Hematopoietic Progenitors and Erythropoietin Affect Osteoblast Function and Lead to Osteoporosis in a Thalassemia Mouse Model

Mesenchymal stem cells (MSCs) from wild type (wt) and th3/+ mice were differentiated into osteoblasts in osteogenic media. MSC co-cultures with various hematopoietic progenitors from wt and th3/+ animals were differentiated to osteoblasts similarly. Osteogenesis was determined by Colony Forming Units for Osteoblasts (CFU-O) numbers and qPCR for osteogenesis-related genes (genes that encode for Runx2, bone sialoprotein, osterix, osteocalcin expressed through osteoblast matuaration), with four experiments performed in triplicates.

CFU-O numbers were decreased by 40% in MSC cultures from th3/+ mice compared to wt, while gene expression was 40 to 60% lower, indicating alterations in MSC fate that led to decreased osteogenesis. Treatment of MSC cultures with erythropoietin (EPO) antibody (EPO Ab) or soluble EPO receptor (s EPOR) reversed the decreased CFU-O numbers and expression of osteogenesis-related genes in th3/+ animals, indicating that EPO signaling guides MSC fate in thalassemia. Consistently, qPCR analysis of MSCs and osteoblasts showed increased EPO and EPOR expression in th3/+ versus wt controls.

To determine the role of hematopoietic progenitors on MSC fate, we isolated various progenitors and co-cultured them with MSC from wt animals. Lin-Sca+ ckit+ (LSK) precursors from th3/+ mice yielded 50% fewer CFU-O and decreased expression of osteogenic genes by 70%. In contrast, thalassemic erythroid precursors (CD71+Ter119- cells) and mature erythroid cells (Ter119+) had little to no effect on osteogenesis respectively. LSK-MSC co-cultures from wt and th3/+ mice were then treated with EPO Ab to determine the role of EPO on LSK-MSC interactions. Indeed, a reversal of the suppressed osteogenesis (determined by CFU-O numbers and qPCR results) was seen in th3/+ with such treatment, indicating a role of EPO signaling in LSK-MSK interactions. In support of these findings, EPOR and EPO expression in thalassemia LSK was increased two fold as compared to wt. Since Bone Morphogenic Proteins (BMPs) potentiate both erythroid expansion and osteogenesis, we examined BMP-2,-4,-6 expression in LSK from wt and th3/+ mice by qPCR. Expression of all three cytokines was decreased by 60–70% in th3/+, and reversed when thalassemic LSK cells were treated with EPO-Ab, suggesting that EPO affects LSK-MSC interaction by regulating BMP expression in LSK.

In summary, we observed decreased osteogenesis as result of altered MSC differentiation in the th3/+ thalassemia mouse. MSC fate is regulated by both hematopoietic progenitors and EPO. Specifically, LSK but not mature erythroid cells interact with MSCs in th3/+ to decrease osteogenesis. Our data support a dual role of EPO on osteogenesis in thalassemia. First, EPO produced by thalassemic MSCs alters osteogenesis via direct autocrine EPO signaling. Second, EPO also guides MSC-LSK interactions by regulating expression of BMPs -2,-4 and 6 in hematopoietic progenitors. Therefore, we conclude that EPO through an extra-hematopoietic action leads to bone loss in thalassemia.

Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.