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Recent developments of surrogate assays for human hematopoietic stem cells (HSC) have facilitated efforts at improving HSC gene transfer efficiency. Through the use of xenograft transplantation models, such as nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, successful oncoretroviral gene transfer to transplantable hematopoietic cells has been achieved. However, because of the low frequency and/or homing efficiency of SCID repopulating cells (SRC) in bone marrow (BM), studies have primarily focused on cord blood (CB). The recently developed extended (> 60 days) long-term culture-initiating cell (ELTC-IC) assay detects an infrequent and highly quiescent candidate stem cell population in BM as well as CB of the CD34(+)CD38(-) phenotype. Although these characteristics suggest that ELTC-IC and SRC might be closely related, attempts to oncoretrovirally transduce ELTC-IC have been unsuccessful. Here, recently developed conditions (high concentrations of SCF + FL + Tpo in serum-free medium) supporting expansion of BM CD34(+)CD38(-) 12 week ELTC-IC promoted efficient oncoretroviral transduction of BM and CB ELTC-IC. Although SRC can be transduced with oncoretroviral vectors, this is frequently associated with loss of reconstituting activity, posing a problem for development of clinical HSC gene therapy. However, previous attempts at expanding transduced HSC posttransduction resulted in compromised rather than improved gene marking. Utilizing conditions promoting cell divisions and transduction of ELTC-IC we show that although 5 days of ex vivo culture is sufficient to obtain maximum gene transfer efficiency to SRC, extension of the expansion period to 12 days significantly enhances multilineage reconstitution activity of transduced SRC, supporting the feasibility of improving gene marking through ex vivo expansion.

Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.

Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34(+)CD38(-) phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34(+)CD38(-) BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34(+)CD38(-) progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.

Fas, a member of the tumor necrosis factor (TNF ) receptor superfamily is a critical downregulator of cellular immune responses. Proinflammatory cytokines like interferon-gamma (IFN-gamma) and TNF-alpha can induce Fas expression and render hematopoietic progenitor cells susceptible to Fas-induced growth suppression and apoptosis. Transforming growth factor-beta1 (TGF-beta1 ) is an essential anti-inflammatory cytokine, thought to play a key role in regulating hematopoiesis. In the present studies we investigated whether TGF-beta1 might regulate growth suppression and apoptosis of murine hematopoietic progenitor cells signaled through Fas. In the presence of TNF, activation of Fas almost completely blocked clonogenic growth of lineage-depleted (Lin-) bone marrow (BM) progenitor cells in response to granulocyte-macrophage colony-stimulating factor (GM-CSF ), CSF-1, or a combination of multiple cytokines. Whereas TGF-beta1 alone had no effect or stimulated growth in response to these cytokines, it abrogated Fas-induced growth suppression. Single-cell studies and delayed addition of TGF-beta1 showed that the ability of TGF-beta1 to inhibit Fas-induced growth suppression was directly mediated on the progenitor cells and not indirect through potentially contaminating accessory cells. Furthermore, TGF-beta1 blocked Fas-induced apoptosis of Lin- BM cells, but did not affect Fas-induced apoptosis of thymocytes. TGF-beta1 also downregulated the expression of Fas on Lin- BM cells. Thus, TGF-beta1 potently and directly inhibits activation-dependent and Fas-mediated growth suppression and apoptosis of murine BM progenitor cells, an effect that appears to be distinct from its ability to induce progenitor cell-cycle arrest. Consequently, TGF-beta1 might act to protect hematopoietic progenitor cells from enhanced Fas expression and function associated with proinflammatory responses.

Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38- human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38- but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38- progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38- long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38- cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.

Thrombopoietin (TPO), the ligand for c-mpl, has recently been demonstrated to be the primary regulator of megakaryocytopoiesis and platelet production. In addition, several studies have demonstrated that c-mpl is expressed on hematopoietic cell populations highly enriched in primitive progenitor cells. Here we summarize and discuss recent studies from our laboratory, as well as others, demonstrating that TPO has effects on primitive hematopoietic progenitor cells. When acting alone, TPO stimulates little or no growth, but promotes viability and suppresses apoptosis of murine multipotent (Lin- Sca-1+) bone marrow progenitor cells in vitro. In addition, TPO directly and potently synergizes with other early acting cytokines (kit ligand, flt3 ligand and interleukin 3) to promote multilineage growth of the same progenitor cell population. Although it remains to be established whether TPO also acts on the long-term reconstituting pluripotent stem cells, these studies combined with progenitor cell studies in c-mpl-deficient mice, suggest that TPO, in addition to its key role in platelet production, might also have an important impact on early hematopoiesis.