Recombinant HoxB4 Fusion Proteins Enhance Hematopoietic Differentiation of Human Embryonic Stem Cells

Abstract

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.

Novel roles for Notch, Wnt and Hedgehog in hematopoesis derived from human pluripotent stem cells

“…NOD/ SCID mice (Dick et al, 1997;Mazurier et al, 2003) and equally contribute to functional reconstitution in human transplant settings (Grewal et al, 2003). While ectopic transcription factors of the Cdx/Hox pathway can modulate the functional behavior of adult HSCs (reviewed in Klump et al, 2005) and confer HSC properties to mouse ESCs (Kyba et al, 2002;Wang et al, 2005a), this is not the case with hESCs where the generation of "putative" HSCs, using various methodologies, animal recipients and injection routes has yielded significant lower levels of reconstitution, as shown by our laboratory (Wang et al, 2005b;Ji et al, 2008) and later others (Lu et al, 2007a;Lee et al, 2008;Narayan et al, 2006;Tian et al, 2006;Ledran et al, 2008).…”

Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4

“…Recombinant protein was purified as previously described, 18 and was desalted using PD-10 columns (GE Healthcare Life Sciences, Pittsburgh, PA For personal use only. on June 7, 2019. by guest www.bloodjournal.org From 2 mM L-glutamine, 1% penicillin/streptomycin, 0.125 mM hemin (Sigma), 20 ng/mL G-CSF (Amgen), c-Kit ligand and IL-3 (Amgen), and 6 U/mL erythropoietin (Amgen) and 1 mg/mL recombinant proteins.…”

“…16,17 Comparable levels of ex vivo murine HSC proliferation were achieved with the protein transduction of recombinant human HOXB4 as well as with retroviral integration, 17 but the short half-life of HOXB4 necessitated frequent replenishing of the recombinant protein, which is impractical for large-scale ex vivo expansion of HSCs. Studies using human embryonic stem cells 18 or bone marrow cells 19,20 also demonstrate the effects of recombinant HOXB4 protein for therapeutic use. Stabilization of the HOXB4 protein through post-translational manipulation would represent a major advance in providing a renewable source of HSCs for human therapeutic use.…”

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

“…Promising results from the mouse model, which demonstrated an improved engraftment in NOD/SCID mice, promoted the use of HoxB4 in human HSCs [10]. In vitro expansion of somatic and embryonic HSCs following exposure to modified HoxB4 protein is possible but does not provide in vivo engraftment advantages [11,12].…”