In vitro erythropoiesis in a 3D bone marrow biomimicry: reproducing physiologic biochemical and microenvironmental factors involved in red blood cell formation

Erythropoiesis studies have been hampered by the lack of culture systems that accurately reproduce the features of the bone marrow (BM). Existing knowledge has been obtained from 2D studies which do not reflect the complexity of the BM microenvironment, where haematopoietic stem cells (HSCs) and differentiating erythroid cells are exposed to complex stimuli, including cell-cell and cell-extra cellular matrix interactions and soluble factors. These limitations have been overcome by addition of high concentrations of exogenous cytokines, serum and/or stroma feeder layers, which introduce non physiologic signals to the cells. In this study, a 3D BM biomimicry was developed, using collagen-coated polyurethane (PU) scaffolds, to expand and differentiate cord blood mononuclear cells (CBMNCs) under serum-free, near-physiologic conditions. This system mimics BM cellular heterogeneity, sustaining complex stromal cell populations and in situ cytokine production. The dynamic evolution of the erythropoiesis was studied with a focus on the role of the hypoxic environment under near-physiologic cytokine concentrations (SCF and EPO only). A combined hypoxia/nomoxia schedule, in this 3D BM biomimicry, was proposed to enable the establishment of erythroid progenitor/precursor populations and a specialized microenvironment, including stroma cells and endogenous cytokine production, and to recreate physiologic erythropoiesis. It was also verified that addition of dexamethasone was not required at any stage of the 3D culture. Furthermore, CBMNC recharge of cultured scaffolds successfully enhanced erythropoiesis with increased production of biconcave shaped enucleated red blood cells (RBCs), simultaneously with the maintenance of a progenitor pool. In parallel, RGD-modification was proposed as a xeno free, defined alternative to collagen coating in PU scaffolds. Preliminary studies using RGD-modified scaffolds did not show a significant improvement of the microenvironment in erythropoiesis when compared with the results of the previously established CBMNCs single cultures. In summary, this thesis reports the development of a reproducible, robust and near physiologic erythropoiesis model that resembles the physiologic microenvironment and allows for in vitro study of erythropoiesis and sustained RBC production.Open Acces