How to Reprogram Fibroblasts into Human Induced Pluripotent Stem (iPS) Cells Using ReproRNA™-OKSGM
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- เผยแพร่เมื่อ 14 มิ.ย. 2016
- This technical video demonstrates how to reprogram fibroblasts in ReproTeSR™ medium using ReproRNA™-OKSGM. ReproRNA™-OKSGM is a non-integrating, self-replicating RNA reprogramming vector for generating human induced pluripotent stem (iPS) cells.
Learn more about ReproTeSR™ medium: bit.ly/38hwZuC
Learn more about ReproRNA™ OKSGM: bit.ly/2I9hoCz
#PluripotentStemCells #CellCulture #Fibroblasts
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thank you for the tutorial now i can make my own stem cells and become immortal
Unfortunately this doesnt work so simple. Yes, is it possible to get IPSC, but as "raw substance" they are useless. To make new tissue or whole you need to reprogram (to different) them back to somatic cells. And besides there are atleast two big problems. First - short telomeres, wich you cant make longe without cancer. And second - IPCS have features from their "parents" - IPCS from skin will have feauters of skin cells; and IPCS from muscles will have feauters of muscles.
The autoimmune disease schleroderma is the hardening of the skin and overproduction of collagen. Could this be used to treat schleroderma?
The overproduction of connective tissue hardens the arteries. Please help if you can provide a resource
Hi, STEMCELL Technologies is not involved with the use of stem cells for clinical applications nor with products for treating humans. We are a biotechnology company that sells products to assist researchers to isolate and culture stem cells from a number of tissue types. We can direct you to a list of clinical trials here: www.clinicaltrials.gov
Hello thank you for the information. Those clinical trials are very helpful thank you :) :)
Now, you can not unfortunately to use this tool as medicament. This is only tool to reprogram "adult" cell to pluripotent cells. If you need to heal something - you need to use genetic engineering and another tools.
May i ask? Unfortunately i have no education in microbiology and also English isnt my first. Am i understanding this correct?: Are you using syntetical RNA wich contains code for producing "Yamanaka-factors" wich used to "back-roll" usual cell back to stem cell and put this RNA into empty virus?
And what means "self-replicating RNA"? Means it, that this RNA can be found in all copies of next generations of stem cells? If not - how long can this RNA exist inside cell?
Is it possible to use for this purpose not artificial RNA, but new artificial chromosome, wich will be able to do the same function and will be able to self-replicate together with cycle of cell division? And also is it possible to control this function (reprogramming to IPS) by light? - firstly add this "thing" to cells and then only later to reprogramm them via turning light on?
Sorry about mistakes - my English is pretty bad :(
I hope for your answer ;)
Indeed, ReproRNA™-OKSGM encodes the Yamanaka factors to reprogram somatic cells to pluripotent stem cells. This is a non-viral method. The vector will persist in cell culture for as long as B18R treatment is maintained. B18R down-regulates the cells innate response against exogenous RNA.
Yoshioka et al., 2013 (pubmed.ncbi.nlm.nih.gov/23910086/) assessed RNA content of cells after the removal of puromycin and B18R selection. They observed that all traces of RNA had cleared by the 8th passage (most had cleared by the 5th). This group failed to detect any integration events in their cell lines, indicating the safety of this system for wild-type genotype iPSC generation. When compared to Sendai virus systems which have been shown to persist for over 11 passages, 5-8 passages are very attractive time savings (see Schlaeger et al., 2015, which also discusses other reprogramming methods:pubmed.ncbi.nlm.nih.gov/25437882/).
We have not tested light-controlled reprogramming in house, but optogenetic-controlled cell reprogramming systems are possible in general, see e.g. Wang et al., 2013 (pubmed.ncbi.nlm.nih.gov/36507552/).
If you have any more questions, feel free to email us at techsupport@stemcell.com.
@@STEMCELLTechnologies Thanks so much for your answer and more thanks for web-links
why genetic instability is caused in ipsc cells
Hi Shivam, human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, acquire recurrent genetic abnormalities during prolonged culture. In vitro, the most commonly seen hPSC genetic changes are those that provide a selective growth advantage due to selection pressures in culture. These karyotypic changes can affect hPSC behaviour in downstream applications. For more information, please consult the following publications by Baker, D. et al, 2016 (www.ncbi.nlm.nih.gov/pmc/articles/PMC5106530/) and Andrews, PW et al., 2017 (www.ncbi.nlm.nih.gov/pmc/articles/PMC5106530/).
If you would like to discuss this topic further, or have questions about our products to support hPSC culture and genetic analysis, please contact us at techsupport@stemcell.com.
what is the difference between this method and the one that used by Shinya Yamanaka? I am kind of confused😅😅
After the original publication on human cell reprogramming by the Yamanaka lab, Takahashi et al., 2007 (www.cell.com/cell/fulltext/S0092-8674(07)01471-7), many different vector types and combinations of reprogramming factors were used and published. So while reprogramming of adult human cells to iPS cells is in principle all based on the original work done by Yamanaka, you will find many variants of methods by now. One improvement over the original retroviral method was using non-integrating vectors to avoid insertional mutagenesis. The system presented in this video, ReproRNA™-OKSGM, is for example a single-stranded RNA replicon vector that contains five reprogramming factors: OCT4, KLF-4, SOX2, GLIS1, and c-MYC, as well as a puromycin-resistance gene. This RNA vector reprograms somatic cells, such as fibroblasts, into induced pluripotent stem (iPS) cells - similarly to the original approach by the Yamanaka lab. However, while the original paper from Takahashi et al., 2007 (www.cell.com/cell/fulltext/S0092-8674(07)01471-7) used retroviral vectors for the insertion of the reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc into the genome, ReproRNA™-OKSGM contains an additional factor, GLIS1, contains a puromycin selection cassette and is not integrating into the genome. If you have any more questions, feel free to email us at techsupport@stemcell.com.
@@STEMCELLTechnologies thank you so much. I am only a student now so thanks for your detailed explanation.
@@STEMCELLTechnologies Sorry to bother you, but can I ask you one more question? How exactly does a cell react to the appearance of such reprogramming factors within itself? Is it simply important that all factors be present simultaneously, as if we had a logical “0” for the absence of factors and a logical “1” for their presence? Or, for example, is it important that the factors be present in a certain number?
@@kerbalfly529introducing simultaneously will do it most likely