Abstract 4234: Translational regulation mediates hematopoietic progenitor cell generation in embryonic stem cell culture

Abstract

The development and maintenance of hematopoietic progenitors (HP) requires modulation of a network of genes that balance self-renewal, expansion and lineage commitment. Regulation by miRNAs is critical for the differentiation of progenitors along defined pathways, and requires the recruitment and coordinate interaction of RNA binding proteins that modulate translation and/or target specific transcripts for sequestration or degradation. Here we provide evidence that translational regulation by the RNA binding proteins HuR and Pum2 are critical to HP generation from murine embryonic stem (ES) cell culture and that translational regulation during HP generation enhance ex vivo proliferative capacity and transplantability of progenitors. Translationally regulated transcript subsets in ES culture were identified by sucrose gradient polysome analysis. Ribonucleases and RNA-binding proteins comprised 12% of transcripts downregulated during ES cell differentiation to hematopoietic lineage (p=2.34 × 10-27 and 7.3 × 10-7 respectively). RNA-immunoprecipitation-sequencing (RIP-seq) using antibodies to several of the identified RNA binding proteins, followed by sequencing (Roche GS-FLX) revealed transcripts regulated by each. Pathway analysis of the transcripts associated with Pum2 in differentiated cells identified p53, p53-interacting proteins, and translation regulation Go (p=2.48×10-11). There was significant overlap with transcripts associated with HuR from undifferentiated ES (p=8.1×10-11), suggesting that the transcripts regulated by HuR in undifferentiated cells become associated with Pum2 with upon differentiation. Confocal imaging of HuR and Pum2 was consistent with this finding. Pathway analysis of DnD-associated transcripts revealed a network centered about Rac signaling and the Abl, Src and MAPK pathway via MAPKKK1–an essential pathway for stem cell self-renewal and maintenance. Consistent with the central role of translational regulation in the generation of HP, inhibition of translation using pharmacologic approaches (rapamycin, wortmannin, or clotrimazole) or siRNA (HuR, Pum2) resulted in up to three log increases in early HP derived from ES cell culture as measured by long term culture initiating cell (LTCIC) assay and the acquisition of competitive transplantability in irradiated hosts as measured by percentage chimeric animals and survival (p<0.05). Our findings reveal a system of broad-based translational regulation of HP differentiation mediated by RNA binding proteins. The findings suggest a methodology for ex vivo expansion of marrow-derived HP and enhanced generation of ES-derived HP by coordinate regulation of specific RNA binding proteins and their associated transcripts to regulate cell differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4234.