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Notch Activation Confers Enhanced Lymphoid Potential in Murine ESC/iPSC-derived HSC and Reconstitutes Adaptive Immunity In Vivo [RNA-Seq]

ABSTRACT: Hematopoietic stem cell (HSC) transplantation has the potential to cure blood disorders but is limited by donor availability. Hence innovative approaches to engineer HSC are critically needed. HoxB4 over-expression in mouse embryonic stem cell-derived HSC (ESC-HSC) confers long-term engraftment, yet lacks efficient lymphogenesis. Transcriptome comparison of ESC-HSC versus embryo-derived HSC showed that ESC-HSC are deficient in expression programs activated by Notch. Therefore, we aim to improve ESC-HSC by further providing Notch signals through Notch1 intra-cellular domain transgene activation or by ligand stimulation. Here, we report that Notch-enhanced ESC-HSC (nESC-HSC) confer clonal multipotentiality with robust lymphopoiesis that endows adaptive immunity. Notably, nESC-HSC further evolve to a hybrid cell-type co-expressing gene regulatory networks of hematopoietic stem/progenitor cells and differentiated lineages at single-cell level that accounts for multipotentiality. Our work reveals a proof-of-concept model of HSC engineering by assembling self-renewing factor and lineage-guiding pathway into one product-cell that functionally recapitulate HSC in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE71794 | GEO | 2016/12/21

SECONDARY ACCESSION(S): PRJNA292097

REPOSITORIES: GEO

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Project description:HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis using enriched adult HSCs expressing inducible HOXB4. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of adult HSCs was enhanced by specific and complete inhibition of FGF-receptors. Based on our results we propose that HOXB4 governs pivotal cell-intrinsic pathways involved in the regulation of cell cycle, differentiation and apoptosis. Our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance towards stem cell self-renewal. Experiment Overall Design: To understand the mechanisms of HOXB4 activity, we wished to identify target genes of HOXB4 in adult hematopoietic stem and progenitor cells (HSC/HPCs). We thus transduced murine HSC/HPCs with a retroviral vector that co-expresses EGFP and a tamoxifen-inducible form of HOXB4 (HOXB4-ER). Upon addition of 4-hydroxytamoxifen (TMX), the HOXB4-ER fusion protein translocates from the cytoplasm to the nucleus, consequently being capable of modulating gene expression. Transduced cell populations were expanded for 14 days in the presence of TMX. Thereafter, HOXB4-ER+LSK (GFP+ , lineage negative, Sca1+, ckit+) cells were flow cytometrically isolated and cultivated either with or without TMX for 1 or 4 hours. Inactivation of HOXB4 activity by TMX withdrawal was intended to mimic the naturally occurring down-regulation of HOXB4 in differentiating stem cells. RNA was prepared after the aforementioned times and the transcriptional profiles of HOXB4-ER+LSK +/- TMX analyzed using the Affymetrixâ¢ platform. As a control, profiling was also performed with LSK cells expressing unmodified constitutively active HOXB4 (HOXB4const) Â± TMX, to exclude changes in gene expression due to unknown effects of tamoxifen itself. RNAs from adult LSK cells were processed for use on Affymetrix GeneChips Mouse Genome 430 2.0. All quality parameters for the arrays were confirmed to be in the recommended range.

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Notch Activation Confers Enhanced Lymphoid Potential in Murine ESC/iPSC-derived HSC and Reconstitutes Adaptive Immunity In Vivo [RNA-Seq]

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