HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells

Abstract

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, "safe" clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo, when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo, based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

Fig. 1.

Experimental design for comparison of embryonic and adult stem cell-derived hematopoiesis and retroviral vectors used for HOXB4 expression. (A) Transduction, culture, and transplantation procedure. ES cells (CCE) and prestimulated primary mouse bone marrow (C57BL/6J, CD45.2) were treated with ecotropic retroviral HOXB4 coexpression (and eGFP control) vector particles (as described in Methods). After differentiation to EBs for 6 days, EBs were dissociated and cultured in medium supportive for hematopoiesis. HOXB4 transduced ES-HCs and BM-HCs were transplanted into six irradiated Rag2^(–/–)γC^(–/–) recipient mice and C57BL/6J mice (CD45.1, 10 mice per vector), respectively. (B) Retroviral expression vector architecture and expression levels of HOXB4. For HOXB4 expression, retroviral backbones of MSCV (30, 31) and SF91 (44) were used, the latter with or without an additional wPRE (45). The coexpression cassette eGFP2AHOXB4 leads to cotranslational separation of eGFP and HOXB4 [with an additional N-terminal hemagglutinin (HA) tag] mediated by the 2A-esterase of foot-and-mouth disease virus, resulting in a constant ratio of both proteins (13). The relative expression levels of HOXB4 as determined by Western blot are indicated (14). The HA tag is symbolized as filled black block.

Fig. 2.

FACS analysis of HOXB4 transduced ES-HCs maintained as bulk or clonal cultures, in vitro. SF-HOX vector (SF91-eGFP2AHOXB4+wPRE) transduced bulk ES-HCs grown in vitro for 18 or 75 days or one representative clone, grown for 48 days, were immunostained against the assigned surface antigens. GFP⁺ and live cells subjected to FACS analysis were gated electronically (Upper) and analyzed for the assigned surface antigen expression. The quantitation of quadrant analysis is shown within each FACS analysis diagram.

Fig. 3.

Influence of HOXB4 expression levels on overall repopulation and lineage distribution in vivo. (A) HOXB4 expression levels affect in vivo repopulation of transduced bone marrow cells. C57BL/6J CD45.2 bone marrow cells were transduced with retroviral expression vectors, which mediate a 28-fold difference in HOXB4 expression levels (14). Thirteen weeks after transplantation, bone marrow and peripheral blood was analyzed for repopulation by GFP⁺ cells. Engraftment by CD45.2 cells was comparable (≈80%). Repopulation by GFP⁺ cells was significantly reduced in bone marrow and the periphery of the SF-HOX+Pre (HOX^high) group (SF91-eGFP vs. SF-HOX+Pre, BM and PB: P < 10^–4). HOXB4 expression levels relative to the reference vector described in ref. are shown in parentheses next to the construct designations. (B) Influence of HOXB4 expression levels on the lineage distribution in bone marrow in vivo. Each column represents the relative contribution of cells transduced with different retroviral vectors to a certain lineage in the competitive repopulation assay (see Methods). For statistical calculations, the proportion of GFP⁺ cells within each lineage was first determined for each mouse group (eGFP^lo, eGFP^hi, HOX^lo, and HOX^hi). These values were summarized for each mouse, set to 100%, and the relative contribution of each of the lineages recalculated. The relative contributions of transduced cells were compared pair-wise by the two-sided Student's t test. The significance level was defined as 0.05. Error bars represent standard deviations. Statistics for CD3⁺ lymphocytes and Ter119⁺ erythroid cells: CD3⁺: eGFP^lo vs. HOX^lo, P = 0.003; eGFP^hi vs. HOX^hi, P = 0.021; Ter119⁺: eGFP^lo vs. HOX^lo, P = 0.920; eGFP^hi vs. HOX^hi, P = 0.0122; HOX^lo vs. HOX^hi: P = 0.005.

Fig. 4.

FACS analysis of bone marrow cells from C57BL/6J and Rag2^(–/–)γC^(–/–) posttransplantation of HOXB4 expressing BM-HCs or ES-HCs, respectively. In the case of C57BL/6J mice, SF-HOX (SF91-eGFP2AHOXB4+wPRE) or SF-eGFP (SF91-eGFP+wPRE, control) transduced bone marrow cells were transplanted into 10 primary recipient mice each, together with SF-dsRed2 (SF91-dsRed2+wPRE) transduced bone marrow cells. After 54 weeks of repopulation, 10³, 10⁴, and 10⁵ cells of each control and HOX mice were serially transplanted each into four secondary recipient animals, which were finally analyzed after 7 months. Rag2^(–/–)γC^(–/–) mice were transplanted with 2.5 × 10⁶ SF-HOX transduced ES-HCs grown for 20 days in vitro. As a control, the same amount of freshly prepared Rag2^(–/–)γC^(–/–) bone marrow cells was transplanted. Analysis was performed 6 months posttransplantation. FACS analysis of bone marrow cells from representative animals from both experimental approaches is shown. x axis, eGFP (= HOXB4) expression height; y axis, surface marker. Quadrant analysis, given as percentages, is shown as Insets within the FACS diagrams. Arrows mark cell populations expressing intermediate amounts of HOXB4.