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FGF9 induces functional differentiation to Schwann cells from human adipose derived stem cells.

ABSTRACT: Rationale: The formation of adipose-derived stem cells (ASCs) into spheres on a chitosan-coated microenvironment promoted ASCs differentiation into a mixed population of neural lineage-like cells (NLCs), but the underline mechanism is still unknown. Since the fibroblast growth factor 9 (FGF9) and fibroblast growth factor receptors (FGFRs) play as key regulators of neural cell fate during embryo development and stem cell differentiation, the current study aims to reveal the interplay of FGF9 and FGFRs for promoting peripheral nerve regeneration. Methods: Different concentration of FGF9 peptide (10, 25, 50, 100 ng/mL) were added during NLCs induction (FGF9-NLCs). The FGFR expressions and potential signaling were studied by gene and protein expressions as well as knocking down by specific FGFR siRNA or commercial inhibitors. FGF9-NLCs were fluorescent labeled and applied into a nerve conduit upon the injured sciatic nerves of experimental rats. Results: The FGFR2 and FGFR4 were significantly increased during NLCs induction. The FGF9 treated FGF9-NLCs spheres became smaller and changed into Schwann cells (SCs) which expressed S100? and GFAP. The specific silencing of FGFR2 diminished FGF9-induced Akt phosphorylation and inhibited the differentiation of SCs. Transplanted FGF9-NLCs participated in myelin sheath formation, enhanced axonal regrowth and promoted innervated muscle regeneration. The knockdown of FGFR2 in FGF9-NLCs led to the abolishment of nerve regeneration. Conclusions: Our data therefore demonstrate the importance of FGF9 in the determination of SC fate via the FGF9-FGFR2-Akt pathway and reveal the therapeutic benefit of FGF9-NLCs.

SUBMITTER: Huang CW

PROVIDER: S-EPMC7052907 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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FGF9 induces functional differentiation to Schwann cells from human adipose derived stem cells.

Huang Chia-Wei CW Lu Shih-Yu SY Huang Tzu-Chieh TC Huang Bu-Miim BM Sun H Sunny HS Yang Shang-Hsun SH Chuang Jih-Ing JI Hsueh Yuan-Yu YY Wu Yi-Ting YT Wu Chia-Ching CC

Theranostics 20200203 6

<b>Rationale</b>: The formation of adipose-derived stem cells (ASCs) into spheres on a chitosan-coated microenvironment promoted ASCs differentiation into a mixed population of neural lineage-like cells (NLCs), but the underline mechanism is still unknown. Since the fibroblast growth factor 9 (FGF9) and fibroblast growth factor receptors (FGFRs) play as key regulators of neural cell fate during embryo development and stem cell differentiation, the current study aims to reveal the interplay of FG ...[more]

PMID: 32194837

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Project description:Schwann cells (SCs) are fundamental for development, myelination and regeneration in the peripheral nervous system. Slow growth rate and difficulties in harvesting limit SC applications in regenerative medicine. Several molecules, including receptors for neurosteroids and neurotransmitters, have been suggested to be implicated in regulating physiology and regenerative potential of SCs. Adipose-derived stem cells (ASCs) can be differentiated into SC-like phenotype (dASC) sharing morphological and functional properties with SC, thus representing a valid SC alternative. We have previously shown that dASC express γ-aminobutyric-acid receptors, which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitters in ASC. In this study, we investigated the expression of purinergic receptors in dASC. Using reverse transriptase (RT)-PCR, western blot analyses and immunocytochemistry, we have demonstrated that ASCs express P2X3, P2X4 and P2X7 purinoceptors. Differentiation of ASCs towards glial phenotype was accompanied by upregulation of P2X4 and P2X7 receptors. Using Ca(2+)-imaging techniques, we have shown that stimulation of purinoceptors with adenosine 5'-triphosphate (ATP) triggers intracellular Ca(2+) signals, indicating functional activity of these receptors. Whole-cell voltage clamp recordings showed that ATP and BzATP induced ion currents that can be fully inhibited with specific P2X7 antagonists. Finally, using cytotoxicity assays we have shown that the increase of intracellular Ca(2+) leads to dASC death, an effect that can be prevented using a specific P2X7 antagonist. Altogether, these results show, for the first time, the presence of functional P2X7 receptors in dASC and their link with critical physiological processes such as cell death and survival. The presence of these novel pharmacological targets in dASC might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dASC for nerve repair.

Project description:Human adipose-derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5-azacytidine (5-aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co-culture with neonatal rat cardiomyocytes. 5-aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5- to 1.9-fold) and the number of cells co-expressing nkx2.5/sarcomeric alpha-actin (27.2% versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11-fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA-treated cells also stained positively for cardiac myosin heavy chain, alpha-actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non-contact co-culture showed no cardiac differentiation; however, ASCs co-cultured in direct contact co-culture exhibited a time-dependent increase in cardiac actin mRNA expression (up to 33-fold) between days 3 and 14. Immunocytochemistry revealed co-expression of GATA4 and Nkx2.5, alpha-actin, TropI and cardiac myosin heavy chain in CM-DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca(2+) transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell-to-cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co-culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.

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FGF9 induces functional differentiation to Schwann cells from human adipose derived stem cells.

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FGF9 induces functional differentiation to Schwann cells from human adipose derived stem cells.

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