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Regulatory mechanisms governing adhesion of hematopoietic progenitor cells to the stromal nische are poorly understood. Growth factors such as stem cell factor (SCF),granulocyte-macrophage colony-stimulating factor,and thrombopoietin were reported to upregulate the adhesion of hematopoietic progenitors to immobilized fibronectin through activation of integrin alpha4beta1 and alpha5beta1. Macrophage inflammatory protein (MIP)-1alpha is a C-C chemokine that suppresses colony formation by stem/progenitor cells in vitro. We asked if MIP-1alpha would modulate the adhesive phenotype of colony-forming cells (CFCs) obtained from healthy donor bone marrow (BM),cord blood (CB),and mobilized peripheral blood (mPB) CD34+ cells,in comparison with SCF,using immobilized fibronectin. SCF significantly increased the level of adhesion of CFCs from BM,CB,and mPB. On the other hand,MIP-1alpha significantly increased the level of adhesion of CFCs from BM and CB,but less so from mPB. The effects of MIP-1alpha were inhibited by blocking antibodies to integrin alpha4,alpha5,or beta1,and polymerization plus rearrangement of F-actin were observed in affected cells by labeling with rhodamine-conjugated phalloidine. These data indicate that the effect of MIP-1alpha on the adhesive phenotype of CFCs is mediated by modulation of the organization of integrin. The amount of MIP-1alpha receptor on mPB was less than for BM or CB,which may explain the distinct characteristics in the adhesive response induced by MIP-1alpha. We suggest that hematopoietic progenitor cells from different sources may be heterogeneous with respect to maturation,integrin affinity,MIP-1alpha receptor expression,and regulation of MIP-1alpha signaling. Our data indicate that MIP-1alpha may affect migration,homing,and mobilization of hematopoietic progenitors by modulating the adhesive phenotype of these cells. View Publication

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted,self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani,K. Hobson,and M. S. Rao,1997,Dev. Biol. 186,202-223; M. S. Rao and M. Mayer-Proschel,1997,Dev. Biol. 188,48-63; M. Mayer-Proschel,A. J. Kalyani,T. Mujtaba,and M. S. Rao,1997,Neuron 19,773-785). We now show that cells identical to rat NEPs,NRPs,and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs,respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive,undergo self-renewal in defined medium,and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus,lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells. View Publication

To study molecular events involved in B lymphocyte development and V(D)J rearrangement,we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here,we show that B lineage cells generated in vitro from ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first,they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second,they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third,they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover,the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably,EAB cells possess functional V(D)J recombinase activity. In particular,the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation. View Publication

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity,we developed a fluorescent substrate for ALDH,termed BODIPY aminoacetaldehyde (BAAA),and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells,40-90% pure for CD34(+)CD38(lo/-) cells,and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together,these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well. View Publication

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation,such as the TCR/CD3 complex and the costimulatory receptor CD28,are capable of mediating T-cell activation resulting in tumor cell killing. In this study,we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2,when used alone rather than in combination,markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore,these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and,in particular,of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients. View Publication

In this report,we sought to optimize gene transfer into primitive human umbilical cord blood (UCB) cells. Initially,we found that fresh UCB isolated with the CD34brCD38 CD33 phenotype were highly enriched for hematopoietic progenitors detected in extended long-term cultures (8-week LTCs). In addition,following ex vivo gene transfer,this population possessed virtually all the 8-week LTC activity of the cultured cells. A multiparameter FACS assay was developed to efficiently screen the effects of alternative retroviral vector gene transfer procedures on the transduction efficiency and maintenance of CD34brCD38 CD33 cells. Proliferation of the CD34brCD38 CD33 cells was found to be a prerequisite for efficient transduction. However,in all conditions tested,proliferation of the CD34brCD38 CD33 cells was associated with a progressive loss of primitive cell properties including a reduction in CD34 expression,an increase in CD38/CD33 expression,and a decline in the ability to sustain 8-week LTCs. These observations indicate that it will be necessary to define conditions that more effectively support the self-renewal capacity of CD34brCD38 CD33 cells to optimize retroviral vector gene transfer in these cells. Evaluating these conditions and reagents will be facilitated by the multiparameter FACS assay described in this report. View Publication

Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source,potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc- eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 × 102,1 × 103,1 × 104,1 × 105 or 1 × 106 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly,in this study,a minimum of 1 × 105 ES cells in the myocardium and 1 × 104 cells in the skeletal muscle was observed to be requisite for teratoma development,suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic. Keywords View Publication

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia associated with a poor prognosis. However,there are relatively few insights into the genetic etiology of AMKL. We developed a screening assay for mutations that cause AMKL,based on the hypothesis that constitutive activation of STAT5 would be a biochemical indicator of mutation in an upstream effector tyrosine kinase. We screened human AMKL cell lines for constitutive STAT5 activation,and then used an approach combining mass spectrometry identification of tyrosine phosphorylated proteins and growth inhibition in the presence of selective small molecule tyrosine kinase inhibitors that would inform DNA sequence analysis of candidate tyrosine kinases. Using this strategy,we identified a new JAK2T875N mutation in the AMKL cell line CHRF-288-11. JAK2T875N is a constitutively activated tyrosine kinase that activates downstream effectors including STAT5 in hematopoietic cells in vitro. In a murine transplant model,JAK2T875N induced a myeloproliferative disease characterized by features of AMKL,including megakaryocytic hyperplasia in the spleen; impaired megakaryocyte polyploidization; and increased reticulin fibrosis of the bone marrow and spleen. These findings provide new insights into pathways and therapeutic targets that contribute to the pathogenesis of AMKL. View Publication

OBJECTIVE: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. METHODS: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients,respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. RESULTS: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (textgreater or = 98%) for CD73,CD105,CD106,CD29,CD13,CD44 and negative (textless or = 1%) for CD34,CD45,CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients,respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. CONCLUSIONS: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units. View Publication

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism,erythropoiesis,angiogenesis,and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene,which encodes the hypoxia-responsive alpha subunit of HIF-1,causes cardiac,vascular,and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos,as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines,such as vascular endothelial growth factor and Epo,but were ameliorated by Fe-SIH,a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis,Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin,Fpn1,Irp1,and frascati. We conclude that dysregulated expression of genes encoding Epo,EpoR,and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis. View Publication