Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation

Abstract

Cord blood (CB) is an attractive alternative to bone marrow or peripheral blood as a source of transplantable hematopoietic tissue. However, because of the reduced volume, the stem cell content is limited; therefore its use as a graft for adult patients might require ex vivo manipulations. Two systems have been described that identify these stem cell populations in vitro in both mice and humans: (1) the long-term culture-initiating cells (LTC-IC), thus named because of their ability to support the growth of hematopoietic colonies (colony-forming cell (CFC)) for 5-6 weeks when co-cultured on stromal layers; (2) the generation of hematopoietic progenitors (CFC) from stroma-free liquid cultures for extended periods of time, which provides further indirect evidence of the presence of primitive stem cells. Both systems detect largely overlapping but not identical populations of stem cells. Thus the identification of the growth factor requirements for the maintenance and amplification of both systems is relevant. On this basis, analysis of the effects of 18 cytokine combinations on stroma-free liquid cultures of CB CD34 + cells, showed that: (1) after 7-and 14 day-incubation periods, several growth factor combinations expanded the LTC-IC pool to a similar extent; as compared to the LTC-IC, the generation of CFC was not impressive; (2) time-course analysis of the LTC-IC expansion demonstrated that, by extending the incubation period, only a few growth factor combinations, containing FL, TPO, KL and IL6, could support a further, increasingly greater LTC-IC expansion (up to 270 000-fold of the initial value). In similar culture conditions, CFC production underwent continuous expansion, which persisted for over 7 months and reached values of one million-fold of the initial value. The simultaneous presence of FL and TPO was both necessary and sufficient to support this phenomenon. The addition of KL ± IL6 did not appear to substantially modify the extent of LTC-IC expansion; nevertheless, it played an important role in sustaining an even more massive and prolonged output of CFU-GM, CFU-Mk and BFU/CFU-GEMM (up to 100 million-fold); (3) the presence of IL3 was found to be negative, in that it inhibited both the extent of LTC-IC expansion and the long-term generation of CFC. Thus, FL and TPO appear as two unique growth factors that preferentially support the self-renewal of primitive stem cells; the additional presence of KL and IL6 seems to enhance the proliferative potential of at least one subpopulation of daughter stem cells, which may follow three differentiation pathways. Far from being definitive, our data demonstrated that massive stem cell expansion, in cord blood, can be obtained in reasonably well-defined culture conditions. This could represent an initial step towards larger scale cultures for transplantation and gene therapy protocols.

Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells

“…This result was not surprising and, based on previous observations, it can be clearly ascribed to the capacity of hematopoietic cytokines to induce the spontaneous differentiation of CD34 þ cells in liquid culture. 11,12 Expression of the CD11b myeloid differentiation marker 10 in MafB transduced cells exhibited a delayed upregulation as compared to CD14 antigen, culminating at day 10 of culture where it raised up to 5276 versus 2976% of control untransduced cells (Figure 5b). Morphological analysis performed at the end of experiment (day 14) evidenced that monocytes-macrophages represented over 90% of purified NGFR þ transduced cells and only 25% of NGFRÀ untransduced cells (Figure 5a, right panel).…”

“…These experimental conditions normally promote a mixed granulo-monocyte differentiation of CD34 þ cells that is generally achieved within 14 days from plating. 11,12,34 Normal human monocytes were selected from Ficoll separated PB mononuclear cells of adult samples by using magnetic microbeads conjugated with mouse monoclonal (Mo) anti-human CD14 antibody (Ab) (Miltenyi Biotec, Auburn, CA, USA). Human lymphocytes were obtained as negative fraction of this last purification procedure.…”

Umbilical cord blood for allogeneic transplantation in children and adults

“…61 These preclinical studies identify further UCB graft engineering questions including the role of accessory lymphoid populations in ex vivo expanded allogeneic grafts, [62][63][64][65][66] as well as the role of stromal elements in maintaining immature stem cells with self-renewal capacity during expansion. [67][68][69][70] In summary, banked unrelated umbilical cord blood (UCB) has emerged as a substitute allogeneic stem cell source, due to the lack of available HLA-matched related donors for patients requiring allogeneic transplantation, and the observed increased incidence and severity of acute and chronic GVHD when alternative HLA-matched unrelated or partially mismatched family member grafts are utilized. Early clinical reports of UCB transplantation have focused primarily on pediatric or small stature adult recipients.…”

Combination of Early and Late Growth Factors to Enrich Transplantable Cord Blood CD34+ Cells in Short Term Cultures

“…SCF and IL-3, FL and Tpo, and IL-3, IL-6 and Epo we re com bi ned in short term cul tu res by Mo o re and Hoskins, 39 Pi a ci bel lo et al 40 and Mad ka i kar et al 41 res pec ti vely. In the se cul tu res ini ti a ted by the men ti o ned cyto ki nes, signi fi cant in cre a ses in to tal nuc le a ted cell co unts were re por ted.…”