ABSTRACT
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.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/virology , Host-Pathogen Interactions , Nuclear Proteins , Picornaviridae Infections/mortality , Respiratory Mucosa/virology , Rhinovirus/physiology , Transcription Factors , Animals , Cell Line , Embryonic Stem Cells/metabolism , Humans , Mice , Mice, Nude , Picornaviridae Infections/pathology , Respiratory Mucosa/metabolism , Thyroid Nuclear Factor 1ABSTRACT
NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.
Subject(s)
Cell Separation/methods , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/metabolism , Myoblasts, Cardiac/cytology , Myocytes, Cardiac/cytology , Transcription Factors/metabolism , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Biomarkers/analysis , Cell Differentiation , Gene Expression Profiling , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Humans , Myoblasts, Cardiac/metabolism , Myocytes, Cardiac/metabolism , Polymerase Chain Reaction , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Transcription Factors/genetics , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.
Subject(s)
Cell Lineage/genetics , Cell Tracking/methods , Embryonic Stem Cells/metabolism , Nuclear Proteins/genetics , Prosencephalon/embryology , Transcription Factors/genetics , Animals , Animals, Newborn , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Embryonic Stem Cells/cytology , Flow Cytometry/methods , Genes, Reporter , Humans , Mice , Mice, Transgenic , Molecular Targeted Therapy/methods , Neurogenesis/genetics , Neurogenesis/physiology , Nuclear Proteins/metabolism , Prosencephalon/cytology , Prosencephalon/physiology , Thyroid Nuclear Factor 1 , Transcription Factors/metabolismABSTRACT
The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.
