Christopher N. Mayhew

Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro

Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal ‘organoids’ consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.

Modelling human development and disease in pluripotent stem-cell-derived gastric organoids

Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signalling and induction of epithelial proliferation. Together, these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.

An in vivo model of human small intestine using pluripotent stem cells

Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies.

In vitro generation of human pluripotent stem cell derived lung organoids

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease. DOI: http://dx.doi.org/10.7554/eLife.05098.001

Liver-Specific pRB Loss Results in Ectopic Cell Cycle Entry and Aberrant Ploidy

The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals.

Wnt11/5a Complex Formation Caused by Tyrosine Sulfation Increases Canonical Signaling Activity

Wnt signaling plays important roles in embryonic development, tissue differentiation, and cancer. In both normal and malignant tissue, Wnt family members are often expressed combinatorially, although the significance of this is not understood. We recently showed that Wnt11 and Wnt5a are both required for the initiation of embryonic axis formation and that the two proteins physically interact with each other. However, little is known about the mechanism or biological significance of Wnt-Wnt protein interaction. Here we show in three assays, with Xenopus oocytes, mouse L cells, and human embryonic stem cells, that secreted Xenopus Wnt11/5a complexes have more canonical Wnt signaling activity than secreted Wnt11 or Wnt5a acting alone. We demonstrate that the sulfation activity of tyrosylprotein sulfotransferase-1 (TPST-1) is required for Xenopus dorsal axis formation and that O-sulfation of specific tyrosine residues is necessary for the interaction of Wnt11 with Wnt5a and for enhanced canonical signaling activity. These findings demonstrate a novel aspect of Wnt biology-Wnt family member interaction that depends on tyrosyl sulfation.

Proliferative Suppression by CDK4/6 Inhibition: Complex Function of the Retinoblastoma Pathway in Liver Tissue and Hepatoma Cells

BACKGROUND & AIMS Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide; current chemotherapeutic interventions for this disease are largely ineffective. The retinoblastoma tumor suppressor (RB) is functionally inactivated at relatively high frequency in hepatocellular carcinoma and hepatoma cell lines. Here, we analyzed the ability of CDK4/6 inhibition to inhibit hepatocyte proliferation and the effect of RB status on this process. METHODS Hepatoma cell lines and xenograft models harboring RB knockdown and mice harboring liver-specific Rb deletion were used to define the role of RB function in response to CDK4/6 inhibition. RESULTS Our study shows that CDK4/6-dependent cell cycle progression in hepatoma cells was readily arrested by inhibition of CDK4/6 by PD-0332991 or p16ink4a irrespective of RB status. Interestingly, upon CDK4/6 inhibition, p107 protein stability was dramatically increased as a function of RB loss. This engagement of compensatory mechanisms was critical for cell cycle inhibition in the absence of RB, because both the E1A oncoprotein and overexpression of E2F proteins were capable of overcoming the effect of CDK4/6 inhibition. These findings were recapitulated in xenograft models. Furthermore, to determine how these findings relate to hepatocyte proliferation in vivo, mice were exposed to carbon tetrachloride to induce liver regeneration followed by treatment with PD-0332991. This treatment significantly inhibited hepatocyte proliferation. Strikingly, this facet of PD-0332991 function was retained even in RB-deficient livers. CONCLUSIONS These data show that CDK4/6 inhibition is a potent mediator of cytostasis and that RB loss can be readily compensated for in the context of both hepatoma cell lines and liver tissue.

Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function

Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of cis-elements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB. Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.

Converting human pluripotent stem cells into beta-cells: recent advances and future challenges.

Purpose of reviewThe transplantation of insulin-producing β-cells derived from human embryonic stem cells and induced pluripotent stem cells (collectively termed pluripotent stem cells or PSCs) holds great promise for therapy of diabetes mellitus. The purpose of this review is to summarize recent advances in this area, emphasizing the importance of studies of endocrine pancreas development in efforts to direct PSC differentiation into endocrine cells, as well as to outline the major challenges remaining before transplantation of PSC-derived β-cells can become a reality. Recent findingsAlthough several protocols to generate glucose-responsive pancreatic β-cells in vitro have been described, the most successful approaches are those that most closely mimic embryonic development of the endocrine pancreas. Until recently, cells generated by these methods have exhibited immature pancreatic endocrine phenotypes. However, protocols that generate more functional β-like cells have now been described. In addition, small molecules are being used to improve protocols to direct differentiation of PSCs into endoderm and pancreatic lineages. SummaryAdvances over the last decade suggest that generating functional β-cells from human PSCs is achievable. However, there are aspects of β-cell development that are not well understood and are hampering generation of PSC-derived β-cells. In particular, the signaling pathways that instruct endocrine progenitor cells to differentiate into mature and functional β-cells are poorly understood. Other significant obstacles remain, including the need for safe and cost-effective differentiation methods for large-scale generation of transplantation quality β-cells, methods to prevent immune rejection of grafted tissues, and amelioration of the risks of tumorigenesis.

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents.

The retinoblastoma (RB) tumor suppressor has been proposed to function as a key mediator of cell cycle checkpoints induced by chemotherapeutic agents. However, these prior studies have relied on embryonic fibroblasts harboring chronic loss of RB, a condition under which compensation of RB functions is known to occur. Here we utilized primary adult fibroblasts derived from mice harboring loxP sites flanking exon 3 of the Rb gene to delineate the action of RB in the chemotherapeutic response. In this system we find that targeted disruption of Rb leads to little overt change in cell cycle distribution. However, these cells exhibited deregulation of RB/E2F target genes and became aneuploid following culture in the absence of RB. When challenged with both DNA damaging and antimetabolite chemotherapeutics, RB was required for primary adult cells to undergo DNA damage checkpoint responses and loss of RB resulted in enhanced aneuploidy following challenge. In contrast, following spontaneous immortalization and the loss of functional p53 signaling, the antimetabolite 5-fluorouracil (5-FU) failed to induce arrest despite the presence of RB. In these immortal cultures RB/E2F targets were deregulated in a complex, gene-specific manner and RB was required for the checkpoint response to camptothecin (CPT). Mechanistic analyses of the checkpoint responses in primary cells indicated that loss of RB leads to increased p53 signaling and decreased viability following both CPT and 5-FU treatment. However, the mechanism through which these agents act to facilitate cell cycle inhibition through RB were distinct. These studies underscore the critical role of RB in DNA-damage checkpoint signaling and demonstrate that RB mediates chemotherapeutic-induced cell cycle inhibition in adult fibroblasts by distinct mechanisms.