The α2β1 Integrin Regulates Hematopoietic Stem Cell Engraftment.
Authors: Sheehan Andrea M. , Ford Laura E. , Link Daniel C. , Zutter Mary M. 
Published: April 2020

Abstract
The α2β1 integrin, a receptor for collagen, laminin, and other matrix and non-matrix ligands, is expressed on some subsets of hematopoietic stem cells (HSC). Previous studies of mobilized HSC suggested that α2β1 integrin expression may distinguish long-term (LT-HSC) from short-term (ST-HSC) HSC. We sought to address the role of the integrin in normal hematopoiesis and following bone marrow transplant. Evaluation of mice in which the α2β1 integrin was genetically deleted demonstrated that peripheral blood counts, bone marrow cellularity, and bone marrow morphology were identical in α2-null mice and their wild type littermate controls, suggesting that α2β1 integrin is not required for hematopoiesis. To further explore the role of the α2β1 integrin in HSC homing and engraftment, we undertook a competitive repopulation assay using a 1:1 mix of α2β1−/− (Ly5.2) and α2β1+/+ (Ly5.1) cells transplanted into lethally irradiated Ly5.1/Ly5.2 mice. Analysis of peripheral blood, bone marrow, spleen, and lymph nodes post-transplant by FACS for the detection of lineage markers (CD3, B220, CD11b, F4/80, Gr-1, NK1.1) and for expression of either CD45.1 (Ly5.1) or CD45.2 (Ly5.2), demonstrated significantly greater reconstitution with α2β1−/− marrow than α2β1+/+ wild type marrow for lymphoid, myeloid, and monocytic lineages. The ratios ranged from 2:1(CD3, p=0.0024) − 5.6:1 (F4/80, p=0.0021). Secondary transplants of bone marrow harvested from primarily reconstituted mice showed similar findings with α2β1−/− (Ly5.2) to α2β1+/+ (Ly5.1) ratios ranging from 3.2:1 (CD3, p=0.0001) to 6.5:1 (F4/80, p&lt;0.0001). The ratio was also altered within the HSC population (Lin−ThyloSca+c-kit+ (LTSK)) after transplant with an α2β1−/− (Ly5.2) to α2β1+/+ (Ly5.1) ratio of 4.63:1 (p=0.0136). To evaluate the molecular mechanisms that resulted in dramatic engraftment advantage, quantitative analysis of resting HSC in α2-null and wild-type bone marrow and spleen by FACS was performed. However, no quantitative difference in the LTSK population, and no difference in LT-HSC (LSK CD34−Flk-2−), ST-HSC (LSK CD34+Flk-2−), or multipotent progenitors (LSK CD34+ Flk-2+) was identified. FACS analysis of α4β1 expression showed no increase in α4β1 expression in a2-null HSC compared to wild-type, indicating that α2-null HSC do not compensate with upregulation of α4β1 expression. Homing assays using CFSE labeled α2-null or wild-type bone marrow transplanted into lethally irradiated α2-null or wild type recipients showed no difference in homing, regardless of the donor or recipient genotype. Although HSC numbers were the same, methylcellulose cultures for hematopoietic progenitors showed significantly greater numbers of CFU-C for α2-null bone marrow compared to wild-type (p=0.0067), suggesting either a proliferative advantage or heightened sensitivity to growth factors present in the media. Immunohistochemical analysis of bone marrow from α2-null and wild-type mice for Ki-67 showed significantly greater proliferation in the wild-type compared to α2-null bone marrow. These studies suggest that although α2β1 integrin is not required for normal hematopoiesis, the integrin may play important roles in regulation of HSC proliferation as well as maintenance of the HSC niche.