Bioengineered iPSC-derived models of human bone marrow for studies of systemic injury and disease

Introduction:: The human bone marrow (BM) is one of the most complex and critical tissues in the adult, functioning as the site for blood and immune cell production in homeostasis, injury, and disease. The marrow acts as an incredibly diverse stem cell niche, containing stromal and blood cells that help support the maintenance and differentiation capacity of hematopoietic stem and progenitor cells (HSPCs). The cell-cell and cell-matrix interactions within the niche help trigger blood cell production in response to injury, and to harbor downstream changes that may persist in the hematopoietic system during disease, such as in cancer metastasis or leukemias. As the development of human organs-on-a-chip (OoC) platforms has emerged over the past decade, there has been an increased relevance of using human BM models to study human- and patient-specific immune interactions in vitro. Here we report the development of a patient-specific bioengineered model of the BM (eBM), derived entirely from induced pluripotent stem cells (iPSCs) and its use in studies of radiation toxicity, injury, and blood malignancies. We demonstrate the feasibility of our system to be altered to include any combination of stromal/hematopoietic cells, allowing for this “plug-and-play” model to incorporate HSPCs from multiple donor sources (e.g., iPSCs, adult BM, or cord blood (CB)), or a combination of genetically modified iPSC-derived cells (e.g., healthy stroma and leukemic HSPCs from the same iPSCs or primary donors). Finally, we show applications of our eBM model in studying systemic responses of the BM in responding to ischemic cardiac muscle injury.

Materials and Methods:: iPSCs were first differentiated into each cell subpopulation prior to integration into the model. First, iPSCs were differentiated into iMSCs and iECs using established protocols, sorted for either CD90+ and CD144+, respectively, and frozen for downstream studies. In addition, iHSPCs were sorted for CD34+ expression and analyzed in comparison to CB-HSPCs in flow cytometry and colony forming functional assays. iMSCs were seeded onto decellularized bone matrix scaffolds, as fabricated in our previous work, and differentiated into matrix-secreting osteoblasts over four weeks. After this stage, iMSCs, iECs, and iHSPCs were combined within a fibrin hydrogel, seeded and polymerized on top of the living osteogenic bone. As a control, iMSCs, iECs, and primary CB-HSPCs were combined into the same tissue format. In addition, we applied a number of applications to the eBM tissue itself, including exposure to ionizing radiation (5 Gy photons) and culture with malignant leukemic blasts from primary human pediatric acute myeloid (AML) and lymphoid (ALL) blood cancers. We also combined our healthy eBM tissues with other engineered human tissues, including engineered cardiac muscle tissues, within a multi-organ platform that is designed to maintain each individual tissue compartment but allow for selective communication between tissues, as previously published1. In these studies, we integrated the eBM with either healthy or cryoinjured cardiac muscle tissues to identify immune infiltration over time. For all studies, we characterized the eBM and integrated tissues with flow cytometry, histological staining, or single cell RNA sequencing over 1-4 weeks.

Results, Conclusions, and Discussions:: The eBM model was developed to be modular, with ability to include either healthy iPSC-, or primary CB- or BM-derived HSPCs, demonstrating maintenance and differentiation of blood progenitors and progeny, and describe here three applications for (1) acute radiation injury, (2) leukemic infiltration, and (3) systemic, multi-tissue interactions. First, we confirmed presence of CD34+ progenitors after 2 weeks post-maturation, primarily in the adherent fraction, indicating the need for the supportive microenvironment to maintain the stemness of HSPCs.

To assess acute injury of eBM tissues, we applied 5 Gy of ionizing radiation to eBM tissues in culture. 48 hours after exposure to radiation, irradiated eBM tissues exhibited significant decreases in total CD45+ cell output in all tissues, regardless of HSPC source. In modeling diseases of the BM, we demonstrated the ability of the eBM to support both AML and ALL blasts in vitro for over 2-4 weeks, with survival of primary leukemic blast samples significantly higher (5-10 fold expansion) than that of 2D controls, which typically cannot remain viable in culture. Acute leukemias are known to alter their microenvironmental niche, and in many cases, use the stroma to protect malignant clones during treatment. Notably, leukemic eBMs were able to recapitulate drug responses of a common Bcl2-inhibitor, Venetoclax, seen clinically and in vivo, as compared to traditional liquid cultures. Finally, we linked the healthy eBM model to engineered cardiac muscle tissues with our previously-established multi-organ platform, allowing for a reservoir of immune cells capable of responding to off-target, systemic injury. As we were interested in modeling immune infiltration seen in cardiac injuries (i.e., myocardial infarction), we connected the eBM tissue to both healthy and cryoinjured cardiac muscle tissues. Over 2 weeks, we were able to visualize recruitment of CD11b+CD14+ monocytes from the eBM in response to the acute cardiac injury.

Here, we present a novel, customizable iPSC-derived BM model capable of modeling healthy and malignant hematopoiesis, as well as acute and systemic injuries of the body in vitro. We propose that this model system can be used to study systemic human- and patient-specific immune responses, leading to the development of personalized therapeutics.


Acknowledgements (Optional): : We gratefully acknowledge funding support by the National Institutes of Health (Grants No. EB025765, EB027062, HL076485, HL120046, CA249799 to G.V.N. & 1F31CA275733-01 to D.N.T.), National Science Foundation (Grant No. 1644869 to D.N.T. & ERC 16478 to G.V.N.), Translational Research in Space Health (TRISH/NASA) agency (Grants No. RAD0104 and NNX16A069A to G.V.-N., D.B.), Columbia SEAS-HICCC Pilot Grant, and Alex’s Lemonade Stand Foundation.


References (Optional): : ¹Ronaldson-Bouchard, et al. (2022). "A multi-organ chip with matured tissue niches linked by vascular flow." Nat. Biomed. Eng 6, 351–371.