
p < 0.01, repeated measures ANOVA). No significant decreases in the percentage of NCC were observed in p75NTR- and ErbB3-negative population comparing to US cells. Nuclei were counter-stained with Dapi. Scale bar = 10 μm.
Figure 4. Sphere-forming capacity of adult bone marrow NCC. (A) When cultured in serum-free medium supplemented with fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF), only X-gal-positive cells formed spheres. (B) Sphere forming efficiency increases from primary (0.3% ± 0.05% of aBMC) to secondary spheres (1.99% ± 0.53% of aBMC; n = 3 independent cultures; *, p < 0.05 paired t-test). (C) Developing sphere from a single BMSC after 0, 4 and 12 days of culture. (D) Sphere-forming cells express the NCC markers Sox10, p75NTR, ErbB3, NrCAM and nestin. Nuclei were counter-stained with Dapi.
Scale bar = 50 μm.
Figure 5. Mesenchymal fate of sphere-dissociated NCC. (A-B) Placed in presence of endothelin-3 and mSCF, cells adopted a melanogenic fate assessed by (A) Trp2 staining and (B) a DOPA reaction. (C-D) Osteogenic differentiation was evaluated by (C) Alizarin Red staining and (D) measurement of alkaline phosphatase activity (APA). Enzyme activity was detected by absorbance variation and read by spectrophotometry at 405 nm. Test was performed in triplicate (n = 3 independent cultures). (E) Chondrocytes were obtained by culturing sphere-derived cells as a pellet for 30 days in chondrogenic induction medium.
Sections of pellet were stained with toluidine blue. Nuclei were counter-stained with Dapi.
Scale bars = 20 μm (A); 30 μm (B-C); 50 μm (E).
Figure 6. Neural differentiation of sphere-dissociated NCC. Sphere-dissociated cells have been cultured in three media permissive for neural differentiation. (A) Cells have been cultured in DEM/F12 supplemented with 10% FBS for 10 days. (B) Cells have been cultured in DMEM/F12 medium supplemented with B27 and BDNF, NGF, NT-3 for 15 days. (C) Cells were co-cultured for 7-17 days with GFP-positive cerebellar granule neurons (CGN;
green). In those tree conditions, some NCC (X-gal-positive) expressed the glial marker GFAP (green in A and B; red in C) or the neuronal marker, -III-tubulin (Tuj1; red) whereas expression of the mature neuron marker Map2ab (red) by NCSC was observed only when co- cultured with CGN. (D) Comparison of media permissive for neural induction. GFAP- expressing cells proportion was also increased in neurotrophin-containing medium by 407 % compared to co-culture and by 300 % compared to serum-containing medium (n = 3).
Neurotrophin-containing medium increased proportion of Tuj1-expressing cells by 248 % compared to co-culture condition and by 339 % compared to serum-containing medium (n = 3). **, p < 0.01; ***, p < 0.001. Nuclei were counter-stained in blue with Dapi. Scale bars = 30 μm.
Figure 7. Electrophysiological analysis of neuronal differentiated NCC isolated as spheres and co-cultured with CGN. Representative traces of Na+ and K+-currents recorded in differentiated NCC in co-culture condition with CGN. (A) The holding potential, transiently and repeatedly stepped from -50 mV to -30 mV in voltage-clamped cells, induced inward current blocked by tetrodotoxin (TTX) 0.5 μM. (B) An holding potential from -50 mV to +20mV also revealed outward current blocked by tetraethylamonium (TEA) 20 mM.