Human induced pluripotent stem cells provide a great route towards personalized medicine and high accuracy drug screening. Allele Biotech has developed the most efficient method of making human iPSCs by using enhanced mRNAs, which have been adopted by leading pharmaceutical companies for clinical trials. The effects from medium-supplemented mRNAs are robust yet transient, and highly specific compared to both miRNAs (off-targets) and small molecules (unknown targets). To repress cellular immune response to introduced RNA molecules, viral protein B18R was previously used during mRNA reprogramming.

B18R is relatively expensive and inconvenient to use because it requires pre-aliquoting and -80C storage. The protocol has recently been dramatically improved at Allele through an NIDA-funded project. In our latest reprogramming run, all we needed to do was to include mRNA complex in the supplement during medium change for just a week without the need of adding any other type of molecules (such as B18R, miRNA, or chemicals) to help the mRNA mix, unlike all other known mRNA-reprogramming protocols. This advancement can make reprogramming human fibroblasts to footprint-free and xeno-free iPSCs a routine experiment for any lab to perform.

Human R-iPSCs were created without the need of B18R, dramatically reduced the cost and inconvenience. Shown is a newly formed iPSC colony.

Picture Blog: More Efficient Reprogramming for Creating Induced Stem Cells (iPSCs)

Researchers at Allele Biotech achieved reprogramming of human fibroblasts into iPSCs within one week, at “bulk conversion”, and with cells seeded at a much wider density range compared to our previous publications. These significant improvements will further facilitate high throughput, large scale iPSC production using Allele’s feeder-free, xeno-free, footprint-free reprogramming, which was already a preferred method for both clinical applications and stem cell banking. The reprogramming project is currently being funded by the NIDA/NIH.

Human skin fibroblasts were reprogrammed into stem cells using a proprietary mRNA cocktail in just a week.

Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming

AlleleBlog:

Induced pluripotent stem cells (iPSCs) can be achieved through introduction of a small group of stem cell specific transcription factors. Ever since this was first demonstrated by Takahashi and Yamanaka, there have been relentless efforts for improving the efficiency of this generally inefficient process. There is also a general opinion that iPSCs are different from each other and from embryonic stem cells (ESCs) in various aspects, depending on the method of the induction. As a result, another focus of the reprogramming field has been to find ways for creating iPSCs that are as close to ESCs as possible. One of the parameters for defining stem cell status is their epigenetic characters; epigenetic changes have been demonstrated to occur during reprogramming of subsequent differentiation.

In fact, it seems that reprogramming can be largely described as a process composed of chromatin remodeling and specific transcription activation. Strong transcription activators are known to effectively recruit multiple chromatin remodeling complexes when exerting their functions. A good example is MyoD, a master transcription factor for skeletal myogenesis that can “single-handedly” switch (transdifferentiate) the fate of differentiated cells. Hirai et al. speculated that since MyoD is such a strong transcription factor, it may be able to increase chromatin accessibility to iPS factors if fused together. When transduced on retroviral vectors, Oct-TAD (Transcription Activation Domain) of MyoD, in combination with Sox2 and Klf4, increased the number of iPSC colonies by 40-fold. Additionally, these iPSCs appeared to quickly adopt stem cell gene expression profiles, days faster than when traditional Oct4, Sox2, c-Myc, and Klf4 were used; and sometimes the levels of pluripotency genes even exceeds those seen in ESCs. Amazingly, when using the fusion assisted method some colonies are formed without the help of feeder cells, a requirement of ESCs grown in similar medium. Does this mean that these iPSCs can even be more “stem-like” than embryonic stem cells?

Like MyoD, VP16, also widely known for its strong transcription activation domain, when fused to iPS factors, was shown to exhibit a similar stimulation effect on reprogramming. Although the details of the fusion arrangements and specificity appear to differ between MyoD and VP16, the fact that two research groups could achieve similar results using comparable strategies provides a good argument that other labs should at least consider this method when creating mouse or human iPSCs. Previously in our blog we have discussed using iPS factor mRNAs, a method originally developed by Warren et al., for substantially shortening the time required for reprogramming and making it more robust across cell types and media conditions. If the new TAD-fusion factors are used also in the mRNA format, then the protocol might be further shortened and simplified. If successful, this non-integrating approach could become a dominant method in the field, even making competitive non-integrating method such as Sendai and plasmid-based miRNA irrelevant.

New product of the week:SurfaceBind RNA purification system, higher capacity and simpler procedure than Qiagen or Ambion’s comparable products, particularly suitable for mRNA cleanup after in vitro transcription.

Promotion of the week: 30% off RNA SurfaceBind Purification kits. To redeem this offer email the code PURIFY to oligo@allelebiotech.com.

Retroviral Vectors with Integrated oriP/EBNA1 for IPSC

The new product of the week of Sep 21-27 is the retrovirus plasmid sets that contain a built-in episomal expression system. As we have discussed previously, OriP/EBNA1 system originated from Epstein-Bar virus, which allows the establishment of stable episomes at 5-20 copies per cell, and duplication once per cell division.

By using the oriP/EBNA1 episomal system, reprogramming cDNAs can be expressed at prolonged time period in reference to plasmid transfection, without integration into chromosomal DNA. A paper published in PLoS One on Sep 18, 2009 by Marchetto et al. showed that by using such a system (on different plasmids) the authors were able to create induced pluripotent stems cells (iPS cells,) effectively from human embryo neural precursor cells.

The Allele pCHAC-EBNA system has dual functions: it can be ready-to-use plasmids for episomal expression of Oct4, Sox2, c-Myc, Klf4, or Nanog and Lin28 by a simple transfection into target cells; it can also be packaged into retroviruses by transfecting into the Allele Phoenix Retrovirus packaging Eco or Ampho cells. This product group is officially launched today. It should become a highly convenient and unique tool for iPSC-related studies.

Difference between hESCs and NSC-Derived Integration-Free iPSCs

By introducing Oct4 and Nanog into human fetal neural progenitor cells, the Muotri lab at UCSD, in collaboration with colleagues from the Yeo and Gage labs of UCSD and Salk, was able to create induced pluripotent stem cells (iPS cells) using the oriP/EBNA1 episomal system to introduce the aforementioned Oct4 and Nanog. While it has been shown by others that with less differentiated cells, such as progenitor cells, it is relatively easy to reprogram using two, or sometimes even one, of the commonly used iPSC generating factors (Oct4, Sox2, Klf4, c-Myc, Nanog, or Lin28) as reported in the AlleleNews article “iPSC generated by using a single reprogramming factor” on August 3rd, 2009, the new publication in PLoS ONE analyzed in detail the differences between embryonic stem cells (ES cells) and iPS cells.



There were three groups of genes that changed significantly between hESC versus iPSC and iPSC versus NSC; genes that are important in early embryonic fate including iPSC-expressed factors that are not sufficiently repressed and genes that are upregulated in iPSCs but are silenced in both NSCs and hESCs which may be downstream to the reprogramming genes during dedifferentiation. Their conclusion is that iPSCs may retain the gene expression signature of donor cells in human reprogrammed cells, even with integration-free gene transfer vehicles such as oriP/EBNA1 element containing plasmids. It should be pointed out that this episomal vector system has been previously reported for iPS cell generation by the Thomson group, see:

(http://allelebiotech.com/blogs/2009/03/episomal-expression-of-ips-inducing-genes-no-trace-of-transgenes-afterwards/). In this paper it was also shown, not unexpectedly, that myc-immortalized cell lines can be efficiently reprogrammed.



Marchetto et al. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007076



Note from Allele: Congratulations to Maria, Gene, Alysson and the Gage lab.