Methods of iPSC Generation Update

Induced pluripotent stem cells can be directly generated from adult cell cultures through the introduction of a group of factors, e.g. Oct4, Sox2, Klf4, and c-Myc (the Yamanaka factors) [Takahashi and Yamanaka, 2006]. Additional factors such as Nanog and Lin28 can either substitute some of the Yamanaka factors or supplement them for higher reprogramming efficiency [Yu et al. 2007].

The original pluripotent stem cells induction methods involved retrovirus or lentivirus that would leave foot-print in the host genome, a concern for clinical use of iPSCs. Several groups have tried to create iPSCs without integrating viruses, such as using small molecules, directly delivering proteins instead of cDNAs, viruses with RNA genomes, episomal systems, or removable elements such as PiggyBac or Sleeping Beauty transposons. From the literature and our first-hand experience in the iPS market, none of these methods has become a widely applicable tool, mostly due to impractically low reprogramming efficiency.

In addition to low efficiency, RNA viruses, such as the sendai virus, are still viruses and have virus-associated risks. Episomal plasmids or removable transposons still involve DNA, so the possibility of genomic integration by recombination remains. In case of some transposons such as PiggyBac, there is an additional question about the degree of removal – whether it is certain that all integrated transposons, often inserted within genes, are deleted; in case of transposons similar to Sleeping Beauty, the small footprints they leave behind may post a concern.

The method of choice for generating zero-footprint iPSCs should clearly be RNA-based without the involvement of virus. Luigi Warren and his former colleagues at Harvard demonstrated that by using in vitro transcribed iPS factor mRNAs with modified CTP and UTP, and 5’-cap can effectively reprogram a number of different human as well as mouse cells. The efficiency even exceeds those by using retrovirus or lentivirus by 10 to 100 fold. Furthermore, the RiPSCs created with mRNAs appear to be closer to hESCs as shown by expression profiling.

Very recently, a few miRNAs that have high expression levels in stem cells were shown to be able to reprogram mouse and human somatic cells when expressed together from a lentivirus [Anokye-Danso et al. 2011]. while that work used lentivirus, thus not directly applicable to the current project, Miyoshi et al. later showed that by using synthesized mature miRNA (overlapping but not the same set of miRNAs as used by Anokye-Danso et al.) reprogramming cold be achieved without viral infection. We believe that this is a promising method and would like to pursue it further and to find out whether these mi-iPSCs relate to hESCs as closely as R-iPSCs. Because transfecting synthetic miRNAs “does operate at considerably lower efficiency” in terms of iPSC creation [Miyoshi et al. 2011],alternative protocols may include transfecting the iPS factor mRNAs together with various miRNAs at different doses and frequencies.

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Blog References: Warren, L. et al. “Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA” 2010 Cell Stem Cell 7(5): 618-30

Anokye-Danso, F. et al. “Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency” 2011 Cell Stem Cell 8(4): 376-88

Miyoshi, N. et al. “Reprogramming of mouse and human cells to pluripotency using mature microRNAs” 2011 Cell Stem Cell 8(6): 633-8

Kim, H. et al. “miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells” 2011 Cell Stem Cell 8(6): 695-706

Takahashi, K. and Yamanaka, S. “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” 2006 Cell 126(4): 663-76

Yu, J. et al. “Induced pluripotent stem cell lines derived from human somatic cells” 2007 Science 318(5858): 1917-20

Tags: 200c, 367s, Episomal plasmids, Luigi Warren, mi-iPSC, miRNA-302s, miRNA-369, mRNA transfection, PiggyBac iPSC, RiPSC, RiPSCs

Our New Website is Complete, 10% off this week

We recently launched our new website, and to celebrate the launch, we’ll be offering a discount of 10 % on all our products and services from July 18th to July 22nd! Come explore a wide variety of products that Allele Biotech has to offer from viral expression to fluorescent proteins. Examples of services offered include custom lentiviral, retroviral, and baculoviral packaging along with cell production and cell line development. This is our effort to enhance your online shopping experience through our improved shopping cart system. Our mission remains the same; to increase accessibility to innovative molecular biology research tools by offering cutting edge products at a reasonable cost. Please visit http://www.allelebiotech.com and use the code NEWSITE to redeem the offer. We thank you for your support!
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Tags: Fluorescent proteins, iPS, Online Shopping, RNAi, viral packaging

Generate mouse and human iPS cells with transfected mature miRNAs

In last week’s blog we discussed generation of induced pluripotent stem cells (iPSCs) with miRNAs expressed from lentivirus. To take it a step further, synthetic, mature miRNAs can be used to avoid the use of viral vectors. Sure enough, Miyoshi et al. published a paper online a few days ago showing that by transfecting 6 miRNAs at 48 hour intervals, they were able to create iPSCs from mouse and human somatic cells. The efficiency is comparable to retrovirus-mediated OSKM factor over-expression (Yoshida et al.), and therefore lower than lentivirus-mediated miR302/369 expression (Anokye-Danso et al.).

In the study of using mature miRNA for obtaining iPSCs, the researchers transfected miRNAs mir200c, mir302s and mir-369 into tissue cultured cells and achieved reprogramming results. Interestingly, only mir302s are common between this study and that with lentivirus-mediated miRNAs by Anokye-Danso et al. There is no current explanation as to why mir-367, which was shown to be required by Anokye-Danso et al., did not seem to be needed in the mature miRNA transfection experiments. Perhaps a level of redundancy among miRNAs, combined with their broad target range and relatively low specificity, allow some of the miRNAs to be interchangeable when used for reprogramming.

Finally, neither of these two recent miRNA-iPSCs works was the first to demonstrate that miRNAs can initiate or facilitate reprogramming. As early as 2008, Lin et al. showed that mir302s could induce pluripotency in a dose-dependent manner by using tet-induced lentivirus expression. They further illustrated that the underlying mechanism is likely through mir302s’ regulation of epigenetic regulators AOFs and other similar factors.

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Tags: Anokye-Danso, iPS cells, iPSCs, mir-367, mir-369, mir200c, mir302s, Miyoshi et al., no foot print iPSCs, pluripotency

Mouse and human cells can both be reprogrammed with one cluster of specific miRNAs

The miRNA302/367 cluster was first found to be a direct target for the stem cell-specific factors Oct4 and Sox2, recently Anokye-Danso et al. showed that by overexpressing this miRNA cluster mouse and human cells can be reprogrammed without the OSKM factors. Moreover, according to the publication in Cell Stem Cell, miRNA-mediated reprogramming is “up to two orders of magnitude” more efficient than OSKM overexpression (but the authors used individual Oct4, Sox2, Klf4, and c-Myc lentiviruses, instead of a polycistronic virus such as Allele’s lenti-iPS-4-in-1).

To reprogram mouse embryonic fibroblasts (MEFs), suppression of chromatin remodeling factor Hdac2 is necessary when using miRNA for iPSC isolation. Surprisingly, the Hdac2 level is low in human fibroblasts, which do not need an Hdac inhibitor such as valproic acid (VPA) for reprogramming. Oct4-GFP positive cells (stem cells) are observed only 7 days post infecting MEFs with the miRNA302/367, and hundreds colonies appear per 10 thousand cells. When using human fibroblasts, iPSCs form at 18 to 26 days, at an efficiency of approximately 10%, which is significantly higher than using individual OSKM viruses.

The high efficiency from using miRNA for reprogramming is likely due to the fact that miRNAs can target hundreds of mRNAs, compared to providing one mRNA at a time. Although this study concluded that the miRNA302/367 expressing lentivirus was eventually silenced post stem cell induction, emphasis must still be placed on finding a non-integrating method to deliver this miRNA cluster.

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Tags: Anokye-Danso, Hdac2, human fibroblasts, iPSC, lentivirus, MEFs, miRNA derived iPSC, miRNA lentivirus, miRNA-mediated reprogramming, OSKM, VPA

mRNA Transfection for Better Transgene Expression

Different approaches have been developed to over-express or ectopically express a protein in cells: peptide or full length recombinant protein transfer, viral gene transfer, non-viral DNA transfer and non-viral mRNA transfer.

1) Peptide transfection can be efficient, yet it is limited to only a small part of the protein, limiting the functional potential. Protein transfection is not consistent enough so far, because of the complicated properties of different proteins. Allele Biotech has tested dozens of proteins with several proprietary reagents, leader peptides, etc. but we have decided not to carry a protein transfection product line due to its instability. Furthermore, protein production is an expensive and laborious process.

2) Viral gene transfer is very effective, such as the HIV-based lentivirus or MMLV-based retrovirus, adenovirus, adeno-like virus or baculovirus, etc. However, the potent side-effect will still need to be considered for certain applications, especially involving clinical studies. Nevertheless, as research tools, viral gene transfer is still a highly preferred method. Allele Biotech has been providing the most effective platform for both MMLV-based and HIV-1-based retrovirus packaging. Check out our product website for details.

3) Non-viral DNA transfer is the most widely used transgene method in the biological research community, due to the simplicity of the procedure. There are many commercial kits on the market. However, the low efficiency for transfecting most primary cells significantly limits their use. In recent years, several leading biotech companies have developed various electroporation systems to improve the transfection efficiency and cell viability; although these improvements help with getting DNA inside the cytoplasm, they hardly help transport it into nucleus where DNA is transcribed.

4) Non-viral mRNA transfer has been around for a long time, but it is not widely used. It made a big splash recently through its use for iPSCs reprogramming. IPSCs factor mRNAs greatly improved the iPSCs induction efficiency and completely avoided the viral integration. Other well-known examples of mRNA transfection include loading special cancer antigens or HIV antigens to dendritic cells (DCs) in vitro for personal immunotherapy. PSA antigen expressing DCs transfected by mRNA has moved on to Phrase I Clinical Trials for this purpose.

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Tags: 5-methylcytidine (5mC), B18R, in vitro transcription, interferon, iPSC factor mRNA, iPSC Warren, modified mRNA, mRNA iPSC, mRNA transfection, mRNA transfer, mRNA transgene, pseudouridine (psi), RiPSC, T7 RNA polymerase, VPA