The goal of our Wet Lab project was to design a functional, physiologically-relevant organoid capable of precisely mimicking the human hematopoietic niche. In order to engineer such an organoid, we followed a protocol involving a four-phase differentiation of human induced pluripotent stem cells iPSCs). Differentiating iPSCs into bone marrow organoids requires mesodermal induction (Phase I), iPSC commitment to stromal and hematopoietic commitment (Phase II), formation of a vascular structure (Phase III), and establishment of the organoids’ individuality (Phase IV).
Phase I is crucial as it forms the very basic characteristics of the hematopoietic niche that will eventually give rise to an organoid. In human physiology, the mesoderm generates HSCs, blood cells, and immune cells during embryonic development and mesodermal progenitors give rise to various lineages in the human bone marrow structure. Directing iPSCs toward mesodermal lineages is critical for the further generation of hematopoietic cells as well as a complete hematopoietic structure. Mesodermal induction involves exposing the iPSC aggregates to a cocktail of developmental signals that promote mesodermal formation.
Phase II takes the mesodermal aggregates and exposes them to various hematopoietic, stromal, and vascular growth factors to induce differentiation and guide the structure toward hematopoietic fates. At this point in time, the organoid has mesodermal, hematopoietic, and stromal populations involved in function and support.
Phase III involves embedding the hematopoietic organoids into a hydrogel structure alongside vascular growth factors to support the formation of a vascular network. This phase allows the organoids to closely mimic bone marrow remodeling as the vascular network is key for nutrient supply, formation of the perivascular network, the site of cell trafficking, and more. This phase is the longest and most involved, as the generation of the vascular network allows for modeling of cell-cell interactions in 3D, endothelial and perivascular support, and paracrine signaling.
Phase IV establishes the organoids as individual structures that do not rely on other organoids for signaling and are capable of self-generation in a nutrient-dense environment. Organoids are physically removed from the hydrogel and placed in maintenance media.