TGF-β/Smad通路对造血干细胞的调控作用

造血干细胞(hematopoietic stem cells,HSCs)是典型的成体干细胞,造血系统的稳定依靠造血干细胞正确的自我更新、增殖和分化。TGF-β超家族包括TGF-β、骨生成蛋白(BMP)和激活素,可通过Smad蛋白对造血干细胞进行调节。TGF-β/Smad通路可通过降低CDK4的表达、增加p21蛋白表达和改变p27分布,将造血干细胞阻断于G1期;通过上调CD34表达,抑制造血干细胞的分化。但也有不同的观点,认为TGF-β对HSCs的调节与Smads无关,TGF-β并非通过调控p21和p27抑制HSCs的增殖,TGF-β/Smad通路对维持HSCs静止状态无关。     

Phosphorylation of RUNX1 by cyclin-dependent kinase reduces direct interaction with HDAC1 and HDAC3

RUNX1 regulates formation of the definitive hematopoietic stem cell and its subsequent lineage maturation, and mutations of RUNX1 contribute to leukemic transformation. Phosphorylation of Ser-48, Ser-303, and Ser-424 by cyclin-dependent kinases (cdks) increases RUNX1 trans-activation activity without perturbing p300 interaction. We now find that endogenous RUNX1 interacts with endogenous HDAC1 or HDAC3. Mutation of the three RUNX1 serines to aspartic acid reduces co-immunoprecipitation with HDAC1 or HDAC3 when expressed in 293T cells; mutation of these three serines to alanine increases HDAC interaction, and mutation of each serine individually to aspartic acid also reduces these interactions. GST-RUNX1 isolated from bacterial extracts bound in vitro translated HDAC1 or HDAC3, and these interactions were weakened by mutation of Ser-48, Ser-303, and Ser-424 to aspartic acid. The ability of RUNX1 phosphorylation and not only serine to aspartic acid conversion to reduce HDAC1 binding was demonstrated using wild-type GST-RUNX1 phosphorylated in vitro using cdk1/cyclinB and by exposure of 293T cells transduced with RUNX1 and HDAC1 to roscovitine, a cdk inhibitor. Finally, RUNX1 or RUNX1(tripleD), in which Ser-48, Ser-303, and Ser-424 are mutated to aspartic acid, stimulated proliferation of transduced, lineage-negative murine marrow progenitors more potently than did RUNX1(tripleA), in which these serines are mutated to alanine, suggesting that stimulation of RUNX1 trans-activation by cdk-mediated reduction in HDAC interaction increases marrow progenitor cell proliferation.     

Prep1 (pKnox1) Regulates Mouse Embryonic HSC Cycling and Self-Renewal Affecting the Stat1-Sca1 IFN-Dependent Pathway

A hypomorphic Prep1 mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to de novo generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs.     

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.     

A small molecule inhibitor of p53 stimulates amplification of hematopoietic stem cells but does not promote tumor development in mice

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could, in theory, contribute to the increased frequency of tumor development observed in p53-deficient mice and humans. In our previous work, we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice, PFTb impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition, we showed that PFTb stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly, however, PFTb treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus, although PFTb administration led to increased numbers of HSCs and HPCs, it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis.     

造血干细胞相关microRNAs的筛选及其促分化研究

为了获得人脐血造血干细胞(HSCs)的microRNAs(miRNAs)表达谱,并对相关miRNAs功能进行初步鉴定.利用免疫磁珠(MACS)和流式细胞仪(FACS)细胞分选技术分离人脐血造血干细胞(HSCs),分别提取细胞总RNA并分离小分子RNA,经荧光标记后与miRNAs基因芯片杂交,获得HSCs的miRNAs表达谱,集落形成实验(CFC)研究在HSC中高表达miR-520h对HSC的促分化作用.成功分离人脐血CD34 细胞和HSC,经基因芯片杂交获得31个造血干细胞相关miRNAs,其中22个为低表达,9个为高表达;经实时定量RT-PCR验证miR-520h显著升高,CFC实验表明其可增加多种集落形成,具有促进HSC向祖细胞分化的作用.上述结果表明,人脐血HSC具有自身特征性miRNAs,参与并调控HSC生物学功能,为深入探讨miRNAs在造血系统发育中的作用打下基础.     

Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells

Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27(kip1). In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27(kip1). KD of Gfer results in enhanced binding of p27(kip1) to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27(kip1) interaction. Furthermore, normalization of p27(kip1) in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27(kip1) pathway in HSCs that functions to restrict abnormal proliferation.     

Differential requirements for Wnt and Notch signaling in hematopoietic versus thymic niches

All blood cells are derived from multipotent stem cells, the so-called hematopoietic stem cells (HSCs), that in adults reside in the bone marrow. Most types of blood cells also develop there, with the notable exception of T lymphocytes that develop in the thymus. For both HSCs and developing T cells, interactions with the surrounding microenvironment are critical in regulating maintenance, differentiation, apoptosis, and proliferation. Such specialized regulatory microenvironments are referred to as niches and provide both soluble factors as well as cell-cell interactions between niche component cells and blood cells. Two pathways that are critical for early T cell development in the thymic niche are Wnt and Notch signaling. These signals also play important but controversial roles in the HSC niche. Here, we review the differences and similarities between the thymic and hematopoietic niches, with particular focus on Wnt and Notch signals, as well as the latest insights into regulation of these developmentally important pathways.     

Gene profiling reveals association between altered Wnt signaling and loss of T‐cell potential with age in human hematopoietic stem cells

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell (HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T‐cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9‐DL1 in vitro co‐culture system to promote T‐cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T‐lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T‐lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T‐cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T‐progenitor cell subset derived during in vitro T‐lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age‐related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T‐cell generation and immune reconstitution following clinical transplantation.