I am going to be inducing pluripotency in adult rat dermal fibroblasts and adult rat bone marrow cells, and eventually the same with adult human peripheral blood CD34+ monocytes. Can anyone provide me with pluripotency induction protocols they have carried out themselves and can therefore attest to their relative costs/efficiencies?
In you case, for Fibroblasts (human or mice) Lentiviralmediated vector were effective. However, for human CD34 cells we used episomal vectors, you can see the details at Induced Pluripotent Stem Cell for the Study and Treatment of Sickle Cell Anemia. Junqueira L., et al. 2017
The Cytotune 2.0 Sendai reprogramming kit is extremely robust. Though the kit is expensive when used as directed for only 5 reprogrammings, I have used it for 50 from a single kit and am confident that I could do at least 96 with it.
Ron Hrstka how do you use the Sendai kit for more than 5 reprogrammings? Do you do it in smaller wells? Do you aliquot the virus and store it at -80?
Julia Draper The most recent time I needed to do a large number of reprogrammings all at once was done on 52 lines, using one viral kit, in 24 wells. So far 24 well is the smallest I've used but it is apparent I can go smaller. The next time I need to do a large number at once, I'm going to use 96 wells.
For high number reprogrammings I use new vials of virus and then I aliquot the remainder for 5 at a time.
Ron Hrstka do you notice any reduction in reprogramming efficiency after the additional freeze-thaw cycle?
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