Discovery of non-genomic drivers of YAP signaling modulating the cell plasticity in CRC tumor lines.

In normal intestines, a fetal/regenerative/revival cell state can be induced upon inflammation. This plasticity in cell fate is also one of the current topics in human colorectal cancer (CRC). To dissect the underlying mechanisms, we generated human CRC organoids with naturally selected genetic mutation profiles and exposed them to two different conditions by modulating the extracellular matrix (ECM). Among tested mutation profiles, a fetal/regenerative/revival state was induced following YAP activation via a collagen type I-enriched microenvironment. Mechanistically, YAP transcription was promoted by activating AP-1 and TEAD-dependent transcription and suppressing intestinal lineage-determining transcription via mechanotransduction. The phenotypic conversion was also involved in chemoresistance, which could be potentially resolved by targeting the underlying YAP regulatory elements, a potential target of CRC treatment.

Quality Control of Stem Cell-Based Cultured Meat According to Specific Differentiation Abilities.

The demand for stem cell-based cultured meat as an alternative protein source is increasing in response to global food scarcity. However, the definition of quality controls, including appropriate growth factors and cell characteristics, remains incomplete. Cluster of differentiation (CD) 29 is ubiquitously expressed in bovine muscle tissue and is a marker of progenitor cells in cultured meat. However, CD29+ cells are naturally heterogeneous, and this quality control issue must be resolved. In this study, the aim was to identify the subpopulation of the CD29+ cell population with potential utility in cultured meat production. The CD29+ cell population exhibited heterogeneity, discernible through the CD44 and CD344 markers. CD29+CD44-CD344- cells displayed the ability for long-term culture, demonstrating high adipogenic potential and substantial lipid droplet accumulation, even within 3D cultures. Conversely, CD29+CD44+ cells exhibited rapid proliferation but were not viable for prolonged culture. Using cells suitable for adipocyte and muscle differentiation, we successfully designed meat buds, especially those rich in fat. Collectively, the identification and comprehension of distinct cell populations within bovine tissues contribute to quality control predictions in meat production. They also aid in establishing a stable and reliable cultured meat production technique.

Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Complement factor D targeting protects endotheliopathy in organoid and monkey models of COVID-19.

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes.

Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells.

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.

Enteral liquid ventilation oxygenates a hypoxic pig model.

The potential of extrapulmonary ventilation pathways remains largely unexplored. Here, we assessed the enteral ventilation approach in hypoxic porcine models under controlled mechanical ventilation. 20 mL/kg of oxygenated perfluorodecalin (O2-PFD) was intra-anally delivered by a rectal tube. We simultaneously monitored arterial and pulmonary arterial blood gases every 2 min up to 30 min to determine the gut-mediated systemic and venous oxygenation kinetics. Intrarectal O2-PFD administration significantly increased the partial pressure of oxygen in arterial blood from 54.5 ± 6.4 to 61.1 ± 6.2 mmHg (mean ± SD) and reduced the partial pressure of carbon dioxide from 38.0 ± 5.6 to 34.4 ± 5.9 mmHg. Early oxygen transfer dynamics inversely correlate with baseline oxygenation status. SvO2 dynamic monitoring data indicated that oxygenation likely originated from the venous outflow of the broad segment of large intestine including the inferior mesenteric vein route. Enteral ventilation pathway offers an effective means for systemic oxygenation, thus warranting further clinical development.

Collagen type I-mediated mechanotransduction controls epithelial cell fate conversion during intestinal inflammation.

BACKGROUND: The emerging concepts of fetal-like reprogramming following tissue injury have been well recognized as an important cue for resolving regenerative mechanisms of intestinal epithelium during inflammation. We previously revealed that the remodeling of mesenchyme with collagen fibril induces YAP/TAZ-dependent fate conversion of intestinal/colonic epithelial cells covering the wound bed towards fetal-like progenitors. To fully elucidate the mechanisms underlying the link between extracellular matrix (ECM) remodeling of mesenchyme and fetal-like reprogramming of epithelial cells, it is critical to understand how collagen type I influence the phenotype of epithelial cells. In this study, we utilize collagen sphere, which is the epithelial organoids cultured in purified collagen type I, to understand the mechanisms of the inflammatory associated reprogramming. Resolving the entire landscape of regulatory networks of the collagen sphere is useful to dissect the reprogrammed signature of the intestinal epithelium. METHODS: We performed microarray, RNA-seq, and ATAC-seq analyses of the murine collagen sphere in comparison with Matrigel organoid and fetal enterosphere (FEnS). We subsequently cultured human colon epithelium in collagen type I and performed RNA-seq analysis. The enriched genes were validated by gene expression comparison between published gene sets and immunofluorescence in pathological specimens of ulcerative colitis (UC). RESULTS: The murine collagen sphere was confirmed to have inflammatory and regenerative signatures from RNA-seq analysis. ATAC-seq analysis confirmed that the YAP/TAZ-TEAD axis plays a central role in the induction of the distinctive signature. Among them, TAZ has implied its relevant role in the process of reprogramming and the ATAC-based motif analysis demonstrated not only Tead proteins, but also Fra1 and Runx2, which are highly enriched in the collagen sphere. Additionally, the human collagen sphere also showed a highly significant enrichment of both inflammatory and fetal-like signatures. Immunofluorescence staining confirmed that the representative genes in the human collagen sphere were highly expressed in the inflammatory region of ulcerative colitis. CONCLUSIONS: Collagen type I showed a significant influence in the acquisition of the reprogrammed inflammatory signature in both mice and humans. Dissection of the cell fate conversion and its mechanisms shown in this study can enhance our understanding of how the epithelial signature of inflammation is influenced by the ECM niche.

Mechanical guidance of self-condensation patterns of differentiating progeny.

Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

NADPH-Oxidase Derived Hydrogen Peroxide and Irs2b Facilitate Re-oxygenation-Induced Catch-Up Growth in Zebrafish Embryo.

Oxygen deprivation induces multiple changes at the cellular and organismal levels, and its re-supply also brings another special physiological status. We have investigated the effects of hypoxia/re-oxygenation on embryonic growth using the zebrafish model: hypoxia slows embryonic growth, but re-oxygenation induces growth spurt or catch-up growth. The mitogen-activated kinase (MAPK)-pathway downstream insulin-like growth factor (IGF/Igf) has been revealed to positively regulate the re-oxygenation-induced catch-up growth, and the role of reactive oxygen species generated by environmental oxygen fluctuation is potentially involved in the phenomenon. Here, we report the role of NADPH-oxidase (Nox)-dependent hydrogen peroxide (H2O2) production in the MAPK-activation and catch-up growth. The inhibition of Nox significantly blunted catch-up growth and MAPK-activity. Amongst two zebrafish insulin receptor substrate 2 genes (irs2a and irs2b), the loss of irs2b, but not its paralog irs2a, resulted in blunted MAPK-activation and catch-up growth. Furthermore, irs2b forcedly expressed in mammalian cells allowed IGF-MAPK augmentation in the presence of H2O2, and the irs2b deficiency completely abolished the somatotropic action of Nox in re-oxygenation condition. These results indicate that redox signaling alters IGF/Igf signaling to facilitate hypoxia/re-oxygenation-induced embryonic growth compensation.

Isolation and Characterization of Tissue Resident CD29-Positive Progenitor Cells in Livestock to Generate a Three-Dimensional Meat Bud.

The current process of meat production using livestock has significant effects on the global environment, including high emissions of greenhouse gases. In recent years, cultured meat has attracted attention as a way to acquire animal proteins. However, the lack of markers that isolate proliferating cells from bovine tissues and the complex structure of the meat make it difficult to culture meat in a dish. In this study, we screened 246 cell-surface antibodies by fluorescence-activated cell sorting for their capacity to form colonies and their suitability to construct spheroid "meat buds". CD29+ cells (Ha2/5 clone) have a high potency to form colonies and efficiently proliferate on fibronectin-coated dishes. Furthermore, the meat buds created from CD29+ cells could differentiate into muscle and adipose cells in a three-dimensional structure. The meat buds embedded in the collagen gel proliferated in the matrix and formed large aggregates. Approximately 10 trillion cells can theoretically be obtained from 100 g of bovine tissue by culturing and amplifying them using these methods. The CD29+ cell characteristics of bovine tissue provide insights into the production of meat alternatives in vitro.

Rapid manipulation of mitochondrial morphology in a living cell with iCMM.

Engineered synthetic biomolecular devices that integrate elaborate information processing and precisely regulate living cell behavior have potential in various applications. Although devices that directly regulate key biomolecules constituting inherent biological systems exist, no devices have been developed to control intracellular membrane architecture, contributing to the spatiotemporal functions of these biomolecules. This study developed a synthetic biomolecular device, termed inducible counter mitochondrial morphology (iCMM), to manipulate mitochondrial morphology, an emerging informative property for understanding physiopathological cellular behaviors, on a minute timescale by using a chemically inducible dimerization system. Using iCMM, we determined cellular changes by altering mitochondrial morphology in an unprecedented manner. This approach serves as a platform for developing more sophisticated synthetic biomolecular devices to regulate biological systems by extending manipulation targets from conventional biomolecules to mitochondria. Furthermore, iCMM might serve as a tool for uncovering the biological significance of mitochondrial morphology in various physiopathological cellular processes.

Mammalian enteral ventilation ameliorates respiratory failure.

BACKGROUND: Several aquatic organisms such as loaches have evolved unique intestinal breathing mechanisms to survive under extensive hypoxia. To date, it is highly controversial whether such capability can be adapted in mammalian species as another site for gas exchange. Here, we report the advent of the intestinal breathing phenomenon in mammalians by exploiting EVA (enteral ventilation via anus). METHODS: Two different modes of EVA were investigated in an experimental model of respiratory failure: intra-rectal oxygen O2 gas ventilation (g-EVA) or liquid ventilation (l-EVA) with oxygenated perfluorocarbon. After induction of type 1 respiratory failure, we analyzed the effectiveness of g-EVA and I-EVA in mouse and pig, followed by preclinical safety analysis in rat. FINDINGS: Both intra-rectal O2 gas and oxygenated liquid delivery were shown to provide vital rescue of experimental models of respiratory failure, improving survival, behavior, and systemic O2 level. A rodent and porcine model study confirmed the tolerable and repeatable features of an enema-like l-EVA procedure with no major signs of complications. CONCLUSIONS: EVA has proven effective in mammalians such that it oxygenated systemic circulation and ameliorated respiratory failure. Due to the proven safety of perfluorochemicals in clinics, EVA potentially provides an adjunctive means of oxygenation for patients under respiratory distress conditions. FUNDING: This work is funded by the Research Program on Emerging and Re-emerging Infectious Diseases, Research Projects on COVID-19 (JP20fk0108278, 20fk0108506h0001), from the Japan Agency for Medical Research and Development (AMED), to T.T.; Strategic Promotion for Practical Application of Innovative Medical Technology, Seeds A (A145), to T.T.; and KAKENHI 19K22657, to T.C.-Y. This research is partially supported by the AMED Translational Research Program; Strategic Promotion for Practical Application of Innovative Medical Technology (TR-SPRINT), to T.C.-Y.; and AMED JP18bm0704025h0001 (Program for Technological Innovation of Regenerative Medicine), to T.T.

High-Fidelity Drug-Induced Liver Injury Screen Using Human Pluripotent Stem Cell-Derived Organoids.

BACKGROUND & AIMS: Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution. METHODS: We first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform. RESULTS: HLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis. CONCLUSIONS: Liver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.

Myoblasts With Higher IRS-1 Levels Are Eliminated From the Normal Cell Layer During Differentiation.

Insulin receptor substrate (IRS)-1 is a major substrate of insulin-like growth factor (IGF)-I receptors. It is well-known that IGF-I and II play essential roles in myogenesis progression. Herein, we report an unexpected phenomenon that IRS-1-overexpressing L6 myoblasts are eliminated from normal cell layers at the beginning of differentiation. Initially, the IRS protein level and apoptosis were examined during myogenic differentiation in L6 myoblasts. We found that the IRS-1 protein level decreased, whereas active caspase 3 increased around 1 day after induction of differentiation. The addition of a pan-caspase inhibitor, Z-VAD-FMK, inhibited differentiation-induced suppression of the IRS-1 protein level. Apoptosis was not enhanced in L6 myoblasts stably expressing high levels of IRS-1 (L6-IRS-1). However, when L6-IRS-1 was cultured with control cells (L6-mock), we observed that L6-IRS-1 was eliminated from the cell layer. We have recently reported that, in L6-IRS-1, internalization of the IGF-I receptor was delayed and IGF signal activation was sustained for a longer period than in L6-mock. When cells stably expressing IRS-1 3YA mutant, which could not maintain the IGF signals, were cultured with normal cells, elimination from the cell layer was not detected. These data suggested that the high level of IRS-1 in myoblasts induces elimination from the cell layer due to abnormal sustainment of IGF-I receptor activation.

Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids.

Human organoid systems recapitulate in vivo organ architecture yet fail to capture complex pathologies such as inflammation and fibrosis. Here, using 11 different healthy and diseased pluripotent stem cell lines, we developed a reproducible method to derive multi-cellular human liver organoids composed of hepatocyte-, stellate-, and Kupffer-like cells that exhibit transcriptomic resemblance to in vivo-derived tissues. Under free fatty acid treatment, organoids, but not reaggregated cocultured spheroids, recapitulated key features of steatohepatitis, including steatosis, inflammation, and fibrosis phenotypes in a successive manner. Interestingly, an organoid-level biophysical readout with atomic force microscopy demonstrated that organoid stiffening reflects the fibrosis severity. Furthermore, organoids from patients with genetic dysfunction of lysosomal acid lipase phenocopied severe steatohepatitis, rescued by FXR agonism-mediated reactive oxygen species suppression. The presented key methodology and preliminary results offer a new approach for studying a personalized basis for inflammation and fibrosis in humans, thus facilitating the discovery of effective treatments.

IRS-2 deubiquitination by USP9X maintains anchorage-independent cell growth via Erk1/2 activation in prostate carcinoma cell line.

Insulin-like growth factors (IGFs) have been shown to induce proliferation of many types of cells. Insulin receptor substrates (IRSs) are major targets of IGF-I receptor (IGF-IR) tyrosine kinase activated by IGFs, and are known to play important roles in the activation of downstream signaling pathways, such as the Erk1/2 pathway. Dysregulation of IGF signaling represents a central tumor promoting principle in human carcinogenesis. Prostate carcinoma is highly dependent on the IGF/IGF-IR/IRS axis. Here we identified the deubiquitinase, ubiquitin specific peptidase 9X (USP9X) as a novel binding partner of IRS-2. In a human prostate carcinoma cell line, small interfering RNA (siRNA)-mediated knockdown of USP9X reduced IGF-IR as well as IRS-2 protein levels and increased their ubiquitination. Knockdown of USP9X suppressed basal activation of the Erk1/2 pathway, which was significantly restored by exogenous expression of IRS-2 but not by IGF-IR, suggesting that the stabilization of IRS-2 by USP9X is critical for basal Erk1/2 activation. Finally, we measured anchorage-independent cell growth, a characteristic cancer feature, by soft-agar colony formation assay. Knockdown of USP9X significantly reduced anchorage-independent cell growth of prostate carcinoma cell line. Taken all together, our findings indicate that USP9X is required for the promotion of prostate cancer growth by maintaining the activation of the Erk1/2 pathway through IRS-2 stabilization.

Serine Phosphorylation by mTORC1 Promotes IRS-1 Degradation through SCFß-TRCP E3 Ubiquitin Ligase.

The insulin receptor substrate IRS-1 is a key substrate of insulin and insulin-like growth factor (IGF) receptor tyrosine kinases that mediates their metabolic and growth-promoting actions. Proteasomal degradation of IRS-1 is induced following activation of the downstream kinase mTOR complex 1 (mTORC1) to constitute a negative feedback loop. However, the underlying mechanism remains poorly understood. Here we report that Ser 422 of IRS-1 is phosphorylated by mTORC1 and required for IRS-1 degradation induced by prolonged IGF stimulation. Phosphorylation of Ser 422 then recruits the SCFß-TRCP E3 ligase complex, which catalyzes IRS-1 ubiquitination. Phosphorylation-dependent IRS-1 degradation contributes to impaired growth and survival responses to IGF in cells lacking TSC2, a negative regulator of mTORC1. Inhibition of IRS-1 degradation promotes sustained Akt activation in IGF-stimulated cells. Our work clarifies the nature of the IRS-1-mTORC1 feedback loop and elucidates its role in temporal regulation of IGF signaling.

IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling.

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling.

Catch-Up Growth in Zebrafish Embryo Requires Neural Crest Cells Sustained by Irs1 Signaling.

Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as "catch-up" growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1-mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activation‒restored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia.