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Homing of hematopoietic stem cells (HSCs) into the bone marrow (BM) is a prerequisite for establishment of hematopoiesis during development and following transplantation. However,the molecular interactions that control homing of HSCs,in particular,of fetal HSCs,are not well understood. Herein,we studied the role of the alpha6 and alpha4 integrin receptors for homing and engraftment of fetal liver (FL) HSCs and hematopoietic progenitor cells (HPCs) to adult BM by using integrin alpha6 gene-deleted mice and function-blocking antibodies. Both integrins were ubiquitously expressed in FL Lin(-)Sca-1(+)Kit(+) (LSK) cells. Deletion of integrin alpha6 receptor or inhibition by a function-blocking antibody inhibited FL LSK cell adhesion to its extracellular ligands,laminins-411 and -511 in vitro,and significantly reduced homing of HPCs to BM. In contrast,the anti-integrin alpha6 antibody did not inhibit BM homing of HSCs. In agreement with this,integrin alpha6 gene-deleted FL HSCs did not display any homing or engraftment defect compared with wild-type littermates. In contrast,inhibition of integrin alpha4 receptor by a function-blocking antibody virtually abrogated homing of both FL HSCs and HPCs to BM,indicating distinct functions for integrin alpha6 and alpha4 receptors during homing of fetal HSCs and HPCs. View Publication

BACKGROUND: Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood,ex vivo transduction of the gene of interest into them,and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor,time and money,while enhancing HSCs viability,transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes,in which reverse transcription of viral DNA is not completed. METHODS: In the present study,we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors,based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction,we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector,developed in our laboratory,that allows targeted transduction to specific cell receptors via antibody recognition. RESULTS: Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. CONCLUSIONS: Overall,the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. View Publication

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma-null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34(+) human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks),human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses,as documented by the presence of CD4(+) CD8(+) T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation,human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses,the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. View Publication

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CREB-binding protein (CREBBP) is important for the cell-autonomous regulation of hematopoiesis,including the stem cell compartment. In the present study,we show that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the BM microenvironment of Crebbp(+/-) mice was unable to properly maintain the immature stem cell and progenitor cell pools. Instead,it stimulates myeloid differentiation,which progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone that was predominantly caused by increased osteoclastogenesis. Although CFU-fibroblast (CFU-F) and total osteoblast numbers were decreased,the bone formation rate was similar to that found in wild-type mice. At the molecular level,we found that the known hematopoietic modulators matrix metallopeptidase-9 (MMP9) and kit ligand (KITL) were decreased with heterozygous levels of Crebbp. Lastly,potentially important regulatory proteins,endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5),were increased on Crebbp(+/-) endothelial cells. Our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and to prevent excessive myeloproliferation. View Publication

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation,which is only partially successful in the absence of an HLA genoidentical donor,thus justifying research to find an alternative therapeutic approach. To this end,RAG-2-deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2-/-Sca-1(+) selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)-based MND (myeloproliferative sarcoma virus enhancer,negative control region deleted,dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2-/- sublethally irradiated mice (3Gy). Two months later,T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens,allogeneic cells,and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy,a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether,this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2-/- mice,constituting a significant step toward clinical application. View Publication

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study,UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin,flt3 ligand,and granulocyte-colony stimulating factor. By week 4-5,we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor,human vascular cell adhesion molecule-1,human intracellular adhesion molecule-1,human CD31,E-selectin,and human macrophage. Furthermore,when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer,better expansion of total nucleated cells,CD34(+) cells,and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells,which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells,we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method,one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors,establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

Successful blood and marrow transplant (BMT),both autologous and allogeneic,requires the infusion of a sufficient number of hematopoietic progenitor/stem cells (HPCs) capable of homing to the marrow cavity and regenerating a full array of hematopoietic cell lineages in a timely fashion. At present,the most commonly used surrogate marker for HPCs is the cell surface marker CD34,identified in the clinical laboratory by flow cytometry. Clinical studies have shown that infusion of at least 2 x 10(6) CD34(+) cells/kg recipient body weight results in reliable engraftment as measured by recovery of adequate neutrophil and platelet counts approximately 14 days after transplant. Recruitment of HPCs from the marrow into the blood is termed mobilization,or,more commonly,stem cell mobilization. In Section I,Dr. Tsvee Lapidot and colleagues review the wide range of factors influencing stem cell mobilization. Our current understanding focuses on chemokines,proteolytic enzymes,adhesion molecules,cytokines and stromal cell-stem cell interactions. On the basis of this understanding,new approaches to mobilization have been designed and are now starting to undergo clinical testing. In Section II,Dr. Michele Cottler-Fox describes factors predicting the ability to mobilize the older patient with myeloma. In addition,clinical approaches to improving collection by individualizing the timing of apheresis and adjusting the volume of blood processed to achieve a desired product are discussed. Key to this process is the daily enumeration of blood CD34(+) cells. Newer methods of enumerating and mobilizing autologous blood HPCs are discussed. In Section III,Dr. John DiPersio and colleagues provide data on clinical results of mobilizing allogeneic donors with G-CSF,GM-CSF and the combination of both as relates to the number and type of cells collected by apheresis. Newer methods of stem cell mobilization as well as the relationship of graft composition on immune reconstitution and GVHD are discussed. View Publication