Effects of quantum dots and functional RGD-chimeric protein on the properties and differentiation of adipose-derived adult stem cells in an alginate three-dimensional culture system

• Main Authors: Jui-Chih Chang, 張瑞芝
• Other Authors: 蘇鴻麟
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/hw9h24

博士 === 國立中興大學 === 生命科學系所 === 98 === Label of human bone mesenchymal stem cells with CdSe/ZnS quantum dots (QDs) had been demonstrated to impair cell functions and activities. In the present study, QDs delivered by two different routes, Pep-1-labeled QDs (LQ) and PolyFect transfected QDs (TQ), were utilized to assess the effects of delivery mechanisms on various cellular responses of the QDs-internalized human adipose-derived adult stem cells (hADAS). Examination of labeled cells by flow cytometry and laser scanning confocal microscopy showed that LQ had higher fluorescence intensity due to the culster formation and their distribution in cytoplama while TQ were preferentially accumulated at peri-nuclear regions. The fluorescence intensity of the LQ group was still higher than that of the TQ group at 28 days after labeling, though cellular LQ were partitioned after initial cell division. Pep-1 but not PolyFect delivery facilitated QDs to escape from lysosome degradation. Pep-1 delivery of QDs rescued the cells from the negative effects caused by the internalized QDs on cell proliferation and on the expressions of CD29 and CD105 as well as osteogenic and chondrogenic associated lineage markers. The same effect was also observed in the expression of alkaline phosphatase activity, calcium deposition and secretion of chondrogenic matrices (GAG and collagen type II) in micromass culture. These indicated that Pep-1-delivered QDs may serve appropriately to track the hADAS employed in cell therapy/tissue engineering applications. A further study was conducted to reveal the associated mechanism. By comparing Pep-1- and PolyFect-mediated QD internalizations, the connexin 43 (Cx43)-mediated gap junction intercellular communication (GJIC) of hADAS was investigated in monolayer and in three-dimensional (3D) culture (alginate hollow spheres). The latter system offered cells more mobility, which was more similar as in vivo. The results showed that Pep-1-coated QDs, which escaped from the endo-/lysosome degradation, could activate the F-actin assembly and the ERK-dependent phosphorylation of Cx43. The consequence was a reduction in Cx43-mediated GJIC. When the cells were grown in high density 3D alginate hollow spheres instead of in monolayer, the decrease of GJIC caused by the QD internalization was restored. These results indicated that the adaptability in QDs-mediated regulation of GJIC with different delivery coatings depended on the culture systems. The study also suggested that the regulation of gap junction may play a key role in QD cytotoxicity. On the other hand, the role of integrin-binding peptides RGD on chondrogenesis of mesenchymal stem cells is controversial. We revealed the feasibility for flexible modification of RGD by embedding a large molecular weight and slightly charged (isoelectric point, 6-6.25) RGD-chimeric protein (CBD-RGD) with cellulose-binding domain (CBD) in three dimensional (3D) alginate beads to evaluate the chondrogenesis of ADAS. The binding of CBD-RGD with cells and its diffusion from alginate beads were studied on fluorescein isothiocyanate (FITC)-conjugated CBD-RGD. The increases in gene expression (Sox9, Aggrecan, fibronectin and collagen II), accumulation of chondrogenic matrice and decrease of collagen X gene expression during TGF-β3 induction were only observed for those beads containing 10 mg/g CBD-RGD initially, with 20.18±0.73% of that released in a week. The contrary was observed for beads with CBD-RGD 20 mg/g initially and having higher persistence (only 8.6±2.17% relased in a week). The 10 mg/g CBD-RGD-meiated enhancement was demonstrated via the activation of integrin α5 and β1-dependent pathway, and especially related to the upregulation of Sox9 gene and the temporary block of fibronectin expression as well as sustained inhibition of RhoA activity in the early differentiation stage. Thus, we speculated that the dynamic mobility of CBD-RGD may account for the enhanced chondrogenesis. It was concluded that the CBD-RGD-alginate culture system promoted the chondrogenesis of mesenchymal stem cells coordinated with TGF-β3 induction in an RGD dose-dependent manner.