FAS-Based Cell Depletion Facilitates the Selective Isolation of Mouse Induced Pluripotent Stem Cells

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues

Protein transduction from retroviral Gag precursors

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes

Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin

Identification of differentially expressed surface markers.

<p>A) A single flow cytometric analysis is shown to exemplify SSEA1 and ITGAV expression properties (n.d.  =  not determined), both of which were previously shown to be differentially expressed between MEF and PSC. B) Expression frequencies of antibody-targeted surface markers were tested by flow cytometry comparing MEFs (CF1), ESC line HM1 and iPSC line LV1-7b (n = 4 for MEFs: mean +/− SD; n = 2 for ESC/iPSC each). Given are the percentages of positive cells for identified candidate markers (6 potential pluripotency associated markers on the left-hand side and 6 potential MEF associated markers on the right-hand side). Expression data of all antibodies tested in the screen can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone.0102171.s004" target="_blank">Table S1</a>, additional expression characteristics on OG2-MEFs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone.0102171.s001" target="_blank">Figure S1</a>. C) Representative histograms are shown for selected markers.</p

Excerpt of the tissue distribution of EPCAM and FAS mRNA in human cell types.

<p>An mRNA analysis based on the Genevestigator software tool was performed reflecting the tissue distribution of investigated mRNAs. Shown are selected tissues that are relatively easily accessible and have already been reprogrammed in previous reports. The “percentage of expression potential” represents the average expression of a gene across all samples of the particular cell type as compared to the sample with the highest expression (maximum level, 100%) for the particular gene [percentage of expression potential  =  average/maximum]. The number of samples that were included to calculate this average is indicated on the right side of the graph. Results are given as logarithmic (log2) heat map.</p

Expression analysis of EPCAM and FAS protein in selected human cell types.

<p>A, B) Flow cytometry analysis of FAS and EPCAM expression on human foreskin fibroblasts (hFF), human umbilical vein endothelial cells (hUVEC) and two hiPSC lines. A) Representative flow cytometry analysis results. B,) Independent quantification of relative expression levels of FAS and EPCAM for the same four cell types. Stain indices (SI) are depicted to enable the comparison of expression levels for cell types displaying different levels of autofluorescence. SI were calculated as follows: (Median of labeled cells – Median of unlabeled cells)/(2× standard deviation of unlabeled cells). p values were calculated with a Student's t test. *p<0.05; **p<0.01; ***p<0.001. Scale bars show the SD for three separate experiments (alpha = 95%).</p

Oct4-GFP expression characteristics.

<p>A) Immunofluorescence of <i>Oct4</i>-GFP transgenic iPSC line LV1-7b cultured in non-differentiating conditions. Depicted are the <i>Oct4</i>-GFP marker, staining for OCT4 protein, a DAPI counterstain and phase contrast images. The overlay displays <i>Oct4</i>-GFP and OCT4 protein. B) The same iPSC line and analysis as in A cultured under differentiating conditions (2 day LIF withdrawal).</p

Expression kinetics of some candidate markers correlate with reprogramming stages.

<p>A) Expression frequencies (mean +/− SD) of candidate markers on reprogramming subpopulations were investigated by flow cytometry over time (n = 3: mean +/− SD; for SSEA1 n = 1) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone.0102171.s002" target="_blank">Figure S2</a>). Reprogramming subpopulations were defined as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone-0102171-g003" target="_blank">Fig. 3D</a>. B) Correlation of ITGAV, SSEA1, EPCAM and FAS with expression of OCT4 protein as analyzed by flow cytometry at day 12 p.t. C) Likewise, correlation of the selected candidate markers with the <i>Oct4</i>-GFP reporter system is shown at day 12 of reprogramming.</p

Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell–derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events