Assistant Professor University of California, Irvine, United States
Introduction:: iPSC-derived conventional liver organoid models lack adult hepatocyte maturity and intrinsic function. Multilineage communication and liver parenchymal-non-parenchymal cell crosstalk play essential roles in organogenesis. Developmentally, the endoderm- and mesoderm-derived lineages co-emerge in the fetus, and previous studies have shown that colonization of the hematopoietic system enhances the functional maturation of hepatocytes. Cellular interactions and signaling mechanisms implicated in liver development could be investigated using a fused assembloid model consisting of endoderm-derived bipotent liver cells and mesoderm-derived heterogenous vascular cells. Furthermore, understanding the dynamic cues of hepatocyte-cholangiocyte fate decisions during liver development and regeneration would enable precise control over these mechanisms.
Materials and Methods:: Hepatic and vascular organoids were generated from three-dimensional human induced pluripotent stem cell (hiPSC) aggregates. To initiate hepatic differentiation, hiPSC aggregates were induced to form endoderm spheroids on Day 5 by employing Activin A and CHIR99021. Subsequently, hepatic lineage commitment occurred on Days 10-15 through supplementation with growth factors such as bFGF and HGF. The resulting organoids were then characterized using immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) to ascertain the expression of specific markers. Similarly, the generation of vascular organoids was initiated by directing iPSC aggregates toward mesodermal differentiation Subsequent commitment to vascular lineages was achieved by exposing the spheroids to VEGF. Notably, modulation of VEGF allowed for the development of arterial and venous organoids, each exhibiting distinct vascular networks. Characterizing these vascular organoids involved similar techniques, including immunofluorescence, qRT-PCR, and flow cytometry, to analyze the expression of relevant markers.
Results, Conclusions, and Discussions::
In the described assembloid organoid model, interactions between mesoderm and septum transversum replicate developmental dynamics, particularly focusing on the interplay between developing vascular and hepatic tissues. These interactions model embryonic processes, potentially yielding early vascular and hepatic structures. This crosstalk likely involves molecular signaling that influences cell fate and tissue organization. This model offers insights into vascular and hepatic development and tissue patterning, informing regenerative medicine and congenital anomaly research. Furthermore, it provides a platform to study diseases affecting these tissues, enabling disease modeling and therapeutic testing.
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