Cultured retinal pigment epithelium (RPE) may become a therapeutic option for transplantation in retinal disease. However maintaining a native RPE phenotype in vitro has proven challenging. The human RPE cell-line ARPE-19 is used widely as an alternative to primary RPE. It is grown in DMEM/F12 medium as standard, but its phenotype is dependent on culture conditions, and many differentiation markers are usually absent.
The purpose of this study was to examine how this sensitive phenotype of ARPE-19 can be modulated by growth media with or without the metabolite pyruvate to elucidate better RPE growth conditions.
ARPE-19 cells at passages p22 to p28 were cultured on filters for up to 3 months in DMEM/F12 or DMEM media with or without pyruvate and 1% fetal calf serum. Assessment of differentiation was performed using pigmentation, immunocytochemistry, protein/mRNA expression, transepithelial resistance, VEGF secretion, and ultrastructure.
Pyruvate, in combination with DMEM, induced dark pigmentation and promoted differentiation markers such as CRALBP and MerTK. Importantly, RPE65 protein was detected by Western blotting and was enhanced by pyruvate, high glucose, and DMEM. ARPE-19 cells maintained in this medium could also phagocytose human photoreceptor outer segments (POS). VEGF secretion was greater in DMEM cultures and was affected by glucose but not by pyruvate. Pigmentation never occurred in DMEM/F12.
This study demonstrated important differentiation markers, including pigmentation and Western blots of RPE65 protein, and showed human POS phagocytosis in ARPE-19 cultures using a simple differentiation protocol. The results favor the use of high-glucose DMEM with pyruvate for future RPE differentiation studies.
Mesenchymal stem cell transplantation and DMEM administration in a 3NP rat model of Huntington’s disease: morphological and behavioral outcomes.
Transplantation of mesenchymal stem cells (MSCs) may offer a viable treatment for Huntington’s disease (HD). We tested the efficacy of MSC transplants to reduce deficits in a 3-nitropropionic acid (3NP) rat model of HD. Five groups of rats (Sham, 3NP, 3NP+vehicle, 3NP+TP(low), 3NP+TP(high)), were given PBS or 3NP intraperitoneally, twice daily for 42 days.
On day 28, rats in all groups except Sham and 3NP, received intrastriatal injections of either 200,000 MSCs (TP(low)), 400,000 (TP(high)) MSCs or DMEM (VH, the vehicle for transplantation).
MSCs survived 72 days without inducing a strong inflammatory response from the striatum. Behavioral sparing was observed on tests of supported-hindlimb-retraction, unsupported-hindlimb-retraction, visual paw placement and stepping ability for 3NP+TP(low) rats and on the unsupported-hindlimb-retraction and rotarod tasks for 3NP+VH rats.
Relative to 3NP controls, all treated groups were protected from 3NP-induced enlargement of the lateral ventricles. In vitro, MSCs expressed transcripts for numerous neurotrophic factors. In vivo, increased striatal labeling in BDNF, collagen type-I and fibronectin (but not GDNF or CNTF) was observed in the brains of MSC-transplanted rats but not in DMEM-treated rats.
In addition, none of the transplanted MSCs expressed neural phenotypes. These findings suggest that factors other than neuronal replacement underlie the behavioral sparing observed in 3NP rats after MSC transplantation.
Proliferation and gene expression of osteoblasts cultured in DMEM containing the ionic products of dicalcium silicate coating.
The medium containing ionic products of dicalcium silicate (Ca(2)SiO(4)) for culturing the osteoblast-like cells (MG63) were prepared by immersing the plasma sprayed Ca(2)SiO(4) coatings in DMEM solution for 24h. The normal DMEM was also used to culture the MG63 cells as the control group.
The results obtained from this work showed that the proliferation of MG63 cell was more significant in the group containing ionic products of Ca(2)SiO(4) coatings than in the control group. The cell cycle distribution indicated that the decreased G(0)/G(1) phase and increased S phase occurred in the cells cultured in the DMEM containing ionic products of Ca(2)SiO(4) coatings.
The analysis of osteogenic genes indicated that the ionic products of Ca(2)SiO(4) coating enhanced the expression of osteoblast-related genes and promote differentiation of MG63 cells at initial period. Moreover, the ionic products of Ca(2)SiO(4) coating differentially regulated osteoclastogenic gene expression by up-regulated OPG and down-regulated RANKL.