CD362+ stromal stem cells(SSCs) stabilize vascular networks through differentiation into pericyte-like cells

Abstract

Purpose : Cell-based therapy provides a promising strategy to address pericyte loss and microvascular insufficiency in early diabetic retinopathy. In our previous study, a sub-population of bone marrow-derived stromal stem cells (SSCs) sorted on CD362 promoted vascular repair in oxygen-induced retinppathy (OIR). The present work focus on the related mechanism to study angiogenesis in 3D culture system.

Methods : SSCs were sorted from human bone marrow mononuclear cells on the basis of the presence or absence of surface marker CD362 (syndecan-2). This created two distinct populations of SSCs described as CD362+ and CD362- cells. In vitro tubulogenesis was conducted under hypoxia(1% O₂) using two inter-related approaches: 1) A Matrigel assay using endothelial colony forming cells (ECFCs ) (7x10⁴) mixed with CD362+SSCs (1X10⁴) were suspended in Matrigel which was spotted onto 24 well plates; 2) A double-layer Matrigel assay in which ECFCs were suspended in Matrigel and spotted onto 24 well plates followed by topping of another Matrigel layer containing CD362+SSCs one day later. Tube-like structures were imaged using confocal microscopy or conducted for immunostaining assay.

Results : The pericyte markers, NG2, PDGFR β, α-SMA were detected in both CD362+ SSCs and CD362-SSCs, in which significant increase of α-SMA expression cells were found in CD362+ SSCs compared to CD362- SSCs (45.7% ±0.04 in CD 362+SSCs and 24.5% ±0.06 in CD362-SSCs, p=0.0306,n=4 ). In the in vitro angiogenesis assay, regression of ECFC tubules occurred at 1 week while the presence of CD362+SSCs stabilized the network with the cells taking on a perivascular location. CD362+SSCs /ECFC co-culture maintained stabilized vascular networks until two weeks by showing significantly enhanced tube area(p=0.005,n=4) and branch points (p<0.0001,n=8) compared to ECFCs alone. In the double-layer Matrigel assay, CD362+SSCs migrated into “pre-formed” ECFC networks and took up a perivascualr position. Preicyte markers were expressed in those CD362+SSCs resident alongside tubes.

Conclusions : CD362+ SSCs appeared to act as pericyte progenitors by showing the capacity to associate with vascular tubes and stabilize the network under hypoxia conditions. These cells have promise for cell-replacement therapy for retinal ischemic diseases such as diabetic retinopathy.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.