Human Umbilical Cord Perivascular (HUCPV) Cells: A Source of Mesenchymal Progenitors

Abstract

We describe the isolation of a nonhematopoietic (CD45−, CD34−, SH2+, SH3+, Thy-1+, CD44+) human umbilical cord perivascular (HUCPV) cell population. Each HUCPV cell harvest (2–5 × 106, depending on the length of cord available) gave rise to a morphologically homogeneous fibroblastic cell population, which expressed α-actin, desmin, vimentin, and 3G5 (a pericyte marker) in culture. We determined the colony-forming unit-fibro-blast (CFU-F) frequency of primary HUCPV cells to be 1:333 and the doubling time, which was 60 hours at passage 0 (P0), decreased to 20 hours at P2. This resulted in a significant cell expansion, producing over 1010 HUCPV cells within 30 days of culture. Furthermore, HUCPV cells cultured in nonosteogenic conditions contained a subpopulation that exhibited a functional osteogenic phenotype and elaborated bone nodules. The frequency of this CFU-osteogenic subpopulation at P1 was 2.6/105 CFU-F, which increased to 7.5/105 CFU-F at P2. Addition of osteogenic supplements to the culture medium resulted in these frequencies increasing to 1.2/104 and 1.3/104 CFU-F, respectively, for P1 and P2. CFU-O were not seen at P0 in either osteogenic or non-osteogenic culture conditions, but P0 HUCPV cells did contain a 20% subpopulation that presented neither class I nor class II cell-surface major histocompatibility complexes (MHC−/−). This population increased to 95% following passage and cryopreservation (P5). We conclude that, due to their rapid doubling time, high frequencies of CFU-F and CFU-O, and high MHC−/− phenotype, HUCPV cells represent a significant source of cells for allogeneic mesenchymal cell-based therapies.