Li, H. J. | Search bioRxiv

bioRxiv2025

Non-integrating direct reprogramming generates therapeutic endothelial cells with sustained vascular regeneration capacity enhanced by nanomatrix delivery

RationaleDirect reprogramming of fibroblasts into endothelial cells (rECs) using ETV2 shows promise for vascular regeneration. However, current approaches using integrating viral vectors pose clinical translation barriers, and poor long-term cell survival limits therapeutic efficacy. ObjectiveTo develop a clinically compatible method for generating rECs using non-integrating adenoviral ETV2 (Ad-ETV2) and enhance their engraftment and therapeutic efficacy through peptide amphiphile (PA) nanomatrix encapsulation. Methods and ResultsHuman dermal fibroblasts were reprogrammed using Ad-ETV2 and characterized by flow cytometry, RNA sequencing, and functional assays. Therapeutic efficacy was evaluated in murine hindlimb ischemia with or without PA-RGDS encapsulation over 12 months. Ad-ETV2 induced robust endothelial gene expression (CDH5, KDR, PECAM1) within 6 days, with 40-50% reprogramming efficiency. KDR+ Ad-rECs demonstrated functional endothelial properties including Ac-LDL uptake, tube formation, and exceptional proangiogenic factor secretion (200-fold higher HGF than HUVECs). RNA sequencing revealed rapid transcriptional reprogramming with fibroblast gene suppression and endothelial/angiogenic gene activation. In hindlimb ischemia, Ad-rECs significantly enhanced blood flow recovery and capillary density versus controls. Long-term analysis revealed sustained vascular contribution through three mechanisms: direct incorporation, perivascular support, and vessel guidance, persisting throughout 12 months--the longest reported follow-up for reprogrammed cells. PA-RGDS encapsulation markedly improved cell retention; while 75% of cells were lost by 3 months, retention stabilized thereafter with minimal additional loss through 12 months. ConclusionsAdenoviral ETV2 delivery enables efficient generation of clinically compatible rECs without genomic integration. These cells demonstrate potent and sustained therapeutic efficacy through multiple vascular regeneration mechanisms. PA-RGDS encapsulation significantly enhances long-term engraftment, establishing this combined approach as a promising platform for treating ischemic cardiovascular diseases.

cell biology↗

bioRxiv2021

Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity

Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype-antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.

immunology↗