Definitive Endoderm Induction Research Articles

In vitro generation of transplantable insulin-producing cells from canine adipose-derived mesenchymal stem cells

Canine mesenchymal stem cells (cMSCs) have potential applications for regenerative therapy, including the generation of insulin-producing cells (IPCs) for studying and treating diabetes. In this study, we established a useful protocol for generating IPCs from canine adipose mesenchymal stem cells (cAD-MSCs). Subsequently, in vitro preservation of pluronic F127-coated alginate (ALGPA)-encapsulated cAD-MSC-derived IPCs was performed to verify ready-to-use IPCs. IPCs were induced from cAD-MSCs with the modulated three-stepwise protocol. The first step of definitive endoderm (DE) induction showed that the cooperation of Chir99021 and Activin A created the effective production of Sox17-expressed DE cells. The second step for pancreatic endocrine (PE) progenitor induction from DE indicated that the treatment with taurine, retinoic acid, FGF2, EGF, TGFβ inhibitor, dorsomorphin, nicotinamide, and DAPT showed the significant upregulation of the pancreatic endocrine precursor markers Pdx1 and Ngn3. The last step of IPC production, the combination of taurine, nicotinamide, Glp-1, forskolin, PI3K inhibitor, and TGFβ inhibitor, yielded efficiently functional IPCs from PE precursors. Afterward, the maintenance of ALGPA-encapsulated cAD-MSC-derived IPCs with VSCBIC-1, a specialized medium, enhanced IPC properties. Conclusion, the modulated three-stepwise protocol generates the functional IPCs. Together, the encapsulation of cAD-MSC-derived IPCs and the cultivation with VSCBIC-1 enrich the maturation of generated IPCs.

Homogeneous Differentiation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (iPSCs) could provide an unlimited source of liver cells for regenerative medicine, disease modeling, drug screening, and toxicology studies. Here we describe a stepwise improved protocol that enables highly efficient, homogeneous, and reproducible differentiation of human iPSCs into functional hepatocytes through controlling all three stages of hepatocyte differentiation, starting from a single cell (non-colony) culture of iPSCs, through homogeneous definitive endoderm induction and highly efficient hepatic specification, and finally arriving at matured HLCs. The final population of cells exhibits morphology closely resembling that of primary human hepatocytes, and expresses specific hepatic markers as evidenced by immunocytochemical staining. More importantly, these HLCs demonstrate key functional characteristics of mature hepatocytes, including major serum protein (e.g., albumin, fibronectin, and alpha-1 antitrypsin) secretion, urea synthesis, glycogen storage, and inducible cytochrome P450 activity.

Capacitation of Human Naïve Pluripotent Stem Cells.

Naïve and primed pluripotent stem cells resemble epiblast cells of the pre-implantation and post-implantation embryo, respectively. This chapter describes a simple experimental system for the efficient and consistent transition of human pluripotent stem cells (hPSCs) from the naïve to the primed state, which is a process called capacitation. Naïve hPSCs after capacitation can be differentiated further to somatic lineages, thus reproducing the order of developmental events in the embryo. Protocols for the induction of neuroectoderm, definitive endoderm, and paraxial mesoderm from hPSCs after capacitation and also from conventionally derived primed hPSCs are included in the chapter. Importantly, hPSC capacitation closely recapitulates transcriptional, metabolic, signaling, and cell polarity changes in the epiblast of primate embryos, and therefore offers a unique in vitro model of human peri-implantation development.

IPSC-Derived Thymic Epithelial Progenitor Cells As Cell-Based Therapy to Restore Thymic Function in Hematopoietic Stem Cell Transplant Recipients

Hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for many malignant and non-malignant diseases to date. The thymus plays a key role in post-transplant immune reconstitution. The outcome of a HSCT and the recipient thymic function are intimately intertwined. In fact, the production of mature T cells occurs in the thymus and depends on the interaction of hematopoietic progenitor cells with non-hematopoietic stromal elements, notably thymic epithelial cells (TECs). Through positive and negative selection, a healthy thymus generates a broad and self-tolerant T cell receptor (TCR) repertoire that protects from infection and prevents autoimmunity. In addition, the thymus gives rise to regulatory T-cells which confine immune responses and further promote central tolerance. At odds with its importance for successful transplant outcomes, the thymus is exquisitely sensitive to a wide range of insults encountered during the peri-transplant period, including cytotoxic agents, steroids and irradiation. In the allogeneic transplant setting, acute graft-versus-host disease (GVHD) can cause further thymic injury. Thymic injury in turn, leads to susceptibility to infections and autoimmune complications. Moreover, it is now widely recognized that thymic injury leading to defective deletion of self-reactive T-cells along with misguided T-helper cell polarization and B-cell activation constitute major drivers of chronic GVHD. Together these represent the leading causes of non-relapse-related transplant mortality and morbidity. As such, HSCT can precipitate a vicious cycle, in which the success of the HSCT procedure hinges on intact thymic function, but the HSCT process itself often causes irrecoverable thymic injury (Figure1). Thymic function naturally declines with age with little to no endogenous potential to regenerate. Current treatment options for thymic injury during HSCT are limited to symptomatic therapy. Therefore, novel approaches to cure irreversible thymic injury are critically needed.To address this need, our goal is to generate fully functional thymic epithelial progenitor cells (TEPCs) from hPSCs as a stem cell-based therapy for thymic regeneration and restoration of immune competence in HSCT recipients. Building on the efficient induction of definitive endoderm from human pluripotent stem cells (hPSCs) by directing signals controlling lineage bifurcations, we have developed a novel platform that drives TEPC development through sequential induction of the intermediaries anterior foregut (AFG) and pharyngeal pouch endoderm (PPE). Here we show that TEPCs derived in vitro from hPSCs or hESCs express the key thymic transcription factor FOXN1 along with Keratin 5 and Keratin 8, the primary cytoskeletal building blocks of medullary and cortical TECs, respectively. In addition, in vitro derived TEPCs express MHC class II to varying degrees (Figure 2). To optimize our in vitro platform, modifications of key-signaling pathways in the differentiation protocol are validated by comparing the transcriptome of hPSC-derived TEPCs to that of primary human medullary and cortical thymic epithelial cells using next generation sequencing. Our ongoing work is testing the capacity of hPSC-derived TEPCs to promote T-cell maturation, positive and negative selection resulting in the development of a broad and self-tolerant TCR repertoire from adoptively transferred human HSPCs in a humanized mouse model.Through sequential validation of fidelity, functionality, transplantability and sustainability of hPSC-derived TEPCs, we strive to develop an experimental model that can be translated by a clinical Good Manufacturing Practice (cGMP) facility for use in bone marrow transplant recipients.Ultimately, the potential applications of in vitro derived TEPCs are not confined to restoring thymic function in the HSCT setting. TEPCs derived from solid organ donors hold the promise of inducing tolerance to donor tissues, thereby preventing rejection of the transplanted donor organ. Leveraging the body's own ability to educate T-cells through regenerating thymic tissues could represent a novel approach to addressing key challenges in transplantation immunology. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The potential for designing urothelial carcinomas using pluripotent stem cell-based systems

Despite a variety of therapeutic options for urothelial carcinoma of the bladder (UCB) in the early stages, the efficacy of treatment options in metastatic disease remains modest. Investigating the influence of different mutational patterns during the complex process of carcinogenesis may enable the development of innovative therapeutic agents. The differentiation of human pluripotent stem cells (hPSCs) into mature bladder urothelial cells offers a distinct platform to study embryonic development and disease modelling in the human urothelium. Given the precisely defined genetic background of those cells, it becomes evident that hPSCs-derived urothelium provides a valuable tool to study the processes during early carcinogenesis upon defined oncogenic stimuli. Differentiation of hPSCs into urothelial cells was performed by mimicking different stages of embryonic development with the induction of definitive endoderm, a hindgut differentiation step and finally, the induction of mature urothelium. To study the influence of different genes on the carcinogenesis of UCB, knockouts of TP53, BRCA2 and ATM were generated by CRISPR-Cas9 mediated gene editing. A protocol for the differentiation of urothelial cells from hPSCs was established by reaching different milestones of embryonic development (1. definitive endoderm, 2. hindgut, 3. urothelium). The induction of urothelium was confirmed by upregulation of uroplakins (UPK2, UPK3a) in the differentiated cells. Functional relevance of the knockouts was confirmed by reduced gene and protein expression (BRCA2, TP53), the presence of truncated proteins (ATM and BRCA2) and enhanced sensitivity to genotoxic stress. An efficient generation of urothelial cells from hPSCs is feasible. The functional relevance of different gene knockouts underlines the validity of the gene-editing strategy. In future studies, the significance of exogenous and endogenous carcinogenic stimuli in the context of distinct mutational signatures during early carcinogenesis of UCB can be investigated in this defined genetic background.

The Expression of Wnt5a Gene Throughout Definitive Endoderm Induction Process in Induced Pluripotent Stem Cells

Background: Induced pluripotent stem cells (iPSCs) have the ability to proliferate indefinitely and differentiate into three germ layers of ectoderm, mesoderm, and endoderm. Definitive induction is the first and the most delicate stage of differentiation of various iPSC-derived organs. It has been found that the Wnt signaling pathway implicates in embryogenesis, organogenesis, and cell communication. Objectives: In the present study, we aimed to investigate the expression pattern of the Wnt5a gene as an indicator of non-canonical Wnt signaling activity during definitive endoderm induction of iPSCs. Methods: Human iPSCs (RSCB0042) were acquired from Royan stem cell bank of Royan Institute (Tehran, Iran). The iPSCs were cultured on a feeder layer of mitomycin-inactivated mouse embryonic fibroblasts (MEF), and iPSC colonies were collected for embryoid body (EB) generation by suspension culture method. Then endoderm induction step was performed using a series of small molecules. The quantitative real-time PCR was used to assess the mRNA expression of wnt5a, Nanog, OCT4, SOX17, and FOXA2 genes. Results: The production of efficient EBs confirmed by a decrease in Nanog and Oct4 gene expression and the success of DE (definite endoderm) induction step was confirmed by a high expression level of DE specific genes, Sox17, and FoxA2. A significant upregulation of Wnt5a in EB samples and a minor decrease at day 4 was observed. However, the differentiation process followed by an incremental fashion in Wnt5a mRNA expression starting from day 4 of differentiation among the samples of days 6 and 8 (DE stage). Conclusions: Our results suggest that Wnt5a is more activated at the later steps of endoderm induction rather than the early steps, which may be due to the stimulation of canonical Wnt signaling. Finding the expression level of Wnt5a could rise insights for developing more efficient differentiation induction protocols.

Integrative protocols for an in vitro generation of pancreatic progenitors from human dental pulp stem cells

Efficiency of the induction protocol is crucial for the generation of insulin-producing cells (IPCs) from human dental pulp stem cells (hDPSCs). Here, we established the integrative induction protocol by merging genetic manipulation technique with our previous published 3-step induction protocol aiming to enhance the pancreatic progenitor commitment and production yield. We found that the overexpression of PDX1 following with 3-step induction protocol were able to generate the 3-dimensional (3D) colony structure of pancreatic progenitors (PPs) with the beneficial trends of pancreatic endoderm commitment and production yield, while other protocols using the prolong maintenance of PDX1-overexpressed hDPSCs and the PDX1 overexpression after definitive endoderm induction were unable to generate and sustain the 3D structure of the colonies. Further Notch signaling manipulation by DAPT treatment showed lesser degree of positive effects on progenitor commitment and production yield. Although the generated PPs from the integrative protocol expressed pancreatic mRNA markers along with pro-insulin and insulin proteins, they still contained the defective glucose-responsive C-peptide secretion. Only basal secreted C-peptide level was observed. In summary, the integrative induction protocol potentially enhanced the PP generation with high colony production yield and could serve as an efficient platform for further hDPSC-derived IPC production and maturation.

High-Dimensional Design-Of-Experiments Extracts Small-Molecule-Only Induction Conditions for Dorsal Pancreatic Endoderm from Pluripotency.

SummaryThe derivation of endoderm and descendant organs, such as pancreas, liver, and intestine, impacts disease modeling and regenerative medicine. Use of TGF-β signaling agonism is a common method for induction of definitive endoderm from pluripotency. By using a data-driven, High-Dimensional Design of Experiments (HD-DoE)-based methodology to address multifactorial problems in directed differentiation, we found instead that optimal conditions demanded BMP antagonism and retinoid input leading to induction of dorsal foregut endoderm (DFE). We demonstrate that pancreatic identity can be rapidly, and robustly, induced from DFE and that such cells are of dorsal pancreatic identity. The DFE population was highly competent to differentiate into both stomach organoids and pancreatic tissue types and able to generate fetal-type β cells through two subsequent differentiation steps using only small molecules. This alternative, rapid, and low-cost basis for generating pancreatic insulin-producing cells may have impact for the development of cell-based therapies for diabetes.

Directed differentiation of human induced pluripotent stem cells into mature stratified bladder urothelium

For augmentation or reconstruction of urinary bladder after cystectomy, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has recently received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs, and no report has demonstrated the stratified structure, which is a particularly important attribute of the barrier function of mature bladder urothelium. In present study, we developed a method for the directed differentiation of hiPSCs into mature stratified bladder urothelium. The caudal hindgut, from which the bladder urothelium develops, was predominantly induced via the high-dose administration of CHIR99021 during definitive endoderm induction, and this treatment subsequently increased the expressions of uroplakins. Terminal differentiation, characterized by the increased expression of uroplakins, CK13, and CK20, was induced with the combination of Troglitazone + PD153035. FGF10 enhanced the expression of uroplakins and the stratification of the epithelium, and the transwell culture system further enhanced such stratification. Furthermore, the barrier function of our urothelium was demonstrated by a permeability assay using FITC-dextran. According to an immunohistological analysis, the stratified uroplakin II-positive epithelium was observed in the transwells. This method might be useful in the field of regenerative medicine of the bladder.

Fine Tuning of Hepatocyte Differentiation from Human Embryonic Stem Cells: Growth Factor vs. Small Molecule-Based Approaches.

Human embryonic stem cells (hESCs) are being utilized in diverse areas of studies such as development and disease modeling, cell replacement therapy, or drug toxicity testing because of their potential to be differentiated into any cell type in the body. The directed differentiation of hESCs into hepatocytes could provide an invaluable source of liver cells for various liver-based applications. Therefore, several protocols have been established in the past for hESC-hepatocyte differentiation based on the knowledge of signaling pathways and growth factors involved in different stages of embryonic hepatogenesis. Although successful derivation of hepatocytes has been achieved through these protocols, the efficiency is not always ideal. Herein, we have tested several combinations of published protocols, for example, growth factor vs. small molecule and different time durations of treatment for definitive endoderm (DE) induction and further hepatocyte differentiation to develop an efficient DE induction and hepatocyte differentiation in a highly reproducible manner based on the stage-specific marker expression and functional analysis.

Establishment of Insulin-Producing Cells From Human Embryonic Stem Cells Underhypoxic Condition for Cell Based Therapy.

Diabetes mellitus (DM) is a group of diseases characterized by abnormally high levels of glucose in the blood stream. In developing a potential therapy for diabetic patients, pancreatic cells transplantation has drawn great attention. However, the hinder of cell transplantation for diabetes treatment is insufficient sources of insulin-producing cells. Therefore, new cell based therapy need to be developed. In this regard, human embryonic stem cells (hESCs) may serve as good candidates for this based on their capability of differentiation into various cell types. In this study, we designed a new differentiation protocol that can generate hESC-derived insulin-producing cells (hES-DIPCs) in a hypoxic condition. We also emphasized on the induction of definitive endoderm during embryoid bodies (EBs) formation. After induction of hESCs differentiation into insulin-producing cells (IPCs), the cells obtained from the cultures exhibited pancreas-related genes such as Pdx1, Ngn3, Nkx6.1, GLUT2, and insulin. These cells also showed positive for DTZ-stained cellular clusters and contained ability of insulin secretion in a glucose-dependent manner. After achievement to generated functional hES-DIPCs in vitro, some of the hES-DIPCs were then encapsulated named encapsulated hES-DIPCs. The data showed that the encapsulated cells could possess the function of insulin secretion in a time-dependent manner. The hES-DIPCs and encapsulated hES-DIPCs were then separately transplanted into STZ-induced diabetic mice. The findings showed the significant blood glucose levels regulation capacity and declination of IL-1β concentration in all transplanted mice. These results indicated that both hES-DIPCs and encapsulated hES-DIPCs contained the ability to sustain hyperglycemia condition as well as decrease inflammatory cytokine level in vivo. The findings of this study may apply for generation of a large number of hES-DIPCs in vitro. In addition, the implication of this work is therapeutic value in type I diabetes treatment in the future. The application for type II diabetes treatment remain to be investigated.

Differences in definitive endoderm induction approaches using growth factors and small molecules.

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.

GATA6 Plays an Important Role in the Induction of Human Definitive Endoderm, Development of the Pancreas, and Functionality of Pancreatic βCells.

SummaryInduced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into β-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient’s disease. GATA6 mutant β-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and β-like cell functionality in humans.

Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used invitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells.

Efficient generation of functional pancreatic β-cells from human induced pluripotent stem cells.

Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells. A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo. Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.

Stem Cell‐based Biliary Organoids and Gallbladder‐like Structures for Disease Modeling of Primary Sclerosing Cholangitis and Alagille Syndrome

Cholangiopathies such as primary sclerosing cholangitis (PSC), Alagille Syndrome (AS), polycystic liver disease and cholangiocarcinoma result in end‐stage liver disease. To understand the cell and molecular basis of these progressive disorders, a cell‐based interface would be very useful. Availability of human primary cholangiocytes is a limiting factor, and pluripotent stem cells provide an exciting option of generating unlimited quantity of cholangiocytes for in vitro disease modeling purposes. Our goal is to develop and in vitro platform using stem cell‐derived cholangiocytes to model the pathogenesis of PSC and AS. We established a three stage differentiation program to differentiate pluripotent stem cells to biliary epithelial cells. The first stage involves hepatoblast formation by definitive endoderm induction; a second phase entails biliary specification prompting cholangiocyte progenitors and the final third stage of cholangiocyte maturation. At 16 days post‐differentiation, the expression of biliary markers SOX9, CFTR, cytokeratin 7 (CK7) and CK19 were verified by RT‐qPCR and immunofluorescence assay. Marker expression was similar to that of human gallbladder tissue. Day 16 cells were induced to form organoids in 3D matrigel culture. The cells were organized to become branched tubular structures. The histology of these organoids showed the formation of duct‐like structures which are cytokeratin 19 and CK 8/18 positive with a defined collagen IV positive basement membrane. The tubules were surrounded by connective tissue‐like structures. By day 33, pear‐shaped gallbladder‐like cystic structures emerged. Immunohistochemistry of these tubular organoids showed polarized columnar cells lining the ductular structures. Zo‐1 tight junction proteins were localized to the apical side of the cells facing the lumen. In conclusion, we have developed a 3D in vitro platform to study the bile duct structure formation based on stem cell‐derived cholangiocytes. This system is currently being investigated for studying the effect of primary sclerosing cholangitis risk genes and the deregulation of NOTCH signaling pathway of Alagille Syndrome model.

Stage-specific regulation of the WNT/β-catenin pathway enhances differentiation of hESCs into hepatocytes

Hepatocytes differentiated from human embryonic stem cells (hESCs) have the potential to overcome the shortage of primary hepatocytes for clinical use and drug development. Many strategies for this process have been reported, but the functionality of the resulting cells is incomplete. We hypothesize that the functionality of hPSC-derived hepatocytes might be improved by making the differentiation method more similar to normal in vivo hepatic development. We tested combinations of growth factors and small molecules targeting candidate signaling pathways culled from the literature to identify optimal conditions for differentiation of hESCs to hepatocytes, using qRT-PCR for stage-specific markers to identify the best conditions. Immunocytochemistry was then used to validate the selected conditions. Finally, induction of expression of metabolic enzymes in terminally differentiated cells was used to assess the functionality of the hESC-derived hepatocytes. Optimal differentiation of hESCs was attained using a 5-stage protocol. After initial induction of definitive endoderm (stage 1), we showed that inhibition of the WNT/β-catenin pathway during the 2nd and 3rd stages of differentiation was required to specify first posterior foregut, and then hepatic gut cells. In contrast, during the 4th stage of differentiation, we found that activation of the WNT/β-catenin pathway allowed generation of proliferative bipotent hepatoblasts, which then were efficiently differentiated into hepatocytes in the 5th stage by dual inhibition of TGF-β and NOTCH signaling. Here, we show that stage-specific regulation of the WNT/β-catenin pathway results in improved differentiation of hESCs to functional hepatocytes.

Alginate encapsulation of chitosan nanoparticles: a viable alternative to soluble chemical signaling in definitive endoderm induction of human embryonic stem cells.

Human embryonic stem cells (hESCs) are characterized by both their pluripotency and ability to self-renew, rendering them attractive for treatment of degenerative diseases. The cues necessary to induce hESC differentiation to a desired lineage can be categorized as either chemical or biophysical in nature. While chemical cues are used as primary inducers of differentiation, biophysical cues have been found to augment the process. In this regard, we have designed a novel chitosan nanoparticle augmented encapsulated alginate (CNPEA) platform which can not only augment, but induce differentiation of hESCs into the definitive endoderm (DE) lineage in the absence of specific soluble chemical inducers. These endoderm cells were comparable in phenotype with chemically driven DE cells and remained amenable to further maturation into pancreatic lineages. This study demonstrates the feasibility of carefully designed and tailored nanomaterials inducing differentiation, and moreover demonstrating the possibility of replacing growth factors by material cues.

Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

Pluripotent embryonic stem cells (ESCs) exert low-traction forces on their niche in vitro whereas specification to definitive endoderm in vivo coincides with force-mediated motility, suggesting a differentiation-mediated switch. However, the onset of contractility and extent to which force-mediated integrin signaling regulates fate choices is not understood. To address the requirement of tractions forces for differentiation, we examined mouse embryonic stem cell (ESC) specification towards definitive endoderm on fibrillar fibronectin containing a deformation-sensitive FRET probe. Inhibiting contractility resulted in an increase in the observed fibronectin FRET intensity ratio but also decreased the amount of phosphorylated nuclear SMAD2, leading to reduced expression of the definitive endoderm marker SOX17. By contrast ESCs maintained in pluripotency medium did not exert significant tractions against the fibronectin matrix. When laminin-111 was added to fibrillar matrices to improve the efficiency of definitive endoderm induction, ESCs decreased their fibronectin traction forces in a laminin-dependent manner; blocking the laminin-binding α3-integrin restored fibronectin matrix deformation and reduced SOX17 expression and SMAD2 phosphorylation, probably because of compensation of inhibitory signaling from SMAD7 after 5 days in culture. These data imply that traction forces and integrin signaling are important regulators of early fate decisions in ESCs.

Hepatic Differentiation of Porcine Embryonic Stem Cells for Translational Research of Hepatocyte Transplantation

Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.