Dolores Baksh

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.

Adult human bone marrow-derived mesenchymal progenitor cells are capable of adhesion-independent survival and expansion

OVERVIEW We show the existence of adult human mesenchymal progenitor cells (hMPCs) that can proliferate, in a cytokine-dependent manner, as individual cells in stirred suspension cultures (SSC) while maintaining their ability to form functional differentiated mesenchymal cell types. MATERIALS AND METHODS Ficolled human bone marrow (BM)-derived cells were grown in SSC (and adherent controls) in the presence and absence of exogenously added cytokines. Phenotypic, gene expression, and functional assays for hematopoietic and nonhematopoietic cell populations were used to kinetically track cell production. Limiting-dilution analysis was used to relate culture-produced cells to input cell populations. RESULTS Cytokine cocktail influenced total and progenitor cell expansion, as well as the types of cells generated upon plating. Flow cytometric analysis of CD117, CD123, and CD45 expression showed that cytokine supplementation influenced SSC output. The concomitant growth of CD45(+) and CD45(-) cells in the cultures that exhibited the greatest hMPC expansions suggests that the growth of these cells may benefit from interactions with hematopoietic cells. Functional assays demonstrated that the SSC-derived cells (input CFU-O number: 1990+/-377) grown in the presence of SCF+IL-3 resulted, after 21 days, in the generation of a significantly greater number (p<0.05) of bone progenitors (33,700+/-8763 CFU-O) than similarly initiated adherent cultures (214+/-75 CFU-O). RT-PCR analysis confirmed that the SSC-derived cells grown in osteogenic conditions express bone-specific genes (Cbfa1/Runx2, bone sialoprotein, and osteocalcin). CONCLUSIONS Our approach not only provides an alternative strategy to expand adult BM-derived nonhematopoietic progenitor cell numbers in a scalable and controllable bioprocess, but also questions established biological paradigms concerning the properties of connective-tissue stem and progenitor cells.

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.

59 – Systematic Approach to the Development of Stem Cell Expansion Cultures

The interactive nature of parameters that affect stem cell growth in vitro suggests the need for a systematic understanding, such as the one starting to be used in other biological applications to optimize stem cell growth. This approach requires culture parameters and networks of parameter interactions governing stem cell self-renewal and differentiation to be defined and characterized; that robust assays of stem cell developmental potential be used; that an assessment of how perturbations in parameter values that affect stem cell growth be performed; and a quantitative understanding of how dynamic changes in biologic and microenvironmental conditions that affect stem cell growth to be developed. Examples of the use of this new systematic approach to the development of bioprocesses to expand hematopoietic stem cell (HSC) and mesenchymal stem cell (MSC) numbers are described in this chapter. Adult tissues are an attractive and readily accepted source of stem cells because such cells have demonstrated efficacy in multiple types of cellular therapeutics and may be directly obtained from individual patients, thereby eliminating difficulties associated with tissue rejection. Designing stem cell-based technologies requires insights into culture parameters that govern stem cell self-renewal and differentiation, an appreciation of how these parameters interact with one another, and an understanding of how dynamic and kinetic changes in biologic and microenvironmental conditions affect stem cell growth.

Culture of mesenchymal stem/progenitor cells in adhesion-independent conditions.

Publisher Summary This chapter describes a novel culture paradigm for the cultivation and expansion of mesenchymal stem/progenitor cells (MSCs/MPCs) that circumvents some limitations, which are characteristic of the traditional method for the culture of MPCs. By applying a systematic experimental design regimen, a novel bioprocess approach to the culture of MPCs is developed. The MPCs generated under these conditions maintain their “stemness” potential and have the ability to form functional mesenchymal tissue types including bone, cartilage, fat, and myoblasts. This approach challenges current biological paradigms surrounding the culture of these MPCs but offers an alternative strategy to generate functional MPCs for a variety of cell-based therapeutic strategies. One of the major goals of therapeutic strategies utilizing stem cells is the delivery of a sufficient number of functional cells. MSCs/ MPCs, one of two types of stem cells found in bone marrow, the other being hematopoietic stem cells, have already demonstrated clinical potential both as a source of cells for cell-based therapies and as a platform for gene therapy. The current and most accepted method for the culture of MPCs relies on the adhesion and subsequent culture of these cells on tissue culture dishes. Expansion is achieved by serial passaging of the adherent cell population. One of the major limitations of the current approach to the culture of MPCs is the loss of developmental potential during culture expansion.

Manipulation of adult bone marrow derived stem and progenitor cell population. Dynamics in suspension culture

Summary form only given. Hematopoietic and non-hematopoietic (mesenchymal) stem and progenitor cells are typically present in vivo in close association with each other. A major bottleneck in the use of these and other adult human-derived stem cells for therapeutic applications is the recognized inability to use them to generate sufficient numbers of functional cells. Manipulation of the culture microenvironment through growth factor supplementation, adherent cell culture, and/or co-culture on adherent feeder cells, have been the predominant approaches in studies attempting to adult derived stem cells. While promising results have been obtained, it is becoming clear that novel methods and approaches must be developed to understand the fundamental mechanisms that regulate adult stem cell proliferation and differentiation in vitro. Ideally, bioprocesses must be designed to specifically target progenitor cell growth while incorporating positive and negative feedback from potentially dynamic mature and maturing cell populations. This presentation reviews our progress towards the design of suspension culture systems for the growth of hematopoietic and non-hematopoietic stem and progenitor cells.