Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.

By T. Brambrink, Ruth K. Foreman, G. Welstead, C. Lengner, Marius Wernig, Heikyung Suh, R. Jaenisch. Cell Stem Cell, 2008

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Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a8db6688da0744df035cb4e6da99363e59ff9e9f

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Citations in the corpus

Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases (2009, methodology): “Vesicular stomatitis virus glycoprotein (VSVG)-coated lentiviruses were generated in 293 cells as described previousl”
Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. (2008, positive): “The transduced factors are required for an initial period of time in the reprogramming process (Brambrink et al., 2008; Stadtfeld et al., 2008) during which they may interact with endogenous pluripotency genes (Boyer et al.”
A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. (2010, background): “Although AP staining is a not-stringent-enough criterion to indicate pluripotency (Brambrink et al., 2008; Silva et al., 2008), these experiments show that TGF-b and Snail mostly abort the reprogramming at an early stage.”
Disease-specific induced pluripotent stem (iPS) cells (2008, positive): “…from a carrier of Lesch-Nyhan syndrome using five doxycycline-inducible lentiviral vectors (OCT4, SOX2, KLF4, c-MYC, and NANOG), a strategy that has been used to isolate murine iPS cells (Brambrink et al., 2008; Stadtfeld et al., 2008) but previously had not been attempted with human somatic cells.”
Facilitators and Impediments of the Pluripotency Reprogramming Factors’ Initial Engagement with the Genome (2012, positive): “…in the First 48 hr of Reprogramming To investigate the initial engagement of O, S, K, and M with chromatin , we transcriptionally induced the factors together in human fibroblasts (hFib) using doxycyline (dox)-inducible lentiviral transduction (Brambrink et al., 2008) (Figure S1A available online).”
Oct4-induced pluripotency in adult neural stem cells. (2009, positive): “Other studies examined the reprogramming timing in more detail by reprogramming mouse fibroblasts using the doxycycline (dox)inducible system (Brambrink et al., 2008; Wernig et al., 2007).”
Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation (2009, background): “The latter system provides the option of a genetically homogeneous background, albeit limiting the study of reprogramming to differentiated human iPSC-derived cells rather than primary cells and subject to instability of generated iPS clones upon doxycycline withdrawal (11, 17).”
Induced pluripotency: history, mechanisms, and applications. (2010, background): “…with doxycycline-inducible vectors, as cells that have entered a self-sustaining pluripotent state can be easily selected for by removal of doxycycline (Brambrink et al. 2008; Stadtfeld et al. 2008b), even though, in rare cases, doxycycline-independent partially reprogrammed cells have been…”
Parkinson’s Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors (2009, methodology): “Lentiviral Infection and hiPSC Derivation Vesicular stomatitis virus G protein (VSVG)-coated lentiviruses were generated in 293 cells as described previously (Brambrink et al., 2008).”
Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells. (2011, neutral): “, 2005) todeterminewhether theexpression levels of genes previously reported to be associated with stem, basal, and luminal differentiationwere specifically enriched in the relevant sorted subpopulations (Assou et al., 2007; Blackman et al., 2005; Böcker et al., 2002; Brambrink et al., 2008; Bussola”
Epigenetic reprogramming and induced pluripotency (2009, background): “3) (Brambrink et al., 2008; Stadtfeld et al., 2008b).”
Direct cell reprogramming is a stochastic process amenable to acceleration (2009, background): “21 Brambrink, T. et al., Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2 (2), 151-159 (2008).”