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Comparison of human retinal progenitor cells cultured in media with or without serum: in vitro and in vivo characteristics and retinal transplantation.

ABSTRACT: Retinal progenitor cells (RPCs) have a potential role in the treatment of retinal degenerative diseases. This study is to investigate in vitro and in vivo characteristics and retinal transplantation of RPCs cultured in media with or without serum. Progenitor cells obtained from the neural retina of human eyes at 6-16 weeks gestation were cultured in serum-free media (SF-hRPCs) or in media containing 10% fetal bovine serum (FBS) (S-hRPCs). The differences were characterized between the cells cultured in vitro and transplanted (retinal transplantation) into Royal College of Surgeons (RCS) rats. The functional status of the rats was examined by flash-electroretinogram recordings. The result was that S-hRPCs exhibited higher proliferative dynamics in vitro. On the basis of outer nuclear layer thickness and flash-electroretinograms, S-hRPCs were more efficacious in slowing the progression of retinal degeneration following transplantation compared with SF-hRPCs. Moreover, retinal mesenchymal-like stem cells were isolated and identified from the S-hRPCs cultures. Our study demonstrated the potential of retinal MSCs for the treatment of retinal degeneration.

SUBMITTER: Qu L

PROVIDER: S-EPMC6963018 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Comparison of human retinal progenitor cells cultured in media with or without serum: in vitro and in vivo characteristics and retinal transplantation.

Qu Linghui L Jin Xin X Chen Nan N Wang Dajiang D

International journal of clinical and experimental pathology 20181101 11

Retinal progenitor cells (RPCs) have a potential role in the treatment of retinal degenerative diseases. This study is to investigate in vitro and in vivo characteristics and retinal transplantation of RPCs cultured in media with or without serum. Progenitor cells obtained from the neural retina of human eyes at 6-16 weeks gestation were cultured in serum-free media (SF-hRPCs) or in media containing 10% fetal bovine serum (FBS) (S-hRPCs). The differences were characterized between the cells cult ...[more]

PMID: 31949597

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Project description:Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions. However, comprehensive identification and quantification of secreted glycoproteins is a daunting task because of their low abundances compared with the high-abundance serum proteins required for cell growth and proliferation. Several studies employed serum-free media to analyze secreted proteins, but it has been shown that serum starvation, even for a short period of time, can alter protein secretion. To overcome these issues, we developed a method to globally characterize secreted glycoproteins and their N-glycosylation sites from cultured cells by combining selective enrichment of secreted glycoproteins with a boosting approach. The results demonstrated the importance of the boosting sample selection and the boosting-to-sample ratio for improving the coverage of secreted glycoproteins. The method was applied to globally quantify secreted glycoproteins from THP-1 monocytes and macrophages in response to lipopolysaccharides (LPS) and from Hep G2 cells treated with TGF-β without serum starvation. We found differentially secreted glycoproteins in these model systems that showed the cellular response to the immune activation or the epithelial-to-mesenchymal transition. Benefiting from the selective enrichment and the signal enhancement of low-abundance secreted glycoproteins, this method can be extensively applied to study secreted glycoproteins without serum starvation, which will provide a better understanding of protein secretion and cellular activity.

Project description:Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model.The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined.We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats.We have successfully differentiated rESCs to glia enriched RPCs and retinal neuron enriched RPCs in vitro. The retinal neuron enriched rESC-RPC2 protected the structure and function of retina in rats with genetic retinal degeneration and could be a candidate cell source for treating some degenerative retinal diseases in human trials.

Dataset Information

Comparison of human retinal progenitor cells cultured in media with or without serum: in vitro and in vivo characteristics and retinal transplantation.

Publications

Comparison of human retinal progenitor cells cultured in media with or without serum: in vitro and in vivo characteristics and retinal transplantation.

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