ABSTRACT
The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor-based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.
Subject(s)
Cell Transdifferentiation , Fibroblasts/cytology , Fibroblasts/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Animals , Cell Lineage , Cell Proliferation , Culture Media , Cytological Techniques , Gene Expression , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/biosynthesis , Mice , Models, Biological , Octamer Transcription Factor-3/biosynthesis , Octamer Transcription Factor-3/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/biosynthesis , SOXB1 Transcription Factors/biosynthesis , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
Yeast vacuoles are very dynamic structures that must respond to changes in extracellular osmolarity by rapidly altering their size, thereby releasing or taking up water and ions. Further, the need to accommodate a constant biosynthetic influx of membrane and to partition vacuoles during cell division necessitates precise regulation of the size and shape of the vacuole. While it is has been shown that the lipid kinase Fab1p and its product phosphatidylinositol 3,5-bisphosphate, and not the mitogen-activated protein kinase Hog1p, are central to this regulatory pathway, key effectors still await identification. Atg18p is the most recently identified candidate for a Fab1p effector mediating the largely uncharacterized processes of vesicle fission and membrane recycling at the vacuole.
Subject(s)
Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vacuoles/metabolism , Intracellular Membranes/metabolism , Ion Transport , Mitogen-Activated Protein Kinases/metabolism , Osmolar Concentration , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
Higher eukaryotic development has traditionally been considered a unidirectional and irreversible process. Beginning in 2006, with Yamanaka and colleagues' report on the first successful generation of induced pluripotent stem cells (iPSCs), the field of stem cell biology has experienced perhaps unprecedented rates of growth and discovery. This review is a summary of recent progress in the field of reprogramming. Advances in small molecule-aided reprogramming and transdifferentiation, currently two of the most intensely studied areas of stem cell biology, are emphasized. The field has collectively covered much ground in the past five years, dramatically increasing reprogramming efficiency and successfully eliminating the need for permanent genetic modification, perhaps the biggest obstacle to eventual clinical use of this strategy. Simultaneously, various transdifferentiation strategies are rapidly expanding the scope of cellular plasticity interconverting unrelated cell types with relative technical ease. While significant challenges remain--such as accomplishing small molecule-only "chemical reprogramming" or ensuring the functional and epigenetic equivalency of reprogrammed or transdifferentiated cells--there is no shortage of enthusiasm in the field.
Subject(s)
Cell Transdifferentiation/physiology , Pluripotent Stem Cells , Animals , Cell Transdifferentiation/drug effects , Humans , Models, Biological , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/physiology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
The lipid kinase Fab1 governs yeast vacuole homeostasis by generating PtdIns(3,5)P(2) on the vacuolar membrane. Recruitment of effector proteins by the phospholipid ensures precise regulation of vacuole morphology and function. Cells lacking the effector Atg18p have enlarged vacuoles and high PtdIns(3,5)P(2) levels. Although Atg18 colocalizes with Fab1p, it likely does not directly interact with Fab1p, as deletion of either kinase activator-VAC7 or VAC14-is epistatic to atg18Delta: atg18Deltavac7Delta cells have no detectable PtdIns(3,5)P(2). Moreover, a 2xAtg18 (tandem fusion) construct localizes to the vacuole membrane in the absence of PtdIns(3,5)P(2), but requires Vac7p for recruitment. Like the endosomal PtdIns(3)P effector EEA1, Atg18 membrane binding may require a protein component. When the lipid requirement is bypassed by fusing Atg18 to ALP, a vacuolar transmembrane protein, vac14Delta vacuoles regain normal morphology. Rescue is independent of PtdIns(3,5)P(2), as mutation of the phospholipid-binding site in Atg18 does not prevent vacuole fission and properly regulates Fab1p activity. Finally, the vacuole-specific type-V myosin adapter Vac17p interacts with Atg18p, perhaps mediating cytoskeletal attachment during retrograde transport. Atg18p is likely a PtdIns(3,5)P(2)"sensor," acting as an effector to remodel membranes as well as regulating its synthesis via feedback that might involve Vac7p.
Subject(s)
Cell Membrane/metabolism , Organelles/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Autophagy-Related Proteins , Binding Sites , Cytosol/metabolism , Enzyme Activation , Flavoproteins/genetics , Flavoproteins/metabolism , Gene Deletion , Genes, Reporter/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation/genetics , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Binding , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/metabolismABSTRACT
B-cell lymphoma is one of the most frequently observed non-cutaneous neoplasms in dogs. For both human and canine BCL, the standard of care treatment typically involves a combination chemotherapy, e.g. "CHOP" therapy. Treatment for human lymphoma greatly benefited from the addition of anti-CD20 targeted biological therapeutics to these chemotherapy protocols; this type of therapeutic has not been available to the veterinary oncologist. Here, we describe the generation and characterization of a rituximab-like anti-CD20 antibody intended as a candidate treatment for canine B-cell lymphoma. A panel of anti-canine CD20 monoclonal antibodies was generated using a mouse hybridoma approach. Mouse monoclonal antibody 1E4 was selected for construction of a canine chimeric molecule based on its rank ordering in a flow cytometry-based affinity assay. 1E4 binds to approximately the same location in the extracellular domain of CD20 as rituximab, and 1E4-based chimeric antibodies co-stain canine B cells in flow cytometric analysis of canine leukocytes using an anti-canine CD21 antibody. We show that two of the four reported canine IgG subclasses (cIgGB and cIgGC) can bind to canine CD16a, a receptor involved in antibody-dependent cellular cytotoxicity (ADCC). Chimeric monoclonal antibodies were assembled using canine heavy chain constant regions that incorporated the appropriate effector function along with the mouse monoclonal 1E4 anti-canine CD20 variable regions, and expressed in CHO cells. We observed that 1E4-cIgGB and 1E4-cIgGC significantly deplete B-cell levels in healthy beagle dogs. The in vivo half-life of 1E4-cIgGB in a healthy dog was â¼14 days. The antibody 1E4-cIgGB has been selected for further testing and development as an agent for the treatment of canine B-cell lymphoma.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antigens, CD20/immunology , Dog Diseases/drug therapy , Lymphoma, B-Cell/veterinary , Animals , Antibody-Dependent Cell Cytotoxicity , Dog Diseases/immunology , Dogs , Humans , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/immunology , Rituximab/therapeutic useABSTRACT
Small molecules have been playing important roles in elucidating basic biology and treatment of a vast number of diseases for nearly a century, making their use in the field of stem cell biology a comparatively recent phenomenon. Nonetheless, the power of biology-oriented chemical design and synthesis, coupled with significant advances in screening technology, has enabled the discovery of a growing number of small molecules that have improved our understanding of stem cell biology and allowed us to manipulate stem cells in unprecedented ways. This review focuses on recent small molecule studies of (i) the key pathways governing stem cell homeostasis, (ii) the pluripotent stem cell niche, (iii) the directed differentiation of stem cells, (iv) the biology of adult stem cells, and (v) somatic cell reprogramming. In a very short period of time, small molecules have defined a perhaps universally attainable naive ground state of pluripotency, and are facilitating the precise, rapid and efficient differentiation of stem cells into somatic cell populations relevant to the clinic. Finally, following the publication of numerous groundbreaking studies at a pace and consistency unusual for a young field, we are closer than ever to completely eliminating the need for genetic modification in reprogramming.
Subject(s)
Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/physiology , Animals , Cell Differentiation , Drug Evaluation, Preclinical , Humans , Signal TransductionABSTRACT
Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11-12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm.
Subject(s)
Cell Culture Techniques , Fibroblasts/cytology , Myocytes, Cardiac/cytology , Animals , Calcium/metabolism , Cell Differentiation , Cell Proliferation , Electrophysiology/methods , Flow Cytometry , Induced Pluripotent Stem Cells/cytology , Kruppel-Like Factor 4 , Mice , Microscopy, Fluorescence/methods , Retroviridae/genetics , Time Factors , TransgenesABSTRACT
Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] regulates several vacuolar functions, including acidification, morphology, and membrane traffic. The lipid kinase Fab1 converts phosphatidylinositol-3-phosphate [PtdIns(3)P] to PtdIns(3,5)P(2). PtdIns(3,5)P(2) levels are controlled by the adaptor-like protein Vac14 and the Fig4 PtdIns(3,5)P(2)-specific 5-phosphatase. Interestingly, Vac14 and Fig4 serve a dual function: they are both implicated in the synthesis and turnover of PtdIns(3,5)P(2) by an unknown mechanism. We now show that Fab1, through its chaperonin-like domain, binds to Vac14 and Fig4 and forms a vacuole-associated signaling complex. The Fab1 complex is tethered to the vacuole via an interaction between the FYVE domain in Fab1 and PtdIns(3)P on the vacuole. Moreover, Vac14 and Fig4 bind to each other directly and are mutually dependent for interaction with the Fab1 kinase. Our observations identify a protein complex that incorporates the antagonizing Fab1 lipid kinase and Fig4 lipid phosphatase into a common functional unit. We propose a model explaining the dual roles of Vac14 and Fig4 in the synthesis and turnover of PtdIns(3,5)P(2).
Subject(s)
Flavoproteins/metabolism , Gene Expression Regulation, Fungal , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Genotype , Lipids/chemistry , Membrane Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Phosphoric Monoester Hydrolases , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Sequence Analysis, DNA , Subcellular Fractions , Vacuoles/chemistrySubject(s)
Cell Transdifferentiation , Cellular Reprogramming , Induced Pluripotent Stem Cells , Animals , Humans , MethodsABSTRACT
Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.
Subject(s)
Bacterial Proteins/genetics , Fungal Proteins/metabolism , Legionella pneumophila/genetics , Legionella pneumophila/pathogenicity , Vesicular Transport Proteins/metabolism , Bacterial Proteins/metabolism , Base Sequence , Carrier Proteins/metabolism , Cyclic AMP/metabolism , Immunoprecipitation , Lysosomes/metabolism , Membrane Proteins/metabolism , Microscopy, Fluorescence , Molecular Chaperones/metabolism , Molecular Sequence Data , Plasmids/genetics , Protein Structure, Tertiary , Protein Transport/genetics , Sequence Analysis, DNA , Virulence , Yeasts , beta-Fructofuranosidase/metabolismABSTRACT
Although the small Arf-like GTPases Arl1-3 are highly conserved eukaryotic proteins, they remain relatively poorly characterized. The yeast and mammalian Arl1 proteins bind to the Golgi complex, where they recruit specific structural proteins such as Golgins. Yeast Arl1p directly interacts with Mon2p/Ysl2p, a protein that displays some sequence homology to the large Sec7 guanine exchange factors (GEFs) of Arf1. Mon2p also binds the putative aminophospholipid translocase (APT) Neo1p, which performs essential function(s) in membrane trafficking. Our detailed analysis reveals that Mon2p contains six distinct amino acid regions (A to F) that are conserved in several other uncharacterized homologs in higher eukaryotes. As the conserved A, E and F domains are unique to these homologues, they represent the signature of a new protein family. To investigate the role of these domains, we made a series of N- and C-terminal deletions of Mon2p. Although fluorescence and biochemical studies showed that the B and C domains (also present in the large Sec7 GEFs) predominantly mediate interaction with Golgi/endosomal membranes, growth complementation studies revealed that the C-terminal F domain is essential for the activity of Mon2p, indicating that Mon2p might also function independently of Arl1p. We provide evidence that Mon2p is required for efficient recycling from endosomes to the late Golgi. Intriguingly, although transport of CPY to the vacuole was nearly normal in the Deltamon2 strain, we found the constitutive delivery of Aminopeptidase 1 from the cytosol to the vacuole to be almost completely blocked. Finally, we show that Mon2p exhibits genetic and physical interactions with Dop1p, a protein with a putative function in cell polarity. We propose that Mon2p is a scaffold protein with novel conserved domains, and is involved in multiple aspects of endomembrane trafficking.