Perivascular Human Umbilical Cord Cells Are Capable of Long Term Culture and Respond to Chemotactic Gradient in an In Vitro-Model of Alveolar Damage.
Authors: Montemurro Tiziana , Andriolo Gabriella , Weissmann Gaia , Colnaghi Mariarosa , Giordano Rosaria , Rebulla Paolo , Mosca Fabio , Lazzari Lorenza 
Published: October 2019

Abstract
Bronchopulmonary dysplasia is a chronic lung disease that occurs in very premature infants and is characterized by impaired alveologenesis. Despite the use of surfactant, antenatal glucocorticoid treatments and new ventilation strategies, mortality in these subjects is still high and the impairment of alveolar formation in the surviving premature infants leads to abnormal lung function that induces neurological and growth dysfunction. In this context innovative cell-based lung repair approaches are highly needed. In particular, it would be important to develop a system that could regenerate the alveolar type I cells (ATI), that cover about 95% of the alveolar surface, and the alveolar type II cells (ATII). ATII cells ensure surfactant production, which decreases the surface tension of the alveolar surface, allowing the alveoli to expand during inspiration and preventing the collapse during expiration. Moreover, a sufficient ATII cell number is important because they serve as stem cells for ATI cells. A very promising, still poorly known and easily available, stem cell source is the human umbilical cord (HUC). We isolated and characterized a cell population within the HUC of pre-term newborns (HUC perivascular cells) to evaluate their capability to induce or improve pneumocyte repair after damage. With this aim we processed the HUCs (n=14) from deliveries of consenting mothers (23–32weeks of gestation). Pieces of cord were dissected to expose the Wharton’s Jelly and the vein and the two arteries were exposed. The ends of each dissected vessel were tied together and digested with Collagenase A for 18 hours. After digestion the cells were extensively characterized by flow cytometry. The median (and range) percentage of the cells with a phenotype consistent with perivascular/pericyte-like cells (CD146+/CD45−/CD34−) was 17.5 (7.6–39). Moreover, these cells were positive for alpha-SMA (median and range: 90.7%, 46.9–94), NG2 (median and range: 23.2%, 13.05–31.8), VeCad (median and range:10.8%, 2.6–32.4), KDR (median and range: 15.25%, 1.5–26.2), CD31 (median and range: 3%, 0.8–8.7) and negative for CD45 and CD105. The HUC perivascular cells were cultured in the presence of EGM2 on a pre-coated gelatin layer for 1 week, and then with DMEM +20%FBS and maintained in long-term culture (11 passages). The cells were then stained with the fluorescent dye PKH26 and co-cultured in transwell with the ATII epithelial rat cellular line damaged by bleomycin. Control cultures were set up with not-damaged cell line. After 4-day co-culture, as a result of the chemiotactic migration, we observed PKH26 positive cells in contact with the damaged alveolar cell layer and not in the control cultures. By immunocytochemistry these cells showed to co-express PKH26 and the typical markers (pro-surfactant protein C, cytokeratin 18) of the ATII cells. Our results suggest that it is possible to isolate and to maintain in long-term culture HUC perivascular cells from pre-term cords. Moreover, this population is able to migrate towards alveolar cell layer previously damaged with bleomycin and to express the markers of the ATII cells. Our data encourage further investigations in order to evaluate the feasibility of therapeutic applications of these cells.