Search

This chapter describes the protocol to derive definitive endoderm cells from epiblast stem cells (EpiSCs) via a process analogous to gastrulation in embryos. The basis of this protocol mimicking the in vivo gastrulation process makes a contrast with those using sequential administration of pharmacological molecules and recombinant signaling proteins even at nonphysiological levels. In the experimental setup, EpiSCs are first freed from the dish-adherent condition to form free-floating aggregates, where endoderm precursor pools are produced. Embedding the EpiSC aggregates in the Matrigel allows the endoderm precursors to interact with the Matrigel mimicking the laminin-rich basement membrane underlying the egg cylinder epiblast in embryos, and let the precursors migrate into the Matrigel-filled external zone and develop into endodermal epithelial tissues., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Human pluripotent stem cells have a wide variety of potential applications, ranging from clinical translation to in vitro disease modeling. However, there is significant variation in the potential of individual cell lines to differentiate towards each of the three germ layers as a result of (epi)genetic background, culture conditions, and other factors. We describe here in detail a methodology to evaluate this bias using short directed differentiation towards neuroectoderm, mesendoderm, and definitive endoderm in combination with quantification by RT-qPCR and immunofluorescent stains., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

In drug discovery, there is an increasing demand for more physiological in vitro models that recapitulate the disease situation in patients. Human induced pluripotent stem (hiPS) cell-derived model cells could serve this purpose. To date, several directed differentiation approaches have been described to generate definitive endoderm (DE) from hiPS cells, but protocols suitable for drug development and high-throughput screening (HTS) have not been reported yet. In this work, a large-scale expansion of hiPS cells for high-throughput adaption is presented and an optimized stepwise differentiation of hiPS cells into DE cells is described. The produced DE cells were demonstrated to express classical DE markers on the gene expression and protein level. The here described DE cells are multipotent progenitors and act as starting points for a broad spectrum of endodermal model cells in HTS and other areas of drug discovery.

Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

Directed differentiation is a powerful cell culture technique where developmental pathways are applied to a pluripotent progenitor in order to generate specific terminally differentiated cell populations. Here, we describe a serum-free protocol using growth factors in defined concentrations to derive iPSC-hepatic cells starting from both feeder and feeder-free conditions. The generated iPSC-hepatic cells are developmentally similar to fetal stage hepatocytes, and when generated from patients with genetic mutations such as alpha-1 antitrypsin deficiency recapitulate pathologic changes associated with clinical disease, such as protein misfolding, intracellular retention of misfolded proteins, and elevated levels of ER stress.

Human iPS cells are capable of differentiation towards all three germ layer lineages. As well as being of use for the study of developmental biology, these cells also provide an excellent resource for disease modeling and cell replacement therapies. iPS cells derived from an individual with a disease type of interest can be differentiated towards the cell type afflicted in that particular disease. Such differentiated cell types can be used to test patient-specific drug responses in vitro, for generation of replacement cells for transplantation, or as a starting material for gene editing to correct disease-causing mutations/deletions. With such a vast array of potential applications, there is a great need to understand the exact mechanisms controlling the maintenance of pluripotency, and the distinct cues signaling these cells to differentiate towards lineages of interest. This chapter focuses on microRNA profiling using TaqMan Human MicroRNA Arrays to examine expression of 754 miRNAs in human iPS cells in the pluripotent state, and iPS derived definitive endoderm.

Over the past several decades, developmental biologists have discovered fundamental mechanisms by which organs form in developing embryos. With this information it is now possible to generate human "organoids" by the stepwise differentiation of human pluripotent stem cells using a process that recapitulates organ development. For the gastrointestinal tract, one of the first key steps is the formation of definitive endoderm and mesoderm, a process that relies on the TGFb molecule Nodal. Endoderm is then patterned along the anterior-posterior axis, with anterior endoderm forming the foregut and posterior endoderm forming the mid and hindgut. A-P patterning of the endoderm is accomplished by the combined activities of Wnt, BMP, and FGF. High Wnt and BMP promote a posterior fate, whereas repressing these pathways promotes an anterior endoderm fate. The stomach derives from the posterior foregut and retinoic acid signaling is required for promoting a posterior foregut fate. The small and large intestine derive from the mid and hindgut, respectively.These stages of gastrointestinal development can be precisely manipulated through the temporal activation and repression of the pathways mentioned above. For example, stimulation of the Nodal pathway with the mimetic Activin A, another TGF-β superfamily member, can trigger the differentiation of pluripotent stem cells into definitive endoderm (D'Amour et al., Nat Biotechnol 23:1534-1541, 2005). Exposure of definitive endoderm to high levels of Wnt and FGF promotes the formation of posterior endoderm and mid/hindgut tissue that expresses CDX2. Mid-hindgut spheroids that are cultured in a three-dimensional matrix form human intestinal organoids (HIOs) that are small intestinal in nature Spence et al., Nature 2011. In contrast, activation of FGF and Wnt in the presence of the BMP inhibitor Noggin promotes the formation of anterior endoderm and foregut tissues that express SOX2. These SOX2-expressing foregut spheroids can be further patterned into posterior foregut by addition of retinoic acid. Once formed, these posterior foregut spheroids can be grown in three-dimensional human gastric organoids (HGOs) that have all of the cell types of antral part of the stomach (Mc Cracken et al. 2014).Here, we describe the detailed methods for generating stomach/human gastric organoids (HGOs) and human intestinal organoids (HIOs) from human pluripotent stem cells. We first present a method for generating definitive endoderm from pluripotent stem cells followed by differentiation of definitive endoderm into either posterior foregut spheroids or mid-hindgut spheroids. We then describe how three-dimensional culturing of these spheroids results in the formation of HGOs and HIOs, respectively.

Differentiation of pluripotent stem cells into cells of the definitive endoderm requires an in vitro gastrulation event. Differentiated somatic cells derived from this germ layer may then be used for cell replacement therapies of degenerative diseases of the liver, lung, and pancreas. Here we describe an endoderm differentiation protocol, which initiates the differentiation from a defined cell number of dispersed single cells and reliably yields in >70-80 % endoderm-committed cells in a short 5-day treatment regimen.