Mesenchymal Cell Associated Fibrosis in Experimental mpl^W515L Mouse Model of Myelofibrosis

Background: Bone marrow fibrosis caused by excessive collagen deposition represents a significant indicator of poor prognosis in myeloproliferative neoplasms (MPNs). Genetic driver mutations have been identified as JAK2, CALR and mpl, but the precise mechanism and cell type leading to fibrosis remains unanswered. TGFβ production by mutant hematopoietic cells is the primary factor responsible for fibrosis initiation. Although effective JAK2 inhibitor therapy has been adopted to manage disease symptoms and decrease abnormal hematopoietic cells, this pharmacological inhibitor does not reverse fibrosis. Our previous observations in patients with Large granular lymphocyte (LGL) leukemia indicate that mesenchymal-derived fibroblasts, distinguished by the capacity to undergo trilineage differentiation into osteoblasts, adipocytes and chondrocytes, are responsible for the fibrosis observed in these patients. In this study, we report the identification of a similar fibrosis-initiating mesenchymal cell population in a mouse model of MPN induced by transplantation of gfp-mpl^W515L retrovirus-expressing hematopoietic cells and indicate a mechanism to reverse the fibrotic phenotype.

Methods: Primary mesenchymal stromal cells (MSCs) were isolated from mice with MPN caused by the transplantation of gfp-mpl^W515L -transduced bone marrow cells (MPN mice) and from control mice that received transplants with gfp-wild-type(wt)-mpl-expressing bone marrow. MSCs were harvested 17 days post-transplant when WBC counts exceeded at least >20,000 cells/μl. Fibrillar collagen I (detected with trichrome stain in bone marrow biopsies) and collagen III (detected with reticulin stain in bone marrow biopsies) were identified as the major proteins produced in the bone marrow and by cultured MSCs in MPN mice consistent with pathologic examination. The amount of collagen was quantified in MSCs grown under reduced oxygen in undifferentiating culture conditions. To determine pluripotency, cells were grown with differentiation-specific culture media and stained with Alizarin Red S (to detect osteoblasts) and Alcian blue (chondrocytes). Collagen production was determined by qRT-PCR and polychromatic immunofluorescence staining of three-dimensional cultures and TGFβ was measured by qRT-PCR and ELISA.

Results: To isolate purified populations of MSCs, gfp-positive hematopoietic cells were removed and MSCs were maintained in long-term culture in the absence of malignant cells. MSCs were isolated in micro-cultures from gfp-wt-mpl (ie, control) and mpl^W515L transplanted mice. Using gfp-wt-mpl-derived MSCs, we first showed that the addition of TGFβ induced collagen I and III expression as expected from previous studies. Original undifferentiated MSCs derived from control and MPN mice grew continuously in undifferentiated culture conditions with a similar life expectancy, and both populations were capable of further differentiation into osteoblasts and chondrocytes. Collagen I (p<0.0001) and III (p=0.0008) mRNA and fiber formation were increased in the MSCs from mpl^W515L -transplanted mice compared to control. Without exogenous cytokines or HSC supernatant, the MSCs from mutant-mpl-mice maintained greater than 2-fold increase in collagen suggesting that intrinsic reprogramming occurred in the MSC population independent of tumor cells that are normally considered the source of TGFβ and other fibrosis-initiating cytokines. TGFβ mRNA (0.9878±0.129, control vs 1.023±0.319, MPN p=ns) and total TGFβ secreted protein detected by ELISA (165.0±22.7pg/ml, control vs 144.1±21.8pg/ml, MPN p=ns) was similar in control and MPN-derived MSCs indicating that excessive TGFβ production does not maintain the fibrotic phenotype in culture. We have shown previously that collagen from fibrotic MSCs in LGL leukemia is inhibited by culturing the cells with fibroblast growth factor-b (FGFb, FGF-2). Similar to LGL leukemia-dervied MSCs, the addition of FGFb stimulated proliferation and restored normal levels of collagen I and III in the MSCs from MPN mice.

Conclusions: This study indicates that expanded MPN-derived MSCs can be re-programmed to produce normal amounts of collagen, which may reverse fibrosis. This study has implications for the development of MSC-based cell therapies in MPN.