Dissecting the genetic signatures of a multipotent hematopoietic progenitor’s subpopulations

Abstract

In hematopoiesis and other developmental systems, there is an active debate regarding the heterogeneity of apparently phenotypically homogeneous progenitors having different lineage potentials. The host laboratory has previously characterized a B220+ c-Kitint CD19− and NK1.1− uncommitted and multipotent hematopoietic progenitor with combined lymphoid and myeloid differentiation capacity that was called Early Progenitor with Lymphoid and Myeloid potential (EPLM). Under physiological conditions, EPLM was mainly described as a B-cell progenitor. More recently, with flow cytometry analysis, EPLM has been fractionated into at least four subpopulations based on the expression of Ly6D, SiglecH and CD11c cell surface markers, thus revealing phenotypic heterogeneity. The question remains whether these subpopulations are still multipotent or, instead, biased towards distinct hematopoietic lineages. In this project, I have further studied the two EPLM subpopulations, namely Ly6D+ and triple negative (TN), that could possess B-cell developmental potential and/or be multipotent. The main goal was to elucidate if the phenotypic heterogeneity (differential expression of Ly6D) would reflect distinct and biologically meaningful molecular signatures that could indicate, for instance, different developmental potentials for the subpopulations. A second goal was to identify a potential EPLM fraction containing most of the B-cell differentiation capacity and being the precursor of the first B-cell committed stage, the pro-B cells. To address the previous goals, I performed population RNA sequencing and carried a detail analysis of the molecular signatures of the two EPLM subsets while comparing them with the transcriptome profile of the first B-cell committed progenitor, the pro-B. The results obtained in this project demonstrate that heterogeneous expression of Ly6D can be used to discriminate among EPLM subpopulations that have distinct genetic signatures. Whereas the Ly6D+ cells are lymphoid primed and have a strong B-cell genetic signature, the TN cells are myeloid primed. Therefore, EPLM is not only phenotypically but also genetically heterogeneous. I speculate that the lympho-myeloid developmental potential observed for the whole EPLM population could be constrained within the Ly6D+ and TN fractions, respectively. Moreover, the Ly6D+ cells, which have a closer transcriptome profile to pro-B than when the TN cells are compared with pro-B, could be the direct precursor of the first B-cell committed stage. Ultimately, this master project sets the basis for further functional experiments to resolve the developmental potentials of the EPLM subsets.