Hypoxic Conditions Promote the Angiogenic Potential of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles

Abstract

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.

Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles

“…EVs (10 µL) were incubated for 48 h at 4 °C on Ibidi slides with 40 µL sodium chloride/HEPES. Staining was performed as previously described [ 15 ], using anti-human CD63-AlexaFluor488 (clone MEM-259, Invitrogen, Waltham, MA, USA, 0.2 mg/mL) and anti-human CD81-AlexaFluor647 (clone 454720, R&D Systems, Minneapolis, MN, USA, 0.2 mg/mL) for staining.…”

“…Stimulation of PBMCs was performed with either 5 µg/mL PHA (Sigma-Aldrich). EVs were added in ratios of 15,000:1, 5000:1, and 1666:1, EVs:PBMCs, respectively, based on previous titration [ 15 , 32 , 33 ]. The precise number of EVs secreted in vivo is not known.…”

“…Hypoxia, as an intrinsic property of certain tumor environments, can promote increased tumor-derived EV secretion [ 13 , 14 ]. Hypoxia can also induce elevated secretion of proangiogenic EVs by pluripotent stem cells indicating a more general mechanism [ 15 ]. In a community approach, minimal information for studies of EVs (MISEV) guidelines were established in 2014 and updated in 2018, recommending stringent reporting of EV isolation and characterization parameters, and encouraging the use of the ‘umbrella term’ EV for the various species of exomeres, exosomes, microvesicles, ectosomes, oncosomes, and apoptotic bodies [ 16 , 17 ].…”

“…Particularly, the EV quantity, but also quality and characterization, remain paramount for preclinical and clinical research [ 8 ]. We took advantage of our protocols for stem cell and stromal cell EV isolation [ 15 , 30 ] and devised a scalable strategy for AML-EV purification.…”

Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production

“…Starting from seven million hiPSCs at research scale in this study, we obtained only 1.64 ± 0.59 × 10 9 platelets in total (mean ± SD). In a recent study, we demonstrated reproducible hiPSC 2D large scale propagation and obtained 9.8 ± 1.2 × 10 8 hiPSCs (mean ± SD) from one million hiPSCs per four-layered cell factory on 2528 cm 2 growth area within eight days [ 34 ]. MK culture using our improved protocol would suffice producing one single unit of platelets for transfusion including 2.5 × 10 11 platelets, in theory, within less than a month.…”