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大学・研究所にある論文を検索できる 「Differentiation of Adipose Derived Stem Cells by Stimulating with Extracellular Vesicles Secreted from Dermal Papilla Cells Activated with Osteogenic and Adipogenic Inducers」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。
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Differentiation of Adipose Derived Stem Cells by Stimulating with Extracellular Vesicles Secreted from Dermal Papilla Cells Activated with Osteogenic and Adipogenic Inducers

Taheruzzaman Kazi 島根大学

2022.09.16

概要

Dermal papilla ceils (DPCs) exist as spheroidal or globular shape m In-vivo condition and play crucial roles in hair regeneration, but they readily lose their hair forming ability during in vitro culture. Although the formation of spheroids partially restores the ability, shrinkage of the spheroids makes it difficult to maintain cellular viability. To address this problem, we stimulated DPCs with factors known to induce adipogenic and/or osteogenic differentiation, because DPCs share unique gene expression profiles with adipocytes and osteocytes. We isolated DPCs from versican (vcan)-GFP mice, in which GFP is expressed under the control of a vcan promoter, which is strongly active in DPCs of anagen hair follicles. In shortly, versican is a proteoglycan sulfate and serve the purpose as ECM (extracellular matrix molecule to cell-cell attachment. Isolated intact DPCs from anagen hair follicle showed intense GFP-fluorescence intensity, but it lost the fluorescence while these spheroids attached in monolayer culture surface. Even though in spheroid culture system, fluorescence getting declined and spheroid shaped became shrink which indicated the lost of hair inductive properties in DPCs. However, we analyze GFP fluorescence was most intense when the spheroids were made from DPCs cultured in a half-diluted combination of adipogenic and osteogenic media (CAO1/2), a Dulbecco's modified Eagle's medium-based medium (DMEM) that contains 10% FBS, 275 nM dexamethasone, 2.5 mM β-glycerol phosphate, 12.5 μg/mL ascorbic acid, 0.125 μM Isobutylmethylxanthine and 2.5 ng/mL insulin. The dose of each additive used was less than the optimal dose for adipogenic or osteogenic differentiation. We observed that, the media named CAOl/2 able to maintain GFP-fluorescence compare with the control DPCs which cultured with only 10% FBS contain DMEM, but the DPCs spheroid shrinkage was unavoidable in both groups.

　The shrinkage of the spheroids was avoided through the addition of fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor-AA (PDGF-AA) to CAOl/2 and named CAO1/2-FP. Addition of FGF-2 and PDGF-AA was administrated in CAOl/2 medium as dose dependent manner. The concentrations of two growth factors FGF-2 and PDGF-AA were 20, 40 and 100 ng/mL, respectively. We noticed that these concentrations elevated the GFP-fluorescence of DPC spheroid which was significant during the culture days but unable to prevent spheroidal shrinkage. Interestingly the GFP-fluorescence and spheroid size were maintained during the 4day spheroid culture while the FGF-2 and PDGF-AA applied jointly in culture condition at higher concentration. In addition, the gene and protein expression of vcan, osteopontin, alkaline phosphatase and α-smooth muscle actin in the spheroids were augmented to levels similar to those of the intact dermal papillae, which exhibited restored hair-forming activity.

　However, propagation and maintenance of DPCs became futile due loss the hair inductive properties over after several passage whereas isolation of DPCs itself laborious. To resolve this problem differentiation of DPCs properties in stem cells via extracellular vesicle (EV) could be an alternative way. Adipose derived stem cells (ASCs) are multipotent stem cells which are differentiated in several types of cells, if they are feed with appropriate condition media, for instance, chondrocyte medium differentiated ASCs into a chondrocyte cell. Recently, extracellular vesicle (EV)-mediated cell differentiation has gained attention in developmental biology due to genetic exchange between donor performed western-blot to scrutinize by specific and non-specific marker antibodies with DPC lysate and DPC-EVs lysate, respectively. Several antibodies are being used to detect EVs such as CD9, CD63, CD81, TSG101 and so on for positive marker, and Tubulin, CYC1 and HSP90 for negative marker. In our study, anti-CD63 antibody and anti-TSG101 antibody were applied to detect DPC-EVs. On the other hand, cytosolic fraction markers (HSP90 and CYC1) were detected only in cell lysate not in EVs lysate, which indicate that isolated EVs pure and were not contaminated by cell debris. In addition, Transmission electron microscope analyze confirmed that the average size of DPC derived EVs were between 80-170 nm.

　To understand the effect of DPC-EVs on cell differentiation, DPC-EVs were characterized and incubated with ASCs, of monolayer and spheroid cell cultures in combination with the CAO1/2-FP medium specialized for DPCs described above. DPC-like properties in ASCs were initially evaluated by comparing several genes and proteins with those of DPCs via real-time PCR analysis and immunostaining, respectively. We also valuated the presence of hair growth-related microRNAs (miRNAs), specifically mir214-5P, mir218-5p, and mir195-5P. Here, we found that miRNA expression patterns varied in DPC-EVs between passage 4 (P4) and P5. We hypothesized that miRNA may enhance the expression of hair-inductive genes in ASCs, as well as increasing cell proliferation. However, it has been reported that miRNA expression changes in vitro-cultured DPCs in a passage-dependent manner, and a high expression of one such miRNA, mir195-5p, inhibited DPC proliferation when it was transfected. In particular, mir214 was significantly higher in P5 DPC-EVs than in P4 DPC-EVs. However in vivo, mir214 overexpression has been reported to be responsible for the reduction in HFs in developing skin, and for a delay in hair cycle progression during postnatal development. As we showed that the lower expression of mir214 in P4 DPC-EVs implies its potential role in providing DP-like properties to ASCs and DPC-EVs derived from the lower passage cells might have increased hair growth activities.

　DPC-EVs in combination with CAO1/2-FP accelerated ASC proliferation at low concentrations. We have observed that DPC-EVs, at concentrations as low as 2 pg/mL in combination with CAO1/-2FP, enhanced the proliferation of ASCs. This strongly suggests that DPC-EVs are biologically active and affect the transformation of ASC properties into DP-like properties and propagated hair inductive gene expression for versican (vcan), alpha-smooth muscle actin (-sma), osteopontin (opn), and N-Cam (ncam). BVs were endocytosed by recipient cells, and the RNA cargo in the EVs was transferred, which in turn altered the recipient cell gene expression profile and function. Comparison between the expression of hair inductive genes (vcan, a-sma, ctnb, and others), the protein VCAN, α-SMA and β-Catenin (CTNB), and hair inductive miRNAs (mir214-5P, mir218-5p and mir195-5p) of DPC-EVs revealed similarities between P4 DPC-EVs- treated ASCs and DPCs.

　In conclusion, a combination of certain adipogenic and osteogenic inducers, together with fibroblast growth factor 2 and platelet-derived growth factor-AA, can promote differentiation toward the DPC lineage and early passage DPC-EVs, in combination with CAO1/2-FP, enabled ASCs to differentiate into DPC-like cells.

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