Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>10). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes and proliferates at the mucosal surface, inducing severe diarrhoea. Short-chain fatty acids (SCFAs) are abundant in the intestine owing to the metabolic activity of microflora, and are important for colonic health. We found that, although a high concentration of SCFAs inhibited the growth of EHEC, at low concentrations, the SCFAs markedly enhanced the expression of the virulence genes required for cell adherence and the induction of attaching and effacing (A/E) lesions. Of the SCFAs tested, butyrate markedly enhanced the expression of these virulence-associated genes, even at the low concentration of 1.25 mM, but acetate and propionate showed only a small effect at concentrations higher than 40 mM. Butyrate enhanced the promoter activity of the LEE1 operon, which encodes a global regulator of the LEE genes, Ler. This enhancement was dependent on a regulator, PchA. Butyrate sensing was completely abrogated by the deletion of lrp, the gene for the leucine-responsive regulatory protein, Lrp. Expression of a constitutively active mutant of Lrp enhanced the expression of the LEE genes in the absence of butyrate, and a response-defective Lrp derivative reduced the response to butyrate. Thus, upon entering the distal ileum, EHEC may respond to the higher butyrate level via Lrp by increasing its virulence expression, leading to efficient colonization of the target niche.

Activating transcription factor (ATF) 5 is a transcription factor belonging to the ATF/cAMP-response element-binding protein gene family. We previously reported that ATF5 mRNA expression increased in response to amino acid limitation. The ATF5 gene allows transcription of mRNAs with at least two alternative 5-untranslated regions (5-UTRs), 5-UTR␣ and 5-UTR␤, derived from exon1␣ and exon1␤. 5-UTR␣ contains highly conserved sequences, in which the upstream open reading frames (uORFs) uORF1 and uORF2 are found in many species. This study was designed to investigate the potential role of 5-UTRs in translational control. These 5-UTRs differentially determined translation efficiency from mRNA. The presence of 5-UTR␣ or 5-UTR␤ represses translation from the downstream ATF5 ORF. Moreover, 5-UTR␣-regulated translational repression is released by amino acid limitation or NaAsO 2 exposure. This release was not seen for 5-UTR␤. Mutation of uAUG2 in the uORF2 of 5-UTR␣ restored the basal expression and abolished the positive regulation by amino acid limitation or arsenite exposure. We demonstrated that phosphorylation of eukaryotic initiation factor 2␣ was required for amino acid limitation-induced translational regulation of ATF5. Furthermore, arsenite exposure activated the exogenously expressed hemeregulated inhibitor kinase and induced the phosphorylation of eukaryotic initiation factor 2␣ in nonerythroid cells. These results suggest that translation of ATF5 is regulated by the alternative 5-UTR region of its mRNA, and ATF5 may play a role in protecting cells from amino acid limitation or arsenite-induced oxidative stress.Activating transcription factor (ATF) 2 -5 (formerly designated ATFx) is a transcription factor of the cAMP-response element-binding protein (CREB)/ATF family that was first identified as a protein that binds to the lipopolysaccharide-response element (GPE-1) on the granulocyte colony-stimulating factor (CSF3) gene along with C/EBP␥ (1). It contains a DNAbinding and dimerization domain (bZIP domain) and regulates processes that are involved in cellular differentiation (2, 3), the cell cycle (4), and apoptosis (5, 6). ATF5 represses cAMP-induced transcription in cultured cells (4) and is shown to inhibit apoptosis (6). Angelastro et al. (2) demonstrated that ATF5 inhibits CRE-mediated expression of neural genes and neural differentiation. Cdc34 is the G 2 checkpoint gene, and ATF5 is a target of Cdc34-dependent ubiquitin-mediated proteolysis (4), expression of which is affected by the cell cycle. Recently, Monaco et al. (7) showed that ATF5 is widely expressed in carcinomas, and interference with its function caused apoptotic cell death of neoplastic breast cell lines. This suggests that ATF5 may be a target for cancer therapy and that studies of the mechanism by which ATF5 expression is regulated might be important in the investigation of treatments for cancer.Mammalian cells have the ability to alter their gene expression to adapt to a variety of environmental stresses, including nutrient limitation, ...

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SummaryFor a new pathogen to emerge, it must acquire both virulence genes and a system for responding to changes in environmental conditions. Starvation of nutrients or growth arrest induces the stringent response in Escherichia coli, via increased ppGpp. We found the adherence capacity of enterohaemorrhagic E. coli (EHEC) and gene expression in the locus of enterocyte effacement (LEE) were enhanced by a downshift in nutrients or by entry into the stationary growth phase, both of which increase the ppGpp concentration. The activation was dependent on relA and spoT, which encode enzymes for the synthesis and degradation of ppGpp, and on dksA, which encodes an RNA polymerase accessory protein required for the stringent response. Upon induction of RelA expression, LEE gene transcription was activated within 20 min, even without starvation. The expression of two LEE transcriptional regulators, Ler and Pch, was activated by ppGpp and essential for the enhancement of LEE gene expression. In addition, the ler and pch promoters were directly activated by ppGpp in an in vitro transcription system. These findings suggest that the regulation of virulence genes in EHEC is integrated with E. coli 's stringent response system, through the regulation of virulence regulatory genes.