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 × 10 6 , 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-fibroblast (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 10 10 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 2.6/10 5 CFU-F, which increased to 7.5/10 5 CFU-F at P2. Addition of osteogenic supplements to the culture medium resulted in these frequencies increasing to 1.2/10 4 and 1.3/10 4 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. Stem Cells 2005;23:220-229

BackgroundMesenchymal progenitor cells (MPCs) have been isolated from a variety of connective tissues, and are commonly called “mesenchymal stem cells” (MSCs). A stem cell is defined as having robust clonal self-renewal and multilineage differentiation potential. Accordingly, the term “MSC” has been criticised, as there is little data demonstrating self-renewal of definitive single-cell-derived (SCD) clonal populations from a mesenchymal cell source.Methodology/Principal FindingsHere we show that a tractable MPC population, human umbilical cord perivascular cells (HUCPVCs), was capable of multilineage differentiation in vitro and, more importantly, contributed to rapid connective tissue healing in vivo by producing bone, cartilage and fibrous stroma. Furthermore, HUCPVCs exhibit a high clonogenic frequency, allowing us to isolate definitive SCD parent and daughter clones from mixed gender suspensions as determined by Y-chromosome fluorescent in situ hybridization.Conclusions/SignificanceAnalysis of the multilineage differentiation capacity of SCD parent clones and daughter clones enabled us to formulate a new hierarchical schema for MSC self-renewal and differentiation in which a self-renewing multipotent MSC gives rise to more restricted self-renewing progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached.

Current sources of mesenchymal cells, including bone marrow, fat and muscle, all require invasive procurement procedures, and provide relatively low frequencies of progenitors. Here, we describe the non-invasive isolation, and characterization, of a rich source of mesenchymal progenitor cells, which we call human umbilical cord perivascular cells (HUCPVCs). HUCPVCs show a similar immunological phenotype to bone marrow-derived mesenchymal stromal cells (BM-MSCs), since they are non-alloreactive, exhibit immunosuppression, and significantly reduce lymphocyte activation, in vitro. They present a non-hematopoietic myofibroblastic mesenchymal phenotype (CD45-, CD34-, CD105+, CD73+, CD90+, CD44+, CD106+, 3G5+, CD146+); with a 1:300 frequency at harvest, a short-doubling time, and a clonogenic frequency of >1:3 in culture. Furthermore, in addition to robust quinti-potential differentiation capacity in vitro, HUCPVCs have been shown to contribute to both musculo-skeletal and dermal wound healing in vivo.

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