AR-42 HDAC br Results br Discussion The self renewal

Results
Discussion The self-renewal capacity of erythroid progenitors is an important component of current protocols to generate cultured RBCs for potential therapeutic purposes. However, a major obstacle in generating the 2.5 × 1012 RBCs that constitutes a single unit of blood is the limited capacity of erythroblasts derived from neonatal and adult sources to self-renew in vitro. Understanding the mechanisms promoting extensive erythroid self-renewal could lead to improved generation of cultured RBCs. Here, we have discovered that Bmi-1 is preferentially expressed in self-renewing compared to differentiating erythroblasts. Importantly, we determined that overexpression of Bmi-1 is sufficient to induce the extensive ex vivo self-renewal of adult erythroblasts that normally are only capable of limited self-renewal. These iESREs have a similar morphology, cell surface phenotype, and cytokine dependence compared to ESREs derived from embryonic sources. Bmi-1 regulates the self-renewal of several adult stem cell populations, including hematopoietic, neural, and cancer stem cells, often through repression of p16Ink4a/p19Arf (Jacobs et al., 1999; Park et al., 2003; Molofsky et al., 2003; Kreso et al., 2014). While we did not detect Cdkn2a in erythroblasts (Kingsley et al., 2013), we did identify several other potential BMI-1 target AR-42 HDAC that are differentially expressed in ESREs/iESREs, studies suggesting that BMI-1 may function independently of p16Ink4a/p19Arf repression in ESREs (Bruggeman et al., 2007; Abdouh et al., 2009). The function of Bmi-1 in ESRE self-renewal may be independent of its role in erythroid maturation, where it was recently shown to positively regulate ribosomal protein genes (Gao et al., 2015). Bmi-1 transduction of self-renewing erythroblasts did not interfere with terminal in vitro maturation of iESREs into reticulocytes. The generation of iESREs from adult Protein 4.1R null mice indicates that this experimental approach can be used to generate large numbers of mutant erythroblasts for the study of red-cell-intrinsic disorders, particularly diseases that affect terminal stages of erythroid maturation such as disorders of the membrane cytoskeleton or of globin gene expression. Intravenous transfusion of iESREs resulted in the emergence of a large wave of reticulocytes 4–8 days later. The timing and the transient nature of this reticulocyte emergence indicate that iESREs rapidly switched in vivo from a self-renewal to a maturation program. This transient wave of iESRE-derived reticulocytes generated a population of mature RBCs that constituted 10%–15% of the total circulating RBC mass from week 2 to week 6 following iESRE transfusion. Consistent with a near normal life span, iESRE-derived RBCs displayed normal shape and deformability when compared to co-circulating endogenous RBCs. Transfusion of 10 × 107 iESREs resulted in the stable circulation of approximately 1.6 × 109 RBCs, suggesting that each iESRE generated 16–32 RBCs, consistent with the differentiation potential of normal ProE/CFU-E. Taken together, these findings indicate that Bmi-1-induced ESREs are not immortalized but rather constitute committed erythroid precursors that rapidly mature, not only in vitro but also in vivo. These findings also distinguish iESREs from immortalized erythroid cell lines generated by genetic perturbation of multiple transcription regulators, including C-MYC, TP53, E6/E7, and SOX2, some of which must be exogenously extinguished before terminal maturation can proceed (Huang et al., 2014; Hirose et al., 2013; Kurita et al., 2013). Our data indicate that Bmi-1 promotes the extensive erythroid self-renewal of adult erythroblasts cultured ex vivo with EPO, SCF, and DEX. iESREs may ultimately provide an alternative source of cultured RBCs to meet the challenge of generating the extremely large numbers of RBCs needed for transfusion therapy. Interestingly, iESREs transfused in vivo result in large numbers of fully mature RBCs, obviating the need to generate and fully mature RBCs in vitro. Importantly, no GFP+ cells in peripheral blood or tumors were detected in recipients 6 months after transfusion (data not shown). However, host conditioning and safety concerns must be evaluated before iESREs can be considered as a potential transfusion product.