Abstract
BackgroundThe ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine, especially for replacing neuronal tissue damaged by different neurological disorders. Reprogramming of ASCs can be induced by the growth medium with neurogenic inductors and specific growth factors. We investigated the neural differentiation potential of canine ASCs using several growth media (KEM, NIMa, NIMb, NIMc) containing various combinations of neurogenic inductors: B27 supplement, valproic acid, forskolin, N2-supplement, and retinoic acid. Cells were first preconditioned in the pre-differentiation neural induction medium (mitogenically stimulated; STIM1), followed by the induction of neuronal differentiation.ResultsAfter 3, 6, and 9 days of neural induction, elongated neural-like cells with bipolar elongations were observed, and some oval cells with light nuclei appeared. The expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H), microtubule-associated protein-2 (MAP2), and glial fibrillary acidic protein (GFAP) was observed using immunocytochemistry, which confirmed the differentiation into neurons and glial cells. Flow cytometry analysis showed high GFAP expression (between 70 and 90% of all cells) after cells had been growing three days in the neural induction medium a (NIMa). Around 25% of all cells also expressed adult neuronal markers NF-H and MAP2. After nine days of ASCs differentiation, the expression of all neural markers was reduced. There were no differences between the neural differentiation of ASCs isolated from female or male dogs.ConclusionsThe differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium, the canine ASCs differentiated into neural lineages and expressed markers of neuronal and glial cells, and also displayed the typical neuronal morphology. Differentiated ASCs can thus be a source of neural cellular lineages for the regenerative therapy of nerve damage and could be useful in the future for therapy or the modelling of neurodegenerative diseases.
Highlights
The ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine, especially for replacing neuronal tissue damaged by different neurological disorders
Pre-differentiation medium STIM2 was highly toxic to the cells, while medium STIM1 was suitable for cell culture
The pre-differentiation was introduced in order to enhance the proliferation of ASCs and to slowly direct the lineage commitment to neuronal cell types
Summary
The ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine, especially for replacing neuronal tissue damaged by different neurological disorders. Multipotent mesenchymal stromal cells (MSCs), commonly referred to as mesenchymal stem cells are self-renewing, multipotent, adult stem cells that have a mesodermal and neuroectodermal origin [1, 2] They are found in many tissues, such as adipose tissue, bone marrow, cord blood, chorionic folds of the placenta, amniotic fluid, blood, lungs, etc., most of which are accessible and represent a potentially significant source of cells. Canine adipose tissue-derived multipotent mesenchymal stromal cells (ASCs) could be induced to express some of these neuronal genes after growing in the presence of the neurogenic inductors valproic acid and forskolin [14, 16], basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) [15], in a commercial neurogenic differentiation medium [18] and in a medium containing N2 supplement, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) [19]. The neural differentiation of human ASCs was induced by a conditioned medium obtained from glial cells [21]
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