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CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect
Susumu Sakimoto, … , Peter D. Westenskow, Martin Friedlander
Susumu Sakimoto, … , Peter D. Westenskow, Martin Friedlander
Published January 26, 2017
Citation Information: JCI Insight. 2017;2(2):e89906. https://doi.org/10.1172/jci.insight.89906.
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Research Article Angiogenesis Stem cells

CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect

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Abstract

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations, since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina, it may be possible to use one cell type to rescue another cell type in the face of severe stress, such as hypoxia or genetically encoded cell-specific degenerations. Here, we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore, we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44, the hyaluronic acid receptor, and IGFBPs (insulin-like growth factor–binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases.

Authors

Susumu Sakimoto, Valentina Marchetti, Edith Aguilar, Kelsey Lee, Yoshihiko Usui, Salome Murinello, Felicitas Bucher, Jennifer K. Trombley, Regis Fallon, Ravenska Wagey, Carrie Peters, Elizabeth L. Scheppke, Peter D. Westenskow, Martin Friedlander

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Figure 1

Isolation and amplification of ECFCs using xeno-free media: in vitro and in vivo characterization.

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Isolation and amplification of ECFCs using xeno-free media: in vitro and...
(A) Representative photomicrographs of individual human ECFC-derived endothelial cell colonies from umbilical cord blood in serum-containing media (SCM) and xeno-free media (XFM). Morphology of ECFCs derived in XFM did not differ from that of those derived in SCM. Colony of ECFCs derived in XFM at day 10 at low (top row) and high (bottom row) magnification. Scale bar: 500 μm (top row); 200 μm (bottom row). (B) No significant difference in the mean number of days before initial colony appeared from mononuclear cells after culture initiation in SCM and XFM (n = 4). (C) No significant difference in the number of initial EC colonies between culture in SCM and XFM at day 10 (n = 7). (B and C) Error bars represent SEM. (D) Average number of colonies in the cloning assay at day 14 passage 4 (*P = 0.0021, Student’s t test) and distribution of colony size for in vitro cloning assay in SCM and XFM. XFM supports the whole hierarchy of ECFCs. ECFCs derived and amplified in XFM maintained their high proliferative capacity (medium and large colony size) in cloning assays. Error bars represent SD. (E) Phenotypic analysis of ECFCs derived in XFM. Representative flow cytometry histograms of ECFCs derived in XFM show reactivity with EC-expressed marker molecules (right-shifted, black-filled curves compared with gray-filled curves of the appropriate isotype controls) and lack of reactivity with hematopoietic (CD14 and CD45) and mesenchymal stem cell–associated (CD90) markers. (F and G) Rescue of oxygen-induced retinopathy (OIR) by intravitreally injected ECFCs from XFM or SCM, normalized to vehicle injection. (F) Representative GS lectin–stained neural retina flat-mount preparations injected at P12 with vehicle or ECFCs from XFM or SCM. Eyes were isolated from P17 OIR mice. In the insets of F, neovascular tufts (NV) and vaso-obliterated regions (VO) are labeled red and yellow, respectively. Scale bar: 500 μm. (G) Total area of NV and VO in vehicle and ECFCs from XFM or SCM injected eyes was calculated and plotted (n = 16 in ECFCs and n = 10 in vehicle. *P < 0.001, Kruskal-Wallis test with Dunn’s multiple comparison test). Error bars represent SEM.

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