Rahul Sarugaser

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

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.

Human Mesenchymal Stem Cells Self-Renew and Differentiate According to a Deterministic Hierarchy

Background Mesenchymal 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 Findings Here 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/Significance Analysis 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.

Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs).

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.

Isolation, Characterization, and Differentiation of Human Umbilical Cord Perivascular Cells (HUCPVCs)

Publisher Summary This chapter presents the techniques necessary to extract and culture human umbilical cord perivascular cells (HUCPVCs) along with the methods to determine their functionality. Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are considered perivascular in origin, and the perivascular niche is the source of mesenchymal progenitors in many organs. Given the increasing promise of the clinical utility of MSCs, together with the need to establish alternative, higher yield, sources of these cells to those currently obtained from marrow. The chapter explores the possibility of harvesting MSCs from the perivascular tissue of the human umbilical cord, a tissue which is discarded at birth. Because the umbilical cord is a rapidly growing organ comprising three blood vessels supported in a specialized connective tissue, it is hypothesized that the cells of the latter, the so-called Whartons Jelly, would be differentiated from an MSC source which, like other MSCs, should be present in the perivascular region of the organ. Thus, the chapter provides improved extraction method for these perivascular cell populations, a method of cryogenically storing the umbilical vessels without the need for prior enzymatic removal of the perivascular cells, methods to demonstrate multilineage differentiation capacity and immunoregulatory phenotype, together with other methods of characterization of this unique MSC population.