βig-h3 Regulates Differentiation and Survival of Human Hematopoietic Stem/Progenitor Cells.

Abstract 2591

Adult hematopoietic stem cells (HSC) reside in dedicated niches in the bone marrow (BM). Within this specialized microenvironment, the various interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are critical for the maintenance of the HSC population and the concomitant development of the distinct blood cell lineages. Comparative gene-expression profiling of purified HSC identified ECM proteins that are differentially expressed in homeostatic and regenerative conditions. The ECM protein βig-h3 was one of the proteins upregulated in regenerative conditions. Therefore, we characterized the role of βig-h3 in the regulation of HSC self-renewal and differentiation.

A comparison between human CD34⁺ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB), revealed the highest βig-h3 expression in BM-HSPC (3.9-fold increased compared to MPB, 1.7-fold increased compared to CB), which may implie a role for βig-h3 in retaining HSC in the BM.

To examine the functional relevance of βig-h3 in HSC, we first increased βig-h3 expression by transducing human HSPC with a lentiviral βig-h3-SIN-GFP expression vector or a control SIN-GFP vector. Over-expression of βig-h3 (80-fold) in HSPC decreased colony-forming-unit-granulocyte-monocyte (CFU-GM) formation from 130 (SEM=47) to 73 (SEM=19, n=3) CFU-GM per 500 plated CD34⁺ cells, while megakaryopoiesis was accelerated and the number of mature megakaryocytic cells increased from 16% (SEM=6%) to 30% (SEM=7%, n=4) at day 14 of culture. Ectopic expression of βig-h3 did not affect differentiation along the erythroid or granulopoietic lineage. The development of megakaryocytes at the cost of pluripotent CFU-GM suggests that βig-h3 drives differentiation.

In addition, βig-h3 expression in HSPC was reduced by two different short-hairpin-RNAs (shRNA) expressed from lentiviral vectors, which resulted in decreased proliferation (from 19.6- to 5.8-fold per input cell at day 13) and increased apoptosis (from 13.5% to 25.3% at day 13) in liquid HSPC cultures, as analyzed by Annexin V staining. Similarly, knock-down of βig-h3 in various cell lines also resulted in a decreased proliferation and increased apoptosis. Knock-down of βig-h3 in primary HSPC dramatically reduced CFU-GM from 73 (SEM=8.7) to 31 (SEM=14.4, n=6) CFU-GM per 500 CD34⁺ cells plated, and reduced colony-forming-unit-erythrocyte (CFU-E) formation from 30 (SEM=6.5) to 9 (SEM=1.6, n=4) CFU-E per 500 CD34⁺ cells plated. This can be explained by increased apoptosis of βig-h3 knock-down cells. Notably, co-culture of βig-h3 knock-down HSPC with stromal feeder cells, known to express high levels of βig-h3, showed no difference compared to control HSPC in cobblestone area formation within two weeks, indicating that stromal cells can counteract apoptosis in βig-h3 knock-down cells. Remarkably, long-term-culture CFU-GM (LTC-CFU) formation of HSPC that were co-cultured with stromal cells during two weeks, was even significantly increased (1.9-fold, n=2) in βig-h3 knock-down cells, indicating that decreased endogenous levels of βig-h3 stimulates the maintenance or expansion of HSPC on stroma.

In conclusion, ectopic expression of βig-h3 decreased CFU-GM in HSPC and accelerated differentiation towards megakaryocytes, suggesting that βig-h3 might drive lineage commitment of HSC. Conversely, knock-down of βig-h3 in HSPC stimulated LTC-CFU formation, indicating that decreased βig-h3 levels in HSPC maintain their undifferentiated state. In absence of stroma, however, knock-down of βig-h3 induces apoptosis, indicating βig-h3 as an essential survival factor, which expression levels regulate differentiation and maintenance of HSC.