Differential Expression of Novel Potential Regulators in Hematopoietic Stem Cells

Received June Differential Expression of Novel Potential Regulators in Hematopoietic Stem Cells E. Camilla Forsberg 0 Susan S. Prohaska 0 Sol Katzman 0 Garrett C. Heffner 0 Josh M. Stuart 0 Irving L. Weissman 0 Derry Roopenian, The Jackson Laboratory, United States of America 0 1 Departments of Pathology and Developmental Biology, Institute of Cancer and Stem Cell Biology and Medicine, Stanford University Medical School, Stanford, California, United States of America, 2 Biomolecular Engineering, University of California at Santa Cruz , Santa Cruz, California , United States of America 1 www.plosgenetics.org The hematopoietic system is an invaluable model both for understanding basic developmental biology and for developing clinically relevant cell therapies. Using highly purified cells and rigorous microarray analysis we have compared the expression pattern of three of the most primitive hematopoietic subpopulations in adult mouse bone marrow: long-term hematopoietic stem cells (HSC), short-term HSC, and multipotent progenitors. All three populations are capable of differentiating into a spectrum of mature blood cells, but differ in their self-renewal and proliferative capacity. We identified numerous novel potential regulators of HSC self-renewal and proliferation that were differentially expressed between these closely related cell populations. Many of the differentially expressed transcripts fit into pathways and protein complexes not previously identified in HSC, providing evidence for new HSC regulatory units. Extending these observations to the protein level, we demonstrate expression of several of the corresponding proteins, which provide novel surface markers for HSC. We discuss the implications of our findings for HSC biology. In particular, our data suggest that cell-cell and cell-matrix interactions are major regulators of long-term HSC, and that HSC themselves play important roles in regulating their immediate microenvironment. - Mature blood cells have a high turnover rate and need to be constantly replaced as well as respond to more acute conditions such as blood loss or infections, requiring the rapid generation of millions of new blood cells. This demand is fulfilled for life by a pool of hematopoietic stem cells (HSC). The long-term repopulating HSC (LT-HSC) thus has to be capable of differentiating without depleting the stem cell pool, thereby satisfying the definition of a stem cell: the ability at the single cell level to both self-renew and differentiate into more mature cell types. LT-HSC normally reside in the bone marrow and have essentially six developmental choices: remain quiescent, differentiate, self-renew, migrate, enter senescence, or undergo apoptosis. Such fate decisions are likely controlled both by HSC-intrinsic mechanisms and by the bone marrow microenvironment or niche. It has proved difficult to define the complex intrinsic and extrinsic mechanisms that govern the balance of these decisions. In hematopoiesis, only LT-HSC are capable of lifelong self-renewal and, therefore, is the operative population in hematopoietic transplantation. Understanding how HSC fate decisions are controlled is therefore of critical importance. The expression profiles of primitive hematopoietic cells defined by various criteria have previously been compared to other hematopoietic and non-hematopoietic cell types [15]. In addition, several molecules and pathways have been implicated in HSC self-renewal, including HoxB4, Bmi1, the Wnt/b-catenin signaling pathway, and Notch [69]. As the bone marrow microenvironment likely provides unique cues necessary for proper HSC function, cell surface proteins mediating these signals should play important roles in HSC fate decisions. While there have been recent advances in defining a potential HSC niche within the bone marrow [10,11], little is known about the specific signals regulating HSC in vivo. A central question is how the interplay of soluble ligands, matrix interactions, and cellcell contacts influence HSC fate. Recent evidence points to a role for the angiopoietin receptor Tek, also known as Tie2, in maintaining transplantable HSC [12]. Likewise, HSC in mice null for Mmp9, a matrix metalloproteinase (Mmp) that facilitates cell migration by proteolytic cleavage, have impaired proliferation and differentiation capabilities [13]. In addition, mice lacking integrin b1, part of the HSC homing receptor a4b1 [14], cannot establish fetal liver hematopoiesis [15], presumably due to a failure of LT-HSC to engraft at this fetal site. Thus, there is increasing evidence that interactions with the environment are important for the maintenance of HSC self-renewal capability. A thorough understanding of cell surface molecules expressed on HSC is an important step in identifying functional interactions with the environment. Here, we have carefully analyzed the transcription profiles of three highly purified subpopulations within the mouse adult bone marrow lineage /c-kit/Sca1 (KLS) fraction: LTHematopoietic, or blood-forming, stem cells (HSC) are responsible for the continual replenishment of all blood cells throughout life. This ability to both renew themselves and give rise to expanded populations of differentiating and mature cells is a hallmark of stem cells and is therefore an area of intense research. The rarity of HSC as well as their location in the bone marrow environment has made it difficult to identify the genes that regulate these properties. The earliest stages of blood development begins with the long-term (LT) repopulating HSC that then differentiate into short-term (ST) repopulating HSC and non-self renewing multipotent progenitors (MPP). The authors investigated the gene expression differences in these highly purified populations that differ mainly in their capacity to self renew, and identified a number of genes specific to each of these populations. Intriguingly, many of these genes code for proteins that are involved in cellcell and cellmatrix interactions that were not previously identified on these populations. These novel discoveries will, together with future experiments, enhance our understanding of the basic biology of stem cells and their clinical uses. HSC (defined as Thy1.1lo/Flk2 KLS), short-term (ST)-HSC (Thy1.1lo/Flk2 KLS), and multipotent progenitors (MPP) (Thy1.1 /Flk2 KLS) [16]. These three populations have the ability to give rise to both lymphoid and myeloid lineages [16] and platelets (E. C. F., E. Passegu e, and I. L. W., unpublished data) when transplanted into irradiated mice. Thus, LT-HSC, ST-HSC and MPP have similar multilineage potential, but differ in their self-renewal and proliferative capacity. All long-term repopulating activity is contained in the LT-HSC fraction; thus, cells within this fraction are the only cells capable of maintaining hematopoiesis for the life of the host. As LT-HSC differentiate to ST-HSC and then to MPP, selfrenewal capability progressively d (...truncated)