scholarly journals Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taina Viheriälä ◽  
Juhana Sorvari ◽  
Teemu O. Ihalainen ◽  
Anni Mörö ◽  
Pyry Grönroos ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Attachment ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Embryonic Stem ◽  
Calcium Signalling ◽  
Surface Protein ◽  
Barrier Properties ◽  
Type Iv ◽  
Nidogen 1

AbstractHuman pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.

scholarly journals Embryonic stem cell microenvironment enhances proliferation of human retinal pigment epithelium cells by activating the PI3K signaling pathway

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiahui Liu ◽  
Liu Yang ◽  
Xiaoran Wang ◽  
Shoubi Wang ◽  
Zheqian Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Signaling Pathway ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Embryonic Stem ◽  
Age Related Macular Degeneration ◽  
Pi3k Signaling ◽  
Age Related ◽  
Pi3k Signaling Pathway

Abstract Background Retinal pigment epithelium (RPE) replacement has been proposed as an efficacious treatment for age-related macular degeneration (AMD), which is the primary cause of vision loss in the elderly worldwide. The embryonic stem cell (ESC) microenvironment has been demonstrated to enable mature cells to gain a powerful proliferative ability and even enhance the stem/progenitor phenotype via activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As the PI3K signaling pathway plays a pivotal role in proliferation and homeostasis of RPE, we hypothesize that the stemness and proliferative capability of RPE can be enhanced by the ESC microenvironment via activation of the PI3K signaling pathway. Methods To investigate whether the ESC microenvironment improves the stem cell phenotype and proliferation properties of human RPE (hRPE) cells by regulating the PI3K signaling pathway, primary hRPE cells were cocultured with either ESCs or human corneal epithelial cells (CECs) for 72 h, after which their proliferation, apoptosis, cell cycle progression, and colony formation were assayed to evaluate changes in their biological characteristics. Gene expression was detected by real-time PCR and protein levels were determined by western blotting or immunofluorescence. LY294002, an antagonist of the PI3K signaling pathway, was used to further confirm the mechanism involved. Results In comparison to hRPE cells cultured alone, hRPE cells cocultured with ESCs had an increased proliferative capacity, reduced apoptotic rate, and higher colony-forming efficiency. The expression of the stem cell-associated marker KLF4 and the differentiation marker CRALBP increased and decreased, respectively, in hRPE cells isolated from the ESC coculture. Furthermore, PI3K pathway-related genes were significantly upregulated in hRPE cells after exposure to ESCs. LY294002 reversed the pro-proliferative effect of ESCs on hRPE cells. In contrast, CECs did not share the ability of ESCs to influence the biological behavior and gene expression of hRPE cells. Conclusions Our findings indicate that the ESC microenvironment enhances stemness and proliferation of hRPE cells, partially via activation of the PI3K signaling pathway. This study may have a significant impact and clinical implication on cell therapy in regenerative medicine, specifically for age-related macular degeneration.

scholarly journals Transcriptomic Profiling of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Over Time

2019 ◽  
Author(s):  
Grace E. Lidgerwood ◽  
Anne Senabouth ◽  
Casey J.A. Smith-Anttila ◽  
Vikkitharan Gnanasambandapillai ◽  
Dominik C. Kaczorowski ◽  
...  
Keyword(s):  
Stem Cell ◽  
Retinal Pigment Epithelium ◽  
Pluripotent Stem Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cell ◽  
Mesenchymal Transition ◽  
Rpe Cells

AbstractHuman pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of disease associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate points in time for RPE cultures to reflect native counterparts, we characterised the transcriptomic profiles of hPSC-derived retinal pigment epithelium (RPE) cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single cell RNA-sequencing of a total of 16,576 cells, and analysed the resulting data to assess the molecular changes of RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial – mesenchymal transition, and with maturing populations of RPE observed with time in culture. Assessment of gene ontology pathways revealed that as cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirmed a maturing transcriptional profile of RPE cells in culture. These results suggest that in vitro long-term culture of RPE cells allow the modelling of specific phenotypes observed in native mature tissue. Our work highlights the transcriptional landscape of hPSC-derived RPE as they age in culture, which provides a reference for native and patient-samples to be benchmarked against.

scholarly journals Phase 1 clinical study of an embryonic stem cell–derived retinal pigment epithelium patch in age-related macular degeneration

2018 ◽  
Vol 36 (4) ◽  
pp. 328-337 ◽  
Author(s):  
Lyndon da Cruz ◽  
Kate Fynes ◽  
Odysseas Georgiadis ◽  
Julie Kerby ◽  
Yvonne H Luo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Clinical Study ◽  
Retinal Pigment Epithelium ◽  
Embryonic Stem Cell ◽  
Pigment Epithelium ◽  
Phase 1 ◽  
Retinal Pigment ◽  
Embryonic Stem ◽  
Age Related Macular Degeneration ◽  
Age Related
Sign in / Sign up

Export Citation Format

Share Document