Project description:Pluripotent stem cell (PSC)-derived organoids are emerging as novel human-based microphysiological models but display immature phenotypes with limited subsets of endothelial or stromal cells. Here we demonstrate that in vitro manipulation of gene regulatory networks (GRNs) in PSC-derived liver organoids selected either through computational analysis or targeted tissue design can advance tissue maturation in vitro. Through an unbiased comparison with the genetic signature of mature livers, we identify downregulated GRNs in fetal liver organoids compared to adult livers. We demonstrate that overexpression of PROX1 and ATF5, together with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, drives maturation in vitro. Single cell analyses reveal hepatobiliary-, endothelial-, and stellate-like cell populations. The engineered organoids demonstrate enhanced vasculogenesis, capture native liver characteristics (e.g. FXR signaling, CYP3A4 activity), and exhibit therapeutic potential in mice. Collectively, our approach provides a genetically guided framework for engineering developmentally advanced multilineage tissues from hiPSCs.
Project description:Intestinal failure (IF), following extensive anatomical or functional loss of small intestine (SI), hasdebilitating long-term consequences on children1. Priority of care is to increase the child’s length of functional intestine, jejunum in particular, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using primary patient biomaterials. We show that organoids derived from patients can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which form optimal biological scaffolds. Remarkably, proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human SI and colon scaffolds, indicating that both can be used interchangeably as platforms for intestinal engineering. Indeed, seeding jejunal organoids onto either scaffold type reliably reconstructs grafts that exhibit several aspects of physiological jejunal function with potential to survive after transplantation. Our findings provide proof-of-concept data for engineering IF patient-specific jejunal grafts, ultimately aiding in restoration of nutritional autonomy.
Project description:To analyze stem/progenitor cell function, we purified hepatocytes derived from adult livers and fetal hepatoblasts derived from embryonic day 13 livers. Compared gene expression in E13 hepatoblasts and adult hepatocytes derived from C57BL/6NCr mice
Project description:We found that hepatocytes isolated from adult mouse livers can dedifferentiate into progenitor-like cells and exhibit a hybrid epithelial/mesenchymal phenotype in monolayer culture. dediHeps can re-differentiate into mature hepatocytes by forming aggregates and fetal intestinal progenitor cells (FIPCs) by forming organoids in three-dimensional culture. We conducted RNA-seq analyses to investigate the characteristincs of hepatocytes, dediHeps, dediHep aggregates, dediHep derived spherical organoids, dediHep derived budding organoids, FIPC derived spherical organoids, intestinal stem cell (ISC) derived budding organoids, and CLiP cells (Katsuda, 2016).
Project description:Understanding gene expression profile and transcriptional regulation of healthy adult human hepatocytes Gene expression was analyzed
Project description:To investigate whether rat adult hepatocytes would exhibit different characteristics dependent on their ploidy statuses, we compared the transcriptome profile of 2c, 4c and 8c hepatocytes by mRNA microarray.
Project description:To analyze stem/progenitor cell function, we purified hepatocytes derived from adult livers and fetal hepatoblasts derived from embryonic day 13 livers.
Project description:Hepatocellular carcinoma (HCC) and cholangiocarcinoma (ICC) are two main forms liver cancers with poor prognosis. Models for studying HCC and ICC development using human liver cells are urgently needed. Organoids serve as in vitro models for cancer studies as it recapitulates in vivo structures and microenvironment of solid tumors. Herein, we established liver cancer organoid models by introducing specific mutations into human induced hepatocyte (hiHep)-derived organoids. c-MYC and hRASG12V overexpression in hiHep organoids with repressed p53 activation by large T led to distinct HCC and ICC signatures. With these oncogenic mutations, the neoplastic hiHep organoids formed cancerous structures and possessed cancer-specific hallmarks. Comprehensive transcriptional analysis of liver cancer organoids revealed genes and pathways with disease-stage-specific alterations. Notably, with RAS mutations, hiHep organoids acquired biliary trans-differentiation, and showed a process of conversion from hepatocytes to ICC. To sum up, we have established a useful and convenient in vitro human organoid systems modeling liver cancer development.