Homeobox-containing transcription factors, particularly HOXB4, and Notch signaling provide 2 important regulatory mechanisms that control proliferation and self-renewal of hematopoietic stem cells, and these pathways may be manipulated to develop protocols for expansion of stem cells.
Expansion of hematopoietic stem cells (HSCs) has remained an important goal to develop advanced cell therapies for blood disorders. During the last 2 decades, ever since the first hematopoietic growth factors were identified and isolated, there have been serious attempts to expand HSCs in vitro using purified growth factors that are known to regulate primitive hematopoietic cells. However, these attempts have met with limited success. For example, the hematopoietic growth factors fetal liver tyrosine kinase (Flt3) ligand, stem cell factor, and interleukins 6 and 11 promoted self-renewal of murine hematopoietic stem cells, although only a limited expansion of stem cells compared with fresh input cells could be obtained.1 More recently, regulatory pathways other than those activated by the traditional hematopoietic growth factors have been investigated. Developmental cues that are important during embryonic development have recently been found to have an impact on the fate of HSCs. Sonic hedgehog was found to induce the proliferation of primitive hematopoietic progenitors by activation of bone morphogenetic protein 4.2 Similarly, Wnt3a stimulates self-renewal of HSCs and the same effect is obtained by overexpression of activated β-catenin.3
Interestingly, the activation of Wnt signaling in HSCs induced increased expression of HoxB4 and Notch1. Activation of Notch1 has been shown to induce self-renewal of HSCs by several groups. Using retroviral vectors to overexpress Notch1, Stier et al4 showed that Notch 1 increases self-renewal of HSCs and favors lymphoid over myeloid lineage outcome. In this issue of Blood, Vercauteren and Sutherland test the role of constitutively active Notch4 in regulating the fate of hematopoietic cells. They find that similar to overexpression of Notch1, self-renewal of primitive hematopoietic progenitors is increased and lineage decisions of hematopoietic progenitors are perturbed. Notch4 increased the repopulation capacity of stem cells but generated a block in B-cell development and accumulation of an immature CD4+/CD8+ T-cell population, causing lymphoid abnormalities in vivo.
When it comes to self-renewal of HSCs and increased regeneration of stem cells following bone marrow transplantation, overexpression of HOXB4 is the most effective method reported to date. Recently, Antonchuk et al5 showed that retroviral overexpression of HOXB4 for 10 to 14 days in vitro could increase the number of repopulating HSCs by 40-fold compared with fresh bone marrow stem cells. Further, it has been shown that HOXB4 protein can stimulate self-renewal of HSCs in culture, eliminating the mutagenesis risk of retroviral integration.6 In this issue of Blood, Beslu and colleagues demonstrate that stimulation of stem cell renewal by HOXB4 overexpression requires intact DNA-binding capacity of HOXB4, whereas binding to pre–B-cell leukemia transcription factor cofactors is not required for this effect. The findings show that the genetic program that needs induction for the HSC proliferative effect involves genes that are direct targets of HOXB4. This mechanistic insight will help identify the genetic targets that induce HSC proliferation upon enforced expression of HOXB4. HOXB4 protein treatment may be a promising avenue for development of stem cell expansion protocols that are applicable in the clinic. However, overexpression of HOXB4 at high levels can, in addition to increased stem cell proliferation, lead to abnormalities in myeloid and lymphoid differentiation.7 The papers by Beslu et al and Vercauteren and Sutherland have contributed toward increased understanding of the mechanisms that regulate stem cell proliferation, but the regulation of stem cell renewal is complex and further work is needed to understand the mechanisms that control stem cell proliferation to allow development of stem cell expansion protocols for the clinic. ▪
