Neuro-glial differentiation of human bone marrow stem cells in vitro
Introduction
Stem cells (SCs) are undifferentiated cells characterized by peculiar gene expression profiles (“stemness” identity) (Ramalho-Santos et al., 2002) and by the capacity to either proliferate indefinitely (self-renewal) or to originate tissue-specific committed progenitors or differentiated cells (Joshi and Enver, 2003). The possibility to differentiate bone marrow (BM) SCs into a neuro-glial phenotype (neuro-glial progenitor cells capable of giving raise to both differentiated glial and neuronal cells) would be an important step towards the cell therapy of patients affected by neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), Parkinson's, or Huntington's diseases (Huttmann et al., 2003, Isacson et al., 2003, Silani et al., 2004). BM, a well known source of both hematopoietic (HSCs) and mesenchymal SCs (MSCs), may represent a valid alternative to embryonic SCs for cell therapy to replace or, better, to protect dying neurons (Clement et al., 2003, Holden and Vogel, 2002, Moore and Quesenberry, 2003, Svensen and Langston, 2004). As a matter of fact, the appealing prospective of obtaining autologous SCs with minor patient distress, expanding/trans-differentiating cells in vitro, and re-transplanting them into the same patient has opened interesting new possibilities (Daley et al., 2003). BM SC “trans-differentiation” (direct fate conversion of a cell type to another) both in vitro and in vivo has been reported by numerous investigators (Brazelton et al., 2000, Kopen et al., 1999, Mezey et al., 2000, Sanchez-Ramos et al., 2000) although no definitive evidence has been given so far that a single purified BM SC can contribute to neural regeneration. As pointed out by several recent reviews (Collas and Håkelien, 2003, Goodell, 2003, Prockop and Gregory, 2003), due to the absence of unambiguous tissue-specific SC markers, alternative explanations including cell fusion, contamination by different stem/progenitor subpopulations, or activation of the native SC compartment can be advocated to explain the functional recovery. Moreover, some authors were unable to obtain BM SC trans-differentiation in vivo, but their failure could be due to differences in experimental protocols (Castro, 2002, Ono et al., 2003, Terada et al., 2002). A subpopulation of MSCs called multipotent adult progenitor cells or MAPCS has been demonstrated to give rise to astrocytes, oligodendrocytes, and neurons in vitro (Jiang et al., 2002). Furthermore, allogeneic BM cells can generate new neurons in human brain in vivo after transplantation for malignancy (Mezey et al., 2003). Best results have been achieved using fetal HSCs, successfully transformed and differentiated into neural SCs and then astrocytes (Hao et al., 2003) but, overall, an unambiguous demonstration of adult SC trans-differentiation is still far from being reached (Liu and Rao, 2003).
The main purposes of this work were to identify putative progenitor cells in the human adult whole BM or in selected cell subpopulations, capable of trans-differentiation into neuro-glial cells, and then to define their in vitro response to definite culture media and conditions, i.e. growth factors and supplements. Cellular interaction influences on the cell fate were further evaluated using conditioned media derived from neuronal or glial cell cultures or using co-culture systems.
Section snippets
Cell populations used
BM cells used in our study were obtained from allogeneic BM transplantation normal donors (mean age 35 years, ranging from 21 to 39 years) and from patients who underwent rib resection during thoracotomy for lung cancer surgery (mean age 63 years, ranging from 53 to 79 years). In both cases, samples were obtained following informed consent according to the Ethic Committee Guidelines of IRCCS Ospedale Maggiore in Milan. To test the neuro-glial trans-differentiation potential, the following
Results
Overall results of the performed experiments and cell populations used in this study are shown in Table 1a in which only positive or negative results after treatments are reported, without any specification regarding the analysis performed (morphological observations, RT–PCR, or immunocytochemistry, see Table 1a-Legend). Results are not quantitative.
In summary, the most effective media inducing neuro-glial differentiation seem to be the NS-A medium (usually used to maintain and expand neural
Discussion
In this study, we focused on the identification of adult BM cell populations capable of a neuro-glial differentiation potential. We compared whole BM, MNCs, MSCs, and immunomagnetically sorted fractions to identify SC populations able to acquire novel neuronal fates in vitro. To our knowledge, this is the first report in which many different adult human BM cell types have been studied in vitro and compared after treatment with different media, additives in several combinations, and conditioning.
Acknowledgments
This work is supported in part by grants from the Italian Ministery of Health (Stem 2001) and Fondazione Italo Monzino. The funding sources had no involvement or influence in the preparation of the paper.
We also thank Prof. Pogliani for providing some of the bone marrow samples used in this study and Dr. Stefania Corti for the Transgenic C57BL/6-TgN (ACTbEGFP)1Osb mouse expressing an “enhanced” GFP.
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Contributed equally to the work.