Tackling fate-bias in hematopoietic stem cells: a focused study on Neogenin-1 and a broader study on the relationship between their transcriptome and proteome

Hematopoietic stem cells (HSCs) supply the blood and immune cells of the body throughout life. A dysfunctional hematopoietic system can lead to a weakened immune system and predisposition to certain hematopoietic malignancies. Gaining a better understanding of HSCs can allow us to combat the weaknesses and disease in the blood system of the human body. Here we study the hematopoietic system of mice to identify Neogenin-1 (NEO1) as a transmembrane protein whose expression is highly specific to true long-term HSCs distinguishable based on the expression level of Hoxb5. In young mice, NEO1+ LT-HSCs demonstrate myeloid-bias, and the same population of LT-HSCs expands with age. In the process of demonstrating that NEO1+ LT-HSCs continued to demonstrate phenotypes of myeloid-bias even in older mice, we also demonstrate that Hoxb5, although previously reported to be a good marker for distinguishing LT-HSCs from ST-HSCs in young mice, is no longer a good marker for distinguishing LT-HSCs from ST-HSCs in old. A mouse model (Hoxb5CreERT2/Neo1fl/fl/Rosa26mTmG) was additionally generated to genetically manipulate Neo1 in the context of HSCs and has been marginally validated thus far. A more detailed, molecular study on Neo1 transcripts in bulk LT-HSCs revealed that both NEO1+ LT-HSCs and NEO1- LT-HSCs contained transcripts of Neo1. The former predominantly had a computationally predicted splice variant, and the latter largely dominated by a completely novel splice variant retaining the intron between exons 16 and 17. This intron retention introduces a premature termination codon that likely leads to the downregulation of surface NEO1 in NEO1- LT-HSCs. This evidence of complex post-transcriptional regulation of gene expression in HSCs motivated a project broadly comparing the proteome and transcriptome of various hematopoietic stem and progenitor cells. This comparison revealed that hematopoietic stem cells from young mice have an unusually low proteomic diversity as well as greater discordance between protein and transcript expression in comparison to other progenitor cell types. Interestingly, the low proteomic diversity is no longer observed in HSCs of old mice. These comparative analyses provide further corroboration of the complex post-transcriptional regulation that may be present in young mouse hematopoietic stem cells (possibly through the control of ribosomes and miRNAs) which either seems to change with age or is hidden by an increase in heterogeneity of what is immunophenotypically defined as “HSCs” that accompanies aging in mice. Put together, this body of work paves the way for new, meaningful research investigating 1) the role of post-transcriptional regulation in hematopoietic stem cell maintenance and cell fate; 2) the interactions between the surface proteins present on HSCs and proteins present on cells comprising the HSC niche; and 3) how those two relate to and can be used to counteract any immunodeficiencies or hematological disorders.

Keywords:  Department of Biology
Biology
Neogenin-1
Hematopoietic Stem Cells
Splice Variants
Proteomics
Transcriptomics
Text
Thesis
Advisor: Weissman Irving L
Jones Patricia P