HOXB4 Enforces Equivalent Fates of ES-Cell-Derived and Adult Hematopoietic Cells.

In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent <i>in vitro</i> selection and clonal expansion are only available for embryonic stem (ES−) cells. Previously, it has been shown that <i>in vitro</i> differentiated ES-cells engraft when ectopically expressing HOXB4, but it remained unclear whether these cells could fully resemble adult bone marrow function after transplantation¹. We here demonstrate for the first time the functional equivalence of <i>in vitro</i> differentiated ES-cells and adult bone marrow cells mediated by HOXB4. Differentiated ES-cells expressing HOXB4 from a retroviral vector and grown <i>in vitro</i> for 20 days, recapitulated the growth and differentiation properties of adult bone marrow cells after transplantation into

Rag2^(−/−)γC^(−/−) and C57Bl/6J recipient mice. Furthermore, we show that the amount of ectopically expressed HOXB4 influences differentiation in both systems in a similar manner. HOXB4 enforced myeloid and suppressed T-lymphoid development over a wide range of expression levels, whereas only high expression levels of HOXB4 were detrimental for erythroid development (P-values for C57Bl/6J mice, Student’s t-test, 2-sided:

CD3⁺ : eGFP^low vs. HOX^low = 0.003; eGFP^high vs. HOX^high = 0.021;

Ter119⁺: eGFP^low vs. HOX^low = 0.920; eGFP^high vs. HOX^high = 0.0122; HOX^low vs. HOX^high = 0.005).

Incompatibility of high levels of HOXB4 expression with erythroid differentiation was also directly demonstrated using a recently described <i>in vitro</i> ES-cell differentiation system². Histological analysis of the “HOXB4-transplanted” mice revealed increased granulopoiesis both in sternal bone marrow and in spleen sections. However, all stages of granulocytic differentiation were present and neither were immature cells detected in the periphery nor was leukemic infiltration detected in other organs. Hence, none of the animals became leukemic during the observation period. In summary, ES-cells should be considered a promising alternative to bone marrow stem cells carrying the potential of safe somatic gene therapy, provided that human ES-cells can be similarly manipulated. Nonetheless, advanced cell therapy will certainly require regulated expression of HOXB4 to avoid unwanted effects such as disturbed lineage differentiation. This work was supported by the German Research Foundation (KL1311/2-3 and 2-4), German Cancer Aid (10-1763-OS5) and the Austrian Industrial Research Promotion Fund (808714).