Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages tend to be slower and generate target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors (primed ground state conditions) as a starting point for directed differentiation. As a proof-of-concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. First, we developed a new protocol that can rapidly generate nearly pure definitive endoderm from EpiSCs. Second, we developed a protocol for generating forebrain organoids that model the development of prethalamic and hippocampal neurons. These significantly improved differentiation models present new possibilities for combining mouse genetic tools and resources with in vitro differentiation to characterize the molecular and cellular mechanisms of embryonic development.

In silico discovery of small molecules for efficient stem cell differentiation into definitive endoderm

Improving methods for human embryonic stem cell differentiation represents a challenge in modern regenerative medicine research. Using drug repurposing approaches, we discover small molecules that regulate the formation of definitive endoderm. Among them are inhibitors of known processes involved in endoderm differentiation (mTOR, PI3K, and JNK pathways) and a new compound, with an unknown mechanism of action, capable of inducing endoderm formation in the absence of growth factors in the media. Optimization of the classical protocol by including this compound achieves the same differentiation efficiency with a 90% cost reduction. The gene expression profile induced by the compound suggests that it is an inhibitor of the MYC pathway. The proposed in silico procedure for candidate molecule selection has broad potential for improving stem cell differentiation protocols.

miR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.

Differentiation of Human Pluripotent Stem Cells Into Definitive Endoderm Cells in Various Flexible Three-Dimensional Cell Culture Systems: Possibilities and Limitations

The generation of human stem cell-derived spheroids and organoids represents a major step in solving numerous medical, pharmacological, and biological challenges. Due to the advantages of three-dimensional (3D) cell culture systems and the diverse applications of human pluripotent stem cell (iPSC)-derived definitive endoderm (DE), we studied the influence of spheroid size and 3D cell culture systems on spheroid morphology and the effectiveness of DE differentiation as assessed by quantitative PCR (qPCR), flow cytometry, immunofluorescence, and computational modeling. Among the tested hydrogel-based 3D systems, we found that basement membrane extract (BME) hydrogel could not retain spheroid morphology due to dominant cell–matrix interactions. On the other hand, we found that nanofibrillar cellulose (NFC) hydrogel could maintain spheroid morphology but impeded growth factor diffusion, thereby negatively affecting cell differentiation. In contrast, suspension culture provided sufficient mass transfer and was demonstrated by protein expression assays, morphological analyses, and mathematical modeling to be superior to the hydrogel-based systems. In addition, we found that spheroid size was reversely correlated with the effectiveness of DE formation. However, spheroids of insufficient sizes failed to retain 3D morphology during differentiation in all the studied culture conditions. We hereby demonstrate how the properties of a chosen biomaterial influence the differentiation process and the importance of spheroid size control for successful human iPSC differentiation. Our study provides critical parametric information for the generation of human DE-derived, tissue-specific organoids in future studies.

Pancreatic Differentiation of Stem Cells Reveals Pathogenesis of a Syndrome of Ketosis-Prone Diabetes

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.

Pancreatic Differentiation of Stem Cells Reveals Pathogenesis of a Syndrome of Ketosis-Prone Diabetes

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.

Continuous Inhibition of Sonic Hedgehog Signaling Leads to Differentiation of Human-Induced Pluripotent Stem Cells into Functional Insulin-Producing β Cells

Human-induced pluripotent stem cell- (iPSC-) derived insulin-producing cells (IPCs) can be used for islet cell transplantation into type 1 diabetic patients and as patient-specific cells for the development of novel antidiabetic drugs. However, a method is needed to generate functional IPCs from iPSCs and simplify the protocol. We compared combinations of small molecules that could induce the differentiation of cells into a definitive endoderm and preferentially into islet precursor cells. When generated using an optimal combination of small molecules, IPCs secreted insulin in response to glucose stimulation. We constructed spheroid IPCs and optimized the culture and maturation conditions. Quantitative PCR revealed that the expression of definitive endoderm-specific markers differed depending on the combination of the small molecules. The small molecule, N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine, induced the differentiation of cells into functional IPCs by inhibiting Sonic hedgehog signaling. Images of the 2D culture showed that IPCs formed spheroids from day 5 and continuously secreted insulin. We developed a simple differentiation method using small molecules that produced functional IPCs that responded to glucose stimulation within a relatively short period. We posit that this method along with further refinement of the differentiation process can be applied to culture IPCs that can be used in clinical trials.