OC 18.1 - CXCL12 abundant reticular (CAR) cells spatially regulate platelet production in the bone marrow

Background: During thrombopoiesis, megakaryocytes (MKs) penetrate bone marrow (BM) sinusoids and release platelets exclusively into the blood stream. We recently identified perivascular CXCL12-abundant reticular (CAR) cells as spatial regulators of proplatelet formation (PPF).

Aims: Deciphering the molecular interactions of MKs with CAR cells that mediate PPF selectively into sinusoids.

Methods: We established a co-culture system using BM-derived MKs and stroma cell lines OP-9, MS-5, or ST-2 (Fig.1A) to assess PPF and MK maturation. Stroma cell surface receptor expressions were analyzed flow cytometrically. We examined MK-CAR cell interactions in 3D-reconstructed thick femur sections from CXCL12DsRed/+ reporter mice using confocal immunofluorescence microscopy (Fig.1B-C).

Results: We identified distinct BM pericyte populations (CXCL12DsRed+PDGFRβ+ and CXCL12DsRed-PDGFRβ+), all in contact with MKs (Fig.1C). MK co-culture with stroma cells decreased the number of mature CD41+CD42a+ MKs by 53.3 ± 14.4% (Fig.1D), suggesting that stroma cell interaction limits MK differentiation. When in direct contact to stroma cells, PPF was reduced by 7.7 ± 2.2% comparable to the known inhibitory effect of collagen I (6.6%, Fig.1E). When MKs and stroma cells were cultured in transwells, PPF remained unaffected (Fig.1F), indicating that the modulatory effect of CAR cells on MKs depends on direct cell-cell contacts. We identified multiple differentially regulated molecular interaction interfaces between MKs and CAR cells. Expression of VCAM-1 on stroma cells and its ligand VLA-4 on MKs was increased, while PDGFRβ expression on stroma cells was decreased, suggesting a mutual influence of stroma cells on MKs. We hypothesize that especially VCAM-1/VLA-4 interactions might act as a molecular break for platelet production.

Conclusion(s): Intriguingly, CAR cells affect MK maturation and PPF. The direct MK-CAR cell interaction represents an additional layer of regulation to mediate mature MKs to release platelets into sinusoids. We expect that modulation of this interface is a putative target in patients with defects in platelet formation.