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The quest for physiologically active human hepatocyte-like cells for in vitro research and drug screening is high. The recent progress in the field of pluripotent stem cell (PSC)-derived hepatic cells within the last decade brings those cells closer to applications in translational medicine. However, the classical two-dimensional (2D) cell culture systems are of limited use, because relevant cell-cell interactions based on cell polarity, which is a major prerequisite for proper hepatic cell metabolisms, are not provided. In this study, we report a scalable 3D suspension culture system, in which PSC-derived hepatic cells can be maintained for up to 3 weeks with stable gene expression profiles and metabolic features in a suspension culture system ranging from a 1.5 mL up to a 15 mL. Adjustments of culture conditions and, most importantly, the size of the organoids resulted in the robust generation of hepatic organoids consisting of a quite homogenous cell population. Importantly, the generation of these hepatic organoids was highly reproducible and allowed, in contrast to hepatic PSC derivatives in 2D culture conditions, a sensitive assessment of acetaminophen-related toxicity, the most common source for drug-induced liver failure.

Pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are of great promise in regenerative medicine, including molecular studies of disease mechanisms, if the affected cell type can be authentically generated during in vitro differentiation. Most existing protocols aim to mimic embryonic development steps by the supplementation of specific cytokines and small molecules, but the involved signaling pathways need further exploration. In this study, we investigated enhanced initial activation of Wnt signaling for definitive endoderm formation and subsequent rapid shutdown of Wnt signaling for proper foregut endoderm specification using 3 μM CHIR99021 and 0.5 μg/mL of secreted frizzled-related protein 5 (sFRP-5) for biphasic modulation of the Wnt pathway. The definitive endoderm and foregut endoderm differentiation capabilities of Wnt pathway-modulated cells were determined based on the expression levels of the endodermal transcription factors SOX17 and FOXA2 and those of the transcription activator GATA4 and the α-fetoprotein (AFP) gene, respectively. Furthermore, the resulting biphasic Wnt pathway modulation was investigated at the protein level by analyzing phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and β-catenin. Finally, Wnt target gene expression was determined using an improved lentiviral reporter construct that enabled robust T-cell transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1)-mediated luciferase expression in differentiating pluripotent stem cells. In conclusion, we demonstrated robust, homogeneous, and efficient derivation of foregut endodermal cells by inducing a biphasic modulation of the Wnt signaling pathway., (© 2016 International Federation for Cell Biology.)

Respiratory progenitors can be efficiently generated from pluripotent stem cells (PSCs). However, further targeted differentiation into bronchoalveolar sublineages is still in its infancy, and distinct specifying effects of key differentiation factors are not well explored. Focusing on airway epithelial Clara cell generation, we analyzed the effect of the glucocorticoid dexamethasone plus cAMP-elevating agents (DCI) on the differentiation of murine embryonic and induced pluripotent stem cells (iPSCs) into bronchoalveolar epithelial lineages, and whether keratinocyte growth factor (KGF) might further influence lineage decisions. We demonstrate that DCI strongly induce expression of the Clara cell marker Clara cell secretory protein (CCSP). While KGF synergistically supports the inducing effect of DCI on alveolar markers with increased expression of surfactant protein (SP)-C and SP-B, an inhibitory effect on CCSP expression was shown. In contrast, neither KGF nor DCI seem to have an inducing effect on ciliated cell markers. Furthermore, the use of iPSCs from transgenic mice with CCSP promoter-dependent lacZ expression or a knockin of a YFP reporter cassette in the CCSP locus enabled detection of derivatives with Clara cell typical features. Collectively, DCI was shown to support bronchoalveolar specification of mouse PSCs, in particular Clara-like cells, and KGF to inhibit bronchial epithelial differentiation. The targeted in vitro generation of Clara cells with their important function in airway protection and regeneration will enable the evaluation of innovative cellular therapies in animal models of lung diseases.

Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application.

In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.