Cellular self-assembly into 3D microtissues enhances the angiogenic activity and functional neovascularization capacity of human cardiopoietic stem cells

Abstract

While cell therapy has been proposed as next-generation therapy to treat the diseased heart, current strategies display only limited clinical efficacy. Besides the ongoing quest for the ideal cell type, in particular the very low retention rate of single-cell (SC) suspensions after delivery remains a major problem. To improve cellular retention, cellular self-assembly into 3D microtissues (MTs) prior to transplantation has emerged as an encouraging alternative. Importantly, 3D-MTs have also been reported to enhance the angiogenic activity and neovascularization potential of stem cells. Therefore, here using the chorioallantoic membrane (CAM) assay we comprehensively evaluate the impact of cell format (SCs versus 3D-MTs) on the angiogenic potential of human cardiopoietic stem cells, a promising second-generation cell type for cardiac repair. Biodegradable collagen scaffolds were seeded with human cardiopoietic stem cells, either as SCs or as 3D-MTs generated by using a modified hanging drop method. Thereafter, seeded scaffolds were placed on the CAM of living chicken embryos and analyzed for their perfusion capacity in vivo using magnetic resonance imaging assessment which was then linked to a longitudinal histomorphometric ex vivo analysis comprising blood vessel density and characteristics such as shape and size. Cellular self-assembly into 3D-MTs led to a significant increase of vessel density mainly driven by a higher number of neo-capillary formation. In contrast, SC-seeded scaffolds displayed a higher frequency of larger neo-vessels resulting in an overall 1.76-fold higher total vessel area (TVA). Importantly, despite that larger TVA in SC-seeded group, the mean perfusion capacity (MPC) was comparable between groups, therefore suggesting functional superiority together with an enhanced perfusion efficacy of the neo-vessels in 3D-MT-seeded scaffolds. This was further underlined by a 1.64-fold higher perfusion ratio when relating MPC to TVA. Our study shows that cellular self-assembly of human cardiopoietic stem cells into 3D-MTs substantially enhances their overall angiogenic potential and their functional neovascularization capacity. Hence, the concept of 3D-MTs may be considered to increase the therapeutic efficacy of future cell therapy concepts.

Cardiopoietic stem cell therapy restores infarction-altered cardiac proteome

“…While a metabolic impact is traditionally observed with stem cellmediated repair 29,30 , the prominent association with provasculogenic induction distinguishes CP cell therapy. In accordance, independent reports highlight the angiogenic potential and functional neovascularization capacity of CP stem cells promoting new vessel formation in treated hearts 4,5,47 . While CP stem cell therapy did not completely restore the normal cardiac proteome, the proportion that was reversed exhibited sufficient reparative action to counteract cardiomyopathic traits and associated adverse outcomes yielding new identity.…”

“…Although various -omics approaches offer a means of addressing underlying modes of action, systems interpretation ultimately relies on probabilistic predictive tools. To increase predictive confidence, iterative analysis was conducted here at both proteome and network levels, yielding complementary support of a pro-vasculogenic transition documented in independent studies 4,5,47 , while predicted functional repair was validated experimentally by randomized multi-parametric functional and structural evaluation. Finally, while delivered stem cells, including cargo that they release, are the presumed active ingredient, confounding factors within the infarcted myocardium have been recently shown to contribute to repair, including the initial extent of disease manifestation 57 and the pro-healing inflammatory processes 58 .…”

SERCA Overexpression Improves Mitochondrial Quality Control and Attenuates Cardiac Microvascular Ischemia-Reperfusion Injury

“…Proteins isolated from sI/R-treated CMECs were also analyzed through western blots. In brief, proteins were separated by SDS electrophoresis and transferred to membranes using standard protocols, 67 after which they were probed with antibodies against p-eNOS (Ser1117) (1:1,000, #ab184154; Abcam), ET-1 (1:1,000, #ab2786; Abcam), ICAM1 (1:1,000, #ab119871; Abcam), VCAM1 (1:1,000, #ab134047; Abcam), Gr1 (1:1,000, #ab25377; Abcam), FUNDC1 (1:1,000, #ab224722; Abcam), ATG5 (1:1,000, #12994, Cell Signaling Technology), and Beclin1 (1:1,000, #3738; Cell Signaling Technology). The blots were visualized by chemiluminescence (ECL, Immunobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, MA, USA), and the signals were quantified by densitometry.…”

Resveratrol Improves Bnip3-Related Mitophagy and Attenuates High-Fat-Induced Endothelial Dysfunction

“…A total of 50,000 cells/well were plated onto a 12-well plate. After 22 h, cells were replenished with fresh growth medium (Wolint et al, 2019), then 2 h later, cells were transfected with siRNA. Twenty-four hours after transfection, cell viability was measured through MTT assay as previously described (Trindade et al, 2019).…”