Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment

Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f(+) cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell-based therapeutics.     

Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches

Upon intravenous transplantation, hematopoietic stem cells (HSCs) can home to specialized niches, yet most HSCs fail to engraft unless recipients are subjected to toxic preconditioning. We provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Administration of ACK2, an antibody that blocks c-kit function, led to the transient removal of >98% of endogenous HSCs in immunodeficient mice. Subsequent transplantation of these mice with donor HSCs led to chimerism levels of up to 90%. Extrapolation of these methods to humans may enable mild but effective conditioning regimens for transplantation.     

Purification and characterization of mouse hematopoietic stem cells

Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.     

Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans

Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.     

The genetic program of hematopoietic stem cells

Blood cell production originates from a rare population of multipotent, self-renewing stem cells. A genome-wide gene expression analysis was performed in order to define regulatory pathways in stem cells as well as their global genetic program. Subtracted complementary DNA libraries from highly purified murine fetal liver stem cells were analyzed with bioinformatic and array hybridization strategies. A large percentage of the several thousand gene products that have been characterized correspond to previously undescribed molecules with properties suggestive of regulatory functions. The complete data, available in a biological process-oriented database, represent the molecular phenotype of the hematopoietic stem cell.     

Transplantation of fetal hematopoietic stem cells in utero: the creation of hematopoietic chimeras

Transplantation of normal, immature, fetal hematopoietic cells into a preimmune fetal recipient with a congenital hemoglobinopathy may allow partial reconstitution of normal hemoglobin production without the complications associated with postnatal bone marrow transplantation (immunosuppression and the occurrence of graft versus host disease). In order to test this hypothesis the naturally occurring polymorphism at the beta-hemoglobin locus of the sheep was used as a marker for engraftment and hematopoietic chimerism. Intraperitoneal injection of allogeneic fetal stem cells into normal fetal lambs resulted in hematopoietic chimerism in three of four surviving recipients. This chimerism has been sustained for 6 months after birth and 9 months after engraftment, without evidence of graft versus host disease, and without the use of immunosuppressive therapy.     

Little evidence for developmental plasticity of adult hematopoietic stem cells

To rigorously test the in vivo cell fate specificity of bone marrow (BM) hematopoietic stem cells (HSCs), we generated chimeric animals by transplantation of a single green fluorescent protein (GFP)-marked HSC into lethally irradiated nontransgenic recipients. Single HSCs robustly reconstituted peripheral blood leukocytes in these animals, but did not contribute appreciably to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Similarly, in GFP+:GFP- parabiotic mice, we found substantial chimerism of hematopoietic but not nonhematopoietic cells. These data indicate that "transdifferentiation" of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all.     

Modulation of adenylate cyclase activity in liver and fat cell membranes by insulin

Insulin depresses both the activity of adenylate cyclase stimulated by glucagon, epinephrine, and sodium fluoride in liver cell membranes and the activity of adenylate cyclase stimulated by epinephrine and adrenocorticotropin in particulate preparations from homogenates of isolated fat cells. Significant inhibition is detected with very low concentrations (10(-11) molar) of insulin but not with unphysiologically high (10(-9)molar) concentrations of the hormone. These direct effects of insulin on an enzymatic system in broken-cell -preparations suggest a fundamental role of adenylate cyclase activity and of cyclic adenosine monophosphate in the mechanism of action of insulin.     

Inhibition of T cell receptor expression and function in immature CD4+CD8+ cells by CD4

Most immature CD4+CD8+ thymocytes express only a small number of T cell receptor (TCR) molecules on their surface, and the TCR molecules they do express are only marginally capable of transducing intracellular signals. TCR expression and function was not intrinsically low in immature CD4+CD8+ thymocytes, but was found to be actively inhibited by CD4-mediated signals. Indeed, release of CD4+CD8+ thymocytes from CD4-mediated signals resulted in significant increases in both TCR expression and signaling function. These results suggest that, in CD4+CD8+ cells developing in the thymus, increased TCR expression and function requires release from CD4-mediated inhibition.     

Wolbachia enhance Drosophila stem cell proliferation and target the germline stem cell niche

Wolbachia are widespread maternally transmitted intracellular bacteria that infect most insect species and are able to alter the reproduction of innumerous hosts. The cellular bases of these alterations remain largely unknown. Here, we report that Drosophila mauritiana infected with a native Wolbachia wMau strain produces about four times more eggs than the noninfected counterpart. Wolbachia infection leads to an increase in the mitotic activity of germline stem cells (GSCs), as well as a decrease in programmed cell death in the germarium. Our results suggest that up-regulation of GSC division is mediated by a tropism of Wolbachia for the GSC niche, the cellular microenvironment that supports GSCs.     

Modulation of cell adhesion and motility in the immune system by Myo1f

Although class I myosins are known to play a wide range of roles, the physiological function of long-tailed class I myosins in vertebrates remains elusive. We demonstrated that one of these proteins, Myo1f, is expressed predominantly in the mammalian immune system. Cells from Myo1f-deficient mice exhibited abnormally increased adhesion and reduced motility, resulting from augmented exocytosis of beta2 integrin-containing granules. Also, the cortical actin that co-localizes with Myo1f was reduced in Myo1f-deficient cells. In vivo, Myo1f-deficient mice showed increased susceptibility to infection by Listeria monocytogenes and an impaired neutrophil response. Thus, Myo1f directs immune cell motility and innate host defense against infection.     

Modulation of the metastatic capability in B16 melanoma by cell shape

The lung colonization of B16-F1 cells grown in flat and spherical configurations was studied. Cells cultivated in vitro as spheroids on a nonadhesive substrate expressed in a reversible fashion a marked increase in their propensity to establish metastases. The altered metastatic capability was accompanied by a reversible reduction in the accessibility of cell surface proteins to external iodination and by a dramatic decrease in the synthesis of vimentin.     

Nanos maintains germline stem cell self-renewal by preventing differentiation

Despite much progress in understanding how extrinsic signaling regulates stem cell self-renewal, little is known about how cell-autonomous gene regulation controls this process. In Drosophila ovaries, germline stem cells (GSCs) divide asymmetrically to produce daughter GSCs and cystoblasts, the latter of which develop into germline cysts. Here, we show that removing the translational repressor Nanos from either GSCs or their precursors, the primordial germ cells (PGCs), causes both cell types to differentiate into germline cysts. Thus, Nanos is essential for both establishing and maintaining GSCs by preventing their precocious entry into oogenesis. These functions are likely achieved by repressing the translation of differentiation factors in PGCs and GSCs.     

Germline stem cell transplantation and transgenesis

The recently developed testis cell transplantation method provides a powerful approach to studying the biology of the male germline stem cell and its microenvironment, the stem cell niche. The technique also is being used to examine spermatogenic defects, correct male infertility, and generate transgenic animals.     

Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-gamma

T cell memory depends on factors that regulate expansion and death of these cells after antigenic stimulation. Mice deficient in perforin and interferon-gamma (IFN-gamma) exhibited increased expansion, altered immunodominance, and decreased death of antigen-specific CD8+ T cells after infection with an attenuated strain of Listeria monocytogenes, which was cleared from these mice. Expansion of CD8+ T cells was controlled by perforin, whereas IFN-gamma regulated immunodominance and the death phase. Thus, perforin and IFN-gamma regulate distinct elements of CD8+ T cell homeostasis independently of their role as antimicrobial effector molecules.     

Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome

Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. Here, we show that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation.     

Regulation of CD8+ T cell development by thymus-specific proteasomes

Proteasomes are responsible for generating peptides presented by the class I major histocompatibility complex (MHC) molecules of the immune system. Here, we report the identification of a previously unrecognized catalytic subunit called beta5t. beta5t is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. Although the chymotrypsin-like activity of proteasomes is considered to be important for the production of peptides with high affinities for MHC class I clefts, incorporation of beta5t into proteasomes in place of beta5 or beta5i selectively reduces this activity. We also found that beta5t-deficient mice displayed defective development of CD8(+) T cells in the thymus. Our results suggest a key role for beta5t in generating the MHC class I-restricted CD8(+) T cell repertoire during thymic selection.     

受体胚龄对鸡原始生殖细胞移植后归巢的影响

【目的】探究受体胚龄对鸡原始生殖细胞(Primordial germ cell,PGC)移植后归巢的影响,确定最佳回注时间窗口,为提高转基因鸡的生产效率提供参考依据。【方法】从广西黄羽鸡胚分离获得PGC,体外培养并进行转基因操作后,移植回注到不同胚龄的受体鸡胚中,移植5 d后分离性腺拍照观察,对比受体鸡胚孵育至14HH~17HH时对PGC迁移归巢到性腺的影响。【结果】分别在14HH~17HH胚龄时移植回注PGC,移植5 d后的鸡胚死亡率为30.0%~60.9%,但不同移植胚龄间的差异不显著(P>0.05)。根据鸡胚性腺中的GFP阳性细胞数可划分为6个等级(I~VI),且随着移植胚龄的增加,PGC迁移效率呈下降趋势。14HH胚龄移植的鸡胚性腺GFP阳性细胞集中在V级(40.0%),15HH胚龄移植的集中在III级(42.1%),16HH和17HH胚龄移植的则集中在II级(45.4%和54.5%),GFP阳性细胞含量最高等级(VI级)仅出现在14HH胚龄移植的鸡胚性腺中。【结论】随着受体胚龄的增加,PGC迁移效率呈下降趋势,但鉴于实际操作的难易程度,确定PGC移植回注的最佳时间窗口为14HH鸡胚(孵育50 h)。