Scientific Resources

Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist,intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression,IFN-λ responsiveness is restricted mainly to epithelial cells. Here,we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially,we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently,HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second,in contrast to the type I IFN system,polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally,we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity. View Publication

Sterol regulatory element-binding protein-2 (SREBP-2) activates transcription of all genes needed for cholesterol biosynthesis. To study SREBP-2 function in intestine,we generated a mouse model (Vil-BP2(-/-) ) in which Cre recombinase ablates SREBP-2 in intestinal epithelia. Intestines of Vil-BP2(-/-) mice had reduced expression of genes required for sterol synthesis,in vivo sterol synthesis rates,and epithelial cholesterol contents. On a cholesterol-free diet,they displayed chronic enteropathy with histological abnormalities of both villi and crypts,growth restriction,and reduced survival that was prevented by supplementation of cholesterol in the diet. Likewise,SREBP-2-deficient enteroids required exogenous cholesterol for growth. Blockade of luminal cholesterol uptake into enterocytes with ezetimibe precipitated acutely lethal intestinal damage in Vil-BP2(-/-) mice,highlighting the critical interplay in the small intestine of sterol absorption via NPC1L1 and sterol synthesis via SREBP-2 in sustaining the intestinal mucosa. These data show that small intestine requires SREBP-2 to drive cholesterol synthesis that sustains the intestinal epithelia when uptake of cholesterol from the gut lumen is not available,and provide a unique example of cholesterol auxotrophy expressed in an intact,adult mammal. View Publication

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells,it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system,aerobic glycolysis,and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells,while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass,increased proton leak,and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference,and highlight the importance of further research concerning energy metabolism of stem cells. View Publication

The current in vitro or in vivo intestinal fibrosis models have many limitations. Recent advancements in the isolation and culturing of organoids has led to development of various three-dimensional (3D) intestinal disease models with in vivo physiology. In this study,we generated an organoid-based epithelial to mesenchymal transition (OEMT) model,which could be used as a novel intestinal fibrosis model. Intestinal epithelial organoids (IEOs) were isolated and cultured from the small intestines of normal mice. IEOs were treated with transforming growth factor- β1 (TGF-β1) or Tumor necrosis factor-α (TNF-α) to evaluate their phenotypic change. Raw 264.7 cells (macrophage) stimulated with lipopolysaccharide were co-cultured with IEOs in growth media with or without TGF-β1. TGF-β1 alone slightly induced epithelial to mesenchymal transition (EMT) in the IEOs but mainly disrupted them. Macrophage released cytokines synergistically induced mesenchymal phenotypic changes in TGF-β1 stimulated intestinal organoids. TNF-α and TGF-β1 synergistically induced proliferation of mesenchymal cells as well as EMT in the IEOs. We generated a novel OEMT model based on our finding that TNF-α and TGF-β synergistically induce type 2 EMT in IEOs. This 3D EMT model with in vivo physiology could be used to study EMT associated intestinal fibrosis. View Publication

iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed,iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro,phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation,validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data,typically by relating the obtained levels of gene expression to the levels of expression of a house keeping gene"� View Publication

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4,KLF4,SOX2,LIN28,cMYC and nuclear GFP over 18 days,then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4,NANOG,TRA-1-60,and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal,ectodermal,and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation,cells contracted spontaneously,expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns,expressed cardiac troponin T and β-myosin heavy chain,showed upregulation of NKX2.5,ISL-1 and cardiac troponin T with downregulation of POU5F1,and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes. View Publication

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine,including individualized,patient-specific stem cell-based treatments. There are,however,few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues,ideally comprising direct-write printing of cells for encapsulation,proliferation,and differentiation. Here,such a method,employing a clinically amenable polysaccharide-based bioink,is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically,There are extrusion printed the bioink including iPSCs,alginate (Al; 5% weight/volume [w/v]),carboxymethyl-chitosan (5% w/v),and agarose (Ag; 1.5% w/v),crosslinked the bioink in calcium chloride for a stable and porous construct,proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm,ectoderm,and mesoderm,or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined,scalable,and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine. View Publication

Genome-wide association studies (GWASs) have identified many disease-associated variant alleles,but understanding whether and how different genes/loci interact requires a platform for probing how the variant alleles act mechanistically. Isogenic mutant human embryonic stem cells (hESCs) provide an unlimited resource to derive and study human disease-relevant cells. Here,we focused on CDKAL1,linked by GWASs to diabetes. Through transcript profiling,we find that expression of the metallothionein (MT) gene family,also linked by GWASs to diabetes,is significantly downregulated in CDKAL1(-/-) cells that have been differentiated to insulin-expressing pancreatic beta-like cells. Forced MT1E expression rescues both hypersensitivity of CDKAL1 mutant cells to glycolipotoxicity and pancreatic beta-cell dysfunction in vitro and in vivo. MT1E functions at least in part through relief of ER stress. This study establishes an isogenic hESC-based platform to study the interaction of GWAS-identified diabetes gene variants and illuminate the molecular network impacting disease progression. View Publication

Human pluripotent stem cells (HPSCs) cultured in conditions that maintain pluripotency via FGF and TGFβ signaling have been described as being in a primed state. These cells have been shown to exhibit characteristics more closely related to mouse epiblast-derived stem cells than to so called naïve mouse PSCs said to possess a more ground state pluripotency that mimics the early mouse embryo inner cell mass. Initial attempts to create culture conditions favorable for generation of naïve HPSCs from primed HPSCs has required the use of mouse embryonic fibroblasts as a feeder layer to support this transition. A protocol for the routine derivation and maintenance of naïve HPSCs in completely defined conditions is highly desirable for stem cell researchers to enhance the study and clinical translation of naïve HPSCs. Here we describe a standard protocol for transitioning primed HPSCs to a naïve state using commercial RSet media and xeno-free recombinant vitronectin. View Publication

Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes,they have not demonstrated scalability,safety,and in vivo engraftment,which are key aspects for their future clinical application. Here we recapitulate one prominent protocol,and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation,the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle,we incorporated the inducible PAX7 lentiviral system into this protocol,which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7(+) myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases. View Publication

Microglia,the immune cells of the brain,are crucial to proper development and maintenance of the CNS,and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology,we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes,expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca(2+) transients,whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines. View Publication

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here,we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9(+)/RUNX2(+) cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium,mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro,but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether,we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues. View Publication