βig-h3, a Novel Regulator of Adhesion and Differentiation of Human Hematopoietic Stem/Progenitor Cells.

Abstract 3640

Poster Board III-576

Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population.

Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation.

First we analyzed the mRNA expression in human CD34⁺ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34⁺ cells as compared to MPB-CD34⁺ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34⁺ population, the expression was compared between sorted sub-populations of BM-CD34⁺ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38⁺/CD110⁺/CD45RA⁻), common myeloid progenitors (CMP: CD38⁺/CD110⁻/CD45RA⁻), granulocyte-monocyte-progenitors (GMP: CD38⁺/CD110⁻/CD45RA⁺) and more immature CD34⁺/CD38⁻ HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34⁺ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34⁺ cells derived from all sources.

Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34⁺ cells were performed. These experiments show a significant (p<0.01) two-fold increased adhesion of MPB-CD34⁺ cells to βig-h3 compared to a BSA coating (mean 40% (SEM ± 9.8%) and 23% (SEM ± 5.0%), respectively, (n=3)). Further experiments showed that adhesion of CD34⁺ cells to βig-h3 is mediated by both β1- and β2- integrins.

The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34⁺ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34⁺ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41⁺) two weeks after transduction.

In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34⁺ subsets. Moreover, ectopic expression of βig-h3 in CD34⁺ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions.