The Impact of CD44 and Osteopontin on Hematopoiesis

Abstract

Hematopoiesis is the process, which enables the replenishment of blood cellular components in mammals throughout their lifespan. Hematopoietic stem cells (HSC), which reside within the bone marrow (BM), are the primary progenitor unit for the production of myeloid and lymphoid blood cell populations within the living organism. Balance disturbances between self-renewal and differentiation in HSCs, which may occur in case of mutations, can turn healthy hematopoiesis into hematological diseases. Mutations in a huge variety of genes in HSCs have been analyzed by scientists over decades, in order to find treatment angles for severe blood disorders. Within this PhD thesis, the impact of two genes (CD44 and osteopontin) on hematopoiesis has been investigated. CD44 encoding a cell-surface glycoprotein involved in cell-cell interactions, is expressed by HSCs while osteopontin (OPN) is abundantly present in the HSC niche as an extracellular matrix protein. Within the pre-experimental work of this thesis, CD44 and OPN double deficient mice (CD44^-/-:OPN^-/-) have been successfully bred by mating of CD44^-/- and OPN^-/- single deficient mice. The resulting offspring being CD44^-/-:OPN^-/-, CD44^+/-:OPN^+/- and OPN^-/- have been examined in their peripheral blood and BM composition in comparison to wild-type mice (CD44^+/+:OPN^+/+), which possess fully functional CD44 and OPN genes, under steady-state and stress-state experimental conditions. Steady-state conditions define analyses of blood and BM composition of mice under unaffected living conditions during their whole lifespan (3-21 months), whereas stress-state conditions are represented by the impact of a serial stem cell transplantation, G-CSF and Poly(I:C) stress. Blood and BM status of mice have been investigated with the BC-5000-Vet blood analyzer (Mindray) and by Fluorescent-activated cell-sorting (FACS; BD FACS Canto II). Steady-state results reveal elevated thrombocyte and leukocyte cell counts in the peripheral blood of CD44^-/-:OPN^-/- and OPN^-/- mice during their lifespan, especially in the older cohorts, while the erythrocyte blood values are only elevated in young OPN^-/- and old CD44^-/-:OPN^-/- mice. <br> CD44^-/-:OPN^-/- mice display higher B^- and T cell levels in their peripheral blood than wild-types in the majority of the observed age cohorts. Furthermore, old CD44^-/-:OPN^-/- mice exhibit reduced granulocyte- and Lin^- cKit⁺ Sca1⁺ (LKS) numbers within the BM, whereas the size of the HSC pool in young and old CD44^-/-:OPN^-/- mice is unaltered referring to controls. OPN-deficiency enhances HSC proliferation in BM of young mice and shows a tendency of enhanced MPP proliferation in BM of old mice. In a serial stem cell transplantation young CD44^-/-:OPN^-/--BM-recipient cells fail to adequately reconstitute thrombocyte, leukocyte and erythroid replenishment in the peripheral blood resulting in hematopoietic failure at 16 weeks after 3^rd- round of transplantation shown by the premature death of 3 out of 10 mice while all 10 out of 10 wild-type-BM-recipient mice (controls) have survived. G-CSF treatment lowers blood T cell levels in wild-type, CD44^-/-:OPN^-/- and OPN^-/- mice, reduces blood monocyte levels in OPN^-/- mice and elevates blood granulocyte levels in wild-type and CD44^-/-:OPN^-/- mice at 6 days after treatment. Thrombocyte levels in the peripheral blood of wild-type and OPN^-/- mice have been reduced under G-CSF stress, while those of CD44^-/-:OPN^-/- mice have not been affected. Therefore, CD44^-/-:OPN^-/- mice might have an impaired thrombocyte function. Furthermore, G-CSF treatment does not alter LKS, hematopoietic progenitor cell (HPC), HSC and MPP numbers in BM of CD44^-/-:OPN^-/- mice. Leukocytes in the peripheral blood of CD44^-/-:OPN^-/- mice react with different intensity to the viral-infection simulation of Poly(I:C) compared to wild-type leukocytes, implicating an impaired immune response capability of CD44^-/-:OPN^-/- mice. Poly(I:C) increases HSC proliferation in BM of OPN^-/- mice and MPP proliferation in BM of CD44^-/-:OPN^-/- mice in comparison to untreated controls, underlining an impaired immune response of the referring mice. Summarizingly, the double-deficiency of CD44 and OPN in HSCs has a profound impact on blood and BM cell populations of the referring mice under steady- and stress-state conditions.