Transdifferentiation of Bone Marrow Pro-B Cells into Bone-Resorbing Osteoclasts- an Unexpected Role for Erythropoietin
Authors: Deshet-Unger Naamit , Hiram-Bab Sahar , Ben-Califa Nathalie , Gilboa Dafna , Liron Tamar , Albert Kolomansky , Oster Howard S , Mittelman Moshe , Gabet Yankel , Neumann Drorit 
Published: October 2019

Abstract
Bone turnover is regulated by the coupled actions of osteoblasts, the bone-forming cells, and monocyte-derived osteoclasts, which mediate bone resorption. B cells were also shown to regulate bone metabolism, chiefly via paracrine signals. Depending on their state and/or mode of activation, B cells may inhibit or enhance osteoclastogenesis. In mammals, B cell development and maturation occurs in the bone marrow (BM), spleen, and other peripheral lymphoid tissues. In the BM, Pro-B cells sequentially differentiate into Pre-B and immature B cells. Whether BM B cells can transdifferentiate into osteoclasts remains controversial, since osteoclast differentiation from residual monocytic precursors in the cultures was not excluded in the earlier studies. Osteoclasts and B cells arise from distinct myeloid and lymphoid progenitors, respectively, which are downstream of a common multipotent progenitor cell. Monocyte differentiation into osteoclasts relies on monocyte-macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). Here, we investigated the possibility that BM B-cells contribute to bone loss by transdifferentiating into bone-resorbing osteoclasts. We found that B220+CD19+ cells can transdifferentiate into multinucleated tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the presence of RANKL and M-CSF. Our results show that Pro-B cells (B220+CD19+CD43HighIGM-), but not Pre-B cells (B220+CD19+CD43LowIGM-), nor immature B cells (B220+CD19+CD43-IGM+), could transdifferentiate into osteoclasts (16%±3.7 vs. 0.79%±0.28 and 0.48%±0.13 osteoclasts area, respectively). Moreover, among the Pro-B cells, only those expressing M-CSF receptor (CD115) could transdifferentiate into functional osteoclasts (18%±6.55 vs. 0.11%±0.05 osteoclasts area, respectively, Figure 1A and B). To unequivocally establish the generation of osteoclasts from B-cells, we next utilized a mouse model in which all B cell lineage-derived progenies express EYFP. We found that B cells isolated from BM of CD19-Cre:EYFP mice differentiated into TRAP+ multinucleated osteoclasts that were also positive for EYFP (Figure1C).
Erythropoietin (EPO) is a crucial kidney-derived hormone responsible for erythropoiesis. Once thought to act solely on the erythroid compartment to potentiate red blood cell production, it became evident that EPO receptors are also found on the monocytic lineage (monocytes, macrophages and dendritic cells). In that respect, we have reported that EPO directly stimulates bone loss via activation of EPO-R signaling in the monocytic lineage (Hiram-Bab et al., 2015). Here we report that B cells express EPO-R, and that EPO enhances Pro-B cell differentiation into osteoclasts by 70% (p<0.05) (Figure 1D).
Conceivably, in other scenarios, e.g., sex hormone deficiency, certain hematological cancers, and treatment with anti-CD20, increased B cell lymphocytes contribute to the bone loss phenotype due to transdifferentiation of B-cell precursors into osteoclasts. Taken together, our data suggest a new physio-pathological role for BM B cell precursors in bone metabolism, via their capacity to differentiate into functional osteoclasts, and a possible role for EPO in this process.
Figure 1 Osteoclastogenesis in vitro from sorted B cells. (A) TRAP staining of osteoclasts derived from the indicated sorted cells originating from BM (10,000 cells per well) and cultured with M-CSF and RANKL. Left - Pro-B cells expressing CD115 (B220+CD19+CD43HighIgM-CD115+). Right - Pro-B cells not expressing CD115 (B220+CD19+CD43HighIgM-CD115-). Data are mean±SEM of osteoclast area, n=5 mice in each group; *p<0.05. (B) Pit resorption area from the indicated sorted cells cultured on calcium phosphate-coated 96-well plates with M-CSF and RANKL. (C) Transdifferentiation of 180,000 cells per well CD19-Cre;R26R-EYFP into osteoclasts. DRAQ5 (blue) and anti-GFP (green) for CD19-Cre;R26R-EYFP and CD19-Cre cells. Bottom - TRAP staining. Confocal images (x20 magnification) (D) EPO increased differentiation of Pro-B cells into osteoclasts in vitro. TRAP staining of osteoclasts derived from sorted B220+CD19+CD43HighIgM- cells (180,000 per well) utilizing same culture conditions as in A ± 5U/ml EPO, n=7 mice in each group. Data are % osteoclasts of a representative image; EPO versus Control, displayed a 70% increase, p<0.05. Figure 1. Osteoclastogenesis in vitro from sorted B cells. (A) TRAP staining of osteoclasts derived from the indicated sorted cells originating from BM (10,000 cells per well) and cultured with M-CSF and RANKL. Left - Pro-B cells expressing CD115 (B220+CD19+CD43HighIgM-CD115+). Right - Pro-B cells not expressing CD115 (B220+CD19+CD43HighIgM-CD115-). Data are mean±SEM of osteoclast area, n=5 mice in each group; *p<0.05. (B) Pit resorption area from the indicated sorted cells cultured on calcium phosphate-coated 96-well plates with M-CSF and RANKL. (C) Transdifferentiation of 180,000 cells per well CD19-Cre;R26R-EYFP into osteoclasts. DRAQ5 (blue) and anti-GFP (green) for CD19-Cre;R26R-EYFP and CD19-Cre cells. Bottom - TRAP staining. Confocal images (x20 magnification) (D) EPO increased differentiation of Pro-B cells into osteoclasts in vitro. TRAP staining of osteoclasts derived from sorted B220+CD19+CD43HighIgM- cells (180,000 per well) utilizing same culture conditions as in A ± 5U/ml EPO, n=7 mice in each group. Data are % osteoclasts of a representative image; EPO versus Control, displayed a 70% increase, p<0.05.

Disclosures
No relevant conflicts of interest to declare.