Identification of Bone Marrow-Derived and Circulating Fibrocytes in Gata1low Mice with Myelofibrosis
Authors: Marquez Ana , George Jeffy , Mehta Hrishikesh M , Day Regina M. , Corey Seth 
Published: October 2019

Abstract
Introduction: It has recently been shown that the population of CD45+ Col1+ CXCR4+ (characteristic fibrocyte markers) cells increases in the bone marrow after bleomycin-induced lung injury. But little is known about their existence in primary or secondary myelofibrosis. Questions also remain about the exact origin of fibrocytes and how their development/differentiation is altered in response to tissue damage. Fibrocytes are believed to be a bone marrow-derived cell type, differentiated from monocytes, with essential roles in both normal tissue repair and multiple fibrotic diseases. Fibrocytes are unique in that they express both stromal and hematopoietic markers. Fibrocytes have been isolated and cultured from murine and human blood PMBCs, however, this method yields a low number of cells per animal. The murine splenic monocyte reservoir has also been shown to be able to give rise to fibrocyte cultures. Methods: Long bones (humeri and femurs) were obtained from adult C57BL/6J mice ± 14 Gy and Gata-1low C57Bl/6J mice bred with CD1 mice. Bones were aseptically flushed with sterile PBS. Collected bone marrow was passed through a 22 gauge needle to further dissociate the cells. Cells were centrifuged at 300 x g for 10 min at room temperature. Cells were washed and red blood cells were lysed. After a wash cells were resuspended in supplemented Fibrolife media (10 mM, 2 x non-essential amino acids, 2 mM sodium pyruvate, 4 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 2× ITS-3, and 50 μM 2-mercaptoethanol) and plated at 1.25x106 cells/mL. Cells differentiated for 7-10 days before analysis. Immunocytochemical staining and qPCR analysis was performed using standard methods. Results: Using this new method to isolate fibrocytes from total murine bone marrow, we found that Fibrocytes differentiated as early as 5 days in serum-free media. In addition to exhibiting the characteristic spindle shape morphology, cultured fibrocytes also expressed both hematopoietic (CD45) and stromal markers (collagen I). Importantly, these cells were negative for the fibroblast specific marker fibronectin, confirming that these cells were not fibroblasts. Cultured fibrocytes also appeared to be terminally differentiated, as they could not be passaged. Bone marrow-derived fibrocytes were found to upregulate collagen I in response to TGFβ treatment, mirroring the response reported in blood-derived fibrocytes in lung fibrosis. We further identified fibrocytes from the peripheral blood of heterozygous Gata-1low mice, which displayed myelofibrosis. Irradiation of shielded mice with 14 Gy resulted in myelofibrosis by 60 days (as opposed to ~12 months in Gata-1low mice). Furthermore, there was associated increased TGFbeta production. This correlated with a marked increase in fibrocytes. Conclusions: Our new culturing method will further facilitate the study of fibrocyte differentiation and response to both pro- and anti-inflammatory signals. We are currently utilizing flow cytometry to characterize fibrocyte kinetics and to quantify these cells in mice and patients with myelofibrosis. Our data also suggest that fibrocytes may play a contributing role to myelofibrosis in part through TGFb pathways. We have also developed an radiation-induced model of myelofibrosis with characteristic TGFbeta and circulating fibrocytes. Because myelofibrosis occurs in a much shorter time period, this model may be useful to more rapidly evaluate novel anti-fibrotic therapies.
This work was supported by a Translational Award from the Leukemia and Lymphoma Society.

Disclosures
No relevant conflicts of interest to declare.