Purchase this article with an account.
Hannah Jane Levis, Rachel Williams, Jessica Jane Eyre; Endothelial colony forming cell repair in an in vitro co-culture model of the diabetic human retinal microvasculature.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2722.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the repair potential of endothelial colony forming cells (ECFCs) isolated from human umbilical cord (UCB) and peripheral blood (PB) in an in vitro co-culture model of the diabetic human retinal microvasculature.
ECFCs were isolated from healthy and gestational diabetic (GD) UCB and PB from healthy volunteers. Cells were expanded in culture and their growth was assessed and protein expression determined using immunostaining (CD31, CD34, vWF, ZO-1, VE-Cad). Cell surface immunophenotyping was also performed using flow cytometry. An in vitro co-culture model using human retinal pericytes (hRP) and human retinal endothelial cells (hRECs; both Cell Systems) was set up using PET transwell inserts (Merck). Cells in co-culture were established in physiological conditions (20% oxygen, 5.5mM glucose) for 3 days before being subjected to diabetic conditions (DC; 2% oxygen 33mM glucose) or maintained in control conditions (CC) for 7 days. ECFCs were transduced with GFP to enable tracking and then added to the endothelial layer of the co-cultures in either DC or CC. Integration of the ECFCs was assessed using ZO-1 staining and confocal microscopy after 24hrs and 7 days in culture.
ECFCs from PB and UCB could be isolated and expanded in vitro with PB displaying a slower expansion rate compared to UCB (3-6 weeks compared to 11-21 days, respectively). Populations were characterised using immunostaining and were seen to express typical markers of ECFCs (CD31, CD34, vWF, ZO-1, VE-Cad) as well as the typical cell surface markers using flow cytometry (CD34 +/-, CD105 +ve, CD146 +ve, CD31 +ve, CD14 -ve and CD45 –ve, CD90 -ve). Healthy UCB ECFCs incorporated into the endothelial layer of the co-culture model maintained in DC and CC as indicated by ZO-1 staining of GFP labelled cells. Gestational diabetic ECFCs also incorporated into the monolayer.
Expanded ECFCs could offer a potential novel cellular therapy for the repair of damaged vessels in the early stages of diabetic retinopathy. Our model of the human diabetic retinal microvasculature is very useful for assessing the potential of these expanded ECFCs. Our results suggest that even cells subjected to diabetic environments, such as in GD blood, are able to integrate into impaired endothelial layers. This is promising for the next stage of testing with peripheral blood from diabetic patients.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only