人胚胎干细胞建系及向神经细胞定向分化的研究

研究和利用人胚胎干细胞(hES)细胞已成为生命科学领域的核心问题之一。当前hES 细胞研究主要集中在hES 的建系和维持其不分化状态；提高hES 细胞定向分化为特定 细胞的比例；ES 细胞自我更新和分化的机制等方面。本论文一方面概述了hES 细胞相 关领域的研究进展；另一方面建立了不同培养体系条件下3 株hES 细胞，并在此基础 上利用G5 和肝生长因子（HGF）诱导hES 细胞定向分化成高纯度的NPs。主要结论如 下：1) 建立人卵体外受精和胚胎培养体系。获得了15 个囊胚，采用了免疫外科法分离 内细胞团，运用含血清以及不含血清的培养体系，在ICR 小鼠胚胎成纤维饲养层上分 别建立了YKh-1、YKh-2 和YKh-3 3 株人胚胎干细胞系，生长良好，核型正常。ES 细 胞表达碱性磷酸酶活性、SSEA-3、SSEA-4、TRA-1-60、TRA-1-81 和Oct-4，但不表达 SSEA-1； ES 细胞在体外能够分化为属于外胚层、中胚层和内胚层的各种分化细胞， 在SCID 小鼠体内能形成畸胎瘤，畸胎瘤包括了所有三个胚层来源的细胞类型。证实了 ES 细胞系的多向分化潜能。2) 对比含血清以及无血清的培养体系的hES 细胞系的特征， 观察了其集落形态、生长速度、分化能力。结果表明，在含血清培养体系的Yhk-2，其 集落形态较致密，含2-3 个核的细胞较多，细胞倍增时间为43.9±5.7h；而在无血清培 养体系的Yhk-3，其集落形态较铺展，细胞较小而圆，倍增时间为34.8±3.8h。细胞免疫 染色和PCR 结果表明，二者在体外都能分化为三个胚层来源的多种细胞，但比例有所 差异。提示二者在向三个胚层来源的细胞的分化能力上有所不同。 3) 以所建立的hES 细胞系为模型，采用HGF 和G5 作为诱导因子添加到神经诱导培养基中，诱导hES 细 胞分化成高纯度的NPs。单独的HGF 或G5 仅能诱导ES 细胞分化成70.9± 5.0%和 72.9±7.2%NPs，而联用HGF 和G5 使NPs 的比率达到91.2±11.2%，进一步纯化后获得 98±3.2%的NPs。获得的NPs 能分化成三个谱系神经细胞，亚克隆实验也进一步证明采 用HGF+G5 获得的单个NPs 具有神经干细胞的特性，也能在体外分化成三个谱系的神 经细胞。用SHH 处理NPs，获得的分化细胞表达不同脑区标志，表明所得到NPs 具有 对脑区信号发生反应，进一步分化为不同脑区神经元细胞的能力。 本实验建立了具有自主知识产权的中国人源胚胎干细胞系，建立了ES 细胞的含血 清以及无血清的培养体系和向神经前体细胞定向分化系统，得到高比例的神经前体细 胞，为进一步研究利用人胚胎干细胞打下良好的基础。

人胚胎干细胞定向分化为神经前体细胞

人胚胎干细胞(human embryonic stem cells, hES细胞)来源于植入前胚胎的内细胞团，具有自我更新能力和发育全能性，能够在体内外分化为代表三个胚层的细胞类型。hES细胞来源的神经前体细胞（neural progenitor）对于研究胚胎早期的神经发育以及药物筛选和神经系统疾病的细胞替代性治疗具有重要意义。然而，许多因素影响了ES细胞的临床应用，如供体细胞不足、纯度低、异源物质污染等。 本研究采用同源饲养层培养的hES细胞在单层培养条件下高效地分化得到了神经前体细胞。主要结论如下：1）hES细胞在同源饲养层HAFi上培养八个月后仍保持ES细胞的各项表型特征和抗原特性。表明HAFi能够支持hES细胞的长期培养，从培养条件上避免了异源物质污染的可能性。2）单层贴壁分化的方法培养成分简单，不含血清和条件培养基，不需繁琐的筛选步骤就可以得到高比例的神经前体细胞（97.5%±0.83%）（P＜0.05）。此外，成分确定的培养基是研究神经分化的分子机制的良好模型。3）hES细胞来源的神经前体细胞具有分化为神经元，星形胶质和少突胶质细胞的能力，并能够模拟体内神经发育的过程和分子表达模式。长期的传代培养中发现，随着培养时间的延长，nestin阳性的神经前体细胞比例下降，同时发育能力也发生了变化。在传代培养的早期，神经前体细胞发育为神经元的比例很高，几乎没有胶质细胞分化出来。随培养时间的延长，胶质细胞的比例逐渐上升。进一步研究发现具有bHLH (basic helix-loop-helix) 结构域的转录因子neurogenein2(Ngn2) 和olig2可能在这一变化中发挥了重要的作用。

Knocking-Down Cyclin A(2) by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.

Origin of the Qinghai-Tibetan Plateau endemic Milula (Liliaceae): further insights from karyological comparisons with Allium

Milula, a monotypic genus endemic to the Qinghai-Tibetan Plateau, was found to be nested deeply within Allium by the molecular phylogeny despite the aberrant morphology. It remains unknown what had contributed to the rapid evolution of morphology and origin of this exceptional species. In contrast to a previous report of its karyotypes with 2n = 16 = 8M+8SM (2SAT), similar to most species of Allium, a rather different karyotype, 2n = 20 = 4M +10SM+6T (2SAT), was found in examined 31 individuals from 6 populations of M. spicata distributed in the central Tibet. Karyotypes of 7 Allium species occurring in the Qinghai-Tibetan Plateau were further reported. The basic number x = 8 was confirmed for all of them and their karyotypes consist mainly of metacentric and submetacentric chromosomes with rare subterminal and terminal chromosomes. The karyotype of M. spicata is distinctly different from that of most Allium species occurring in the plateau through a complete comparison of all available species in this region and adjacent areas. However, the same chromosome number and similar karyotypic structure were found in A. fasciculatum of Sect. Bromatorrhiza, indicating a possible close relationship between them. But this similarity is contradictory to the preliminary molecular phylogenetic analysis that Milula was closely related to A. cyathophorum of Sect. Bromatorrhiza with x=8, but the other species with x=10 and 11 in this section were clearly placed in the other clade. We therefore suggested that the paralleling evolution from x=8 to x=9, 10 and 11 with increasing asymmetry of karyotype possibly due to the chromosomal Robertsonian translocation might occur separately in the two recognized phylogenetic lineages of Allium. In addition to aneuploidy and following change of the chromosomal structures, the habitat isolation due to the recent uplift of the Qinghai-Tibetan Plateau and the Quaternary climatic oscillation, plays a greater role in origin of Milula and other endemic species (genera) with aberrant morphology from their progenitors.

Molecular phylogeny and biogeography of the Qinghai-Tibet Plateau endemic Nannoglottis (Asteraceae)

All taxa endemic to the Qinghai-Tibet Plateau are hypothesized to have originated in situ or from immediately adjacent areas because of the relatively recent formation of the plateau since the Pliocene, followed by the large-scaled biota extinction and recession caused by the Quaternary ice sheet. However, identification of specific progenitors remains difficult for some endemics, especially some endemic genera. Nannoglottis, with about eight species endemic to this region, is one such genus. Past taxonomic treatments have suggested its relationships with four different tribes of Asteraceae. We intend to identify the closest relatives of Nannoglottis by evaluating the level of monophyly, tribal delimitation, and systematic position of the genus by using molecular data from ndhF gene, trnL-F, and ITS region sequences. We find that all sampled species of Nannoglottis are a well-defined monophyly. This supports all recent taxonomic treatments of Nannoglottis, in which all sampled species were placed in one broadly re-circumscribed genus. Nannoglottis is most closely related to the Astereae, but stands as an isolated genus as the first diverging lineage of the tribe, without close relatives. A tentative relationship was suggested for Nannoglottis and the next lineage of the tribe was based on the ITS topology, the "basal group," which consists of seven genera from the Southern Hemisphere. Such a relationship is supported by some commonly shared plesiomorphic morphological characters. Despite the very early divergence of Nannoglottis in the Astereae, the tribe must be regarded to have its origin in Southern Hemisphere rather than in Asia, because based on all morphological, molecular, biogeographical, and fossil data, the Asteraceae and its major lineages (tribes) are supposed to have originated in the former area. Long-distance dispersal using Southeast Asia as a steppingstone from Southern Hemisphere to the Qinghai-Tibet Plateau is the most likely explanation for this unusual biogeographic link of Nannoglottis. The 23-32-million-year divergence time between Nannoglottis and the other Astereae estimated by DNA sequences predated the formation of the plateau. This estimation is further favored by the fossil record of the Asteraceae and the possible time of origin of the Astereae. Nannoglottis seems to have reached the Qinghai-Tibet area in the Oligocene-Eocene and then re-diversified with the uplift of the plateau. The molecular infragenetic phylogeny of the genus identifies two distinct clades, which reject the earlier infrageneric classification based on the arrangement of the involucral bracts and the length of the ligules, but agree well with the habits and ecological preferences of its current species. The "alpine shrub" vs. "coniferous forest" divergence within Nannoglottis was estimated at about 3.4 million years ago when the plateau began its first large-scale uplifting and the coniferous vegetation began to appear. Most of the current species at the "coniferous forest" clade of the genus are estimated to have originated from 1.02 to 1.94 million years ago, when the second and third uprisings of the plateau occurred, the climate oscillated and the habitats were strongly changed. The assumed evolution, speciation diversity, and radiation of Nannoglottis based on molecular phylogeny and divergence times agree well with the known geological and paleobotanical histories of the Qinghai-Tibet Plateau. (C) 2002 Elsevier Science (USA). All rights reserved.

C-Kit(+) Cells Isolated from Developing Kidneys Are a Novel Population of Stem Cells with Regenerative Potential

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. STEM Cells 2013;31:1644-1656

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex.

The human neocortex differs from that of other great apes in several notable regards, including altered cell cycle, prolonged corticogenesis, and increased size [1-5]. Although these evolutionary changes most likely contributed to the origin of distinctively human cognitive faculties, their genetic basis remains almost entirely unknown. Highly conserved non-coding regions showing rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers [6-14], but none have linked an expression difference to a specific organismal trait. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) of FZD8, a receptor of the Wnt pathway implicated in brain development and size [15, 16]. Using transgenic mice, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving early and robust expression at the onset of corticogenesis. Similar to HARE5 activity, FZD8 is expressed in neural progenitors of the developing neocortex [17-19]. Chromosome conformation capture assays reveal that HARE5 physically and specifically contacts the core Fzd8 promoter in the mouse embryonic neocortex. To assess the phenotypic consequences of HARE5 activity, we generated transgenic mice in which Fzd8 expression is under control of orthologous enhancers (Pt-HARE5::Fzd8 and Hs-HARE5::Fzd8). In comparison to Pt-HARE5::Fzd8, Hs-HARE5::Fzd8 mice showed marked acceleration of neural progenitor cell cycle and increased brain size. Changes in HARE5 function unique to humans thus alter the cell-cycle dynamics of a critical population of stem cells during corticogenesis and may underlie some distinctive anatomical features of the human brain.

Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation.

Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of β-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate.

Visualization of the earliest steps of gammadelta T cell development in the adult thymus.

The checkpoint in cell development that controls successful T cell receptor (TCR) gene rearrangements remains poorly characterized. Using mice expressing a reporter gene 'knocked into' the Tcrd constant region gene, we have characterized many of the events that mark the life of early cells in the adult thymus. We identify the developmental stage during which the Tcrd locus 'opens' in early T cell progenitors and show that a single checkpoint controls cell development during the penultimate CD4-CD8- stage. Passage through this checkpoint required the assembly of TCR heterodimers on the cell surface and signaling via the Lat adaptor protein. In addition, we show that selection triggered a phase of sustained proliferation similar to that induced by the pre-TCR.

Continuous MLL-ENL expression is necessary to establish a "Hox Code" and maintain immortalization of hematopoietic progenitor cells

The t[(11;19)(p22;q23)] translocation, which gives rise to the MLL-ENL fusion protein, is commonly found in infant acute leukemias of both the myeloid and lymphoid lineage. To investigate the molecular mechanism of immortalization by MLL-ENL we established a Tet-regulatable system of MLL-ENL expression in primary hematopoietic progenitor cells. Immortalized myeloid cell lines were generated, which are dependent on continued MLL-ENL expression for their survival and proliferation. These cells either terminally differentiate or die when MLL-ENL expression is turned off with doxycycline. The expression profile of all 39 murine Hox genes was analyzed in these cells by real-time quantitative PCR. This analysis showed that loss of MLL-ENL was accompanied by a reduction in the expression of multiple Hoxa genes. By comparing these changes with Hox gene expression in cells induced to differentiate with granulocyte colony-stimulating factor, we show for the first time that reduced Hox gene expression is specific to loss of MLL-ENL and is not a consequence of differentiation. Our data also suggest that the Hox cofactor Meis-2 can substitute for Meis-1 function. Thus, MLL-ENL is required to initiate and maintain immortalization of myeloid progenitors and may contribute to leukemogenesis by aberrantly sustaining the expression of a "Hox code" consisting of Hoxa4 to Hoxa11.

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis

Background The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. Methods We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. Results We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. Conclusions JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

The VLT-FLAMES survey of massive stars: atmospheric parameters and rotational velocity distributions for B-type stars in the Magellanic Clouds

Aims.We aim to provide the atmospheric parameters and rotational velocities for a large sample of O- and early B-type stars, analysed in a homogeneous and consistent manner, for use in constraining theoretical models. Methods: Atmospheric parameters, stellar masses, and rotational velocities have been estimated for approximately 250 early B-type stars in the Large (LMC) and Small (SMC) Magellanic Clouds from high-resolution VLT-FLAMES data using the non-LTE TLUSTY model atmosphere code. This data set has been supplemented with our previous analyses of some 50 O-type stars (Mokiem et al. 2006, 2007) and 100 narrow-lined early B-type stars (Hunter et al. 2006; Trundle et al. 2007) from the same survey, providing a sample of ~400 early-type objects. Results: Comparison of the rotational velocities with evolutionary tracks suggests that the end of core hydrogen burning occurs later than currently predicted and we argue for an extension of the evolutionary tracks. We also show that the large number of the luminous blue supergiants observed in the fields are unlikely to have directly evolved from main-sequence massive O-type stars as neither their low rotational velocities nor their position on the H-R diagram are predicted. We suggest that blue loops or mass-transfer binary systems may populate the blue supergiant regime. By comparing the rotational velocity distributions of the Magellanic Cloud stars to a similar Galactic sample, we find that (at 3s confidence level) massive stars (above 8 M?) in the SMC rotate faster than those in the solar neighbourhood. However there appears to be no significant difference between the rotational velocity distributions in the Galaxy and the LMC. We find that the v sin i distributions in the SMC and LMC can modelled with an intrinsic rotational velocity distribution that is a Gaussian peaking at 175 km s-1 (SMC) and 100 km s-1 (LMC) with a 1/e half width of 150 km s-1. We find that in NGC 346 in the SMC, the 10-25 M? main-sequence stars appear to rotate faster than their higher mass counterparts. It is not expected that O-type stars spin down significantly through angular momentum loss via stellar winds at SMC metallicity, hence this could be a reflection of mass dependent birth spin rates. Recently Yoon et al. (2006) have determined rates of GRBs by modelling rapidly rotating massive star progenitors. Our measured rotational velocity distribution for the 10-25 M? stars is peaked at slightly higher velocities than they assume, supporting the idea that GRBs could come from rapid rotators with initial masses as low as 14 M? at low metallicities.

The frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels

Idiopathic erythrocytosis (IE) is characterized by erythrocytosis in the absence of megakaryocytic or granulocytic hyperplasia, and is associated with variable serum erythropoietin (Epo) levels. Most patients with IE lack the JAK2 V617F mutation that occurs in the majority of polycythemia vera patients. Four novel JAK2 mutant alleles have recently been described in patients with V617F-negative myeloproliferative disorders presenting with erythrocytosis. The aims of this study were to assess the prevalence of JAK2 exon 12 mutations in IE patients, and to determine the associated clinicopathological features.

Hoxa6 potentiates short-term hemopoietic cell proliferation and extended self-renewal.

Hemopoietic progenitor cells express clustered homeobox (Hox) genes in a pattern characteristic of their lineage and stage of differentiation. In general, HOX expression tends to be higher in more primitive and lower in lineage-committed cells. These trends have led to the hypothesis that self-renewal of hemopoietic stem/progenitor cells is HOX-dependent and that dysregulated HOX expression underlies maintenance of the leukemia-initiating cell. Gene expression profile studies support this hypothesis and specifically highlight the importance of the HOXA cluster in hemopoiesis and leukemogenesis. Within this cluster HOXA6 and HOXA9 are highly expressed in patients with acute myeloid leukemia and form part of the "Hox code" identified in murine models of this disease. We have examined endogenous expression of Hoxa6 and Hoxa9 in purified primary progenitors as well as four growth factor-dependent cell lines FDCP-Mix, EML, 32Dcl3, and Ba/F3, representative of early multipotential and later committed precursor cells respectively. Hoxa6 was consistently higher expressed than Hoxa9, preferentially expressed in primitive cells and was both growth-factor and cell-cycle regulated. Enforced overexpression of HOXA6 or HOXA9 in FDCP-Mix resulted in increased proliferation and colony formation but had negligible effect on differentiation. In both FDCP-Mix and the more committed Ba/F3 precursor cells overexpression of HOXA6 potentiated factor-independent proliferation. These findings demonstrate that Hoxa6 is directly involved in fundamental processes of hemopoietic progenitor cell development.