急性白血病干细胞研究进展

白血病干细胞很可能是起源于发生了突变的正常造血干细胞。除与正常造血干细胞具有相同的CD34~ 、CD38~-、HLA-DR~-等细胞表型外,急性髓系白血病干细胞还具有自己特有的CD90~-、CD117~-、CD123~ 细胞表型、对热更敏感、持续活化的核因子kB以及比正常造血干细胞具有更高的自我更新和分化增殖能力等特征。核因子kB的异常活化、白细胞介素受体3以及PI3K信号传导等通路可能参与白血病干细胞生长和生存的调节,在白血病的发生与发展中起着重要的作用。     

白血病干细胞的起源、表型及其调控机制

白血病细胞群是由不同分化阶段的白血病细胞组成,其中最原始的细胞为白血病干细胞(LSC),LSC可能起源于定向祖细胞或造血干细胞,也可能是更原始的细胞群.白血病干细胞有着与正常干细胞不同的表型标志,它的形成也有着复杂的分子调控机制,尤其是核因子κB(NF-κB)和γ连环蛋白相关的机制,使它们有可能成为白血病治疗的分子靶点.     

白血病干细胞的起源、表型及其调控机制

白血病细胞群是由不同分化阶段的自血病细胞组成，其中最原始的细胞为白血病干细胞(LSC)，LSC呵能起源于定向祖细胞或造血干细胞，也可能是更原始的细胞群。白血病干细胞有着与正常干细胞不同的表型标志，它的形成也有着复杂的分子调控机制，尤其是核因子κB(NF-κB)和γ连环蛋白相关的机制，使它们有可能成为白血病治疗的分子靶点。     

造血B祖细胞与急性B淋巴细胞白血病细胞的流式细胞术免疫表型分析与鉴别

目的 分析造血B祖细胞与急性B淋巴细胞白血病（B-ALL）细胞在形态学、免疫表型等方面的差异,为造血B祖细胞、微小残留白血病细胞的正确鉴定提供参考.方法 采用流式细胞术对临床确诊的58例B-ALL患者初诊、缓解、复发期的132份骨髓样本进行细胞免疫表型分析,通过CD34/CD10/CD19/CD45或CD34/CD10/CD45/CD19/CD20/CD38抗体组合鉴定造血B祖细胞群.结果 132份骨髓样本中,45份（34％）检出造血B祖细胞群,检测范围0～36％,其中3份样本出现在初诊病例,1份样本出现在复发病例,41份样本出现在缓解病例.66份缓解病例样本中,造血B祖细胞检出率为62％（41/66）.白血病细胞在初诊、复发病例中均占有核细胞群体5％以上,24份缓解病例样本可检出低于5％的残留白血病细胞.造血B祖细胞与白血病细胞共存在28份样本中,其中3份出现在初诊病例,1份出现在复发病例,24份出现在缓解病例.造血B祖细胞CD45呈阴性至弱阳性连续分布,侧向散射信号（SSC）较低,早期造血B祖细胞群CD34＋,随着细胞成熟度的增加,CD34消失.CD19、CD10在造血B祖细胞整个阶段均为阳性,早期CD10表达强度更高.白血病细胞呈“发育阻滞”模式,呈现为较均一的荧光细胞群;SSC值较正常B祖细胞偏高.白血病细胞抗原表达过强或过低的表型在正常造血B祖细胞均未见到,且造血B祖细胞不会出现交叉系列抗原标记,CD20呈阴性至弱阳性的连续分布.结论 造血B祖细胞在B-ALL患者不同阶段均不同程度地存在,尤其在化疗后的骨髓恢复期有一过性增长,分析此阶段的样本鉴定B系群体来源需谨慎.了解正常细胞的发育背景、认知白血病细胞表型的漂移变化模式及掌握多色分析优化抗体组合的流式细胞技术,对白血病细胞的准确鉴定及微小残留病检测的准确性提升具有重要意义.     

慢性粒细胞白血病CD+34细胞的生物学特性

慢性粒细胞白血病(CML)是一种起源于骨髓多能造血干细胞的恶性克隆性疾病,其中90%以上的病例具有ph染色体,其分子基础是bcr/abl基因重排.CD34抗原是一种表达于造血干/祖细胞的膜表面糖蛋白,正常CD+34细胞亚群中90%以上为祖细胞和干细胞,骨髓中约1.5%的单个核细胞表达CD34抗原,未经刺激的外周血中0.1%～0.2%的单个核细胞为CD+34,体内单独或联合应用造血生长因子能够提高外周血中CD+34细胞的数量,并有效地应用于外周血干细胞移植(PBSCT).     

C D44与抗白血病效应的研究进展

白血病是一种造血干细胞的恶性克隆性疾病。其发病机制与白血病细胞增殖失控、分化障碍、凋亡受阻等有关。研究证实白血病细胞表达CD44,它通过参与细胞黏附、增殖、迁移、浸润等行为促进白血病的发生发展。体外试验表明,以CD44为靶点的治疗能有效杀伤白血病细胞,并降低对正常组织的细胞毒性。因此,针对CD44抗癌药物的设计有望用于白血病的临床治疗。     

rHuG-CSF动员健康供者外周血造血干细胞的效果观察

目的:观察重组人粒细胞集落刺激因子(recombinant human granulocyte-colony stimulating factor,rHuG-CSF)动员健康供者外周血造血干细胞的效果及影响因素。方法:将本研究中心异基因造血干细胞移植健康供者163例,分别采用3种不同厂家生产的rHuG-CSF进行外周血造血干细胞动员,对其动员效果、受者移植后造血重建等情况进行比较。结果:不同种rHuG-CSF动员后采集的单个核细胞(MNC)及CD34 细胞均能满足临床造血干细胞移植的需要,130例人类白细胞抗原(human leucocyte antigens,HLA)配型相合的同胞受者移植后均获得顺利植入。动员的单个核细胞数及CD34 细胞数与性别无关,而CD34 细胞数在41~60岁年龄组中明显减少(P<0.05);对采集时机的分析显示:第5天采集的单个核细胞数及CD34 细胞数最高(P<0.05),此后逐渐下降。结论:糖基化的及两个非糖基化的rHuG-CSF制剂均能有效地在异基因移植中作为外周血造血干细胞动员剂。     

正常供者外周血造血干细胞动员效果及影响因素

 目的　探讨粒细胞集落刺激因子（G-CSF）动员正常供者外周血造血干细胞的效果、毒副作用及影响因素。方法　对59名异基因造血干细胞正常供者采用G-CSF 皮下注射3 ~ 5 d,使用COBE Spectra血细胞分离机采集外周血干细胞,流式细胞术检测采集物中CD+34细胞数。结果　所有供者第一次采集的单个核细胞（MNC）及CD+34细胞量平均值分别为4.4（1.12～13.06）×108／kg供者体重及3.78（1.14～12.92）×106／kg供者体重。患者不良反应轻微。男性供者、年龄小于45岁者及采集前白细胞计数高者采集所得CD+34细胞数较高。结论　G-CSF作为正常供者动员剂安全有效。患者性别、年龄及采集前白细胞计数可作为预测因素。     

急性髓系白血病患者来源白血病干细胞的分选与鉴定

 目的　分选急性髓系白血病患者白血病干细胞并进行鉴定,为白血病干细胞的进一步研究奠定基础。方法　采用Ficoll密度梯度离心法从患者骨髓中分离单个核细胞。采用流式细胞术从单个核细胞中分选CD+34 CD+123白血病干细胞,通过集落形成能力和鹅卵石形成能力检测分选后白血病干细胞的自我更新与分化能力,同时检测CD+34 CD+123 细胞纯度,观察其细胞形态。结果　分选后的CD+34 CD+123 白血病干细胞比例占总分选细胞的10.7 %,具有集落形成能力和鹅卵石形成能力,CD+34 CD+123 细胞纯度为62.1 %。结论　成功分选与鉴定了白血病干细胞,可用于其后的进一步研究工作。     

来源不同的三种树突状细胞介导白血病免疫杀伤效应比较研究

树突状细胞(dendritic cells,DC)是迄今为止发现的递呈功能最强的专职抗原呈递细胞(antigen presenting cells,APC),通过启动细胞毒性T淋巴细胞(cytotoxic T lympho-cytes,CTL)而发挥特异性抗肿瘤免疫效应.本研究利用流式细胞技术及体外细胞培养方法,观察脐血、健康人及慢性粒细胞白血病(chronic myeloid leukemia,CML)患者外周血等不同来源的单个核细胞在细胞因子(GM-CSF,TNF-a,IL-4)培养条件下诱导DC,观察DC细胞形态、产率、免疫表型的差别,同时观察了Ph染色体阳性的CML细胞可溶性抗原刺激后的DC所诱导的CTL特异性抗白血病效应,为临床最终治愈白血病提供实验依据.结果表明:3种来源的DC均表现出典型的镜下DC形态,DC产率分别为74.43%、61.23%和60.48%,脐血DC产率高于其它2组(P＜0.001),这可能由于:①脐带血中含有较丰富和更纯的造血干/祖细胞(与外周血比),其分化增殖的潜能更强;②脐血血浆中含有丰富的造血因子如GM-CSF、干细胞生长因子(SCF)等DC生长所必须的因子.培养第7天后,3组DC均高表达HLA-DR,CD83,CD1a,CD80等特征性DC分子.     

Targeting leukemic stem cells by breaking their dormancy

Transient or long‐term quiescence, the latter referred to as dormancy are fundamental features of at least some adult stem cells. The status of dormancy is likely a critical mechanism for the observed resistance of normal HSCs and leukemic stem cells (LSCs) to anti‐proliferative chemotherapy. Recent studies have revealed cytokines such as Interferon‐alpha (IFNα) and G‐CSF as well as arsenic trioxide (As2O3) to be efficient agents for promoting cycling of dormant HSCs and LSCs. Most interestingly, such cell cycle activated stem cells become exquisitely sensitive to killing by different chemotherapeutic agents, suggesting that dormant LSCs in patients may be targeted by a sequential two‐step protocol involving an initial activation by IFNα, G‐CSF or As2O3, followed by targeted chemotherapy.     

TIM-3 in normal and malignant hematopoiesis: Structure,function, and signaling pathways

Acute myeloid leukemia (AML) is hierarchically organized by self-renewing leukemic stem cells (LSCs). LSCs originate from hematopoietic stem cells (HSCs) by acquiring multistep leukemogenic events. To specifically eradicate LSCs, while keeping normal HSCs intact, the discrimination of LSCs from HSCs is important. We have identified T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) as an LSC-specific surface molecule in human myeloid malignancies and demonstrated its essential function in maintaining the self-renewal ability of LSCs. TIM-3 has been intensively investigated as a “coinhibitory” or “immune checkpoint” molecule of T cells. However, little is known about its distinct function in T cells and myeloid malignancies. In this review, we discuss the structure of TIM-3 and its function in normal blood cells and LSCs, emphasizing the specific signaling pathways involved, as well as the therapeutic applications of TIM-3 molecules in human myeloid malignancies.     

Enrichment of N-Cadherin and Tie2-bearing CD34/CD38/CD123 leukemic stem cells by chemotherapy-resistance

Acute myeloid leukemia (AML) arises from genetic changes at the level of stem cell, various mutations have been elucidated, including AML1–ETO fusion gene has been shown as the representative target of cellular transformation for LSCs originating from hematopoietic stem cells (HSCs) compartment. LSCs resemble HSCs with respect to self-renewal capacity and chemotherapy-resistance. However, LSCs possess specific cell-surface markers, they are proposed to reside within the CD34⁺/CD38⁻/CD123⁺ compartment. And the interaction mediated by adhesion molecules between LSCs and niche played a role in chemoresistance of LSCs. Therefore, study on the LSCs surface makers related to niche is helpful for the potential target therapy in the future. In this study, the proportions of CD34⁺/CD38⁻/CD123⁺ LSCs compartment co-expressing the three adhesion molecules, N-Cadherin, Tie2 and CD44, respectively, from AML patients before and after chemotherapy were analyzed. We demonstrated N-Cadherin and Tie2 positive CD34⁺/CD38⁻/CD123⁺ LSCs populations could be enriched by chemotherapy. Furthermore, AML1/ETO fusion signals and MDR1 expression were detected on the CD34⁺/CD38⁻/CD123⁺ LSCs populations expressing N-Cadherin and Tie2. Therefore, N-Cadherin and Tie2 are probably the potential markers for identification of LSCs.     

The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.     

Elevated TIM3+ hematopoietic stem cells in untreated myelodysplastic syndrome displayed aberrant differentiation,overproliferation and decreased apoptosis

TIM3, as a negative regulator of anti-tumor immunity, is highly expressed on LSCs, but not on normal HSCs. TIM3 on HSCs in MDS patients has not been clarified. Here, both the percentage of TIM3 on HSCs and the MFI of TIM3+ HSCs were higher in untreated MDS than control and were closed to AML, and excessive TIM3+ HSCs was closely related to clinical parameters: WPSS score, karyotype analysis, morphologic blasts, the number of cytopenia involving hematopoietic lineages, anemia and granulocytopenia. TIM3+ HSCs expressed lower CD11b, TpoR, EpoR, G-CSFR and Annexin V, and higher CD71 and GATA2. TIM3+ HSCs displayed aberrant differentiation, overproliferation and decreased apoptosis. TIM3 might be a promising marker for identifying malignant clone cells in MDS and a candidate for targeted therapy.     

The effect of cantharidins on leukemic stem cells

To identify an agent with specific activity against leukemic stem cells (LSCs), we evaluated compounds that targeted hepatic leukemia factor (HLF), a gene implicated in hematopoietic stem cell (HSCs) regulation, that we found overexpressed in LSCs. Cantharidin, a natural toxin from blister beetles, used as medicinal agent since antiquity, has been described to modulate the HLF competitor NFIL3 and is under clinical evaluation as an antitumor and antimetastatic agent. The molecule is not a substrate for multidrug resistant pumps and does not cause myelosuppression, and therefore it represents a promising compound for selective ablation of LSCs. Cantharidin and norcantharidin, a derivative with reduced toxicity, decreased HLF protein levels and induced apoptosis in the AML cell line MV4‐11 by modulating the expression of several molecules that govern survival pathway, including HLF, SLUG, NFIL3 and c‐myc, thereby inducing p53 and the mitochondrial caspase cascade. In vitro, cantharidin readily targeted primary AML stem and progenitor cells in contrast to conventional chemotherapeutic agents, such as Ara‐C and daunorubicin, that mainly targeted more differentiated leukemic cells. In vitro the compound did not exhibit a therapeutic window, being equally toxic to normal HSCs and LSCs. In vivo cantharidin did not produce myelosuppression. Because of dose‐limiting toxicity in vivo, neither cantharidin nor norcantharidin proved therapeutical benefit in AML xenograft models as a single agent. However, its potent in vitro LSC activity and pathway targeting may still be exploited clinically with a new generation of cantharidin derivatives or formulations and with appropriate drug combinations. © 2008 Wiley‐Liss, Inc.     

Leukemia stem cells

Leukemia stem cells (LSCs) might originate from malignant transformation of normal hematopoietic stem cells (HSCs), or alternatively, of progenitors in which the acquired mutations have re-installed a dysregulated self-renewal program. LSCs are on top of a hierarchy and generate leukemia cells with more differentiated characteristics. While most leukemia cells are initially sensitive to chemo- and radiotherapy, LSCs are resistant and are considered to be the basis for disease relapse after initial response. Albeit important knowledge on LSC biology has been gained from xenogeneic transplantation models introducing human leukemia cells into immune deficient mouse models, the prospective identification and isolation of human LSC candidates has remained elusive and their prognostic and therapeutic significance controversial. This review focuses on the identification, enrichment and characterization of human LSC derived from patients with acute myeloid leukemia (AML). Experimental data demonstrating the clinical significance of estimating LSC burden and strategies to eliminate LSC will be summarized. For long-term cure of AML, it is of importance to define LSC candidates and to understand their tumor biology compared to normal HSCs. Such comparative studies might provide novel markers for the identification of LSC and for the development of treatment strategies that might be able to eradicate them.     

The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.

Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34+/CD38- or CD34+/HLA-DR-. Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3Ralpha or CD123) was strongly expressed in CD34+/CD38- cells (98 +/- 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34+/CD38- cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3Ralpha positive cells, purified CD34+/CD123+ leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123+ cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.