1Stemgent, 2Whitehead Institute for Biomedical Research, MIT - Massachusetts Institute of Technology
Hamilton, B., Feng, Q., Ye, M., Welstead, G. G. Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System. J. Vis. Exp. (33), e1447, doi:10.3791/1447 (2009).
Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.
1. Viral Transduction of MEFs
2. Doxycycline Induced Reprogramming
3. Immunocytochemical Analysis of Transduction Efficiency
48 hours post-doxycycline induction, determine transduction efficiency by immunohistochemistry(ICC). Carry out ICC testing on cells replated in 4 well plates. All volumes listed in the following protocol should be adjusted according to the cell culture plate size.
4. Isolating and expanding iPS Cell Colonies
5. Immunocytochemical Analysis for Pluripotency
Our ICC testing was carried out on cells expanded in 4 well plates. All volumes listed in the following protocol should be adjusted according to the cell culture plate size.
6. Alkaline Phosphatase (AP) Staining of iPS Cell Colonies
Part 7. Representative Results
The Stemgent DOX Inducible Mouse TF Lentivirus Set can be used to reprogram MEFs to iPS cells. After transduction of the MEFs, expression of transcription factors Oct4, Sox2, Klf4 and c-Myc can be detected in cells treated with doxycycline (DOX+), but little or no expression can be detected in untreated (DOX-) cells (figure 1). Morphological changes will progress over time (12 days of DOX treatment in this example) to generate larger, more ES cell-like colonies with defined colony edges and three dimensional growth (figure 2a). When DOX is removed, there is a noticeable reversion of cellular morphology for some ES cell-like colonies, however, many of the colonies maintained their iPS morphology (figure 2a). These iPS colonies, when picked and passaged, display typical pluripotency marker expression of Alkaline Phosphatase (AP), Nanog, Oct4 and SSEA-1 (figure 3). The type of MEF cells used in this experiment (Nanog-GFP/rtTA MEF cells) express GFP from the endogenous Nanog locus when reprogrammed to the pluripotency state. GFP expression can therefore be used as a preliminary indicator for successful reprogramming (figure 3).
Figure 1: Immunocytochemistry (ICC) analysis 48 hours post-DOX induction. The far left panel (-DOX) is a representative negative control for expression of the four transduction factors without DOX induction. Correctly expressed transcription factors were confirmed by corresponding antibodies (shown in red), stained with DAPI to visualize the nucleus.
Figure 2: Morphological conversion of Nanog-GFP/rtTA MEFs to the iPS cell state. A) 100x phase contrast imaging of cells demonstrating the compaction and conversion of MEFs into iPS colonies over time from day 3 (D3+Dox) to day 22 (D22). DOX was withdrawn on day 12. Upper left panel: 20x negative control image from –DOX plate. B) 200x phase-contrast and GFP fluorescence images of highlighted day 18 post-DOX induction iPS colony. C) 200x phase-contrast and GFP fluorescence images of additional day 18 post-DOX induction iPS colony.
Figure 3: Analysis of iPS colonies. (A) Phase contrast microscopy and AP staining of an iPS colony (200x). (B) Pluripotency marker analysis: Left panel shows phase contrast overlay with GFP reprogramming reporter expression. GFP expression reflects the endogenous Nanog expression level. Middle panel shows ICC staining for pluripotency markers. Right panel shows DAPI staining to visualize the nuclei (100x).
These results demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set can be used to efficiently generate iPS colonies by inducing the ectopic expression of transduced transcription factors in mouse embryonic fibroblasts. When designing reprogramming experiments, several variables should be considered to optimize the efficiency of reprogramming. First, it is possible to modify the active virus-to-target cell ratio (i.e. M.O.I.) during the primary infection step to increase or decrease the transduction efficiency, thereby affecting the number of integrated viruses in the target cell population. Second, adjusting the length of time the cells are exposed to DOX can affect the efficiency of iPS cell colony generation. Third, the proliferative capacity of the target cells can impact reprogramming, as cells which are actively growing and dividing are more amenable to reprogramming. Lastly, when modifying the protocol for different cell numbers or different size tissue culture dishes, it is recommended that target cell numbers be adjusted proportionally to the surface area of the culture dish.
The author, Brad Hamilton, is employed by Stemgent that produces reagents and instruments used in this article.
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