Medullary adipocytes and age related osteoporosis

132 Medullary adipocytes and age- related osteoporosis Background: Our findings suggested that bone marrow adipocytes may contribute to bone loss. Indeed, coculture and conditioned medium (CM) experiments based on commercial human bone marrow stromal cells showed that adipocyte-secreted factors induced the conversion of osteoblasts towards an adipocyte like phenotype. This event was confirmed by microarray analysis showing an enrichment in the adipocyte gene signature in cocultured osteoblasts. Furthermore, double immunofluorescence staining demonstrated the co- expression of adipocyte and osteoblast specific markers in individual cells, providing evidence for a transdifferentiation event. Purpose: The goals were to (1) find the molecules and pathways involved in these events and (2) develop cellular models closer to the physiological environment to confirm the transdifferentiation event. Methods: (1) one dimensional LC-MS/MS and label-free quantification were used to compare stimulatory and non-stimulatory adipocyte culture supernatants and identify adipocyte-secrete proteins potentially regulators of osteoblast differentiation. In parallel, osteoblast gene expression changes induced by adipocyteCM were identified by transcriptional analysis. Physical interactions between adipocyte proteins and osteoblast membrane protein coding genes were examined using StringDB. Significant pathways leading to osteoblast transdifferentiation were then identified using gene set enrichment analysis. Some selected pathways and statistically relevant proteins were investigated through functional assays. (2) human femoral heads were collected during hip replacement surgery and processed to (2a) isolate osteoblasts through outgrowth of primary cells, followed by their incubation in CM, (2b) develop organ culture, which viability was tested with functional and histological assays, and (2c)isolate adipocytes, through collagenase treatment followed by ceiling and 2D culture. Results: (1) Our analysis identified a total of 271 physical interactions between adipocyte-secreted proteins and osteoblast membrane protein coding genes and proposed three pathways for their potential contribution to osteoblast transdifferentiation, the PI3K-AKT, JAK2- STAT3 and SMAD pathways. Western Blot experiments confirmed the modulation of STAT3 pathway in the transdifferentiation event. (2a) We used two rounds of outgrowth from trabecular bone fragments to isolate primary cells that displayed a specific osteoblast phenotype, characterized by osteocalcin mRNA expression and mineralization. We confirmed the transdifferentiation of these primary osteoblasts following their incubation with CM from commercialadipocytes as evidenced by the increase in the levels of adipogenic mRNA markers (PPARG and Leptin) and the decrease of osteocalcin mRNA level. (2b) The suitability of the human organ culture model with no appreciable loss of viability, unchanged structure and presence of functional bone cells was demonstrated. The next step will be to culture the explants in adipocyte CM to examine its effect on bone cells. (2c) Oil red O staining confirmed the isolation of primary adipocytes from bone biopsies. Experiments revealed that their CM induced osteoblast transdifferentiation as the one from commercial adipocytes. Conclusions: (1) We provided new insights into the crosstalk between adipocytes and osteoblasts in the bone marrow and demonstrated the powerfulness of our integrative Omics strategy to decipher cell- cell communication events. (2) Our new models based on primary cells isolated from bone biopsies confirmed the role played by adipocyte-secreted factors in the transdifferentiation of osteoblasts. In the future, we plan to compare inter- donor phenotype variations and bone microarchitecture data obtained by X- ray microtomography to assess the existence of a correlation between the osteoblast response to adipocyte secretions and the quality of the bone from which the cells are derived. Key words : Human bone marrow stromal cells; osteoblasts ; adipocytes