Stromal cells selectively reduce the growth advantage of human committed CD34+ hematopoietic cells ectopically expressing HOXB4.

Key players in self-renewal of hemopoietic stem cells are homeobox (HOX) transcription factors. In murine cells, overexpression of HOXB4 results in expansion of hematopoietic stem- and committed progenitor cells in vitro without obvious hematopoietic alterations. In vivo, HOXB4 induced HSC expansion continued until stem cell regeneration reached pretransplantation levels. HOXB4 is thus an attractive candidate for amplification of stem cells provided that human HOXB4 overexpressing cells can also be restricted to normal growth in vivo. The stromal microenvironment provides the regulatory mechanisms controlling the balance of stem cell self-renewal and differentiation. Here, we compared the response of HOXB4- and GFP-control vector transduced human CD34(+) cells to stroma encoded signals in vitro. In serum-sustained cocultures MS-5 stroma contact reduced the output of late CD34- HOXB4(+) cells in relation to GFP-controls 9-fold whereas the expansion of early CD34(+)HOXB4(+) cells remained unchanged as compared to liquid cultures. In presence of insulin HOXB4 overexpressing cells do not react to stroma encoded growth-restricting signals. Our results show that ectopic expression of HOXB4 in combination with MS-5 stroma exerts different effects in early and late human cord blood CD34(+) cells resulting in an enhanced proliferation of early CD34(+) cells in absence or presence of MS-5 stroma and an impaired output of late committed CD34(+) cells on MS-5 stroma.

Involvement of CSF-1 in generating a stroma-independent hematopoietic stem cell line.

The hematopoietic stem cell line, Myl-D7, is maintained by a self-renewing stem cell population that spontaneously generates myeloid, lymphoid, and erythroid progeny. MS-5 stromal cells are necessary for the growth of Myl-D7 cells. One component of the Myl-D7 cells proliferation activity released by MS-5 stromal cells was enriched by Q sepharose fractionation and shown to be colony stimulating factor-1 (CSF-1) by Western blotting, BAC1.2F5 cell bioassay and inhibition of Myl-D7 proliferation by CSF-1 antibody. The requirement of Myl-D7 cells for CSF-1 was also demonstrated independently by selecting for rare, stroma-independent Myl-D7 mutant clones able to grow without stroma and additional factors. Eighty-nine stroma-independent mutant clones were obtained and belonged to two classes. The majority of mutants did not secrete any growth promoting activity. The second, rarer class of mutants releases a factor that stimulates proliferation/survival for up to several months and approximately half of the secretors express high levels of CSF-1 mRNA. Wild type Myl-D7 grown with supernatants from the secretor cells retained the stem cell phenotype. These data suggest that CSF-1 may act as a key factor in stroma-regulated hematopoiesis and cell-cell interaction.

Diverse isoforms of colony-stimulating factor-1 have different effects on the development of stroma-dependent hematopoietic cells.

Maintenance and differentiation of hematopoietic stem and progenitor cells are controlled by complex interactions with the stroma microenvironment. Stroma-cell interactions can be supported by locally expressed membrane-spanning cell-surface (cs) growth factors. CSF-1 is expressed by stroma as a soluble glycoprotein, as proteoglycan, or as a membrane-spanning cs glycoprotein. CSF-1 regulates the survival, proliferation, and differentiation of mononuclear phagocytes. Whereas the biological role of soluble CSF-1 is well characterized, the function of the membrane-spanning cell-surface CSF-1 (csCSF-1) remains unclear. To analyze the biological significance of csCSF-1 in vitro, we used an epithelial cell line to ectopically express the different CSF-1 isoforms. In co-cultures of CSF-1 transduced epithelial cells with primary, early hematopoietic progenitor cells we examined whether interaction between csCSF-1 and its receptor mediates cell proliferation, self-renewal, or differentiation. csCSF-1 induces long-lasting proliferation of stimulated cells and furthermore supports self-renewal. Ectopic secretion of soluble CSF-1 does not permit long-term growth of progenitor cells but induces differentiation of monocytes into macrophages. Previously, we showed that the soluble and cs isoforms of stroma-encoded SCF differently affect the development of hematopoietic cells. Cell-surface SCF (csSCF) promotes self-renewal of stimulated cells whereas soluble SCF causes clonal extinction. These results and those presented here for CSF-1 provide evidence for diverse functions of the isoforms of the ligands SCF and CSF-1 for two tyrosine kinase receptors of the subclass III both regulating hematopoiesis on stroma.

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation.

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P <.03) and in vivo (P =.01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P <.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

AML1-ETO inhibits maturation of multiple lymphohematopoietic lineages and induces myeloblast transformation in synergy with ICSBP deficiency.

The translocation (8;21), generating the AML1-ETO fusion protein, is one of the most frequent chromosomal abnormalities associated with acute myelogenous leukemia (AML). To elucidate its role in oncogenesis, bone marrow (BM) cells were infected with a retroviral vector carrying AML1-ETO and transplanted into mice. In contrast to previous transgenic mouse models, we show that AML1-ETO directly stimulates granulopoiesis, suppresses erythropoiesis, and impairs the maturation of myeloid, B, and T lymphoid cells in vivo. To determine the significance of earlier findings that expression of the tumor suppressor ICSBP is often downregulated in AML myeloblasts, AML1-ETO was introduced into BM cells derived from mice lacking the interferon regulatory factor ICSBP. Our findings demonstrate that AML1-ETO synergizes with an ICSBP deficiency to induce myeloblastic transformation in the BM, reminiscent of AML.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.