Allele Biotechnology Announces New advance in production of human stem cells

This week in the journal Scientific Reports (Nature Publishing Group) scientists from Allele Biotechnology describe an important advance in the generation of stem cells capable of producing all the different tissues of the human body. In an article entitled “Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA,” Allele’s scientists present the fastest and safest method yet for converting ordinary human skin cells into “induced pluripotent stem cells” (iPSCs).

The scientific efforts were led by Dr. Luigi Warren, whose pioneering work on “footprint-free” reprogramming using messenger RNA was the foundation for Allele’s breakthrough. Through the united efforts of Dr. Warren and the scientists at Allele Biotechnology, his technique was re-engineered to increase cell conversion efficiency and eliminate any use of potentially unsafe reagents, while substantially reducing the time and effort needed to make stem cells. Dr. Warren believes that because of its advantages this technology “should become the method of choice for iPSC cell banking.”

According to Dr. Jiwu Wang, corresponding author on the paper and CEO of Allele Biotechnology, “This advance in stem cell derivation will enable both fundamental scientific research and clinical applications which has been the mission of Allele Biotechnology from its inception.”

Allele Biotechnology and Pharmaceuticals Inc. is a San Diego-based biotechnology company that was established in 1999 by Dr. Jiwu Wang and colleagues. A research based company specializing in the fields of RNAi, stem cells, viral expression, camelid antibodies and fluorescent proteins; Allele Biotechnology has always striven to offer products and services at the cutting edge of research.

Allele Biotechnology and Pharmaceuticals Inc.
Jiwu Wang, Ph.D., 858-587-6645 Ext 3
President and CEO
iPS@allelebiotech.com
fax: 858-587-6692
www.allelebiotech.com
Press release by BusinessWire. Also see Yahoo!News, Reuters, The Herald, etc.

Tags: flourescent proteins, human fibroblasts, iPS, iPSC, iPSCs, Lin28, MO3, mRNA reprogrammin, Myc, Nanog, Oct4, reprogrammin, Sox2, stem cells

Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming

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.

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Tags: chromatin remodeling, epegenetic markers, footprint free, iPS cells, iPSCs, mRNA iPSCs, MyoD-Oct, reprogramming, TAD-Oct, VP16-Oct

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

Expression of iPS Factors from Transfected mRNA

Differentiated cells can be reprogrammed to pluripotency by enforced expression of certain combinations of stem cell-specific protein factors in them. The power of this method was first demonstrated by Yamanaka’s group using retroviruses carrying Oct3/4, Sox2, c-Myc, and Klf4. Alternative factors such as Lin28 and Nanog, and additional factors such as the human telomerase gene hTert and shRNA against p53 were also shown to contribute to reprogramming. From the very beginning it was realized that viral integration would pose a major problem in using the induced pluripotent stem cells (iPSCs) for clinical purposes. There have been multiple attempts to circumvent this problem by using non-integrating vectors such as plasmid, minicircle DNA, adenovirus, baculovirus, removable transposons, episomal DNA, or by introducing recombinant proteins with a transmembrane domain into target cells. From reports in the field and customer feedbacks it seems that retroviral or lentiviral systems are still the most efficient in reprogramming. mRNA is about the only option left unreported, until an article by Warren et al was published in Cell Stem Cell online recently.

From that report, it is clear that the reason that it took so long for RNA-induced iPSCs (RiPSCs) to appear in the literature was because synthetic mRNAs activate interferon responses in mammalian cells, reminding us of the early days of RNAi. The authors took a number of steps to reduce interferon responses, including adding a 5’-cap (actually a fairly standard step in in vitro transcription), using a phosphatase to remove 5’ triphosphates on uncapped mRNAs, and using modified C and U bases (5-methucytidine or 5mC and pseudouridine or psi) during T7 promoter-driven in vitro transcription. The prepared mRNA was then administered everyday for 17 days at an amount not clearly defined in the paper. The main benefit of this method is of course that there is no gene integration to alter the chromosome. The efficiency of the new method was also compared to using viral vectors and it was shown that 1.4% conversion efficiency was achieved vs retroviral systems’ 0.01% (although we have experienced better results using lentivirus, at least the 4-in-1 version).

The DNA templates used for in vitro transcription of the iPS factors were created by multiple PCR reactions and bridged ligation; it could also be done by other cloning strategies. For those excited about trying this new way of making iPSCs, the major hassle would be preparing modified mRNAs good and abundant enough for 17 consecutive transfections. Allele Biotech would like to provide custom services, before offering shelf products, for creating such mRNAs as the method sounds potentially very helpful to many researchers in the iPSC field.

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Tags: c-myc, in vitro transcription, integration-free, iPSC, iPSCs, Klf, lentivirus, modified synthetic mRNA, mRNA iPSCs, mRNA transfection, Oct, retrovirus, RiPSC, RiPSCs, Sox

From iPSC to induced beta-cells, iN and iCM: dedifferentiation vs direct reprogramming

The success of inducing pluripotency in primary fibroblasts and other cells with a combination of only a small number of transcription factors suggested that fully differentiated cells might change fate following similar treatments. Since the demonstration of induced pluripotent stem cells (iPSCs), at least three examples have been published where 3 cell type-specific factors were selected from a pool of 10-20 candidates that, when expressed from viral vectors, could induce beta-cells, neurons, or cardiomyocytes.

Induced beta-cells [1]: Ngn3, Pdx1, and Mafa, adenovirus injected to in vivo targets

Induced neurons (iN) [2]: Ascl1, Brn2, and Myt1l, lentivirus infecting mouse embryonic fibroblasts (MEF) or tail tip fibroblasts (TTF)

Induced cardiomyocytes (iCM) [3]: Gata4, Mef2c, and Tbx5, lentivirus infecting cardiac fibroblasts or TTF

In all 3 cases, the change of fate seemed to be via direct conversion, without passing through a progenitor cell fate before further differentiation. Like iPSC reprogramming, direct reprogramming also requires a transient supply of inducing factors. Unlike generating iPSCs, the percentage of cells getting reprogrammed is much higher in direct reprogramming, ~20% in the cases of iN and iCM vs 0.1-1% in iPSC. It is likely that a transient, inductive expression of essential factors jump-starts endogenous factors to establish cell fate specific programs; it has also been illustrated that chromatin remodeling through DNA methylation, histone modifications, etc. accompanies the direct reprogramming events.

The requirement of the full complement of inducting factors may vary depending on how close the original cell type is to the new cell type. iPSCs are typically created by using 4 genes, but can be created with just Sox2, Oct3/4 particularly when the cells to be reprogrammed are less differentiated, such as tissue progenitor cells. Instead of a more “complete” direct reprogramming from unrelated cells to iN and iCM, the induced beta-cells come from exocrine cells, which share parental cells with beta-cells.

Looking into the near future, it should be expected that cell type-specific gene expression profiles are being re-examined or created right this moment to look for candidate gene pools specific to other cell types, starting from those with cell therapy relevance. Lentivirus, retrovirus, adenovirus, or baculovirus for mammalian expression are being constructed to carry them into fibroblasts or cells that are close to the end product of direct reprogramming. In a few months, many of these inducing gene-expressing viruses will become shelf products as high titer viruses from suppliers like Allele Biotech, incorporating tools in viral packaging, fluorescent proteins, and polycistronic gene expression systems.

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1. Zhou, Q., J. Brown, A. Kanarek, J. Rajagopal, and D.A. Melton, In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature, 2008. 455(7213): p. 627-32.
2. Vierbuchen, T., A. Ostermeier, Z.P. Pang, Y. Kokubu, T.C. Sudhof, and M. Wernig, Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 463(7284): p. 1035-41.
3. Ieda, M., J.D. Fu, P. Delgado-Olguin, V. Vedantham, Y. Hayashi, B.G. Bruneau, and D. Srivastava, Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 142(3): p. 375-86.

Tags: direct reprogramming factors, iCM, iN, induced beta-cells, induced cardiomyocytes, induced neurons, iPS, iPS lentivirus, iPSCs, MEF, TTF