Transformation of a human iPSC-derived bone marrow organoid for the study of hematopoiesis

This proposal is for the transformation of a human-induced pluripotent stem cell (iPSC)-derived bone marrow organoid for the study of hematopoiesis. The scientific premise of this project is to help overcome the limitations of current hematopoiesis research models, such as two-dimensional cell cultures and animal models, by utilizing a human bone marrow organoid. Indeed, organoids have the potential to mimic the microenvironment and functions of tissues or organs more accurately, bridging the gap between in vitro and in vivo systems. We have created a bone marrow organoid using iPSCs that replicates key marrow elements like sinusoidal vessels, stroma, megakaryocyte/endothelial interactions, and myeloid cells. This organoid has already shown promise in studying hematologic cancers and therapeutic interventions. However, the potential of the bone marrow organoid extends beyond malignant samples. In the proposed project, we aim to revolutionize the organoid for the study of hematopoiesis in three specific aims. The first aim is to perform cellular mapping of the bone marrow organoid over its development using advanced flow cytometry and microscopy techniques. This will provide valuable information about the changes that occur in the organoid over time and serve as a resource for the wider hematopoiesis field. The second aim is to optimize the bone marrow organoid for the study of hematopoietic development. The current protocol, which focuses on the differentiation of hematopoietic stem and progenitor cells (HSPCs), leads to a progressive loss of HSPCs over time. By incorporating knowledge from animal models and cell culture systems, we will develop an iPSC differentiation protocol that maintains a stable HSPC population over a 60- day period. This optimization is crucial for the organoid model to effectively study hematopoiesis. The third aim is to culture the organoid under flow using a microfluidics platform. The lack of interstitial flow in the current model limits its ability to replicate physiological conditions. By culturing the organoid in a 3-channel microfluidic bioreactor, we will introduce interstitial flow and enhance vascular maturation and maintenance. This setup will also allow the efficient delivery of small molecules, proteins, and cells into the organoid, mimicking in vivo blood flow. Further, it will present opportunities for testing therapeutic strategies and studying physiological mechanisms relevant to hematopoietic diseases. In sum, this project aligns with the goals of the Catalytic Tool and Technology Development FOA and aims to transform the bone marrow organoid into a novel biological tool that advances translational hematopoiesis research.