Macrophage inflammatory protein 1 alpha, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro.

Factors that induce proliferation of the human hematopoietic stem cell are ill-defined. Primitive hematopoietic progenitors can be maintained and differentiate in stroma-dependent, long-term bone marrow cultures (LTBMC), originally described by Dexter et al. (Dexter, T. M., L. H. Coutinho, E. Spooncer, C. M. Heyworth, C. P. Daniel, R. Schiro, J. Chang, and T. D. Allen. 1990. Molecular Control of Haemopoiesis). However, 70-80% of primitive progenitors capable of reinitiating secondary stromal cultures (LTBMC-initiating cells [IC]) are lost over a period of 5 wk in such cultures. We have recently described a novel "stroma-noncontact" culture system, in which hematopoietic progenitors are separated from the stromal layer by a 0.4-micron microporous filter membrane. Primitive progenitors in such cultures can not only differentiate into committed progenitors, but are also maintained to a greater extent than in "Dexter" cultures. However, still only 50% of the originally seeded LTBMC-IC are recovered at week 5. Since maintenance of primitive progenitors may depend not only on growth-promoting factors but also on factors that inhibit differentiation and/or proliferation, we evaluated the effect of macrophage inflammatory protein 1 alpha (MIP-1 alpha) or "stem cell inhibitor" in combination with the growth-inducing factor interleukin 3 (IL-3) on the recovery of LTBMC-IC from stroma-noncontact cultures. We demonstrate that addition of MIP-1 alpha alone to stroma-noncontact cultures does not change the number of LTBMC-IC present after 8 wk, indicating that this factor may not directly inhibit or stimulate proliferation of primitive progenitors. Addition of the growth stimulatory cytokine, IL-3, alone results in exhaustion of LTBMC-IC after 8 wk of culture, possibly as a result of their terminal differentiation. However, LTBMC-IC can be maintained for at least 8 wk when grown in stroma-noncontact cultures supplemented with both MIP-1 alpha plus IL-3. This effect depends on soluble (ill-defined) stromal factors, and results from a direct interaction of these cytokines with the progenitor population or its progeny, but not the stroma.

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CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Blood ◽

10.1182/blood.v84.5.1442.1442 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1442-1449 ◽

Cited By ~ 23

Author(s):

CM Verfaillie ◽

JS Miller

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Antigen Expression ◽

Hematopoietic Stem ◽

Interleukin 3 ◽

Inflammatory Protein ◽

Hla Dr ◽

Macrophage Inflammatory Protein

Abstract Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

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Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽

10.1182/blood.v81.6.1497.1497 ◽

1993 ◽

Vol 81 (6) ◽

pp. 1497-1504 ◽

Cited By ~ 33

Author(s):

VF Quesniaux ◽

GJ Graham ◽

I Pragnell ◽

D Donaldson ◽

SD Wolpe ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Inhibitory Effect ◽

In Vivo Model ◽

Hematopoietic Progenitors ◽

Inflammatory Protein ◽

Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

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Negative Control Region Is a Critical Element Of Insertional Oncogenesis After Gene Transfer Into Hematopoietic Progenitors With Moloney Murine Leukemia Viruses

Blood ◽

10.1182/blood.v122.21.164.164 ◽

2013 ◽

Vol 122 (21) ◽

pp. 164-164

Author(s):

Yasuhiro Ikawa ◽

Toru Uchiyama ◽

Guridevi Jayashree Jagadeesh ◽

Fabio Candotti

Keyword(s):

Stem Cell ◽

Gene Transfer ◽

Viral Vectors ◽

Negative Control ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Cell Immortalization ◽

The Difference ◽

Murine Leukemia

Abstract Gene transfer into hematopoietic stem cells has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the successes of clinical trials have been tempered by adverse events in which the use of gamma-retroviral vectors (GV) containing full-length long terminal repeats (LTRs) with strong enhancer activity increased transcription of cancer-related genes, and thereby contributed to development of leukemia. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance. The negative control region (NCR) is a particularly well-conserved sequence among mammalian gamma-retroviruses with demonstrated regulating a transcription activity of GV in hematopoietic cells. This suggests that the NCR might play a crucial role of insertional oncogenesis after gene transfer into hematopoietic progenitors. In a series of safety studies of viral gene transfer constructs, we used an in vitro assay of murine bone marrow (BM) cell immortalization and compared the consequences of hematopoietic stem cell transduction with three different kinds of viral vectors, including Moloney murine leukemia virus- (MMLV), lentivirus- (LV), and foamy virus (FV)-based constructs. To evaluate critical elements for cell immortalization by MMLV vectors, we also tested four different MMLV LTR variants deleted of either 1) most of the two 75-bp repeats associated with the viral enhancer (delE1), 2) all of the two 75-bp repeats and the NCR (delE2), 3) only the NCR (delNCR), or 4) carrying a deleterious mutation of the NCR NFAT motif (ΔNFAT). All vectors carried an internal expression cassette including the eGFP gene under the control of a UCOE (ubiquitously acting chromatin opening element) promoter. In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than six weeks in the presence of interleukin-3 and stem cell factor. Real-time PCR was performed to verify comparable transduction efficiency of bone marrow cells by different vectors. In our analysis of MMLV LTR mutants, full-MMLV and delE1 transduction of 92 and 108 cultures, respectively, resulted in 37 and 37 immortalized lines (40% and 34% immortalization rate, respectively). The difference in immortalization rate between full-MMLV and delE1 was not statistically significant. Transductions using delE2-, delNCR- and ΔNFAT-carrying vectors of 60, 36 and 35 cultures resulted in 10, 3 and 10 immortalized lines (17%, 8.3% and 29% immortalization rate, respectively). The difference between the immortalization caused by delE1 and delE2 vectors was statistically significant (p<0.05). Moreover, the difference between the immortalization caused by full-MMLV and delNCR vectors was statistically significant (p<0.01), while there was no significant difference between the immortalization induced by full-MMLV and ΔNFAT vectors. Transduction of 57 and 34 cultures with LV and FV vectors, respectively, resulted in no immortalized lines. Transductions of 128 cultures with a LV construct carrying the U3 region from the murine stem cell virus LTR as an internal promoter (LV-U3) resulted in 2 immortalized lines which was not statistically different from the results obtained with LV vectors carrying the UCOE internal promoter. These results confirm that GV are prone to causing immortalization of hematopoietic cells and indicate that deletion of the whole viral enhancer sequences may not be adequate to eliminate the insertional oncogenesis risk. Importantly, our data point to the NCR as a crucial element for immortalization and justify additional studies to evaluate its specific role in MMLV-mediated insertional oncogenesis. Finally, our results suggest that vectors based on LV and FV backbones are safer alternatives for clinical gene transfer into hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation

Blood ◽

10.1182/blood.v82.7.2045.bloodjournal8272045 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2045-2053 ◽

Cited By ~ 3

Author(s):

CM Verfaillie

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Expansion ◽

Terminal Differentiation ◽

Hematopoietic Progenitors ◽

Soluble Factors ◽

Bone Marrow Cultures ◽

Stromal Layer

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer (“stroma-free”) or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane (“stroma-noncontact”). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a “stroma-free” culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in “stroma-noncontact” cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in “stroma-noncontact” cultures supplemented with or without IL-3 than in “stroma-free” cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.

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Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture

Blood ◽

10.1182/blood.v78.4.914.bloodjournal784914 ◽

1991 ◽

Vol 78 (4) ◽

pp. 914-920 ◽

Cited By ~ 4

Author(s):

DM Bodine ◽

PS Crosier ◽

SC Clark

Keyword(s):

Stem Cell ◽

Growth Factors ◽

Cell Function ◽

Hematopoietic Growth Factors ◽

Gm Csf ◽

Liquid Suspension ◽

Inflammatory Protein ◽

Macrophage Inflammatory Protein ◽

Stimulating Factor

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G- CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

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Developing a Model of Human Pluripotent to Hematopoietic Stem Cell Development in Mistrg Mice

Blood ◽

10.1182/blood.v126.23.4755.4755 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4755-4755

Author(s):

John Astle ◽

Yangfei Xiang ◽

Anthony Rongvaux ◽

Carla Weibel ◽

Henchey Elizabeth ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Biology ◽

De Novo ◽

Conflicts Of Interest ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Immunodeficient Mice

Abstract De novo generation of HSCs has been described as a "holy grail" of stem cell biology, however the factors required for converting human pluripotent stem cells (PSCs) to true hematopoietic stem cells (HSCs) capable of robust long-term engraftment have yet to be fully characterized. Two groups have shown that injection of PSCs into immunodeficient mice leads to teratomas containing niches producing hematopoietic progenitors capable of long-term engraftment. Once these hematopoietic progenitors and their microenvironments are better characterized, this system could be used as a model to help direct in vitro differentiation of PSCs to HSCs. Toward this end, we have injected human PSCs into immunodeficient mice expressing human rather than mouse M-CSF, IL-3, GM-CSF, and thrombopoietin, as well as both human and mouse versions of the "don't eat me signal" Sirpa (collectively termed MISTRG mice). These cytokines are known to support different aspects of hematopoiesis, and thrombopoietin in particular has been shown to support HSC maintenance, suggesting these mice may provide a better environment for human PSC-derived HSCs than the more traditional mice used for human HSC engraftment. The majority of teratomas developed so far in MISTRG contain human hematopoietic cells, and the CD34+ population isolated from over half of the teratomas contained hematopoietic colony forming cells by colony forming assay. These findings further corroborate this approach as a viable method for studying human PSC to HSC differentiation. Disclosures No relevant conflicts of interest to declare.

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Interleukin-3-dependent hematopoietic stem cell lines capable of osteoclast formation in vitro

Journal of Bone and Mineral Research ◽

10.1002/jbmr.5650060908 ◽

2009 ◽

Vol 6 (9) ◽

pp. 947-954 ◽

Cited By ~ 18

Author(s):

C.E. Hagenaars ◽

E.W.M. Kawilarang-de Haas ◽

A.A.M. van der Kraan ◽

E. Spooncer ◽

T.M. Dexter ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Lines ◽

Osteoclast Formation ◽

Hematopoietic Stem ◽

Interleukin 3 ◽

Stem Cell Lines

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MACROPHAGE INFLAMMATORY PROTEIN (MIP)-1-ALPHA STEM-CELL INHIBITOR (SCI) DOES NOT AFFECT CLONAL GROWTH OF HUMAN SOLID TUMOR-CELL LINES IN-VITRO

International Journal of Oncology ◽

10.3892/ijo.4.2.353 ◽

1994 ◽

Author(s):

A KORFEL ◽

Z VONMARSCHALL ◽

M KOENIGSMANN ◽

D OBERBERG ◽

B REUFI ◽

...

Keyword(s):

Stem Cell ◽

Tumor Cell ◽

Cell Lines ◽

Solid Tumor ◽

Clonal Growth ◽

Tumor Cell Lines ◽

Inflammatory Protein ◽

Human Solid Tumor ◽

Macrophage Inflammatory Protein

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Lymphoid, Long-Term Repopulating, and Endothelial Potential of the Embryonic Hemangioblast.

Blood ◽

10.1182/blood.v108.11.1657.1657 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1657-1657

Author(s):

Mitsujiro Osawa ◽

Michael Kyba

Keyword(s):

Endothelial Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Embryoid Bodies ◽

Endothelial Differentiation ◽

Hematopoietic Progenitors ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hematopoietic and endothelial cells are thought to arise from a common progenitor termed the hemangioblast. Direct evidence for the hemangioblast was first obtained from embryonic stem cells differentiated in vitro as embryoid bodies (EBs). Flk-1+ cells from early EBs generate colonies in response to VEGF and SCF (the BL-CFC assay) which can be replated to give secondary hematopoietic or endothelial cells. Bipotent BL-CFCs have also recently been derived from the posterior primitive streak of neural plate stage mouse embryos. However, a direct relationship between the early embryonic hemangioblast defined by the BL-CFC assay and the hematopoietic stem cell remains unproven. Hemangioblast-derived hematopoiesis in vitro is transient and restricted to myelo-erythroid differentiation. Lymphoid potential and long-term repopulation, two hallmarks of the definitive hematopoietic stem cell, have eluded detection to date. Previous work has shown that the homeodomain transcription factor, HoxB4, by enhancing self-renewal in vitro, can reveal latent definitive HSC activity of transient embryonic hematopoietic progenitors. Using an ES cell line with doxycycline-inducible HoxB4 expression, we have investigated the definitive hematopoietic and endothelial potential of individual hemangioblast colonies. BL-CFC numbers were unaffected by HoxB4 expression during EB differentiation, however they were increased threefold by induction during the BL-CFC assay. By replating one half of the cells from an individual blast colony in endothelial medium and the other half on an OP9 monolayer with hematopoietic cytokines, we show that the majority (60%) of HoxB4-induced BL-CFCs are bipotent. HoxB4 expression was compatible with endothelial differentiation and allowed exponential expansion of hematopoietic progenitors on OP9 cocultures. When switched to OP9-DL1 with lymphoid cytokines, T-lymphopoiesis was observed characterized by CD25 expression followed by CD4, CD8, and CD3epsilon expression. To assay long-term repopulation, individual blast colonies were picked and divided into endothelial medium and OP9 monolayers. The hematopoietic arms of colonies defined retrospectively to have been bipotent (endothelial differentiation was observed in vitro) were transplanted into sublethally irradiated Rag2; gamma-c; CD45.1 immunodeficient mice. Mice with long-term hematopoietic engraftment were identified by the presence of CD45.2 cells in peripheral blood 3 months post-transplant. Lymphoid and myeloid contribution was evaluated by costaining with Gr-1, B220, CD19, CD4, and CD8. The donor-derived component of these hematopoietic chimeras, including their entire lymphoid arm (approximately 1/3 of engrafted mice showed lymphoid differentiation) is by definition clonally derived from a single hemangioblast. These results clearly show that the embryonic hemangioblast is not intrinsically limited in its hematopoietic potential. Under conditions that favor self-renewal, lymphoid differentiation and long-term repopulation become evident, revealing the link between endothelial development and definitive hematopoiesis at the clonal level.

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Cdx4 Differs from Cdx2 in Its Oncogenic Potential and Its Regulation of Hox Gene Expression in the Murine Bone Marrow Transplantation Model.

Blood ◽

10.1182/blood.v112.11.1187.1187 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1187-1187

Author(s):

Silvia Thoene ◽

Vijay P.S. Rawat ◽

Vegi M. Naidu ◽

Wolfgang Hiddemann ◽

Michaela Feuring-Buske ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Expression Profile ◽

Ectopic Expression ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Integration Sites ◽

The Impact

Abstract Cdx4 is known to be of importance for specification of cell fate in embryonic hematopoiesis with defects leading to severe perturbation of blood formation. When overexpressed in a murine hematopoietic stem cell line, Cdx4 is capable to enhance progenitor formation in vitro and promote lymphoid reconstitution of lethally irradiated, transplanted mice in vivo. In line with this important function of Cdx4 in early hematopoiesis, we analyzed expression of Cdx4 in highly purified subpopulations isolated from murine bone marrow (BM) cells by TaqMan qPCR. Cdx4 showed an expression profile known from other stem cell regulatory genes with high expression in early hematopoietic progenitors followed by decreasing expression towards the more differentiated stages of hematopoiesis, with a more than 1200-fold lower expression in total BM cells compared to progenitor enriched 5-FU BM cells (n=3). To test the impact of Cdx4 on murine progenitors, we retrovirally transduced 5-FU BM cells with Cdx4. Overexpression of Cdx4 induced growth of BM cells in liquid expansion assay (Cdx4 5.7×108±2.2×108 SEM, EGFP 2.6×106±9×105 SEM, p=0.020; cell numbers after 14 days in cytokine supplemented medium, n=5). In addition, expression of Cdx4 conferred serial replating capacity to murine BM progenitors compared to empty vector control (CFU total after 3rd replating: 4.5×109±1.3×109 SEM/500 input cells in 1st CFC, n=5). This effect was significantly stronger compared to hematopoietic progenitors overexpressing the leukemogenic Cdx2 (p=0.008). Immunophenotyping of cells after 3rd replating showed expression of mainly myeloid antigens and cytospin preparation revealed a mature myeloid morphology. Interestingly, these colonies were able to engraft lethally irradiated mice and showed multilineage engraftment (lymphoid:myloid ratio week 16 after transplantation: 0.5:1, n=2), indicating the ability of Cdx4 expressing colonies to maintain stem cell properties in vitro. In contrast to Cdx2-transplanted mice which showed a severe myeloid bias, regular peripheral blood analysis of mice transplanted with Cdx4 overexpressing BM cells showed multilineage engraftment confirmed by immunophenotyping and normal hematological parameters (RBC 6.7×109±4.2×108, WBC 5.8×106±5.19×105; lymphoid:myeloid ratio 1.4:1; week 8–28). Of note, with a median latency of 309 days after transplantation, nine out of ten mice transplanted with Cdx4-transduced BM cells died of transplantable leukemia. In six out of seven cases we found single retroviral integration sites, indicating a monoclonal origin of the disease. We could determine three different integration sites located between 200 and 700 bp upstream of coding sequences (n=4; Opa3, Akap1, Sema4d). The integration sites of two other mice were located intragenic (Zfyve2, Zfp407), indicating that insertional mutagenesis might be a necessary factor for Cdx4 induced leukemogenesis. Moreover, qRT-PCR revealed that Cdx4 in contrast to Cdx2 did not induce ectopic expression of the leukemogenic Hoxb8 and was associated with a significant lower (7.8-fold) expression of the leukemogenic Hoxb6 in transduced murine BM cells. Taken together, these data indicate that Cdx4 plays a major role in the regulation of early hematopoiesis. Its expression profile and its hematopoietic activity in different hematopoietic assays clearly differs from Cdx2, which was shown to be highly leukemogenic in mice and to be ectopically expressed in human AML. Murine models analyzing the impact of Cdx4 and Cdx2 expression on hematopoietic development will help to delineate critical differences between the two related genes.

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