Background: 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. 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. Results: After 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. Conclusions: The 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.
Background: 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 damaged neuronal tissue in different neurological disorders. Reprogramming of ASCs can be induced by supplying growth medium with chemical neurogenic inductors and/or 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. First the cells were preconditioned in proliferation medium, followed by induction of neuronal differentiation. Six canine ASCs cell lines were assessed, half from female and half from male donors. The cell morphology, growth dynamics, viability were observed along with expression of neuron and astrocyte specific markers, which were assessed by immunocytochemistry and flow cytometry. Results: After 3, 6 and 9 days, elongated neural-like cells with bipolar elongations were observed and some oval cells with light nuclei appeared. After three and nine days of neural induction, differentiation into neurons and glial cells was observed. Expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H) and glial fibrillary acidic protein (GFAP) was observed by immunocytochemistry. High GFAP expression (between 70 and 90% of all cells) was detected after three days of growth in neural induction medium a (NIMa) by flow cytometry, and expression of adult neuronal markers NF-H and microtubule associated protein-2 (MAP2) was detected in around 25% of cells. After nine days of ASCs differentiation a drop in expression rates of all markers was detected. There were no differences between neural differentiation of ASCs isolated from female or male dogs. Conclusions: The differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium the canine ASCs were able to transform to neural lineages, bearing markers of neuronal and glial cells and also displayed the typical neuronal morphology. Differentiated ASCs can be a source of neural cellular lineages for regenerative therapy of nerve damage and also could be applicable for modeling of neurodegenerative diseases.
Background: 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 damaged neuronal tissue in different neurological disorders. Reprogramming of ASCs can be induced by supplying growth medium with chemical neurogenic inductors and/or 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. First the cells were preconditioned in proliferation medium, followed by induction of neuronal differentiation. Six canine ASCs cell lines were assessed, half from female and half from male donors. The cell morphology, growth dynamics, viability were observed along with expression of neuron and astrocyte specific markers, which were assessed by immunocytochemistry and flow cytometry. Results: After 3, 6 and 9 days, elongated neural-like cells with bipolar elongations were observed and some oval cells with light nuclei appeared. After three and nine days of neural induction, differentiation into neurons and glial cells was observed. Expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H) and glial fibrillary acidic protein (GFAP) was observed by immunocytochemistry. High GFAP expression (between 70 and 90% of all cells) was detected after three days of growth in neural induction medium a (NIMa) by flow cytometry, and expression of adult neuronal markers NF-H and microtubule associated protein-2 (MAP2) was detected in around 25% of cells. After nine days of ASCs differentiation a drop in expression rates of all markers was detected. There were no differences between neural differentiation of ASCs isolated from female or male dogs. Conclusions: The differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium the canine ASCs were able to transform to neural lineages, bearing markers of neuronal and glial cells and also displayed the typical neuronal morphology. Differentiated ASCs can be a source of neural cellular lineages for regenerative therapy of nerve damage and also could be applicable for modeling of neurodegenerative diseases.
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