Antonella Severino

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

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.

Megakaryocyte growth and development factor (MGDF)-induced acute leukemia cell proliferation and clonal growth is associated with functional c-mpl

The effects of human recombinant megakaryocyte growth and development factor (MGDF) (also known as thrombopoietin (TPO)), alone or in combination with other growth factors, on the proliferation and on the clonal growth of clonogenic progenitors from 24 acute myeloblastic leukemia (AML) patients were evaluated. A significant proliferative response to MGDF alone (proliferation index >1.5) was observed in nine of 23 cases; the responding cases belonged to all FAB subtypes. However, the greatest response (proliferation index >7) was found in one M6 and in one M7 case. MGDF also enhanced interleukin 3 (IL-3), granulocyte–macrophage colony-stimulating factor (GM-CSF), c-kit ligand (KL) and FLT3 ligand (FL) stimulated blast cell proliferation. MGDF as a single factor induced or significantly enhanced colony formation by clonogenic precursor cells in 12 of 14 AML cases. MGDF strongly increased KL-induced leukemic colony growth in seven cases, whereas it only moderately enhanced IL-3- or GM-CSF-induced colony growth. The analysis of tyrosine phosphorylated protein(s) upon MGDF stimulation in fresh AML cells was also performed. The results demonstrated a band of approximately 90 kDa phosphorylated protein(s) upon MGDF stimulation in AML responsive cases, but not in unresponsive ones. Taken together the present findings suggest that, in a consistent proportion of AML cases, MGDF stimulates blast cell growth and induces tyrosine protein phosphorylation.

Ex Vivo Expansion of Cord Blood Progenitors

Human umbilical cord blood contains abundant primitive and committed hematopoietic progenitors; in addition, the general availability and the ease of procurement make cord blood a very attractive alternative source of transplantable hematopoietic tissue. However, the major limitation to a widespread use of cord blood for transplantation lays in its limited volume. For such a reason, until now, cord blood transplant has been mainly restricted to children and small size adults. Ex vivo expansion of cord blood stem cells could make the use of cord blood transplant feasible also for adult patients. Recently we developed a stroma‐free culture system in which a progressive, increasingly greater production of hemopoietic progenitors belonging to all the hematopoietic lineages was sustained for over six months. A similar sustained and prolonged expansion of the most primitive stem cells that can be detected in vitro (LTC‐IC), was also documented.

Effect of Hemopoietic Growth Factors on the Proliferation of Acute Myeloid and Lymphoid Leukemias.

The effect of G-CSF, GM-CSF, IL-1 and IL-3 on the proliferation of acute leukemia cells (evaluated as (3)HTdR uptake) was investigated in short-term liquid cultures and compared with that observed on normal bone marrow (BM) populations enriched for immature cells. In acute myeloid leukemias (AML), a marked leukemic proliferation was induced in 10/18 cases by IL-3, in 9/18 by GM-CSF, in 7/18 by IL-1 and in 4/18 by G-CSF. In acute lymphoid leukemias (ALL), marked stimulation was observed in 7/11 cases with IL-3 and in 5/11 with GM-CSF, whereas IL-1 and G-CSF were ineffective. Both in AML and ALL, the combination of several factors did not result in an additive synergistic effect. Purified normal BM cells responded to all four growth factors and their combinations produced an additive effect on cell proliferation which probably relates to the heterogeneity of the cell populations studied. The effect of a G-CSF, GM-CSF, IL-1 and IL-3 on the proliferation of acute leukemia cells by different growth factors suggests that caution should be exercised in their clinical use in these diseases.

Role of Hematopoietic Growth Factors on the ex Vivo Expansion of Primitive Cord Blood Stem Cells

Ex vivo expansion of hematopoietic cells has recently been suggested for clinical application.1.2 It has been demonstrated that hematopoietic compartments derived from human bone marrow (BM), mobilized peripheral blood (PB) or umbilical cord blood (CB) cells can be maintained and expanded in liquid culture or stroma coculture systems by the provision of combinations of growth factors.3–5 The potential benefits of such studies include accelerated engraftment, reduced risk of infection, smaller stem cell harvests, and improved effectiveness of genetically modified stem cells.1.2 Although controversies remain concerning what defined populations may or may not be useful for improving in vivo hematologic recovery, ex vivo expansion can only be considered successful when progeny receptor cells retain both pluripotent differentiation and self-renewal capacities of the original stem cells.4 In other words, are the various growth factor combinations able to amplify the late progenitor reservoir without exhausting the stem cell pool, in order to ensure long-term post-transplantation engraftment after a myeloablative conditioning regimen? Human culture systems employing Interleukin 3 (IL3) or Granulocyte-Colony Stimulating Factor (G-CSF) maintain at least some primitive cells;6–9 however, achieving a net expansion of early cells has proven elusive, although some laboratories have reported encouraging results.10–12 With the discovery of new growth factors, specific for early progenitor cells, investigators have re-examined the possibility of expanding stem cells in vitro. In particular, three cytokines discovered in the early 1990’s, c-kit ligand or Stem Cell Factor (KL), flt3 ligand (FL), and c-mpl ligand also termed Megakaryocyte Growth and Development Factor (MGDF) or Thrombopoietin (TPO), appear to have unique activities on primitive progenitor/stem cells.