In vitro biomimetic engineering of a human hematopoietic niche with functional properties
Proceedings of the National Academy of Sciences of the United States of America
In adults, human hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment. Our understanding of human hematopoiesis and the associated niche biology remains limited, due to human material accessibility and limits of existing in vitro culture models. The establishment of an in vitro BM system would offer an experimentally accessible and tunable platform to study human hematopoiesis. Here, we develop a 3D engineered human BM analog by recapitulating some of
... pitulating some of the hematopoietic niche elements. This includes a bone-like scaffold, functionalized by human stromal and osteoblastic cells and by the extracellular matrix they deposited during perfusion culture in bioreactors. The resulting tissue exhibited compositional and structural features of human BM while supporting the maintenance of HSPCs. This was associated with a compartmentalization of phenotypes in the bioreactor system, where committed blood cells are released into the liquid phase and HSPCs preferentially reside within the engineered BM tissue, establishing physical interactions with the stromal compartment. Finally, we demonstrate the possibility to perturb HSPCs' behavior within our 3D niches by molecular customization or injury simulation. The developed system enables the design of advanced, tunable in vitro BM proxies for the study of human hematopoiesis. hematopoiesis | bone marrow niche | 3D culture | tissue engineering | hematopoietic stem cell T he bone marrow (BM) microenvironment is responsible for the maintenance of hematopoietic stem cell (HSC) activity, enabling the lifelong production of mature blood cells (1, 2). The regulation of HSC self-renewal and differentiation is achieved by complex cellular (3) , molecular (4, 5), structural (6), and physical (7, 8) cues defining the HSC niche (2, 9) . The components of the human HSC niche, and how these elements interact to modulate stem cell fate, remain poorly understood. The field is hampered by the limited possibilities to access and harness information from human specimens. Chimeric animal models (10) most closely recapitulate in vivo human physiology, but, in this setting, the niche has remained inaccessible to experimental manipulation and optical observation (11, 12) . In addition, the interspecies-chimerism in both hematopoietic cells and their environment makes interpretation of experimental results difficult. The development of in vitro substitutes is a promising alternative with superior tunability and throughput (13, 14) . Previous studies have described the combination of different stromal and hematopoietic progenitors using a variety of culture substrates (9-11), resulting in the phenotypic preservation of specific blood phenotypes. However, the recapitulation of the structural organization of BM, including essential cell-cell and cell-matrix interactions (15) (16) (17) (18) (19) , and the associated functional preservation of HSCs (20) are still elusive. The need for advanced culture systems of higher biological complexity has gained increasing recognition (21) to study the fundamental biology of stem cells. Similarly to the "organogenesis in a dish" proposed for complex organs [e.g., lung (22), breast (23), kidney (24), and liver (25) ], the in vitro engineering of human BM environments (21, (26) (27) (28) capable to sustain HSCs (28, 29) would enable their study in xeno-free settings. Here, we report an in vitro system supporting the development and maintenance of a human BM analog. Our approach consists in the use of porous hydroxyapatite scaffolds with structural and compositional features of bone (30), functionalized by human mesenchymal stromal cells (hMSCs) and the extracellular matrix (ECM) deposited during their progressive maturation into the osteoblastic lineage. The hMSC culture is performed under direct perfusion flow (31), offering efficient nutrient supply/waste removal, while mimicking interstitial flow and associated shear stress. The blood compartment was introduced into the resulting 3D stromal tissue by perfusion of human purified cord blood (CB)-derived CD34 + cells. This engineered organoid partially recapitulates structural and functional features of the human BM in defined and tunable settings.