LoxP 4-in-1 iPS Factor on Lentiviral Vectors for Efficient Reprogramming

Putting 4 iPS factors on one lentiviral vector, separated by 2A peptides, has appeared to be more efficient in generating iPS cells than having all 4 factors on individual viruses, at least in a number of cases. Stem cell-like colonies start to appear in about 2 weeks using Allele Biotech’s 4-in-1 lentivirus. In addition to the concerted effects from Oct3/4, Sox2, c-Myc, Klf4, it is also believed that the coordinated silencing of these factors after reprogramming help forming iPS colonies.

The 4-in-1 lentivirus from Allele Biotech contains loxP sites that can be used to remove the 4 cDNAs if so desired. For convenience, a new product kit is offered starting this week to include lenti-nCre in a kit with the 4-in-1 iPS viral products.

New Product of the Week 04-11-10 to 04-18-10: 4-In-One-Vector: Human OSKM Lentiviral Paticles (Oct3/4, Sox2, Klf4 and c-Myc) and Cre Lentiviral Particle kits, Cat # ABP-SC-LVI4IN1C1 or ABP-SC-LVI4IN1C5

Promotion of the Week 04-11-10 to 04-18-10: Single vial 4-in-1 is offered only this week. This product has been well established and validated, one of the reasons smaller packages are not normally offered. As a matter of fact, every batch of the 4-in-1 iPS lentivirus has been sold out.

Update note: Lentivirus inserts into the host chromosome, and is gradually being replaced by footprint-free reprogramming reagents, the best being Allele Biotech’s enhanced mRNA reprogramming factors that feature a patent-pending fusion gene.

Tags: 4-in-1, c-myc, cre, iPS, iPSCs, Klf4, lentivirus, loxP, Oct3, repgramming, silencing, Sox2

Using 2A “self-cleaving” peptide in bicistronic mammalian expression

Multiple promoters or internal ribosomal entry sites (IRES) have been used for the production of multiple proteins from the same vector. Potential drawbacks with multiple promoters on viral vectors include unstable genome and interference between promoters. IRES is a relatively large sequence that can cause problems in virus packaging, especially for viruses with very limited genome size such as AAV. In addition, it is required that the start of the second ORF is fairly close to the IRES, adding difficulties to cloning.

2A or 2A-like peptide (collectively called 2A peptide here) is used by several families of viruses, the best known foot-and-mouth disease virus of the Picornaviridae family, for producing multiple polypeptides. Although called a “self-cleaving” peptide or protease site, the mechanism by the 2A sequence for generating two proteins from one transcript is by ribosome skipping–a normal peptide bond is impaired at 2A, resulting in two discontinuous protein fragments from one translation even.

The 2A-based bicistronic expression has been used for several years, but recently gained much more popularity due to its successful use in iPSC generation that required 2 to 4 factors working in concert. Even expression of all factors can be achieved when 2A peptides are used for multiple protein production, due to near 100% efficiency of the 2A “cleavage” at each site, and no interference between multiple 2A sites. Early work used a 36 amino acid sequence as 2A peptide, which was later reduced to about half that size from mutation and screening. Commercial vectors utilizing 2A for co-expression of cDNA and fluorescent protein and/or drug resistance genes have not been available until now. Allele Biotech has introduced a number of such plasmids, establishing another First-to-the-market as it has done many times previously in its 10 year history.

New product of the week 03-29-10 to 04-04-10:

Alleleustrious pmTFP1-2A Bicistronic mammalian expression vector, ABP-FP-T2A10, $399, http://www.allelebiotech.com/shopcart/index.php?c=215&sc=34

Promotion of the week 03-29-10 to 04-04-10:

Buy any GFP-Trap beads or kits, get polyclonal anti-GFP (ABP-PAB-PAGFP10) at half size for FREE!  ***GFP-Trap has been replaced with GFP-nAb, an improved version for the same purposes in the form of agarose beads, magnetic beads, polyacrylamide beads, spin column kits, etc.  Come and take a look at the most comprehensive list of nearly all FPs before purchasing.

Tags: 2A peptide, anti-GFP, bicistronic, drug resistance, GFP-Trap, iPS, iPSCs, IRES, polycistronic, ribosome skip, self-cleaving, transcription, translation

Monitoring the Undifferentiated Stage of Stem Cells—the Pluripotency Markers

Human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells promise to serve as an unlimited source for transplantation or tissue-specific differentiation. However, obtaining and maintaining stem cells are very difficult tasks for multiple reasons. For instance, most stem cell lines tend to spontaneously differentiate in culture, and even if the cells form stem cell-like colonies, they may be of a heterogeneous population.

To identify pluripotency of stem cells, expression of stem cell-specific marker genes (i.e. Oct-3/4, Sox2, Nanog, Rex-1) is monitored by RT-PCR. Alkaline phosphatase activity and methylation profiles of promoters of pluripotency-relevant genes are often analyzed as well. Compared to murine cells, it is noticeably more difficult to obtain human iPSCs, of which stem cell-like colonies sometimes turn out not to be pluripotent cells. We highly recommend testing iPSCs, especially human iPSCs, with antibodies against stage-specific embryonic antigens such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

However, all of these methods require cell destruction or fixation for analysis, therefore, are inconvenient and costly. Furthermore, many studies using ES or iPS cells involve differentiation of stem cells into different lineages, a method for observing live cells to know their undifferentiation/differentiation stages would be very helpful. There have been a number of publications using murine Oct-4, Nanog, and Rex-1 promoter driven fluorescent proteins as markers for pluripotency tests [1-3]. Allele Biotech provides, under its iPS product line, packaged and validated lentiviral particles that would insert these 3 promoter-FP reporters into the stem cells. Although currently these promoters are of mouse sequences, their use in human stem cells have been reported.

New product of the week 01-25-10 to 01-31-10:

All-In-One-Vector: Human OSKM Lentiviral Paticles, with Oct-4, Sox-2, Klf, and c-Myc all expressed from a single virus, ready-to-use.

• Promotion of the week:

human iPS cell detection primer set, the same as the landmark Yamanaka paper [4] on creating human iPS for the first time.

1. Da Yong WU, Zhen YAO (2005). Isolation and characterization of the murine Nanog gene promoter. Cell Research, 15 (5): 317–324.
2. Rachel Eiges, Maya Schuldiner..et.al (2001). Establishment of human embryonic stem cell?transfected clones carrying a marker for undifferentiated cell. Current Biology 11: 514–518.
3. Guangjin Pan, Jun Li, Yali Zhou, Hui Zheng, and Duanqing pei (2006). A negative feedback loop of transcription factors that control stem cell pluripotency and self?renewal. ASEB Journal 20: E1094? E1102
4. Takahashi et al, Induction of Pluripotent Stem Cell from Adult Human Fibroblasts by Defined Factors (2007). Cell 131, 861-872

Tags: differentiation, fluorescent protein, iPS, iPS lentivirus, iPSC, iPSCs, lentivirus, pluripotency reporter, promoter, promoter-FP, stem cells, undifferentiation stage

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