Neuro-glial differentiation of human bone marrow stem cells in vitro

https://doi.org/10.1016/j.expneurol.2004.12.013Get rights and content

Abstract

Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT–PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.

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.

References (57)

  • R.F. Castro

    Failure of bone marrow cells to transdifferentiate into neural cells in vivo

    Science

    (2002)
  • J.R. Chamberlain et al.

    Gene targeting in stem cells from individuals with osteogenesis imperfecta

    Science

    (2004)
  • A.M. Clement et al.

    Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice

    Science

    (2003)
  • J.L. Cordell et al.

    Immunoenzymatic labeling of monoclonal antibody using immune complexes of alkaline phosphotase and monoclonal anti-alkaline phosphatase (APAAP complexes)

    J. Histochem. Cytochem.

    (1984)
  • G.Q. Daley et al.

    Realistic prospects for stem cell therapeutics

    Hematol. (Am. Soc. Hematol. Educ. Program)

    (2003)
  • W.H. Fleming et al.

    Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells

    J. Cell Biol.

    (1993)
  • R. Galli et al.

    Skeletal myogenic potential of human and mouse neural stem cells

    Nat. Neurosci.

    (2000)
  • M.A. Goodell

    Stem-cell “plasticity”: befuddled by the muddle

    Curr. Opin. Hematol.

    (2003)
  • A. Gritti et al.

    Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain

    J. Neurosci.

    (1999)
  • K. Guan et al.

    Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells

    Cell Tissue Res.

    (2001)
  • H.N. Hao et al.

    Fetal hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro

    J. Hematother. Stem Cells Res.

    (2003)
  • C. Holden et al.

    Plasticity: time for a reappraisal?

    Science

    (2002)
  • A. Huttmann et al.

    Bone marrow-derived stem cells and “plasticity”

    Ann. Hematol.

    (2003)
  • T. Imura et al.

    The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP

    J. Neurosci.

    (2003)
  • O. Isacson et al.

    Toward full restoration of synaptic and terminal function of the dopaminergic system in Parkinson's disease by stem cells

    Ann. Neurol.

    (2003)
  • Y. Jiang et al.

    Pluripotency of mesenchymal stem cells derived from adult marrow

    Nature

    (2002)
  • C. Joshi et al.

    Molecular complexities of stem cells

    Curr. Opin. Hematol.

    (2003)
  • G.C. Kopen et al.

    Marrow stromatal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal brains

    Proc. Natl. Acad. Sci. U. S. A.

    (1999)
  • Cited by (203)

    View all citing articles on Scopus
    1

    Contributed equally to the work.

    View full text