128 Abstracts/Experimental Hematology 28 (2000) 31–131

303 Sunday, July 9, 2000 (14:15–16:00)Session II-5: Stem Cell Biology II

LYMPHOHEMATOPOIETIC STEM CELLS FROM DONOR ORIGIN ARE PRESERVED IN BONE MARROW IMPLANTS BENEATH THE RENAL CAPSULE OF NON-CONDITIONED HOST MICET. Grande*, F. Varas*, A. Ramírez*, and J. A. BuerenDpto. Biología Molecular y Celular. Ciemat. Madrid. Spain

Renal ossicles are ossified structures developed beneath themurine kidney capsule after the implantation of a bone marrow(BM) plug. We have investigated the origin of the hematopoieticcells lodged in renal ossicles by using sex-mismatched BM im-plants into Ly5 congenic male mice. Female BM plugs were ob-tained from two different transgenic mouse lines carrying neomy-cin gene sequences which provided additional genotypic tracers.Flow cytometry analyses from long-term cultures established withossicles excised between the 17th and the 40th week post-implan-tation showed that 5% to 70% of the non-adherent myeloid cellswere of donor origin. Genetic analysis of 12 day-spleen coloniesgenerated by ossicles excised between the 10th and the 40th weekpost-implantation revealed a donor contribution which ranged be-tween 16% and 93% within the CFU-S12 population. Moreover,the preservation of donor long-term repopulating lymphohemato-poietic cells was evidenced in ossicles excised between the 10thand the 19th week post-implantation. Our results indicate that re-nal ossicles contain a significant proportion of hematopoietic pro-genitors and long-term repopulating cells from donor origin.

304 Sunday, July 9, 2000 (16:15–18:15)Plenary Symposium III: Molecular Mechanisms of Leukemogenesis and Lymphomagenesis

ROLE OF C/EBPa IN GRANULOCYTIC DIFFERENTIATION AND MYELOID LEUKEMOGENESIS Thomas Pabst,* Tracey Lodie,* and Daniel G. Tenen*Harvard Institutes of Medicine, Boston, MA

C/EBPa plays a critical role in myeloid differentiation. Specifi-cally, induced expression of C/EBPa in early myeloid cells di-rected differentiation along the granulocytic pathway. In addition,C/EBPa knockout mice demonstrate a block in granulocyte differ-entiation. For this reason, we investigated abnormalities in C/EBPain human acute myelogenous leukemia (AML). We identifiedeleven heterozygous mutations in C/EBPa in ten patients, mostlyAML-M2 subtype without the t(8;21). The mutations resulted inproduction of mutant alleles which blocked DNA binding, transac-tivation of granulocyte target genes, and induction of granulocyticdifferentiation mediated by the wild type allele in a dominant neg-ative manner. This is the first report of mutations in C/EBPa in hu-man neoplasia, and such mutations are likely to induce the differ-entiation block found in AML. The hallmark of acutepromyelocytic leukemia (APL), another subtype of AML, is thetranslocation t(15;17) and a block in promyelocytic differentiation.Treatment with All-Trans-Retinoic Acid (ATRA) induces blastdifferentiation and clinical remission. In primary human APLcells, we did not find mutations in C/EBPa, but the fusion proteinPML/RARa physically interacts with C/EBPa to block its func-tion in an ATRA reversible manner. These studies implicate dis-ruption of C/EBPa DNA binding and function by physical interac-

tion with PML/RARa as a mechanism contributing to the block indifferentiation following expression of the fusion protein in APL.In summary, these results implicate abnormalities in function of C/EBPa as a major target leading to the block in myeloid differentia-tion found in AML.

305 Monday, July 10, 2000 (8:00–9:30)Plenary Symposium IV: Stem Cell Plasticity

HEMATOPOIETIC POTENTIAL OF ADULT VASCULAR TISSUEW.H. FlemingDivision of Hematology and Medical Oncology, BMT Program, Oregon Health Sciences University, Portland, OR, U.S.A.

Similar patterns of gene expression are observed during bothvasculogenesis and hematopoiesis. In addition, the close proximityof developing blood vessels and early sites of hematopoiesis dur-ing development is consistent the existence of a common precursorcell. To determine whether hematopoietic progenitor/stem cellspersist in adult blood vessels, we transplanted explants of normaladult vena cava or thoracic aorta under the kidney capsule of le-thally irradiated or unirradiated recipient mice.The spleens of trans-planted animals contained macroscopic colonies (CFU-S) with afrequency similar to that observed in mice transplanted with 105

bone marrow cells. In contrast, irradiated recipients transplantedwith 100 ml of unfractionated peripheral blood (PB) demonstratedvery few CFU-S. When explants of vena cava or aorta were trans-planted ino lethally irradiated mice, a mean of 85% of these recipi-ents were protected while none of the recipients transplanted of100 ml of PB survived. Histologic evaluation of vascular explants14 days after transplant revealed a marked accumulation of cellsand the formation of vascular channels within and around the vas-cular tissue. Transplantation of ROSA-26 donor vascular tissuedemonstrated that this accumulation of cells was primarily donorderived. To identify the earlist proliferative events in these vascu-lar explants, recipient mice were treated with BrdU and trans-planted with vascular tissue. These studies revealed the presenceof proliferating cells within the intima of the vascular grafts asearly as 48 hours after transplantation. Although donor cells weredetected in blood of radioprotected mice, high levels of host he-matopoiesis recovery were found in most recipients. These find-ings indicate that cell populations within adult vascular tissue havethe capacity to both develop into hematopoietic progeny and to ra-dioprotect host hematopoietic stem cells.

306 Saturday, July 8, 2000 (17:15–19:15)Plenary Symposium I: Hemopoietic Stem Cell Biology

ROLE OF RAC 2 IN HEMOPOIETIC STEM CELL MOTILITYD.A. Williams, F.-C. Yang, J.B. Borneo, J. Pennington, A.J. KingHoward Hughes Medical Institute, Herman B Wells Center for Pediatric Research, Section of Pediatric Hematology/Oncology, Departments of Pediatrics and Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

Rho GTPases (Rho, Rac, and Cdc42) act as molecular switchesto regulate cellular functions. We have recently generated a mousedeficient in Rac 2, a GTPase which is expressed only in hemato-poietic cells (Roberts et al. Immunity, 1999), and shown defects inPMN movement and other actin-based functions in vivo and invitro. We now demonstrate that Rac 2 is expressed in purified he-

Abstracts/Experimental Hematology 28 (2000) 31–131 129

matopoietic progenitor/stem cells (HSC) (lin2,c2kit1Sca-11).Analysis of the function of Rac 22/2 HSC shows multiple abnor-malities in vitro and in vivo. Adhesion to the extracellular matrixmolecule fibronectin via integrin VLA-4 is significantly reduced.Surprisingly, movement of progenitor/stem cells in response toSDF-1 is increased in both modified Boyden chamber assays andin quantitative time-lapsed video imaging. This increased move-ment is associated with increased size of filopodia, and increasedf-actin content, reminiscent of enhanced activity of Cdc42, sug-gesting cross-talk between GTPase pathways. Importantly, theseabnormalities are mirrored by changes in cell movement in vivo.G-CSF-induced mobilization of hematopoietic progenitor cells isenhanced nearly 3-fold in Rac 22/2 mice, while the augmentationof G-CSF mobilization by concurrent administration of antibodiesto VLA-4 is reduced, further supporting the role of Rac 2 in VLA-4 function in stem/progenitor cells localization in the bone marrowmicroenvironment. These data demonstrate that Rac 2 is criticalfor normal adhesion and movement of HSC, and suggest that defi-ciency of Rac 2 in these cells may lead to altered activity of otherGTPases, particularly Cdc42.

307 Sunday, July 9, 2000 (8:00–10:00)Plenary Symposium II: Translational Research in Blood and Marrow Transplantation

TREATMENT OF AUTOIMMUNE DISORDERS BY HEMOPOIETIC CELL TRANSPLANTATIONSusumu Ikehara1st Dept. of Pathology, Transplantation Center, Kansai Med. Univ., Osaka, Japan

We have previously found that allogeneic BMT can be used totreat autoimmune diseases using various animal models for au-toimmune diseases (Proc. Natl. Acad. Sci. USA 82:2483, 1985,etc.). However, in MRL/lpr mice that are radiosensitive (less than8.5Gy), conventional BMT was found to have transient effects; theautoimmune diseases recur 3 months after BMT. However, wehave found that the combination of BMT plus bone grafts (to re-cruit donor stromal cells) completely prevents the recurrence ofautoimmune diseases in these mice (J. Immunol. 152: 3119, 1994).In addition, we have found that this strategy has no effect on thetreatment of autoimmune diseases in MRL/lpr mice, since thesemice become more radiosensitive after the onset of lupus nephritis,due to uremic enterocolitis. We have very recently discovered anew safe strategy for the treatment of autoimmune diseases whichincludes fractionated irradiation (5.5 Gy 3 2) (day21) followedby portal venous (P.V.) injection (day 0) plus intravenous (I.V.) in-jection (day 5) of donor bone marrow cells. We have succeeded intreating autoimmune diseases in MRL/lpr mice by this strategy;100% of thus-treated MRL/lpr mice survive more than one year af-ter the treatment (Blood 95: 1862, 2000). We have also found thatthis strategy is applicable to organ allografts in mice; all the skinand pancreas allografts survive more than one year after transplan-tation without using immunosuppressants. We clarify the mecha-nisms underlying the advantages of this strategy, and also show us-ing cynomolgus monkeys that this strategy will become applicableto humans.

308 Saturday, July 8, 2000 (17:15–19:15)Plenary Symposium I: Hemopoietic Stem Cell Biology

INTERACTIONS OF ENDODERM AND ECTODERM INEMBRYONIC HEMATOPOIESISMargaret H. Baron*Mount Sinai School of Medicine, New York

During embryonic development in mammals, hematopoiesisand vasculogenesis begin in the yolk sac from newly induced me-soderm. Little is known about the molecules involved in these pro-cesses in the gastrulating embryo. We devised a novel transgenicembryo explant culture system to examine the possibility that epi-thelial-mesenchymal interactions play an important role in yolksac hematopoiesis and vasculogenesis in the mouse. Embryostransgenic for an e-globin/lacZ reporter gene were harvested priorto the formation of blood, stripped of their surrounding primitiveendoderm (epithelium), and grown in collagen drop cultures aloneor together with the isolated endoderm tissue. These studies dem-onstrated that primitive (visceral) endoderm signaling is essentialfor activation of primitive hematopoiesis and embryonic vasculo-genesis. The endodermal signals act at short range and are medi-cated by diffusible molecules. Remarkably, prospective neural ec-toderm can be reprogrammed to develop along hematopoietic andvascular lineages when anterior embryonic ectoderm is recom-bined with primitive endoderm. Primitive endoderm expresses anumber of signaling molecules that are strong candidates for thehematopoietic mesoderm-inducing signal(s), and we have used theembryonic explant culture to assay for their ability to substitute forendodermal tissue. These studies may have important implicationsnot only for our understanding of hematopoietic development inthe mammalian embryo but may also suggest new and more effec-tive stem cell-based therapeutic approaches for hematopoietic andvascular diseases.

309 Sunday, July 9, 2000 (16:15–18:15)Plenary Symposium III: Molecular Mechanisms of Leukogenesis and Lymphomagenesis

INTERACTION BETWEEN PLZF AND ETO-A POSSIBLE LINK IN THE MOLECULAR MECHANISMS OF M2 AND M3 LEUKEMIAAM Melnick, JJ Westendorf, A Polinger, GW Carlile, S Arai, HJ Ball, B Lutterbach, SW Hiebert, JD Licht

PLZF is a gene disrupted in patients with t(11;17)-associatedAPL. PLZF encodes a sequence specific DNA binding transcrip-tional repressor that PLZF interacts with a transcriptional repres-sion complex that includes co-repressors and histone deacetylases(HDACs). ETO, involved in the (8;21) AML-M2 translocation, as-sociates with the same transcriptional repression complex. Usingthe yeast-two-hybrid system, co-immunoprecipitation and immu-nofluorescence assays we show that PLZF and ETO interact. TheN-terminal portion of ETO, which also binds mSin3A, interactswith PLZF while the second repression domain and not the POZ/BTB domain of PLZF is critical for interaction with ETO. ETO en-hanced the ability of PLZF to repress transcription, while HDACinhibitors abrogated this effect, suggesting that ETO helps recruitHDAC complexes to PLZF. AML1-ETO product of the t(8;21)translocation associate with M2 leukemia was characterized as adominant negative form of AML that represses genes normally ac-tivated by AML1. We now find that AML1-ETO is a dominant in-