OBJECTIVE: By using opportunely supplemented culture media, adipose derived mesenchymal stem cells (ASCs) may transdifferentiate into several cellular lines, including those of ectodermal origin. The aim of the present study was to test whether a neural differentiation of ASCs could be induced by conditioned media (CM) obtained from cultures of Olfactory Ensheathing Cells (OECs) or Schwann Cells (SCs). MATERIALS AND METHODS: After informed consent, adipose tissue was obtained from patients undergoing aesthetic surgery interventions. Following collagenase digestion and centrifugation of lipoaspirate, ASCs contained in the stromal vascular fraction were expanded for 2-3 passages in basal medium for stem cells (DMEM added with 10% FBS and 1% mesenchymal stem cell growth supplement). A population of ASCs was used for their characterization as stem cells; to this purpose the presence of CD44, CD90 and CD105 were verified. Two other populations were cultured in either CM for 6 days. At day 1, 3 and 6, they were tested by mmunocytochemistry and flow cytometry for the expression of some neuronal markers such as nestin, PGP 9.5 and glial marker GFAP. The expression of Connexins (Cx) 32 and 36, strongly expressed in neural cells, was also tested. In all cultures, the presence of cells was assessed by DAPI staining. RESULTS: Stem cell nature of ASCs was determined by their positive immunostaining to CD44, CD90 and CD105; consistently, cells were immunonegative to hematopoietic markers such as CD14, CD34 and CD45. Immunocytochemistry and flow cytometry show that, when compared to controls, all neural markers were overexpressed in cultures with either OECs-CM or SCs-CM, although some markers could be better induced by using OEC-CM (PGP 9.5 and Cx32) or SCs-CM (Nestin and Cx36). Normally, results were clearly present already at day 1, and were more evident after 3 and 6 days. No evident difference was observed for GFAP overexpression when using either CM. It is worth noting that, as the neurogenic differentiation progresses, DAPI staining shows that cell proliferation progressively decreases. CONCLUSION: This study demonstrates that environmental features can induce differentiation of ASCs toward a neural phenotype, either as neuronal or glial elements. This culture strategy provides might be useful in the development of cell-based therapy for central nervous system degenerative diseases.
INFLUENCES OF CONDITIONED MEDIA ON NEURAL DIFFERENTIATION OF HUMAN ADIPOSE MESENCHYMAL STEM CELLS
R Pellitteri;
2016
Abstract
OBJECTIVE: By using opportunely supplemented culture media, adipose derived mesenchymal stem cells (ASCs) may transdifferentiate into several cellular lines, including those of ectodermal origin. The aim of the present study was to test whether a neural differentiation of ASCs could be induced by conditioned media (CM) obtained from cultures of Olfactory Ensheathing Cells (OECs) or Schwann Cells (SCs). MATERIALS AND METHODS: After informed consent, adipose tissue was obtained from patients undergoing aesthetic surgery interventions. Following collagenase digestion and centrifugation of lipoaspirate, ASCs contained in the stromal vascular fraction were expanded for 2-3 passages in basal medium for stem cells (DMEM added with 10% FBS and 1% mesenchymal stem cell growth supplement). A population of ASCs was used for their characterization as stem cells; to this purpose the presence of CD44, CD90 and CD105 were verified. Two other populations were cultured in either CM for 6 days. At day 1, 3 and 6, they were tested by mmunocytochemistry and flow cytometry for the expression of some neuronal markers such as nestin, PGP 9.5 and glial marker GFAP. The expression of Connexins (Cx) 32 and 36, strongly expressed in neural cells, was also tested. In all cultures, the presence of cells was assessed by DAPI staining. RESULTS: Stem cell nature of ASCs was determined by their positive immunostaining to CD44, CD90 and CD105; consistently, cells were immunonegative to hematopoietic markers such as CD14, CD34 and CD45. Immunocytochemistry and flow cytometry show that, when compared to controls, all neural markers were overexpressed in cultures with either OECs-CM or SCs-CM, although some markers could be better induced by using OEC-CM (PGP 9.5 and Cx32) or SCs-CM (Nestin and Cx36). Normally, results were clearly present already at day 1, and were more evident after 3 and 6 days. No evident difference was observed for GFAP overexpression when using either CM. It is worth noting that, as the neurogenic differentiation progresses, DAPI staining shows that cell proliferation progressively decreases. CONCLUSION: This study demonstrates that environmental features can induce differentiation of ASCs toward a neural phenotype, either as neuronal or glial elements. This culture strategy provides might be useful in the development of cell-based therapy for central nervous system degenerative diseases.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
