Converting fully differentiated mouse or human somatic cells into embryonic-like cells (so called induced Pluripotent Stem Cell: iPSC) has attracted enormous attention in stem cell research. Multiple reports have demonstrated that iPS cells were generated by using a set of transcription factors or stem cell factors that delivered as expression virus or expressed proteins. Although the combination of reprogramming factors may slightly different, the main stem cell factors are: OCT3/4, SOX2, NANOG, LIN28, c-Myc and KLF4.  iPSC holds the promise of curing many human diseases and accelerates the stem cell research.

Utilizing its Inducible lentiviral vector system (see vector scheme below), Gentarget generate high-titer inducible lentiviral particles for all six human or mouse stem cell factors. Each factor was fully sequencing verified and matched to the CDs in NCBI database (see details from each product). High titer lentiviral particles / supernatant were produced in 293T packaging cells (Cat# TLV-C) in DMEM with 10% heat-inactivated FBS or in serum-free medium. They are psudotyped with VSV-G glycoprotein.

  

Pre-made Lentiviral particles for iPS provide an easy tool to generate your own iPS cells. Each Ready-To-Use particles was provided at 200ul/per vial at >1 x 10⁷ IFU/ml, shipped on dry-ice and should be stored in a -80ºC freezer until use. The virus is stable for one year.

Please see details in product pages for our premade iPS particles for:

A) Human iPS set (NANOG, OCT4, SOX2, KLF4, Myc, LIN28).

B) Mouse iPS set (NANOG, OCT4, SOX2, KLF4, Myc, LIN28).

Those ready-to-use particles can be used for constitutive high expression. The same particles can also be used as tetracycline inducible expression only when a repressor regulator protein (TetR) is present (see How tet inducible works.)

Transduction protocols for generation of iPSC:

1. Seed the desired parent cells at 1 × 10⁵ cells/well in 24-well plate, incubated overnight;
2. Remove medium and add 100ul completed medium (optional), add 50ul of each lentivirial particle for iPSC (Oct3/4, Sox2, NANOG, LIN28, c-Myc and Klf4) (Note: you can set up your own set by change the factor combination dependent upon your cell types. But reported showed this full set was successfully induced mouse adult fibroblast into iPSC cell). Incubate cells for 6~12 hours (Note: longer transuction time resulted more cell dead because our data showed over expressed Klf4 and SOX2 cause cell death);
3. Change medium with 0.5ml compelete medium and incubated overnight;
4. Check cell viability: you will observe cells are dying (floating up). From now on change medium every two days with completed meddium (be sure to use ES verified serum).
5. Check transduction efficiency (at 72 hours after transduction) by vitalizing RFP signal under florescent microscope using RFP filter (filer: Ex~550 / Em620nm). You will observe majority cell are dead, but there are some living cells showing good RFP signal. (Note: our data showed the LIN28 tend to be sub-localized only in cytoplasm.)
6. At day 8~10, split transduced live cells into feeder cells using your defined medium for the feeder cells, continue incubate for 24~48hrs with completed medium.
7. Change medium with hES medium, continue incubate and change hES medium everyday;
8. At day 13 ~ 18: you will observe cell's morphologic change, the ES like colonies forming (see sample image below).

        

References:

1. NIH stem cell training program (Link).
2. Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.
3. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.
4. Park, I.H., et al., Reprogramming of human somatic cells to pluripotency with defined factors. Nature, 2008. 451(7175): p. 141-6.
5. Shao, L., et al., Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame. Cell Res., 2009. 19(3): p. 296-306.