275 IN VITRO DIFFERENTIATION OF PORCINE MESENCHYMAL STEM CELLS INTO NEURON-LIKE CELLS

Abstract

The unique self-renewal ability for multilineage differentiation makes mesenchymal stem cells (MSCs) a promising cell source for regenerative medicine. However, before adapting in clinical trials, there is a need for a systematic evaluation of the in vitro differentiation process and the functional identities of cells generated using animal model systems. We have previously reported that MSCs derived from porcine umbilical cord blood (pUCB), post-natal bone marrow, and young adult bone marrow with typical phenotypic properties differentiate in vitro into various cells types of mesodermal lineage (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells, in press). In the present study, we examined whether porcine MSCs (pMSCs) obtained from different origins, such as UCB, post-natal (immediately after birth) bone marrow, and young adult (6 months old) bone marrow are capable of neuronal differentiation in vitro. Subconfluent cultures of pMSCs (2–5 passages) were induced to become neuroectodermal cells following the protocol described earlier (Woodbury et al. 2000 J. Neurosci. Res. 61, 364–370), with minor modifications. After induction, 75 80% of cells exhibited substantial morphological changes including appearance of a more spherical shape, with multipolar and extending processes arranged into a network-like structure. Besides acquiring a neuronal phenotype, pMSCs derived from different origins displayed the expression pattern of characteristic neural-specific markers including early, intermediate, and late neural cell types. Differentiated cells expressed markers such as nestin, microtubule-associated protein-2 (MAP-2), neuronal nuclei (NeuN), βIII-tubulin, neurofilament-M (NF-M), glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), and myelin basic protein (MBP) by immunofluorescence analysis. Expression pattern was further confirmed by analyzing selected neuronal transcripts by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Neurally differentiated pMSCs from different origins showed significantly (P < 0.05) higher relative abundance of nestin, βIII-tubulin, NF-M, and GFAP, compared to cells in control. In conclusion, the morphological observations and expression of neuronal-specific markers support the fact that pMSCs from different origins possess the ability to differentiate in vitro into neuron-like cells upon exposure to appropriate stimuli. Furthermore, these findings provide additional support for the emerging concept that the establishment of cell lines from pMSCs can have a pivotal role in standardizing cell replacement strategies.