Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

Abstract

Background: Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC) could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes.

Methods: BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin). By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3.

Results: Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin). Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin). Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3), NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate receptor channel (AMPA3) in the presence of two agonist glutamate, AMPA or CNQX antagonist.

Conclusion: Our results demonstrate that BM-MSC have the potential to differentiate in neuronal cells with specific gene expression and functional properties. BM-MSC are thus promising candidates for cell-based therapy of neurodegenerative diseases.

Figure 1

Characterization and differentiation of BM-MSC. (A) Reprentative flow cytometric analysis of cultured mesenchymal stomal cells. Solid white represente the isotype control. (B) A. BM-MSC, displayed an homogeneous morphology of fibroblastic cells. Cells were stained with May Grunwald Giemsa staining (40×). Under specific induction BM-MSC were differentiated into (B) adipocytes (lipid vacuoles were colored by Oil Red O, ×40), (C) osteocytes (calcium deposits were revealed by Von Kossa method, ×40), (D) chondrocytes (cell pellet was sectioned and stained by toluidine blue, ×4), and (E) neuron-like cells derived from BM-MSC upon treatment with neurogenic medium (×40). Expression of nestin and MAP-2 after 10 days induction, detected by immunofluorescence (F and G, 100×).

Figure 2

Flow cytometric analysis of neurone like cells. Expression of nestin, MAP-2 and TH neural proteins before and after 10 day exposure with neurogenic medium. (A) Typical FACS analysis of neuronal markers expressed by MSC before (gray histogram) and after (black histogram) differentiation. White histogram represents the isotype control. (B) Percentage of positive cells for nestin, MAP-2 and TH before (gray) and after (black) specific neurogenic induction. The mean (SEM) was obtained from three independent experiments.

Figure 3

The synatptic transmission gene expressed. Schematic representation of localization and role of genes increased after neurogenic induction and involved in the synaptic transmission.

Figure 4

Quantitative real-time PCR analysis. Quantitative real-time PCR analysis using β-actin as a housekeeping gene and calibrated with the PANOMICS: Human brain total RNA (invitrogen, Brussels, Belgium). Results are expressed in fold change signal between differentiated and non-differentiated BM-MSC after the neurogenic induction. Selected neuronal genes were NEGR1, EphA4, NEF3, GRIA3 (AMPA), PENK (Grey column). Selected mesenchymal genes were HAPLN1, VIM, CSPG4, CALD1, DSC96, MEST, TUFT1 (black column).

Figure 5

Intracellular Calcium influx. Representative variation of intracellular Calcium influx in differentiated MSC treated with AMPA (40 μM, ---×---), glutamate (100 μM, ) or CNQX (10 μM, ). Results are expressed as fluorescence intensity at 4 second-intervals using the Fluo-4 NW tracer. Agonists (AMPA or Glutamate) and antagonist CNQX were compared with spontaneous level of calcium influx in presence of Ca2+ alone ().