A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

Stem cells expand potency and alter tissue fitness by accumulating diverse epigenetic memories.

Immune and tissue stem cells retain an epigenetic memory of inflammation that intensifies sensitivity to future encounters. We investigated whether and to what consequence stem cells possess and accumulate memories of diverse experiences. Monitoring a choreographed response to wounds, we found that as hair follicle stem cells leave their niche, migrate to repair damaged epidermis, and take up long-term foreign residence there, they accumulate long-lasting epigenetic memories of each experience, culminating in post-repair epigenetic adaptations that sustain the epidermal transcriptional program and surface barrier. Each memory is distinct, separable, and has its own physiological impact, collectively endowing these stem cells with heightened regenerative ability to heal wounds and broadening their tissue-regenerating tasks relative to their naïve counterparts.

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease.

BACKGROUND & AIMS: There are few in vitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. METHODS: We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. RESULTS: Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. CONCLUSIONS: We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

A Chemically Defined Feeder-free System for the Establishment and Maintenance of the Human Naive Pluripotent State.

The distinct states of pluripotency in the pre- and post-implantation embryo can be captured in vitro as naive and primed pluripotent stem cell cultures, respectively. The study and application of the naive state remains hampered, particularly in humans, partially due to current culture protocols relying on extraneous undefined factors such as feeders. Here we performed a small-molecule screen to identify compounds that facilitate chemically defined establishment and maintenance of human feeder-independent naive embryonic (FINE) stem cells. The expression profile in genic and repetitive elements of FINE cells resembles the 8-cell-to-morula stage in vivo, and only differs from feeder-dependent naive cells in genes involved in cell-cell/cell-matrix interactions. FINE cells offer several technical advantages, such as increased amenability to transfection and a longer period of genomic stability, compared with feeder-dependent cells. Thus, FINE cells will serve as an accessible and useful system for scientific and translational applications of naïve pluripotent stem cells.

Skin and Its Regenerative Powers: An Alliance between Stem Cells and Their Niche.

Tissues have a natural capacity to replace dying cells and to heal wounds. This ability resides in resident stem cells, which self-renew, preserve, and repair their tissue during homeostasis and following injury. The skin epidermis and its appendages are subjected to daily assaults from the external environment. A high demand is placed on renewal and regeneration of the skin's barrier in order to protect the body from infection and dehydration and to heal wounds. This review focuses on the epithelial stem cells of skin, where they come from, where they reside, and how they function in normal homeostasis and wound repair.

Looping around Reprogramming: The Topological Memory of Induced Pluripotency.

Genome architecture is associated with cellular identity, but how this organization changes during reprogramming is not well understood. Now in Cell Stem Cell, Krijger et al. (2016) and Beagan et al. (2016) report 3D chromatin interaction maps before and after reprogramming, providing evidence for topological memory in induced pluripotent stem cells.

Transcriptomic profiling of human embryonic stem cells upon cell cycle manipulation during pluripotent state dissolution.

While distinct cell cycle structures have been known to correlate with pluripotent or differentiated cell states [1], there is no evidence on how the cell cycle machinery directly contributes to human embryonic stem cell (hESC) pluripotency. We established a determinant role of cell cycle machineries on the pluripotent state by demonstrating that the specific perturbation of the S and G2 phases can prevent pluripotent state dissolution (PSD) [2]. Active mechanisms in these phases, such as the DNA damage checkpoint and Cyclin B1, promote the pluripotent state [2]. To understand the mechanisms behind the effect on PSD by these pathways in hESCs, we performed comprehensive gene expression analysis by time-course microarray experiments. From these datasets, we observed expression changes in genes involved in the TGFß signaling pathway, which has a well-established role in hESC maintenance [3], [4], [5]. The microarray data have been deposited in NCBI's Gene Expression Omnibus (GEO) and can be accessed through GEO Series accession numbers GSE62062 and GSE63215.

Biological Networks Governing the Acquisition, Maintenance, and Dissolution of Pluripotency: Insights from Functional Genomics Approaches.

The repertoire of transcripts encoded by the genome contributes to the diversity of cellular states. Functional genomics aims to comprehensively uncover the roles of these transcripts to reconstruct biological networks and transform this information into useful knowledge. High-throughput functional screening has served as a powerful genetic discovery tool by enabling massively parallel implementation of biological assays. In recent years, high-throughput screening has unearthed crucial players in the regulation of different aspects of pluripotency, which is a unique property that enables a cell to differentiate into multiple cell types of the three major lineages. Pluripotency thus represents an interesting biological paradigm for studying the acquisition, maintenance, and dissolution of cellular states. In this review, we highlight the major findings of high-throughput studies to dissect these three aspects of pluripotency for the mouse and human systems. Collectively, they provide new insights into cell fate maintenance and transition. In addition, we also discuss the opportunities and challenges awaiting high-throughput screening in the future.

Deterministic Restriction on Pluripotent State Dissolution by Cell-Cycle Pathways.

During differentiation, human embryonic stem cells (hESCs) shut down the regulatory network conferring pluripotency in a process we designated pluripotent state dissolution (PSD). In a high-throughput RNAi screen using an inclusive set of differentiation conditions, we identify centrally important and context-dependent processes regulating PSD in hESCs, including histone acetylation, chromatin remodeling, RNA splicing, and signaling pathways. Strikingly, we detected a strong and specific enrichment of cell-cycle genes involved in DNA replication and G2 phase progression. Genetic and chemical perturbation studies demonstrate that the S and G2 phases attenuate PSD because they possess an intrinsic propensity toward the pluripotent state that is independent of G1 phase. Our data therefore functionally establish that pluripotency control is hardwired to the cell-cycle machinery, where S and G2 phase-specific pathways deterministically restrict PSD, whereas the absence of such pathways in G1 phase potentially permits the initiation of differentiation.

Klf2 is an essential factor that sustains ground state pluripotency.

The maintenance of mouse embryonic stem cells (mESCs) requires LIF and serum. However, a pluripotent "ground state," bearing resemblance to preimplantation mouse epiblasts, can be established through dual inhibition (2i) of both prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. While Gsk3 inhibition has been attributed to the transcriptional derepression of Esrrb, the molecular mechanism mediated by Mek inhibition remains unclear. In this study, we show that Krüppel-like factor 2 (Klf2) is phosphorylated by Erk2 and that phospho-Klf2 is proteosomally degraded. Mek inhibition hence prevents Klf2 protein phosphodegradation to sustain pluripotency. Indeed, while Klf2-null mESCs can survive under LIF/Serum, they are not viable under 2i, demonstrating that Klf2 is essential for ground state pluripotency. Importantly, we also show that ectopic Klf2 expression can replace Mek inhibition in mESCs, allowing the culture of Klf2-null mESCs under Gsk3 inhibition alone. Collectively, our study defines the Mek/Erk/Klf2 axis that cooperates with the Gsk3/Tcf3/Esrrb pathway in mediating ground state pluripotency.

Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast.

Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. Despite sharing the common property of pluripotency, hESCs are notably distinct from epiblast cells of the preimplantation blastocyst. Here we use a combination of three small-molecule inhibitors to sustain hESCs in a LIF signaling-dependent hESC state (3iL hESCs) with elevated expression of NANOG and epiblast-enriched genes such as KLF4, DPPA3, and TBX3. Genome-wide transcriptome analysis confirms that the expression signature of 3iL hESCs shares similarities with native preimplantation epiblast cells. We also show that 3iL hESCs have a distinct epigenetic landscape, characterized by derepression of preimplantation epiblast genes. Using genome-wide binding profiles of NANOG and OCT4, we identify enhancers that contribute to rewiring of the regulatory circuitry. In summary, our study identifies a distinct hESC state with defined regulatory circuitry that will facilitate future analysis of human preimplantation embryogenesis and pluripotency.

Driving pluripotency and reprogramming: nuclear receptors at the helm.

The identity of a cell is determined by the concerted interplay of multiple molecular modulators such as transcription factors, chromatin modifiers and signalling mediators. Among these, the transcriptional circuitry holds great influence on the specification and maintenance of a cellular state, and its perturbation can trigger a transition to another cell state. This is particularly striking in the field of pluripotency, where tempering the expression levels of one or few transcription factors is sufficient to induce the loss or acquisition of the pluripotent state. Recently, nuclear receptors, a class of transcription factors, have emerged as major players in the molecular network governing pluripotency. In this review, we discuss the importance of nuclear receptors in embryonic stem cell self-renewal, differentiation and cellular reprogramming, highlighting recent discoveries as well as providing an outlook in stem cell and nuclear receptor research.

FoxO: a new addition to the ESC cartel.

The forkhead box O (FoxO) family is involved in diverse cellular processes such as tumor suppression, stress response, and metabolism. In a recent Nature Cell Biology Letter, Zhang et al. (2011) uncover a novel role for FoxO proteins in regulating the identity of human ESCs.

Choreographing pluripotency and cell fate with transcription factors.

The cellular identity of both pluripotent and differentiated cells is defined by the concerted interplay of transcriptional factors as well as other modulators such as epigenetic and signaling mediators. Therefore, the manipulation of a cell's transcriptional network directly facilitates inter-conversion between cellular identities. Understanding the molecular regulation of cell fate changes, including those involved in pluripotency, is crucial in realizing the practical potential of pluripotent and induced cell types. Here we review the advancements in the role of transcription factors in pluripotency, as well as in the conversion between and within pluripotent and somatic cell types.