Induced pluripotent stem cell macrophages present antigen to proinsulin-specific T cell receptors from donor-matched islet-infiltrating T cells in type 1 diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes is an autoimmune disorder characterised by loss of insulin-producing beta cells of the pancreas. Progress in understanding the cellular and molecular mechanisms underlying the human disease has been hampered by a dearth of appropriate human experimental models. We previously reported the characterisation of islet-infiltrating CD4+ T cells from a deceased organ donor who had type 1 diabetes. METHODS: Induced pluripotent stem cell (iPSC) lines derived from the above donor were differentiated into CD14+ macrophages and tested for their capacity to present antigen to T cell receptors (TCRs) derived from islet-infiltrating CD4+ T cells from the same donor. RESULTS: The iPSC macrophages displayed typical macrophage morphology, surface markers (CD14, CD86, CD16 and CD11b) and were phagocytic. In response to IFNγ treatment, iPSC macrophages upregulated expression of HLA class II, a characteristic that correlated with their capacity to present epitopes derived from proinsulin C-peptide to a T cell line expressing TCRs derived from islet-infiltrating CD4+ T cells of the original donor. T cell activation was specifically blocked by anti-HLA-DQ antibodies but not by antibodies directed against HLA-DR. CONCLUSIONS/INTERPRETATION: This study provides a proof of principle for the use of iPSC-derived immune cells for modelling key cellular interactions in human type 1 diabetes.

An INSULIN-GFP/GLUCAGON-mCherry reporter line for the study of human pancreatic endocrine cell development.

Insulin expressing beta cells and glucagon expressing alpha cells are the two most abundant endocrine cell types of the human pancreatic islet. Both alpha and beta cells can be generated in vitro via the differentiation of pluripotent stem cells (PSCs), affording the opportunity to study their ontogeny and to examine their developmental inter-relationship. To aid these analyses, we have generated a PSC line in which insulin expression is reported by GFP and glucagon expression is reported by mCherry. This cell line enables viable isolation of cells expressing each hormone and optimisation of methods that lead to their generation.

Generation of a SOX9-tdTomato reporter human iPSC line, MCRIi001-A-2, using CRISPR/Cas9 editing.

To develop an iPSC SOX9 reporter line for monitoring differentiation into SOX9 expressing cells such as chondrocytes, cranial neural crest and Sertoli cells, we used gene editing to introduce sequences encoding the tdTomato fluorescent protein into the SOX9 locus. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. Endogenous SOX9 expression was undisturbed and the tdTomato fluorescent reporter mirrored SOX9 mRNA expression. This iPSC line will be useful for assessing iPSC differentiation into SOX9-expressing cells and enrichment by cell sorting.

Long-Term Maintenance of Human Pluripotent Stem Cells on cRGDfK-Presenting Synthetic Surfaces.

Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.

NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that ß-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

UNLABELLED: Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. SIGNIFICANCE: This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold.