Hematopoietic stem cell and progenitor cell (HSPC) niches are provided by the bone marrow (BM) tissue, from where HSPCs ensure the blood cells production throughout individual’s life. In contrast to the murine counterparts, human HSPC niches remain poorly understood due to accessibility and ethical difficulties. In order to bypass these limitations, our lab has engineered different 3D in vitro models of human BM niches combining patient-derived cells, scaffolding materials and perfusion bioreactors1-3. However, primary cells are subjected to donor variability and are not suitable to model pathophysiological hematopoiesis in highly reproducible settings. Our goal is to engineer 3D standardized HSPC niches by replacing primary cells by human induced pluripotent stem cells (hiPSCs).
Prior to engineer a 3D standardized vascularized osteoblastic niche, we first assessed in static conditions if the WTC hiPSC line4 could differentiate into osteoblasts, perivascular cells and endothelial cells. We cultured these hiPSCs to form aggregates and induced a mesoderm and vascular differentiation for 5 days. These aggregates were embedded into collagen I-Matrigel, and cultured for 11 days more in three different conditions: vascular medium, osteogenic medium and mix of both (1:1). At the end, aggregates were dissociated to analyze their nature by flow cytometry and qPCR. To generate the standardized hematopoietic compartment, we used CD34+ HSPCs that had been differentiated from hiPSCs during 21 days6. The potential of these standardized HSPCs was then assessed by culturing them for 1 and 3 weeks in 3D niches engineered with primary BM mesenchymal stromal cells within perfusion bioreactors1-3. At the end of the culture, tissues were harvested and processed for histological analyses and flow cytometry.
For the stromal compartment, the 1:1 mix of vascular and osteogenic medium allowed the formation of 3D aggregates with CD31+ endothelial cells, CD146+ perivascular cells and CD31-CD146- mesenchymal cells. Gene expression analysis confirmed the expression of vascular-related genes, but we did not observe osteoblastic gene expression, suggesting the lack of osteoblastic differentiation.
For the hematopoietic compartment, a fraction of hiPSC-derived HSPCs were still detected in 3D niches at the end of the culture by flow cytometry, while most of them differentiated into monocytes/macrophages, granulocytes, megakaryocytes, mast cells and erythroid cells. This cellular diversity was confirmed at histological level through hematoxylin-eosin staining and immunofluorescence stainings.
Although 3D structures with vascular cells can be generated with the WTC hiPSC line, the differentiation protocol requires optimization to achieve the osteoblastic differentiation. In contrast, hiPSC-derived HSPCs could be successfully maintained and differentiated into different lineages in 3D engineered niches. Our future research aims to combine the standardized stroma with standardized hematopoietic cells to generate a fully standardized hematopoietic niche model that might be exploited to study human hematopoiesis and/or as drug testing platform.
1. Bourgine, P.E. et al., PNAS 115, 5688—5695 (2018).
2. Born, G. et al., J. of Tiss. Eng. 12, 1—11 (2021).
3. Garcia-Garcia, A. et al., PNAS 118 (2021).
4. Kreitzer, F.R, et al., Am J Stem Cell 2, 119–131 (2013).
5. Toledo, M.A.S. et al., Blood (2021).