Adult Bone Marrow: Which Stem Cells for Cellular Therapy Protocols in Neurodegenerative Disorders?

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crests (NCSCs) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSCs). In this paper, we will review all information available concerning NCSC from adult tissues and their possible use in regenerative medicine. Moreover, as multiple recent studies showed the beneficial effect of bone marrow stromal cells in neurodegenerative diseases, we will discuss which stem cells isolated from adult bone marrow should be more suitable for cell replacement therapy

Correction: In Vivo Tumorigenesis Was Observed after Injection of In Vitro Expanded Neural Crest Stem Cells Isolated from Adult Bone Marrow.

[This corrects the article DOI: 10.1371/journal.pone.0046425.]

Caractérisation du potentiel neurogénique des cellules de la crête neurale et des cellules mésenchymateuses de le moelle osseuse.

Notre travail s’est orienté vers l’étude de sources potentielles de cellules immatures, capables d’adopter un destin neuronal et qui, de ce fait, pourraient constituer la base de protocoles de greffe autologue dans un contexte de médecine régénérative pour des pathologies neurologiques. Notre laboratoire étudie depuis plusieurs années les cellules stromales de la moelle osseuse comme une source de cellules immatures capables, pour certaines d’entre elles, d’adopter un phénotype nerveux. Les premiers résultats ont laissé supposer que les cellules stromales de la moelle osseuse étaient bien plus hétérogènes qu’il n’y paraissait. Dès lors, avant d’envisager tout usage de ces cellules à des fins thérapeutiques, une meilleure caractérisation s’imposait. L’objectif principal de ce travail était donc de mieux appréhender les propriétés fonctionnelles des cellules stromales. Plus particulièrement, nous avons voulu évaluer dans quelle mesure l’origine embryonnaire de ces cellules pouvait influer sur leur capacité à former des neurones.Dans un premier temps, nous avons confirmé la présence de CCN au sein de la moelle osseuse et démontré leur caractère souche. Plus encore, nous avons séparé les CCN des CSM en utilisant leur capacité à former des sphères et caractérisé leur potentiel de différenciation neuronale. Par la suite, des cultures clonales, tant de CSM que de CCN ont été obtenues à partir de cellules stromales de la moelle osseuse. A l’aide de ces clones, nous avons pu comparer les propriétés intrinsèques des deux types cellulaires et caractériser leur potentiel de différenciation. Enfin, nous avons démontré la capacité des facteurs Wnt1 et BMP2 à recruter des cellules neurogéniques parmi les cultures stromales

Validation of Adult Bone Marrow Stromal Cells in Cellular Therapy Protocols, using a Mouse Model for Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder in industrialized countries. Its main characteristic relies in a progressive loss of dopaminergic (DA) neurons in the Substantia Nigra pars compacta (SNpc), resulting in a deficient dopamine release in the striatum and then promoting important defects in motility regulation. Unfortunately, motor symptoms are generally diagnosed once 80% of nigrostriatal neurons are already lost. The emergence of neuroprotective/-restorative strategies is then increasingly raising hope, and a lot of people now focus on cell therapy experiments. Adult bone marrow stromal stem cells (BMSCs) have already been demonstrated as ideal candidates for cell therapy in nervous lesions, regarding their high multipotency and the fact they can be easily harvested in the patient himself. After it has been demonstrated that some BMSCs arise from the embryonic neural crest (NC), we compared NC-BMSCs and mesenchymal (M)-BMSCs in vitro, in terms of differenciation abilities and more particularly in terms of neural fate. We then wanted to investigate and compare the potential usefulness of both populations in the context of a neurological pathology. We have validated a MPTP mouse model, mimicking the specific loss of nigral neurons, and started setting up a cell therapy experiment using stereotaxic brain injection of the two types of BMSCs. The survival rate of grafted cells was analyzed as well as their migration or differentiation, and their ability to restore neuronal loss was also observed. Our first results showed that once grafted inside the brain of MPTP mice, NC-BMSCs survive for about a week, staying tightly close to each other the injection track with no visible sign of migration. Afterwards, cells begin to disappear and we only observe a mean survival rate of 1% after 28 days. Looking at the nigrostriatal pathway integrity, neural crest-BMSCs don’t seem to induce any improvement: they don’t differenciate into neural cells, neither replace lost DA cells, and they do not induce any sprouting of surviving DA neurons. While the M-BMSCs graft experiment has to be completed, these first results showed that NC-BMSCs at the stem cell state are not able to restore the lesioned system, and maybe a pre-differenciation step would be required to trigger those cells into a neuronal fate before grafting them in a MPTP-mouse brain

In vivo tumorigenesis was observed after injection of in vitro expanded neural crest stem cells isolated from adult bone marrow

Bone marrow stromal cells are adult multipotent cells that represent an attractive tool in cellular therapy strategies. Several studies have reported that in vitro passaging of mesenchymal stem cells alters the functional and biological properties of those cells, leading to the accumulation of genetic aberrations. Recent studies described bone marrow stromal cells (BMSC) as mixed populations of cells including mesenchymal (MSC) and neural crest stem cells (NCSC). Here, we report the transformation of NCSC into tumorigenic cells, after in vitro long-term passaging. Indeed, the characterization of 6 neural crest-derived clones revealed the presence of one tumorigenic clone. Transcriptomic analyses of this clone highlighted, among others, numerous cell cycle checkpoint modifications and chromosome 11q down-regulation (suggesting a deletion of chromosome 11q) compared with the other clones. Moreover, unsupervised analysis such as a dendrogram generated after agglomerative hierarchical clustering comparing several transcriptomic data showed important similarities between the tumorigenic neural crest-derived clone and mammary tumor cell lines. Altogether, it appeared that NCSC isolated from adult bone marrow represents a potential danger for cellular therapy, and consequently, we recommend that phenotypic, functional and genetic assays should be performed on bone marrow mesenchymal and neural crest stem cells before in vivo use, to demonstrate whether their biological properties, after ex vivo expansion, remain suitable for clinical application

Chromowave profile of <i>Asclepios</i>, displays the first eigenvector that explains 84% of the variance between <i>Asclepios</i> and NCSC mix.

<p>Each chromosomal signal has been wavelet transformed. Under and over expressions are respectively below or above 0 on the y-axis. The x-axis displays the chromosomal position labeled with cytoband names. A large part of the chromosome 11 is under expressed in <i>Asclepios</i>.</p

<i>In vivo</i> characterization of neural crest derived cells.

<p>To characterize neural crest-derived clones <i>in vivo</i>, we stereotaxically injected 50,000 cells of each NCSC clones (separetly) in mice striatum (<b>A</b>). <i>Asclepios</i> induced massive tumors after 4 weeks as attested by the beta-galactosidase expression of the tumor cells. (<b>B</b>). Immunological characterization of those tumors revealed that they were GFAP (<b>c</b>), beta-III-tubulin (<b>D</b>), nestin (<b>E</b>), N-cadherin (<b>F</b>) and NrCAM-positive (G). Lectin labeling (<b>H</b>) confirmed the presence of blood vessels in the tumors. Finally, no vimentin (<b>I</b>) or Sox2 (<b>J</b>) expressions were observed. Nuclei were counterstained with Dapi (blue). Scale bars = 50 µm.</p