Creating ground-state human iPSCs

AlleleBlog

Murine pluripotent stem cells can exist in two distinct states, blastocyst-derived LIF-dependent embryonic stem cells (ESCs) and epiblast-derived bFGF-dependent stem cells (EpiSCs). Murine ESCs and similar iPSC lines are more of the “ground-state” in terms of developmental status, as reflected by the lack of X chromosome inactivation in female cells and their abilities to pass as single cells. Human iPSCs, like human ES cells, are more similar to mouse EpiSCs. Unfortunately these human pluripotent stem cells are difficult to genetically manipulate, e.g. knockin or knockout. They also grow slowly, with doubling time averaging 36 hours. In order to create ground-state human iPSCs, several approaches have been tested, including reprogramming iPSC-derived fibroblasts, continuously expressing 5 iPS factors (Oct4, Sox2, Nanog, c-Myc, and Klf4), or using chemicals to inhibit chromatin modifying enzyme HDAC. While these approaches succeeded to certain degrees, the resulting cell lines seem to have some limitations, such as limited passage numbers.

Retinoic acid (RA) signaling is involved in many aspects of embryonic development. RA receptor (RAR), together with one of its heterodimerization partners, steroid hormone receptor Lrh-1, was recently found to be able to synergize with the 4 common iPS factors (Oct4, Sox2, Klf4, and c-Myc) to induce mouse and human fibroblasts into ground-state iPSCs. The pluripotent cells created by the so-called F6 factor combination show no X chromosome inactivation if from female origin, can fully activate the endogenous Oct4 promoter, express Rex1 (which is specific to mouse ESCs, not EpiSCs), and grow with a 16 hour doubling time. All these mouse ESC-like features were achieved without detectable expression of the exogenous factors once iPSC colonies formed, indicating transient F6 expression is capable of effectively initiating endogenous stem cell factors. Remarkably, these stem cells can maintain their undifferentiated status in mouse ESC medium for 50 passages or more. This work, published this month in Proceedings of National Academy of Science USA [1], provided the stem cell research and application field with a very desirable choice of human stem cells.

As opposed to ~16 days with F4, it appears that the time required to induce adult fibroblasts into pluripotent stem cells is as short as 4 days if F6 factors are introduced on a murine stem cell virus (MSCV) vector with an integrated piggyback transposon. As the authors noted in their discussion, the speed-up benefit should be particularly advantageous for transient transfection approaches such as mRNA reprogramming. The bottom line from this paper and the engineered factor papers (see the previous AlleleBlog article under “iPS and other Stem Cells”) is that iPSC reprogramming is only going to get faster, which means that hopefully in the near future creating iPSCs will become a routine experiment as easy as a simple transfection.

Wang, W., J. Yang, et al. (2011). “Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.” Proc Natl Acad Sci U S A.

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ASCB Abstract: Increased rate of reprogramming of induced pluripotent stem cells using high-titer lentiviral vectors encoding multiple cell growth and survival regulatory genes

Objective: Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSC) with enforced expression of multiple transcription factors. We aim to improve the reprogramming efficiency using high titer lentiviral vectors encoding additional cell growth and survival regulatory genes.

Methods: Lentiviral vectors encoding multiple cell cycle and apoptosis genes in addition to c-Myc, Klf4, Oct4 and Sox2 were constructed and used to generate iPSC. The iPSC were extensively characterized by immunohistochemical staining and flow cytometry.

Results: Human mesenchymal stem cells can be efficiently transduced and reprogrammed into iPSC using high-titer lentiviral vectors encoding the four known transcription factors. The addition of siRNA suppressing p53 and cell cycle and survival genes including telomerase and BclXL significantly increased the efficiency and rate of iPSC generation. Human iPSC colonies were formed within a week after lentiviral gene transfer.

Conclusions: The protocol for iPSC generation has been improved with high titer lentiviral vectors encoding additional immortalization cellular factors regulating cell cycle progression, senescence and apoptosis. Deletion of the integrated lentiviral genomes using Cre-loxP recombination could increase the safety profile of the reprogrammed iPSC.

more at http://allelebiotech.com/blogs/2009/12/ascb-abstract-increased-rate-of-reprogramming-of-induced-pluripotent-stem-cells-using-high-titer-lentiviral-vectors-encoding-multiple-cell-growth-and-survival-regulatory-genes/

Protocols for Using Human Fibroblasts Expressing Human bFGF as Feeder Cells for iPSCs

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Allele Biotech has introduced the highly efficient GFP-Trap for GFP fusion protein pull-down, and a monoclonal anti-GFP antibody for detecting GFP-fusion proteins after Immunoprecipitation with GFP-Trap. Just launched this week, the anti-GFP polyclonal antibodies provide an alternative method for analyzing the isolated proteins.

Pre-announcement: Allele Biotech will launch a FAQ and a User Forum online where you can also find common protocols in focus areas and exchange ideas with us or others.

1. Thaw one vial of irradiated feeder cells by swirling gently in 37oC water bath until all of the contents are thawed. One vial of 2×106 cells is sufficient to prepare two10-cm dishes, or two 6-well or 12-well plates (about 3-4×104/cm2).
2. Spray vial with 70% ethanol and wipe dry before placing in tissue culture hood.
3. Gently add 1 ml prewarmed feeder cell medium (alphaMEM or DMEM/F12 with 10% FBS), mix with contents of cryovial and transfer into 15-ml conical tube containing 4 ml prewarmed feeder cell medium.
4. Centrifuge the cells at 200g at room temperature for 5 min and discard the supernatant.
5. Resuspend the feeder cells in 12 ml feeder cell medium. If using a 6-well plate: add 1 ml of feeder cell suspension to each well of the 6-well plate containing 1 ml fresh feeder cell media per well. If using a 10-cm tissue culture dish: add 6 ml of feeder cell suspension to 10-cm tissue culture dish containing 6 ml fresh feeder cell media. If using a 12-well plate: add 0.5 ml feeder cell suspension to each well of 12-well plate containing 1 ml fresh feeder cell media per well. Gently shake the dish left/right and up/down 10-20 times without swirling the plate to evenly distribute the cells across the plate.
6. Incubate the cells in 37 1C, 5% CO2, overnight.
CRITICAL STEP When moving the feeder cell plates from the tissue culture hood to incubator, do not swirl the medium, as this tends to cause the cells to accumulate in the center. Immediately after placing the plates in the incubator, slide the plates forward and backward (2–3 cm) two times, then left to right (2–3 cm) two times to ensure equal distribution of the cells. Use within 5–7 days.
7. Split stem cells (~2.5 x 105 to 5 x 105 cells, or ~10% confleuce) into plate with feeder cells. Aspirate medium from ESC or iPSC, wash with PBS and add 0.5 ml of 0.05% trypsin. Incubate at 37oC, 5% CO2, for 5 min.
8. Inactivate trypsin with 3 ml stem cell medium, and collect cell clumps in 15-ml conical tube avoiding making single cell suspension because ESC tends to die in single cell form.
9. Centrifuge at 200g at room temperature for 4 min.
10. Aspirate feeder medium from feeder plates (cells incubated in Step 6), rinse with one ml of stem cell medium and add 5 ml of stem cell medium and return to incubator.
11. Aspirate and discard supernatant from the conical tube in Step 8, resuspend cells in 5 ml stem cell medium, gently dispense the cell pellet three times, add to feeder cell wells or dishes.
12. Incubate stem cells grown on feeder cells at 37oC, 5% CO2, for 48 h.
13. Aspirate medium and replace with stem cell medium every day; if iPSC colony number is low, replace medium every two days.

iPS Cells: Feeder Cells

Allele’s entire iPSC product line is designed for the ease of the researcher. Each component in our iPSC catalog will shave priceless time off your protocol by eliminating the tedious steps in iPS induction so you can get down to work.

Allele is adding a major component to its iPSC line: pre-irradiated, ready-to-use, system specific, bFGF-Producing Feeder Cells for iPSC propagation!

Using Allele’s bFGF-Producing Feeder Cells avoids the usual problems associated with MEF cell lines. They are maintained at low passages, come pre-irradiated and ectopically express bFGF so there is no need to supplement your medium with additional growth factors.

Additionally, Allele Biotech is introducing human fibroblasts to the market for iPSC work. MEF is good for mouse iPSC reprogramming but human fibroblast feeders are preferred when creating human iPSCs due to their secreted factors. Propagate human iPSC with greater efficiency while eliminating non-human cells for therapeutic use of human iPSCs!

As always we encourage customer feed back. We are interested to hear about your stem cell work, needs, and requests for new products. We also welcome those who have new ideas and potential products to collaborate with us. We are here to help advance your research and get your technologies to the public.

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