Lentiviral-Mediated HoxB4 Expression in Human Embryonic Stem Cells Initiates Early Hematopoiesis in a Dose-Dependent Manner but Does Not Promote Myeloid Differentiation

Abstract

The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epitheliumcadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developing embryoid bodies. Notably, a high HoxB4 expression in hESCs also seemed to interfere with the formation of germ layers after xenografting into immunodeficient mice. These data suggest that HoxB4-induced effects on hESC-derived HSCs are concentrationdependent during in vitro development and reduce proliferation of other cell types in vitro and in vivo. The application of the transcription factor HoxB4 during early hematopoiesis from hESCs might provide new means for regenerative medicine, allowing efficient differentiation and engraftment of genetically modified hESC clones. Our study highlights the importance of HoxB4 dosage and points to the need for experimental systems allowing controlled gene expression.

Role of DNMT3B in the regulation of early neural and neural crest specifiers

“…Dysregulated expression of these genes has been shown to affect specification of precursors into certain lineages, especially during hematopoiesis. As previously suggested for the regulation of HOXB4 during hematopoiesis, 57,58 precocious differentiation into terminal cell types could prematurely deplete the progenitor population and result in the perturbations of gene expression, the alteration of overall timing, and development of NC lineages that we observe here.…”

A critical role for phosphatase haplodeficiency in the selective suppression of deletion 5q MDS by lenalidomide

“…To investigate the relationship between Cdc25C and the PP2Ac␣ gene expression level and apoptotic response to lenalidomide, we used a recombinant lentiviral-based gene delivery system carrying short hairpin RNAs (shRNA) to reduce both Cdc25C and PP2Ac␣ gene expression. Recombinant lentiviruses have proven to be an effective tool to deliver genes into human cells (6,27). Lentiviral infection in U937 cells significantly reduced corresponding gene transcript levels after 48 h as depicted in Fig.…”

Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells

“…Cumulatively, the data suggest that the level of HoxB4 expression significantly affects the degree of hematopoietic specification, with moderate levels of HoxB4 expression optimal for hematopoietic induction. [35][36][37][38] Significantly, hematopoietic repopulating cells derived from murine ESC via HoxB4 overexpression can induce tolerance in recipient mice, allowing for the acceptance of cardiac allografts. 16 This system also allows for the derivation of repopulating cells from induced pluripotent cells, revealing that HoxB4 overexpression can effect the derivation of ESC-HSCs from multiple pluripotent cell types.…”