The AML1-ETO fusion gene promotes extensive self-renewal of human primary erythroid cells

The t(8;21) translocation, which encodes the AML1-ETO fusion protein (now known as RUNX1-CBF2T1), is one of the most frequent translocations in acute myeloid leukemia, although its role in leukemogenesis is unclear. Here, we report that exogenous expression of AML1-ETO in human CD34(+) cells severely disrupts normal erythropoiesis, resulting in virtual abrogation of erythroid colony formation. In contrast, in bulk liquid culture of purified erythroid cells, we found that while AML1-ETO initially inhibited proliferation during early (erythropoietin [EPO]-independent) erythropoiesis, growth inhibition gave way to a sustained EPO-independent expansion of early erythroid cells that continued for more than 60 days, whereas control cultures became growth arrested after 10 to 13 days (at the EPO-dependent stage of development). Phenotypic analysis showed that although these cells were CD13(-) and CD34(-), unlike control cultures, these cells failed to up-regulate CD36 or to down-regulate CD33, suggesting that expression of AML1-ETO suppressed the differentiation of these cells and allowed extensive self-renewal to occur. In the early stages of this expansion, addition of EPO was able to promote both phenotypic (CD36(+), CD33(-), glycophorin A(+)) and morphologic differentiation of these cells, almost as effectively as in control cultures. However, with extended culture, cells expressing AML1-ETO became refractory to addition of this cytokine, suggesting that a block in differentiation had been established. These data demonstrate the capacity of AML1-ETO to promote the self-renewal of human hematopoietic cells and therefore support a causal role for t(8;21) translocations in leukemogenesis.     

AML1-ETO fusion protein up-regulates TRKA mRNA expression in human CD34+ cells, allowing nerve growth factor-induced expansion

The AML1-ETO fusion protein, generated by the t(8;21) in acute myeloid leukemia (AML), exerts dominant-negative functions and a variety of gains of function, including a positive effect on the growth of primary human CD34+ hematopoietic stem/progenitor cells. We now show that AML1-ETO expression up-regulates the level of TRKA mRNA and protein in these cells and that AML1-ETO-expressing CD34+ hematopoietic cells grown in the presence of five early-acting hematopoietic cytokines further proliferate in response to nerve growth factor (NGF). These cells also show a unique response to NGF and IL-3; namely, they expand in liquid culture. To determine the biological relevance of our findings, we analyzed 262 primary AML patient samples using real-time RT-PCR and found that t(8;21)-positive AML samples express significantly higher levels of TRKA mRNA than other subtypes of AML. NGF, which is normally expressed by bone marrow stromal cells, could provide important proliferative or survival signals to AML1-ETO-expressing leukemic or preleukemic cells, and the NGF/TRKA signaling pathway may be a suitable target for therapeutic approaches to AML.     

Ex vivo expansion of human hematopoietic stem cells

There has been great interest in the ex vivo expansion of human long-term repopulating hematopoietic stem cells (LTR-HSCs) for a variety of clinical applications such as umbilical cord blood transplantation. The glucoprotein130 signal, activated by a complex of interleukin 6 (IL-6) and soluble IL-6 receptor (IL-6/sIL-6R), acts dramatically in synergy with the c-Kit or Flk2/Flt3 signal to expand immature human HSCs. We demonstrate a significant ex vivo expansion of human LTR-HSCs capable of repopulating in newly discovered nonobese diabetes/Shi-severe combined immunodeficient (NOD/Shi-SCID) mice. The proportion of human CD45+ cells in recipient marrow was 10 times higher in animals receiving the cultured cells with stem cell factor, Flk2/Flt3 ligand, thrombopoietin, and IL-6/sIL-6R than in those receiving comparable numbers of fresh cord blood CD34+ cells. The expansion rate provided by this combination was estimated to be 4.2-fold by a limiting dilution method. Addition of IL-3 to the culture with the cytokine combination abrogated the repopulating ability of the expanded cells. The culture method with the IL-6/sIL-6R complex and other cytokines may pave the way for ex vivo expansion of human transplantable HSCs suitable for clinical applications.     

丙戊酸钠逆转AML1-ETO融合蛋白转录抑制作用的研究

目的:探讨组蛋白脱乙酰化酶(HDAC)抑制剂丙戊酸钠(valproic acid,VPA)逆转AML1-ETO融合蛋白转录抑制作用.方法:VPA处理t(8;21)急性白血病细胞株Kasumi-l细胞后,应用流式细胞术检测髓系分化抗原CD11b表达水平的变化,DNA凝胶电泳检测细胞的凋亡变化,分子生物学半定量RT-PCR的方法检测粒-单核细胞刺激因子(GM-CSF)和半胱氨酸天冬氨酸酶7(Caspase7)mRNA表达水平的变化.结果:①VPA诱导Kasumi-1细胞的髓系分化抗原 CD11b表达的阳性率增加,并呈现时间及剂量依赖性;②VPA诱导Kasumi-1细胞凋亡,出现的典型梯形DNA条带;③VPA诱导AML1靶基因GM-CSF和凋亡相关因子Caspase7的mRNA表达水平的增加,呈时间和剂量依赖性.结论:丙戊酸钠可以通过抑制脱乙酰化酶的活性,消除AML1-ETO融合蛋白转录抑制,诱导细胞分化和凋亡.     

Inhibition of p38 MAPK activity promotes ex vivo expansion of human cord blood hematopoietic stem cells

Ex vivo expansion of hematopoietic stem cells (HSCs) depends on HSC self-renewing proliferation and functional maintenance, which can be negatively affected by HSC differentiation, apoptosis, and senescence. Therefore, inhibition of HSC senescence may promote HSC expansion. To test this hypothesis, we examined the effect of inhibition of p38 mitogen-activated protein kinase (p38) on the expansion of human umbilical cord blood (hUCB) CD133⁺ cells because activation of p38 has been implicated in the induction of HSC senescence under various physiological and pathological conditions. Our results showed that ex vivo expansion of hUCB CD133⁺ cells activated p38, which was abrogated by the p38 specific inhibitor SB203580 (SB). Inhibition of p38 activity with SB promoted the expansion of CD133⁺ cells and CD133⁺CD38⁻ cells. In addition, hUCB CD133⁺ cells expanded in the presence of SB for 7 days showed about threefold increase in the clonogenic function of HSCs and engraftment in non-obese diabetic/severe combined immunodeficient mice after transplantation compared to the input cells. In contrast, the cells expanded without SB exhibited a significant reduction in these HSC functions. The enhancement of ex vivo expansion of hUCB HSCs is primarily attributable to SB-mediated inhibition of HSC senescence. In addition, inhibition of HSC apoptosis and upregulation of CXCR4 may also contribute to the enhancement. However, p38 inhibition had no significant effect on HSC differentiation and proliferation. These findings suggest that inhibition of p38 activation may represent a novel strategy to promote ex vivo expansion of hUCB HSCs.     

A secreted and LIF-mediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells

The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic stem cells (HSCs) is vital to stem cell research. Establishment of such culture systems will have significant impact on ex vivo manipulation and expansion of transplantable stem cells in clinical applications such as gene therapy, tumor cell purging, and stem cell transplantation. We have recently developed a stromal-based culture system that facilitates ex vivo expansion of transplantable human HSCs. In this stromal-based culture system, 2 major contributors to the ex vivo stem cell expansion are the addition of leukemia inhibitory factor (LIF) and the AC6.21 stromal cells. Because the action of LIF is indirect and mediated by stromal cells, we hypothesized that LIF binds to the LIF receptor on AC6.21 stromal cells, leading to up-regulated production of stem cell expansion promoting factor (SCEPF) and/or down-regulated production of stem cell expansion inhibitory factor (SCEIF). Here we demonstrate a secreted SCEPF activity in the conditioned media of LIF-treated AC6.21 stromal cell cultures (SCM-LIF). The magnitude of ex vivo stem cell expansion depends on the concentration of the secreted SCEPF activity in the SCM-LIF. Furthermore, we have ruled out the contribution of 6 known early-acting cytokines, including interleukin-3, interleukin-6, granulocyte macrophage colony-stimulating factor, stem cell factor, flt3 ligand, and thrombopoietin, to this SCEPF activity. Although further studies are required to characterize this secreted SCEPF activity and to determine whether this secreted SCEPF activity is mediated by a single factor or by multiple growth factors, our results demonstrate that stromal cells are not required for this secreted SCEPF activity to facilitate ex vivo stem cell expansion. (Blood. 2000;95:1957-1966)     

Ex vivo expansion of human hematopoietic stem and progenitor cells

Despite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood-derived stem cell. Finally, we discuss some of the challenges still facing this field.     

Long-term expansion of transplantable human fetal liver hematopoietic stem cells

Hematopoietic stem cells (HSCs), with their dual ability for self-renewal and multilineage differentiation, constitute an essential component of hematopoietic transplantations. Human fetal liver (FL) represents a promising alternative HSC source, and we previously reported simple culture conditions allowing long-term expansion of FL hematopoietic progenitors. In the present study, we used the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenotransplantation assay to confirm that human FL is rich in NOD/SCID-repopulating cells (SRCs) and to show that these culture conditions repeatedly maintained short- and long-term SRCs from various FL samples for at least 28 days. Quantitative limited dilution analysis in NOD/SCID mice demonstrated for the first time that a 10- to over a 100-fold net expansion of FL SRCs could be achieved after 28 days of culture. The efficiency of this culture system may lead to an increase in the use of FL as a source of HSCs for transplantation in adult patients, as previously demonstrated with umbilical cord blood under different culture conditions.     

The ETO domain is necessary for the developmental abnormalities associated with AML1-ETO expression in the hematopoietic stem cell compartment in vivo

Translocation of the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO) is a recurrent cytogenetic abnormality associated with approximately 12% of acute myelogenous leukemia (AML) cases. To understand the contribution of the t(8;21) to AML, we transduced purified hematopoietic stem cells (HSC) with a retroviral vector that coexpressed AML1-ETO or just the AML1 portion (AML1d) of the translocation along with a green fluorescent protein reporter gene. Animals reconstituted with AML1-ETO-expressing cells exhibited many of the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21), although the animals did not develop acute leukemia. We noted a gradual increase in primitive myeloblasts that accounted for approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. In addition, accumulation of late stage metamyelocytes was observed in bone marrow along with an increase in immature eosinophil myelocytes that showed abnormal basophilic granulation. There was also a gradual increase in both the frequency and absolute number of AML1-ETO-expressing HSC so that by 10 months posttransplant, there were 29-fold greater HSC numbers than in transplant-matched control mice. These phenotypes were not observed in animals reconstituted with cells expressing only the DNA-binding domain of AML1, suggesting that the ETO domain is necessary to establish the developmental abnormalities associated with AML1-ETO expression in HSC.     

Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells.

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34(+) thy-1(+) cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34(+) thy-1(+) cells on AC6.21 stroma, a murine bone marrow-derived stromal cell line, caused expansion of cells with CD34(+) thy-1(+) phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34(+) thy-1(+) phenotype. The ex vivo-expanded CD34(+) thy-1(+) cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34(+) thy-1(+) cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34(+) CD38(-) cells, shows a similar pattern of proliferative response. This suggests that ex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34(+) thy-1(+) cells.     

Direct evidence for ex vivo expansion of human hematopoietic stem cells

To characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.     

The p21Waf1 pathway is involved in blocking leukemogenesis by the t(8;21) fusion protein AML1-ETO

The 8;21 translocation is a major contributor to acute myeloid leukemia (AML) of the M2 classification occurring in approximately 40% of these cases. Multiple mouse models using this fusion protein demonstrate that AML1-ETO requires secondary mutagenic events to promote leukemogenesis. Here, we show that the negative cell cycle regulator p21(WAF1) gene is up-regulated by AML1-ETO at the protein, RNA, and promoter levels. Retroviral transduction and hematopoietic cell transplantation experiments with p21(WAF1)-deficient cells show that AML1-ETO is able to promote leukemogenesis in the absence of p21(WAF1). Thus, loss of p21(WAF1) facilitates AML1-ETO-induced leukemogenesis, suggesting that mutagenic events in the p21(WAF1) pathway to bypass the growth inhibitory effect from AML1-ETO-induced p21(WAF1) expression can be a significant factor in AML1-ETO-associated acute myeloid leukemia.