Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3620-3627 ◽  
Author(s):  
Graça Almeida-Porada ◽  
Christopher D. Porada ◽  
Nam Tran ◽  
Esmail D. Zanjani

Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3620-3627 ◽  
Author(s):  
Graça Almeida-Porada ◽  
Christopher D. Porada ◽  
Nam Tran ◽  
Esmail D. Zanjani

Abstract Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.


Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5014-5014
Author(s):  
Irina N. Shipounova ◽  
Nataliya A. Petinati ◽  
Nina J. Drize ◽  
Aminat A. Magomedova ◽  
Ekaterina A. Fastova ◽  
...  

Introduction. Stromal microenvironment of the bone marrow (BM) is essential for normal hematopoiesis; the very same cells are involved in the interaction with the leukemic stem cells. The aim of the study was to reveal the alterations in stromal microenvironment of patients in debut and after the therapy using multipotent mesenchymal stromal cells (MSC) as a model. Methods. MSC of patients with acute myeloid leukemia (AML, N=32), acute lymphoblastic leukemia (ALL, N=20), chronic myeloid leukemia (CML, N=19), and diffuse large B-cell lymphoma without BM involvement (DLBCL, N=17) were isolated by standard method from the patients' BM. Each BM sample was acquired during diagnostic aspiration after the informed signed consent was obtained from the patient. Groups of BM donors comparable by age and gender were used as controls for each nosology. Gene expression was analyzed with real-time RT-PCR. The significance of differences was evaluated with Mann-Whitney U-test. Results. The results of gene expression analysis are summarized in Table. The expression of genes regulating hematopoietic stem and precursor cells (JAG1, LIF, IL6) was significantly upregulated in MSC of the patients in debut, except for DLBCL. The latter was characterized with upregulation of osteogenic marker gene SPP1 and downregulation of FGFR1 gene. The upregulation of the expression of genes regulating proliferation of stromal cells (PDGFRA, FGFR1) and adipogenic marker gene (PPARG) was common for AML and CML. Both acute leukemias were characterized by the upregulation of genes associated with inflammation and regulation of hematopoietic precursors (CSF1, IL1B, IL1BR1) and by the downregulation of chondrogenic differentiation marker gene (SOX9). CML and DLBCL demonstrated the upregulation of FGFR2. BM of the DLBCL patients did not contain any malignant cells; nevertheless, stromal precursors from the BM were significantly affected. This indicates the distant effects of DLBCL malignant cells on the patients' BM. Myeloid malignancies seem to affect MSC more profoundly then lymphoid ones. Effect of leukemic cells on stromal microenvironment in case of myeloid leukemia was more pronounced. The treatment significantly affected gene expression in MSC of patients. In all studied nosologies the IL6 gene expression was upregulated, which may reflect the inflammation processes ongoing in the organism. The expression of LIF was upregulated and ICAM1, downregulated in MSCs of AML, ALL, and CML patients. In the MSC of patients with AML, who had received the highest doses of cytostatic drugs to achieve remission, a significant decrease in the expression of most studied genes was found. In patients with ALL with long-term continuing treatment in combination with lower doses of drugs, IL1B expression was increased, while the decrease in expression was detected for a number of genes regulating hematopoietic stem cells (SDF1, TGFB1), differentiation and proliferation (SOX9, FGFR1, FGFR2). Treatment of CML patients is based on tyrosine kinase inhibitors in doses designed for long-term use, and is less damaging for MSC. The upregulation of TGFB1, SOX9, PDGFRA genes and downregulation of IL1B gene was revealed. MCS of DLBCL patients, unlike the other samples, were analyzed after the end of treatment. Nevertheless, significant upregulation of IL8 and FGFR2 genes was found. Thus, both the malignant cells and chemotherapy affect stromal precursor cells. The changes are not transient; they are preserved for a few months at least. MSCs comprise only a minor subpopulation in the BM in vivo. When expanded in vitro, they demonstrate significant changes between groups of patients and healthy donors. Conclusions. Leukemia cells adapt the stromal microenvironment. With different leukemia, the same changes are observed in the expression of genes in MSC. MSC of patients with acute forms have a lot of changes which coincide among these two diseases. MSC of AML patients are most affected both in debut and after the therapy. Treatment depends on the nosology and in varying degrees changes the MSC. This work was supported by the Russian Foundation for Basic Research, project no. 17-00-00170. Disclosures Chelysheva: Novartis: Consultancy, Honoraria; Fusion Pharma: Consultancy. Shukhov:Novartis: Consultancy; Pfizer: Consultancy. Turkina:Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; fusion pharma: Consultancy.


Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 939-948 ◽  
Author(s):  
Y Tomita ◽  
DH Sachs ◽  
M Sykes

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.


Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1966-1968 ◽  
Author(s):  
J Laver ◽  
SC Jhanwar ◽  
RJ O'Reilly ◽  
H Castro-Malaspina

Abstract The origin of marrow stromal cells post allogeneic bone marrow transplantation (BMT) was studied. Two groups of patients receiving HLA- identical marrow grafts from sex mismatched siblings were included in the study: the first group (eight patients) received conventional marrow grafts and the second group (ten patients) received stromal cell and T cell depleted grafts. All patients showed hematopoietic engraftment with donor cells. Marrow aspirates obtained from these patients were used to establish stromal layers in long-term marrow cultures (LTMC) for 4 to 6 weeks. In both groups, karyotype analysis of nonhematopoietic cultured stromal cells showed host origin even as late as day 760 posttransplantation. Immunofluorescence methods using monoclonal antibodies against components of fibroblasts, macrophages, and endothelial cells, showed that the composition of stromal layers was similar to those obtained from normal controls. Our data indicate that marrow stromal progenitors capable of proliferation are nontransplantable and do not originate from a hematopoietic-stromal common progenitor.


Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2515-2522 ◽  
Author(s):  
Esmail D. Zanjani ◽  
Alan W. Flake ◽  
Graça Almeida-Porada ◽  
Nam Tran ◽  
Thalia Papayannopoulou

The mechanisms by which intravenously (IV)-administered hematopoietic cells home to the bone marrow (BM) are poorly defined. Although insightful information has been obtained in mice, our knowledge about homing of human cells is very limited. In the present study, we investigated the importance of very late activation antigen (VLA)-4 in the early phases of lodgment of human CD34+progenitors into the sheep hematopoietic compartment after in utero transplantation. We have found that preincubation of donor cells with anti–VLA-4 blocking antibodies resulted in a profound reduction of human cell lodgment in the fetal BM at 24 and 48 hours after transplantation, with a corresponding increase of human cells in the peripheral circulation. Furthermore, IV infusion of the anti–VLA-4 antibody at later times (posttransplantation days 21 to 24) resulted in redistribution or mobilization of human progenitors from the BM to the peripheral blood. In an attempt to positively modulate homing, we also pretreated human donor cells with an activating antibody to β1 integrins. This treatment resulted in increased lodgment of donor cells in the fetal liver, presumably for hemodynamic reasons, at the expense of the BM. Given previous involvement of the VLA-4/vascular cell adhesion molecule (VCAM)-1 adhesion pathway in homing and mobilization in the murine system, our present data suggest that cross-reacting ligands (likely VCAM-1) for human VLA-4 exist in sheep BM, thereby implicating conservation of molecular mechanisms of homing and mobilization across disparate species barriers. Thus, information from xenogeneic models of human hematopoiesis and specifically, the human/sheep model of in utero transplantation, may provide valuable insights into human hematopoietic transplantation biology.


Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2844-2844
Author(s):  
Yang Jo Chung ◽  
Suntae Kim ◽  
Zhenhua Zhang ◽  
Peter D. Aplan

Abstract Bone marrow (BM) contains both hematopoietic cells and non-hematopoietic, stromal cells, which support hematopoiesis. In mice and humans, CD45 is a pan-hematopoietic marker universally expressed on nucleated hematopoietic cells. Recent studies have shown that CD45-, non-hematopoietic stromal cells present in the BM of patients with MDS can display the same cytogenetic abnormalities found in the CD45+ MDS clone, suggesting that stromal cells may be part of the malignant clone in patients with MDS. Although CD45- , non-hematopoietic BM cells (such as multipotent adult progenitor cells) can produce CD45+, hematopoietic stem cells, there is little evidence that CD45+ hematopoietic cells can de-differentiate into CD45-, non-hematopoietic stromal cells. To study the possible conversion of hematopoietic (CD45+) to non-hematopoietic (CD45-) cells, we used the NUP98-HOXD13 (NHD13) mouse model of myelodysplastic syndrome (MDS). Two immortal cell lines (251 and 63B) were established from NHD13 mice with AML. These cell lines grow as a combination of adherent and suspension cells; although the suspension cells are almost entirely (>98%) CD45+, only 50% of the adherent cells are CD45+. Surprisingly, sub-culture of CD45+ suspension cells, generates CD45- adherent cells, leading to the suggestion that CD45+ hematopoietic cells can de-differentiate and produce CD45-, non-hematopoietic cells. Further immunophenotype analysis demonstrates that the CD45+ cells are also positive for myelo-monocytic markers such as CD11b, CD16/32, and F4/80, whereas the CD45- fraction is negative for these markers. To verify the conversion of CD45+ to CD45- cells at a clonal level, we sorted highly purified (>99.99%) CD45+ cells and sub-cultured CD45+ cells at a density of 1 or 10 cells per well in 96 well plates. Although there was no growth in the wells seeded at 1 cell/well, 23 out of 96 wells seeded at 10 cells/well expanded; at least 5 of these clones produced CD45- cells. Expression of the NHD13 transgene (driven by the pan-hematopoietic Vav1 promoter) was 100-fold higher in the CD45+ fraction compared to the CD45- fraction, and gene expression arrays identified differential expression of a number of hematopoietic markers, such as Pu.1 and Csf2rb. Finally, preliminary bisulfite sequencing experiments suggest that the CD45 promoter is heavily methylated in the CD45- fraction, but un-methylated in the CD45+ fraction. Taken together, these results indicate that the cell lines can cycle between non-hematopoietic and hematopoietic cells. To determine if the cell lines have retained malignant potential, we transplanted lethally irradiated recipients via intravenous injection. At 6 and 16 weeks post-transplant, there was no engraftment detected in the peripheral blood (PB). However, at 24 weeks, one of three recipients showed low level engraftment (1.7% of PB). Remarkably, this mouse became frankly leukemic 2 weeks later, with splenomegaly, anemia, monocytosis, and donor engraftment of 17-53% in the PB, BM, and spleen. To determine which cell fraction was responsible for the disease phenotype, we transplanted purified CD45+ and CD45- cells via intrafemoral (IF) injection. Interestingly, all recipients of the CD45+ cells died due to profound pancytopenia (hgb, 3.9±1.0 g/dL; WBC, 0.24±0.30 K/uL) by day 13 post-transplant; in contrast recipients of CD45- cells were healthy, with normal CBCs. Although we could not detect donor cells in recipients of CD45- cells by FACS at any time point up to 34 weeks post-transplant, engraftment of donor cells in the BM of these recipients could be detected by transgene specific genomic DNA PCR. These results indicate that the disease entity resides in the CD45+ fraction, and that long term engraftment of quiescent CD45- cells occurs. In addition, these results suggest the intriguing possibility that quiescent CD45- cells, which do not express the NHD13 transgene, can be transformed by demethylation of the CD45 promoter (and subsequent activation of the Vav promoter, which drives the NHD13 transgene). Experiments to test this hypothesis are currently in progress. Taken together, these findings suggest that malignant cells can cycle between stromal and hematopoietic cells, based at least in part on epigenetic events such as cytosine methylation, and support the hypothesis that non-hematopoietic BM cells may be an important part of the malignant clone in patients with MDS and AML. Disclosures Aplan: NIH Office of Technology Transfer: Patents & Royalties.


Sign in / Sign up

Export Citation Format

Share Document