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.

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.

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.

Promotion of the week: Promotion of the week: 10% off on all fluorescent proteins. To redeem, email oligo@allelebiotech.com along with PROMO code: JELLYFISH. See weekly promotions on Facebook.

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

Allele Custom Services for Drug Screening Companies

Many target discovery and validation programs can benefit from RNA interference, fluorescent proteins, stem cells, and viral delivery systems. However, applications of these technologies require special reagents and laboratory know-how. Even when available, many generic reagent kits are not tailored for your particular needs in screening or validation.

At Allele, we accelerate your discovery efforts with custom RNAi screening, fluorescence based assays, and cell model development services.

1) Our RNAi platform, based on our patented shRNA/miRNA technologies, use DNA linear template, plasmid, lentivirus, retrovirus, or baculovirus vectors that prompt cells to endogenously express RNAi. As a result, our screens offer advantages over synthetic siRNAs:
• Higher levels of consistency
• Greater delivery and gene silencing efficiencies
• Accessibility to difficult-to-transfect cells, including primary cells
• Potential for inducible RNAi expression
• More persistent silencing with shRNA under Allele’s own IP–you may not need to license siRNA patents!

2) Fluorescent proteins (FPs), which can span the entire visual spectrum, have become some of the most widely used genetically encoded tags. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Allele Biotech is one of a few companies that develop and improve FPs through fundamental research. We have so far achieved:
• The brightest cyan and green FPs, true monomers for minimum artifact or cytotoxicity
• The brightest yellow and red FPs from lancelet, only FPs from vertebrate
• mTFP1 as the best FRET donor by 3 independent reports
• Photoconvertible FPs for super imaging or kinetic labeling
• Delivery on plasmid, retrovirus, lentivirus, baculovirus vectors

3) As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors. These induced stem cells (iPSCs) provide unprecedented resources of cells of different differentiation stages for functional testing and drug screening. Allele Biotech develops and provides state-of-the-art reagents in convenient forms for iPSC production
• iPS factors carried on lentivirus, retrovirus, baculovirus for different cell types
• Availability in combination with fluorescent proteins under own IP, and drug resistant genes
• 4-in-1 or 2-in-1 effective use of iPS factors on one viral vector
• Feeder cells of human origin expressing factors essential for stem cell culturing

4) Introduction of protein factors, miRNA, promoter-reporter, and virtually any other genetic element of interest via the most efficient viral packaging systems.
• Introducing protein-FP fusion, promoter-FP reporter, photoactivatable factors for cell-based assays
• Introducing critical factors for cell immortalization
• Episomal or integrated expression using baculoviral vectors
• High throughput, systematic expression of whole class of molecules in any type of cell
• High titer viral packaging at low cost for delivery to animal tissues

In addition, the Allele team can provide custom-designed assays that can be used for assaying enzyme activities in almost any pathway, such as the EGF pathway, TNF response/apoptosis pathway, nuclear receptors, etc. We utilize technically advanced methods to provide our partners with advantages over alternative methods or other services.

New Product of the Week 06-28-10 to 07-03-10: Eco-friendly mammalian tissue culture plates, 40% less plastic to the environment, 40% less cost to your budget, contact our sales rep today for quotes and details.

Promotion of the Week 06-28-10 to 07-03-10: Oct3/4 iPS lentivirus with RFP as marker, new to the market, this week only all kits containing Oct3/4-RFP same price as the original, non-RFP versions, save ~$50!

Tags: cell assays, cell based assays, cell culture, drug screening, FP assays, FRET, iPS cells, kinase assasy, pathway, RNAi

Highly Efficient and Non-Integrating Vectors for Generating iPSC

The Challenge and Potential Impact

The proliferative and developmental potential of human stem cells offer virtually unlimited access to the differentiated cells that make up the human body [1]. Stem cell has become one of fastest moving areas biomedical research has ever seen. Tests at all stages ranging from cell differentiation to animal models to human clinical trials have begun within a very short period of time aiming at treating a host of diseases. The excitement generated by the vast potential of stem cells is not only felt by members of the health research community but also has caused great interest and awareness from the general population. Until recently, embryonic stem (ES) cells, derived from the inner mass of blastocysts, have been used for these studies. The use of human embryos in creating human ES cells, however, faces ethical controversies.

As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors [2-4]. The availability of induced pluripotent stem cells (iPS cells or iPSCs) could provide an ethically acceptable and relatively easy-to-access alternative to human ES cells. Furthermore, it is anticipated that therapies developed with iPSCs could circumvent the problem of tissue rejection following transplantation in patients by creating patient- or even tissue-specific pluripotent stem cells.

Still, great challenges stand between the current methods for generating iPSCs and their therapeutic potential. The use of integrating viral vectors has limited therapeutic potential due to the increased risk of tumor formation. It is therefore important to develop safe, effective and efficient targeting and delivery systems to produce iPSCs.

Because of this importance, multiple methods have been published within the last year that used delivery systems other than the retroviral or lentiviral vectors employed in the original iPSC publications. However, these newly reported methods have not addressed all of the known difficulties facing iPSCs creation. For instance, very low efficiency of transient transfection of selected cDNAs plasmids into primary cells lowers the already abysmal percentage of adult cells that can be reprogrammed with retroviral vectors [5]. Non-integrating adenovirus has a very short cDNA expression time period and thus requires repeated deliveries [6]. The application of episomal expression element such as oriP/EBNA1 helps sustaining longer expression time, but presently it is carried on a plasmid vector and does not improve transfection efficiency. Transposase and the Cre recombinase were used to remove integrated transgenes after the induction is completed, but the integration nevertheless occurred at multiple sites and requires careful and stringent analysis to make sure the reversion is complete; even so, elements of the vector may remain in the iPSCs genome [7, 8].

We wish to take this challenge and use it as an opportunity to develop a synthetic targeting and delivery system that will have the advantages of safe handling, no integration, prolonged and efficient reprogramming gene expression, and high transduction efficiency into broad cell types.

Baculovirus has been used in mammalian cells for many years (e.g. the BacMam system). Engineered baculovirus expression vectors (EBEV) will be exploited as a carrier for reprogramming genes for deriving induced pluripotent cells (iPSCs) from human adult cells. It has been well established that baculovirus can infect mammalian cells with broad tropism yet are very safe for regular laboratory handling.The elements planned for Allele Biotech’s new iPS generating system will be novel in the following aspects:

a) Promoter and mRNA structures for maximum level of cDNA expression in mammalian cells
b) Extended presence in the nucleus for sustained cDNA expression for weeks
c) Cleavable fluorescent protein for cell tracking and sorting
d) Auxiliary packaging constructs for increasing tropism to infect a broad range of human adult cells

Allele Biotech’s Design of Baculovirus for iPSCs

A) Mammalian Expression: In order to express reprogramming cDNAs in human cells, a mammalian promoter cassette will be inserted into the transfer vector to be used with Allele Biotech’s Sapphire Baculovirus genomic DNA. This system, derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), has been provided commercially for 10 years by our team and proved to be one of the best baculovirus systems because of its ease to use and high efficiency. Heterologous promoters have been used by us and others to create baculoviral vectors with dual or triple promoter for protein expression in insect, mammalian, and even bacterial cells such as Novagen’s pTriEx vectors and Allele’s pMBEVS. For the purpose of this proposal, we will remove insect expression cassette altogether and use a CAG promoter for driving expression solely in human cells.

B) Broader Tropism: Baculoviruses has been used for gene delivery to various hepatic and nonhepatic mammalian cells albeit the transduction of certain nonhepatic human cells is about 10-100 fold less effectively than hepatic cells [9]. Incorporating vesicular stomatitis virus G protein (VSVG) in the viral particle surface can greatly increase their effective transduction in a much broader range of mammalian cells [10]. For pseudotyping baculovirus, we will use a coinfection protocol with a recombinant baculovirus expressing VSVG. We expect that incorporating VSVG will ensure that human skin fibroblasts would be infected efficiently for Aim 1; and that different human cells could also be reprogrammed to become iPSCs via the proposed pathway in Aim 3 (see below). Different proteins or peptides might be tried as backup methods for pseudo-typing should VSVG not provide satisfactory results.

C) Fluorescent Marker: We will insert a fluorescent protein (FP) either expressed on a separate cassette or driven by an IRES downstream of reprogramming genes. This feature will facilitate tracking the infected cells, monitoring transgene expression, and enriching infected cells. Allele Biotech is one of the few suppliers/developers of fluorescent proteins. Our exclusive mTFP1 (cyan) and mWasabi (green) proteins are about 3 times brighter than EGFP and true monomers with great photostability and pH insensitivity, which should make them great choices for EBEV.

D) Promoter: The CAG promoter, which has been shown to be a strong promoter in mammalian cells and preferred for BacMam expression, will also be used in the EBEV vectors. We have previously designed pMBVES, a baculovirus vector for glycoprotein expression in mammalian cells that contains such a CAG enhancer/promoter. The CAG composite promoter also encompasses an exon1-intron-exon2 segment that will help mRNA processing and export to cytoplasm for translation. We will clone this fragmentfrom pMBVES into the proposed EBEV vectors.

E) RNA Elements: The 5’ UTR region on the mRNA will be examined and any hairpin structures with ??G < 30 kcal/mol or even <20 kcal/mol but with >65% GC will be disrupted. This step will ensure that maximum production be achieved at the translational level [11]. Post-transcriptional regulatory element (PRE) will be included to further boost gene expression. It has been shown that Woodchuck hepatitis virus (WPRE) increases transgene expression by many folds for various viral vectors, and there has been at least one case for a BacMam vector [12]. WPRE will be cloned from Allele Biotech’s existing HiTiter Lentiviral Vectors and inserted in the 3’ UTR upstream of the SV40 polyA signal.

F) Episomal Expression: Originally derived from Epstein-Barr virus (EBV), phophoprotein nuclear antigen 1 (EBNA1) ensures that oriP-containing DNA replicate once per S-phase during cell circle and is maintained in the nucleus as episomes. Although mostly applied to plasmid vectors, such as in one of the iPSC reports [13], the oriP/EBNA1 system can be incorporated into baculovirus systems for sustained mammalian expression [14]. Aside from episomal maintenance, oriP/EBNA1 could further up-regulate transcription of adjacent genes [14]. For these reasons, we will clone the oriP sequence together with the EBNA1 expression cassette from Allele Biotech’s Phoenix Retrovirus vector pBMN-GFP to EBEV vector.

Bibliography and Reference Cited
1. Thomson, J.A., J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, and J.M. Jones, Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7.
2. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
3. Takahashi, K., K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, and S. Yamanaka, Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
4. Yu, J., M.A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J.L. Frane, S. Tian, J. Nie, G.A. Jonsdottir, V. Ruotti, R. Stewart, Slukvin, II, and J.A. Thomson, Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
5. Okita, K., M. Nakagawa, H. Hyenjong, T. Ichisaka, and S. Yamanaka, Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008. 322(5903): p. 949-53.
6. Stadtfeld, M., N. Maherali, D.T. Breault, and K. Hochedlinger, Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell, 2008. 2(3): p. 230-40.
7. Woltjen, K., I.P. Michael, P. Mohseni, R. Desai, M. Mileikovsky, R. Hamalainen, R. Cowling, W. Wang, P. Liu, M. Gertsenstein, K. Kaji, H.K. Sung, and A. Nagy, piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 2009. 458(7239): p. 766-70.
8. Kaji, K., K. Norrby, A. Paca, M. Mileikovsky, P. Mohseni, and K. Woltjen, Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 2009. 458(7239): p. 771-5.
9. Stanbridge, L.J., V. Dussupt, and N.J. Maitland, Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer. J Biomed Biotechnol, 2003. 2003(2): p. 79-91.
10. Tani, H., M. Nishijima, H. Ushijima, T. Miyamura, and Y. Matsuura, Characterization of cell-surface determinants important for baculovirus infection. Virology, 2001. 279(1): p. 343-53.
11. Babendure, J.R., J.L. Babendure, J.H. Ding, and R.Y. Tsien, Control of mammalian translation by mRNA structure near caps. Rna, 2006. 12(5): p. 851-61.
12. Mahonen, A.J., K.J. Airenne, S. Purola, E. Peltomaa, M.U. Kaikkonen, M.S. Riekkinen, T. Heikura, K. Kinnunen, M.M. Roschier, T. Wirth, and S. Yla-Herttuala, Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells. J Biotechnol, 2007. 131(1): p. 1-8.
13. Yu, J., K. Hu, K. Smuga-Otto, S. Tian, R. Stewart, Slukvin, II, and J.A. Thomson, Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science, 2009.
14. Shan, L., L. Wang, J. Yin, P. Zhong, and J. Zhong, An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression. J Gene Med, 2006. 8(12): p. 1400-6.

Tags: bacmam, baculovirus, BVES, iPS cells, iPSC, iPSCs, non-integration, stem cells, therapeutic iPS cells

Protocols for Using Human Fibroblasts Expressing Human bFGF as Feeder Cells for iPSCs

New Product of the Week: Anti-GFP Polyclonal Antibody 100ug ABP-PAB-PAGFP10 $175.00.

Allele Biotech has introduced the highly efficient GFP-Trap for GFP fusion protein pull-down, and a monoclonal anti-GFP antibody for detecting GFP-fusion proteins after Immunoprecipitation with GFP-Trap. Just launched this week, the anti-GFP polyclonal antibodies provide an alternative method for analyzing the isolated proteins.

Pre-announcement: Allele Biotech will launch a FAQ and a User Forum online where you can also find common protocols in focus areas and exchange ideas with us or others.

1. Thaw one vial of irradiated feeder cells by swirling gently in 37oC water bath until all of the contents are thawed. One vial of 2×10^6 cells is sufficient to prepare two10-cm dishes, or two 6-well or 12-well plates (about 3-4×10^4/cm2).
2. Spray vial with 70% ethanol and wipe dry before placing in tissue culture hood.
3. Gently add 1 ml prewarmed feeder cell medium (alphaMEM or DMEM/F12 with 10% FBS), mix with contents of cryovial and transfer into 15-ml conical tube containing 4 ml prewarmed feeder cell medium.
4. Centrifuge the cells at 200g at room temperature for 5 min and discard the supernatant.
5. Resuspend the feeder cells in 12 ml feeder cell medium. If using a 6-well plate: add 1 ml of feeder cell suspension to each well of the 6-well plate containing 1 ml fresh feeder cell media per well. If using a 10-cm tissue culture dish: add 6 ml of feeder cell suspension to 10-cm tissue culture dish containing 6 ml fresh feeder cell media. If using a 12-well plate: add 0.5 ml feeder cell suspension to each well of 12-well plate containing 1 ml fresh feeder cell media per well. Gently shake the dish left/right and up/down 10-20 times without swirling the plate to evenly distribute the cells across the plate.
6. Incubate the cells in 37 1C, 5% CO2, overnight.
CRITICAL STEP When moving the feeder cell plates from the tissue culture hood to incubator, do not swirl the medium, as this tends to cause the cells to accumulate in the center. Immediately after placing the plates in the incubator, slide the plates forward and backward (2–3 cm) two times, then left to right (2–3 cm) two times to ensure equal distribution of the cells. Use within 5–7 days.
7. Split stem cells (~2.5 x 10^5 to 5 x 10^5 cells, or ~10% confluence) into plate with feeder cells: aspirate medium from ESC or iPSC, wash with PBS and add 0.5 ml of 0.05% trypsin. Incubate at 37oC, 5% CO2, for 5 min.
8. Inactivate trypsin with 3 ml stem cell medium (e.g. DMEM + 20% knockout serum replacement), and collect cell clumps in 15-ml conical tube avoiding making single cell suspension because ESC tends to die in single cell form.
9. Centrifuge at 200g at room temperature for 4 min.
10. Aspirate feeder medium from feeder plates (cells incubated in Step 6), rinse with one ml of stem cell medium and add 5 ml of stem cell medium and return to incubator.
11. Aspirate and discard supernatant from the conical tube in Step 8, resuspend cells in 5 ml stem cell medium, gently dispense the cell pellet three times, add to feeder cell wells or dishes.
12. Incubate stem cells grown on feeder cells at 37oC, 5% CO2, for 48 h.
13. Aspirate medium and replace with stem cell medium every day; if iPSC colony number is low, replace medium every two days.

Tags: bFGF, DMEM/F12, FAQ, feeder cells, forum, human fibroblasts, iPS cells, iPS protocol, iPSCs, New Product of the week, protocol, stem cells