Project description:In this study, we utilize a scaffolding tray for organoid development. Conventional organoids are compared to those generated using the scaffolding tray.
Project description:The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate perfusable vessels in sufficiently close proximity and at sufficient small scale to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating perfusable synthetic capillary-scale 3D channels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.
Project description:Engineering an inducible leukemia-associated transcription factor enables large-scale ex vivo production of functional human phagocytes
Project description:Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible transcription factor to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) transcription factor to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 years of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated towards CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established a novel transcription factor that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable transcription factors have the potential to be applied as molecular tools to produce functional immune cells for cell therapy.