As far as I know, the efficiency for iPS via retroviral transduction was up to 1%. Some publications mentioned a rate of 5%, but I am not sure about these data sources. Does anyone know of a publication that states the iPS rate in human cells? Does this differ between species? If so, to what degree?
Up to now the best way to induce pluripotency in human fibroblasts is by retroviral transduction. You can use only three transcription factors instead of 4 as in the beginning, but Sox2 and Oct3/4 are essential. The addition of cMyc improve the efficiency of the reprogrammation but it is an oncogene so it depends on the goal. Then the efficiency of reprogrammation depends on the type of cells and the state of maturation of the cells. For exemple keratinocytes are more easily re-programmable than fibroblasts but their culture is difficult!
That's a great question. I like to look at this issue a bit differently, rather than efficiency, I like to use a time coefficient called time to first colony, and time to first passage. The first recognizes the appearance of pro to-colonies, the second refers to the time to subculture the colonies. Now, I have a really efficient all in one poly-cistronic system that has Oct4, Sox2, KLF4, and L-Myc together with a fusion of RFP and Blast. Once the cells are infected, a short selection procedure allows enrichment and finally plating on matrigel and using KOSR based medium with small molecules leads to appearance of many proto-colonies (10 - 100 per 25,000 cells plated) within 10 days. Without adding blasticidin, and without small molecules, I rarely get IPSC colonies. This is most likely due to over-growth of non transformed cells, and sequestration of growth factors. I've done this in fibroblasts only, but I am sure this should work in other cell models.
The rate at the NIMH Center is well over 90% using hFibs & Sendai vectors. Its lower with retrovirus, but at least 50%. We do this as a service. No idea for other species.
Thanks a lot for that great comments.
I am very surprised about that high rates... Would you be so kind and tell me a publication, which states it?
This is not published. We do this as a service for NIMH. see www.nimhstemcells.org, nimhgenetics.org and rucdr.org
Make sure you are taking a close look at your oxygen levels, it can make all the difference from what I understand. A standard oxygen incubator is not the best solution for precise control. Either a small subchamber or a dedicated mini incubator is always better. Also maybe consider using a culturing device with better than standard equilibration.
Look up the work of E. Zambidis
PLoS One. 2012;7(8):e42838. doi: 10.1371/journal.pone.0042838. Epub 2012 Aug 8.
Growth factor-activated stem cell circuits and stromal signals cooperatively accelerate non-integrated iPSC reprogramming of human myeloid progenitors.
Park TS, Huo JS, Peters A, Talbot CC Jr, Verma K, Zimmerlin L, Kaplan IM, Zambidis ET.
They co-cultured human hematopoietic precursors on stromal cells and really increased the efficiency to produce iPSCs.
Thanks a lot for this PLoS One paper. As I am no expert on that field, I really appreciate any help to inform myself on this hot topic.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line