Reconstruction of the erythroblastic microenvironment in a 3D bone marrow analogue

innovative approaches on EPO-driven culture models for the generation of red blood cells in vitro

authored by: Timna Claire Bergmann
supervised by: Cornelia Lee-Thedieck
Abstract: Global access to safe red blood cell (RBC) products remains a significant challenge due to insufficient supply, inadequate screening, and increasing demand. In vitro generation of RBCs from human haematopoietic stem and progenitor cells (HSPCs) offers a potential solution, yet current protocols fail to replicate the complex environment of the haematopoietic stem cell (HSC) niche required for efficient and cost-effective in vitro production of RBCs, which often require excessive supplementation of expensive cytokines, such as erythropoietin (EPO). To address this, innovative approaches on EPO-dependent culture systems were investigated. Therefore, a physiologically relevant 3D platform using RGD-functionalised poly(ethylene glycol) diacrylate (PEG-DA) hydrogels was developed as a mimic of the human trabecular bone to support the in vitro differentiation of cord blood-derived CD34⁺ HSPCs into mature RBCs. This system recapitulated key features of the erythroid niche, promoting cell survival, proliferation, and differentiation. Incorporation of unpolarised M0 and M2c-polarized macrophages further refined the erythroid microenvironment by forming erythroblastic islands (EbIs), contributing to a supportive and non-inflammatory milieu. The 3D cultures maintained high viability over 5 and 12 days and significantly accelerated erythroid maturation compared to conventional 2D systems, with a higher frequency of mature CD71⁻/CD235a⁺ cells observed by day 5, while prolonged culture led to the depletion of the stem cell pool. While incorporation of M0 and M2c-like macrophages increased cell proliferation compared to single cultures, the 3D microenvironment had a greater influence on erythroid maturation than macrophage inclusion alone. This may be due to the spontaneous emergence of a CD14med/CD86hi/CD163lo/CD169lo macrophage subpopulation in 2D and 3D erythroid single cultures, which is likely to enhance erythropoiesis. Furthermore, to assess the potential for cell-autonomous EPO production under normoxic conditions, which could reduce costs in complex EPO-dependent culture models, the ability of tropolone sesquiterpenoid (TS) compounds to induce EPO was tested using the murine renal EPO-producing (REP) cell model FAIK3-5. While the tested TS compounds showed consistent bioactivity, including morphological changes, cytotoxicity, and proliferation inhibition, none induced EPO expression, likely due to the model’s known limitations such as myofibroblastic transdifferentiation and epigenetic silencing of EPO. In conclusion, while their ability to induce cytotoxicity and proliferation inhibition limits the integration of putative EPO-inducing compounds such as TS into erythroid culture systems, the developed 3D erythroid differentiation model provides a promising platform for mimicking the erythroid niche and advancing sustainable in vitro RBC production for transfusion medicine.
Organisation(s): Department of Cell Biology
Type: Doctoral thesis
No. of pages: 230
Publication date: 17.07.2025
Publication status: Published
Sustainable Development Goals: SDG 3 - Good Health and Well-being
Electronic version(s): https://doi.org/10.15488/19278 (Access: Open)