ABSTRACT
With the aim of producing ß cells for replacement therapies to treat diabetes, several protocols have been developed to differentiate human pluripotent stem cells to ß cells via pancreatic progenitors. While in vivo pancreatic progenitors expand throughout development, the in vitro protocols have been designed to make these cells progress as fast as possible to ß cells. Here, we report on a protocol enabling a long-term expansion of human pancreatic progenitors in a defined medium on fibronectin, in the absence of feeder layers. Moreover, through a screening of a polymer library we identify a polymer that can replace fibronectin. Our experiments, comparing expanded progenitors to directly differentiated progenitors, show that the expanded progenitors differentiate more efficiently into glucose-responsive ß cells and produce fewer glucagon-expressing cells. The ability to expand progenitors under defined conditions and cryopreserve them will provide flexibility in research and therapeutic production.
Subject(s)
Insulin-Secreting Cells , Pluripotent Stem Cells , Cell Differentiation , Fibronectins/pharmacology , Humans , Pancreas , PolymersABSTRACT
Human organogenesis remains relatively unexplored for ethical and practical reasons. Here, we report the establishment of a single-cell transcriptome atlas of the human fetal pancreas between 7 and 10 post-conceptional weeks of development. To interrogate cell-cell interactions, we describe InterCom, an R-Package we developed for identifying receptor-ligand pairs and their downstream effects. We further report the establishment of a human pancreas culture system starting from fetal tissue or human pluripotent stem cells, enabling the long-term maintenance of pancreas progenitors in a minimal, defined medium in three-dimensions. Benchmarking the cells produced in 2-dimensions and those expanded in 3-dimensions to fetal tissue identifies that progenitors expanded in 3-dimensions are transcriptionally closer to the fetal pancreas. We further demonstrate the potential of this system as a screening platform and identify the importance of the EGF and FGF pathways controlling human pancreas progenitor expansion.
Subject(s)
Cell Culture Techniques/methods , Organogenesis , Pancreas/embryology , Pluripotent Stem Cells/physiology , Tissue Culture Techniques/methods , Aborted Fetus , Animals , Cell Communication , Cell Differentiation , Cell Line , Datasets as Topic , Embryo, Mammalian , Epidermal Growth Factor/metabolism , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental , Humans , Mice , Pancreas/cytology , RNA-Seq , Signal Transduction/physiology , Single-Cell Analysis , Spheroids, Cellular , TranscriptomeABSTRACT
Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.
Subject(s)
Chromosomes, Artificial, Bacterial , Mutagenesis, Insertional/methods , Transgenes , Cell Line , Exons , Genetic Engineering , Homologous Recombination , Humans , Introns , Phenotype , Point MutationABSTRACT
Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.
