Induced pluripotent reprogramming from promiscuous human stemness related factors.

Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Rochester, MN, USA.
Clinical and Translational Science (Impact Factor: 2.11). 04/2009; 2(2):118-26. DOI: 10.1111/j.1752-8062.2009.00091.x
Source: PubMed

ABSTRACT Ectopic expression of pluripotency gene sets provokes nuclear reprogramming in permissive somatic tissue environments generating induced pluripotent stem (iPS) cells. The evolutionary conserved function of stemness orthologs was here tested through interspecies transduction. A spectrum of HIV-based lentiviral vectors was designed, and point mutations in the HIV-1 capsid region identified for efficient infectivity and expanded trans-species tropism. Human pluripotent gene sequences, OCT3/4, SOX2, KLF4 and c-MYC, packaged into engineered lentiviral expression vectors achieved consistent expression in non-human fibroblasts. Despite variation in primary amino-acid sequence between species, introduction of human pluripotent genes produced cell lines with embryonic stem cell-like morphology. Transduced fibroblasts differentiated in vitro into all three germ layers according to gastrulation gene expression profiles, and formed in vivo teratoma with multi-lineage potential. Reprogrammed progeny incorporated into non-human morula to produce blastomeres capable of developing into chimeric embryos with competent organogenesis. This model system establishes a prototypic approach to examine consequences of human stemness factors induced reprogramming in the context of normal embryonic development, exploiting non-human early stage embryos. Thus, ectopic xeno-transduction across species unmasks the promiscuous nature of stemness induction, suggesting evolutionary selection of core processes for somatic tissue reprogramming.

  • [Show abstract] [Hide abstract]
    ABSTRACT: -Cardiac development is a complex process resulting in an integrated, multi-lineage tissue with developmental corruption in early embryogenesis leading to congenital heart disease. Interrogation of individual genes has provided the backbone for cardiac developmental biology, yet a comprehensive transcriptome derived from natural cardiogenesis is required to gauge innate developmental milestones. -Stage-specific cardiac structures were dissected from eight distinctive mouse embryonic time points to produce genome-wide expressome analysis across cardiogenesis. In reference to this native cardiogenic expression roadmap, divergent iPSC-derived cardiac expression profiles were mapped from pro-cardiogenic 3-factor (SOX2, OCT4, KLF4) and less-cardiogenic 4-factor (plus c-MYC) reprogrammed cells. Expression of cardiac-related genes from 3F-iPSC differentiated in vitro at days 5 and 11 recapitulated expression profiles of natural embryos at days E7.5-E8.5 and E14.5-E18.5, respectively. In contrast, 4F-iPSC demonstrated incomplete cardiogenic gene expression profiles beginning at day 5 of differentiation. Differential gene expression within the pluripotent state revealed 23 distinguishing candidate genes among pluripotent cell lines with divergent cardiogenic potentials. A confirmed panel of 12 genes, differentially expressed between high and low cardiogenic lines, was transformed into a predictive score sufficient to discriminate individual iPSC lines according to relative cardiogenic potential. -Transcriptome analysis attuned to natural embryonic cardiogenesis provides a robust platform to probe coordinated cardiac specification and maturation from bioengineered stem cell-based model systems. A panel of developmental-related genes allowed differential prognosis of cardiogenic competency, thus prioritizing cell lines according to natural blueprint to streamline functional applications.
    Circulation Cardiovascular Genetics 09/2013; 6(5). DOI:10.1161/CIRCGENETICS.113.000045 · 5.34 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The value of induced pluripotent stem cells (iPSCs) within regenerative medicine is contingent on predictable and consistent iPSC differentiation. However, residual influence of the somatic origin or reprogramming technique may variegate differentiation propensity and confound comparative genotype/phenotype analyses. The objective of this study was to define quality control measures to select iPSC clones that minimize the influence of somatic origin on differentiation propensity independent of the reprogramming strategy. Over 60 murine iPSC lines were derived from different fibroblast origins (embryonic, cardiac, tail tip) via lentiviral integration and doxycycline-induced transgene expression. Despite apparent equivalency according to established iPSC histologic and cytomorphologic criteria, clustering of clonal variability in pluripotency-related gene expression identified transcriptional outliers that highlighted cell lines with unpredictable cardiogenic propensity. Following selection according to a standardized gene expression profile calibrated by embryonic stem cells, the influence of somatic origin on iPSC methylation and transcriptional patterns was negated. Furthermore, doxycycline-induced iPSCs consistently demonstrated earlier differentiation than lentiviral-reprogrammed lines using contractile cardiac tissue as a measure of functional differentiation. Moreover, delayed cardiac differentiation was predominately associated with up-regulation in pluripotency-related gene expression upon differentiation. Starting from a standardized pool of iPSCs, relative expression levels of two pluripotency genes, Oct4 and Zfp42, statistically correlated with enhanced cardiogenicity independent of somatic origin or reprogramming strategy (R2=0.85). These studies demonstrate that predictable iPSC differentiation is independent of somatic origin with standardized gene expression selection criteria, while the residual impact of reprogramming strategy greatly influences predictable output of tissue-specification required for comparative genotype/phenotype analyses. Stem Cells 2014
    Stem Cells 09/2014; 32(9). DOI:10.1002/stem.1734 · 7.70 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cell transplantation is a potential treatment for the many liver disorders that are currently only curable by organ transplantation. However, one of the major limitations of hepatocyte transplantation is an inability to monitor cells longitudinally after injection. We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to visualize transplanted hepatocytes in a rodent model of inherited liver disease, hereditary tyrosinemia type 1. Wildtype C57Bl/6J mouse hepatocytes were transduced ex vivo using a lentiviral vector containing the mouse Slc5a5 (NIS) gene under the control of the thyroxine-binding globulin promoter. NIS-transduced cells could robustly concentrate radiolabeled iodine in vitro, with lentiviral transduction efficiencies greater than 80% achieved in the presence of dexamethasone. Next, NIS-transduced hepatocytes were transplanted into congenic fumarylacetoacetate hydrolase knockout (Fah(-/-) ) mice, resulting in prevention of liver failure. NIS-transduced hepatocytes were readily imaged in vivo by single-photon emission computed tomography, demonstrating for the first time noninvasive 3D imaging of regenerating tissue in individual animals over time. We also tested the efficacy of primary hepatocyte spheroids to engraft in the liver. Using the NIS-reporter, robust spheroid engraftment and survival could be detected longitudinally after direct parenchymal injection, thereby demonstrating a novel strategy for hepatocyte transplantation. Conclusion: This work is the first to demonstrate the efficacy of NIS-imaging in the field of hepatocyte transplantation. We anticipate that NIS-labeling will allow non-invasive and longitudinal identification of hepatocytes and stem cells in future studies related to liver regeneration in small and large preclinical animal models. This article is protected by copyright. All rights reserved. © 2014 American Association for the Study of Liver Diseases.
    Liver Transplantation 03/2015; 21(4). DOI:10.1002/lt.24057 · 3.79 Impact Factor

Full-text (2 Sources)

Available from
Jun 4, 2014