A Systems Biology Approach to Study the Acquisition of Adult Repopulating Potential During Hematopoietic Stem Cell Ontogeny.

Abstract 1479

Poster Board I-502

Hematopoietic progenitors and stem cells emerge at distinct times and anatomical locations during ontogeny. The first definitive hematopoietic progenitors appear in the yolk sac (YS) around day 8.5 of development (E8.5) while the first cells to meet the functional criteria of hematopoietic stem cell (HSC; cells capable of life-long restoration of the hematopoietic compartment of a recipient whose own hematopoietic system has been destroyed by chemicals or irradiation) can be detected in the aorta-gonads-mesonephros (AGM) at E10 of development. HSC can later be detected in large numbers in the E12.5 placenta, E14.5 fetal liver (FL) and adult bone marrow (BM). Induction of ectopic Cdx4 and HoxB4 expression during murine embryonic stem cell (ESC) differentiation followed by OP9 co-culture yields cells with in vivo hematopoietic repopulating potential (ESC-HSC). We have recently defined the phenotype of ESC-HSC relative to that of HSCs that arise in the embryo during development. Using cell fractionation followed by transplantation into irradiated Rag-2-/-γc-/- mice, we found that ESC-HSC display cell surface markers representative of both embryonic and adult HSC compartments: they express high levels of CD41, are heterogeneous for CD45, express CD150, and lack CD34 expression. We also found that CD150 is a developmentally regulated molecule on the surface of HSC, absent on the earliest HSC compartments of the AGM and placenta, but present on FL and bone marrow HSC. Although hematopoietic cells of the early YS do not display HSC potential when transplanted into the periphery of adult recipients, these cells can acquire this potential if infected with the homeobox gene HoxB4 and cultured on OP9 stroma, similarly to ESC. These data suggest that activation of specific molecular pathways can induce adult repopulating potential. To identify the critical genetic networks regulating the acquisition of adult hematopoietic repopulating potential, we are exploiting prospective purification via flow cytometry and microarray technology to assess the global gene expression profiles of HSC and progenitors from throughout murine ontogeny and embryonic stem cell differentiation. By comparing populations that lack adult repopulating potential (c-kit+CD41+CD34+ E9 YS cells, c-kit+CD41+CD45- ESC-derived cells and VE-cadherin+CD41- ESC-derived cells) with those that possess this activity (VE-cadherin+CD45+ E11.5 AGM, c-kit+CD34medCD45+ E12.5 placenta, Lin-c-kit+Sca-1+CD150+CD48- E14.5 FL, Lin-c-kit+Sca-1+CD150+CD34- BM, and CD41highCD45-CD34- cells from HoxB4 infected ESC expanded on OP9 stroma) we can use a novel gene network inference algorithm dubbed “mode-of-action by network identification” (MNI) to elucidate the key mediators for this functionality. The MNI algorithm predicts the key mediators under a particular perturbation by using a microarray compendium to infer a gene regulatory network that assesses how gene transcripts change in relation to one another and the net external influence. A large compendium with diverse experimental scenarios helps MNI to reverse engineer a comprehensive network model. We are currently using around 11,000 murine gene expression data sets acquired using 430 2.0 Affymetrix array chips to infer a regulatory network model, through which our HSC and progenitors microarray data will be filtered to identify the mode of action of the specific profiles accordingly. By comparing repopulating and non-repopulating populations from mouse ontogeny and ESC, we hope to identify the critical mediators and gene networks regulating the acquisition of adult repopulating potential during development.