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We use TWO lentiviral systems to efficiently generate IPSCs in the lab and reliably during the preparation for Stem Cell Training Course . Please note that optimized starting material (we use human foreskin fibroblasts) and media schema are equally important as viral tools for successful IPSC generation. If you need help with protocols, we can suggest our refinements which have produced presumptive IPSCs colony formation within 10 days.
1) Millipore/EMD produces a single novel lentiviral vector, with L-Myc, to induce pluripotency in human fibroblastic cells. While this system is listed at $1,378, you need very little optimization for successful and rapid reprograming. Even though it contains Cre sites to remove lentiviral genetic material, EMD Millipore as of 01/07/13 did not sell any appropriate Cre reagent (?)
Human STEMCCA Cre-Excisable Constitutive Polycistronic
(OKS/L-Myc) Lentivirus Reprogramming Kit
2) We developed a convenient Binary C-Myc Replacement Kit (even though we offer c-Myc), which produces RFP/Blast and Puro resistant transduced cells that can be enriched with Puro (3 days) or Blasticidin (5 days), and once successfully reprogrammed into well-formed colonies, loose their RFP signal. We now routinely use only L-Myc, and will be optimizing our SIN lenti-backbone to be an all-in-one. For now, this system offers the user a chance to seek out c-Myc replacements, either small molecules, or new genes, and test them in a Pluripotency Induction Assay. Without c-myc or equivalent, many of the cells aren't fully reprogrammed and in our hands become neural in nature.
This system is available upon request.
b) Choice of...
c-Myc (rapid, many false positives)
L-Myc (intermediate kinetics, but almost only properly formed colonies)
Glis1 (slower, very small granular, longer kinetics with some propely formed colonies)
Sample iPSC colony with self-forming feeder layers.
Please note, these photographs are property of Rutgers University, and Rick Cohen, Ph.D., and may not be used without expressed written permission.