test was used to determine statistical significance. abolished recruitment of hiPSCs in the OIR model. These findings suggest superior angiogenic potential of hiPSC-ECs under hypoxia and underscore the importance of SDF1a/CXCR4 in Psoralen the reparative function of hiPSC-ECs in ischemic diseases. = 30, = 3). Box extends from the 25th to 75th percentile with median line in the middle; whiskers represent min to max range. (D) qRT-PCR for and = 3). Data are presented as mean SD. test was used to determine statistical significance. Significance levels are set at * 0.05, ** 0.01, and **** 0.0001. Scale bars: 200 m. and and an upregulation in mRNA at comparable levels (Physique 1D). Hypoxic network formation on hiPSC-ECs and HRECs in vitro. To determine the effect of hypoxia on network formation, we next analyzed the vasculogenic potential of hiPSC-ECs and HRECs by using an in vitro 3D collagen hydrogel assay. hiPSC-ECs and HRECs were encapsulated in collagen hydrogel and exposed to hypoxia for 48 hours. In atmospheric conditions, hiPSC-ECs formed strong and complex vascular networks, whereas HRECs displayed some sprouting, with many cells remaining globular after 48 hours (Physique 2A). In hypoxic conditions, hiPSC-EC networks were elongated, with a higher number of vascular tubes quantified (Physique 2B), whereas HRECs formed shorter, less thick tubes (though not statistically significant). In addition, there was a statistically significant reduction in total tube volume of HRECs under hypoxia, highlighting the contrast in hypoxic network formation between hiPSC-ECs and HRECs. Open in a separate window Physique 2 In hypoxia, hiPSC-ECs form robust vascular networks compared with HRECs.(A) Representative confocal images of human-induced pluripotent stem cellCderived endothelial cells (hiPSC-ECs) or human retinal endothelial cells (HRECs) in collagen type I hydrogels after 48 hours of culture in atmospheric or hypoxic conditions; left panel is a top view and right panel is usually a side view (phalloidin in green, nuclei in blue, scale bar represents 100 m). (B) Quantification of common Psoralen tube length, and tube volume (= 3). Tukeys multiple comparisons test was conducted to determine statistical significance. Significance levels are set at * 0.05, ** 0.01, and *** 0.001. Data are Psoralen presented as mean SD. Scale bar: 200 m. hiPSC-ECs integrate into mouse host vasculature in the OIR model. To determine if hiPSC-ECs can successfully integrate into host vasculature in an ischemic setting, we used the OIR model in NOD/SCID mice. We reasoned that the mouse OIR model is an excellent model for studying homing and incorporation of hiPSC-ECs in ischemic/hypoxic retina. Given the translational implications, we used the intravitreal injection approach because intravitreal injections are now the most commonly performed ophthalmic procedure (9). The hiPSC-ECs were injected via the intravitreal route on P12, immediately after removal of pups from 75% O2, and retinas were collected on P17 (Figure 3A). PRKD3 We found that hiPSC-ECs showed successful colocalization with host vasculature as early as P14 (Supplemental Figure 2), with continued colocalization on P17. In contrast, eyes injected with HRECs resulted in the deposition of cells anterior to the retinal vasculature (Figure 3, B and C). High-magnification images further demonstrated that hiPSC-ECs showed incorporation into regenerating retinal vessels, whereas HRECs showed limited integration at the same time point (Figure 3C). With the results demonstrating incorporation of hiPSC-ECs, but not HRECs, into retinal vasculature, vascular rescue effects of the hiPSC-ECs were evaluated. For this, VO (avascular retinal area) and pathological retinal NV were measured on P17. Intravitreal injection of hiPSC-ECs significantly reduced VO and NV compared with the contralateral eyes injected with PBS (Figure 3D), indicating a marked degree of reparative angiogenesis. In association with this vascular recovery and reduction in ischemic retinal area, treatment with hiPSC-EC.