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.

New Product of the Week 100410-101010:

pLICO-mWasabi (Promoterless FP Reporter Vector ), listed as product-on-demand, now available, ABP-HL-PE40010 $395.00.

Promotion of the Week 100410-101010:

Barrier too high to start using virus? Allele lowers it for starters, $500 for bactulo virus protein production, and $300 retrovirus packaging. Code 100310VIVEC, email vivec@allelebiotech.com

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

Telling Good iPSCs from Bad iPSCs

Since its discovery pluripotent stem cells (iPSCs) have been known to differ somewhat from embryonic stem cells (ESCs) in term of gene expression profiles. It also appears that only a small percentage of iPSCs have the full potential of stem cells defined by being able to develop into adult animals. Instead of a global pattern of variations, surprisingly, the difference between iPSC and ESC was found to localize in a small region of one chromosome in mouse, 12qF1, which could account for most iPS cells’ lack of complete pluripotency (Stadtfeld et al, Nature 2010). In this region resides an imprinted gene cluster that includes 2 non-coding genes, Gtl2 and Rian, that remain silenced in most iPSCs. The underlining mechanism is hypermethylation and hypoacetylation, resulting in “paternalizaition” of the region. The effects are manifested around the mid-gestation stage.

By adding histone deacetylase inhibitor valproic acid (VPA) the silenced gene cluster may be reactivated and the iPSCs so treated show increased Gtl2 expression and ability to give rise to normal embryos. Expression of other imprinted genes showed clone-to-clone variations, as was previously reported by a number of groups, but no consistent differences between ESCs cells and iPSCs. Therefore, by analyzing the expression levels of just two genes, Gtl2 and Rian, the potential of iPSCs to be fully pluripotent can be assessed.

The relationships between stem cell status and epigenetic repressions also include the recent finding that Oct4 and Sox2, which are both germ cell-specific and critical reprogramming factors, may be implicated in the regulation of Xist and Tsix RNAs that control epigenetic silencing of X chromosome in female embryos.

New Product of the Week 05-17-10 to 05-23-10: RT-PCR primer set, ABP-SC-iPSh4NX $49, for identifying exogenous iPS factor expression from 4-in-1 iPS lentivirus

Promotion of the Week 05-17-10 to 05-23-10: $85 off IceCube dry bath 0-75C variable temp

Tags: 12qF1, ES, ESC, Gtl2, Gtl2off, Gtl2on, imprinted, impriting, iPS, iPSC, pluripotency, Rian, silenced, VPA

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

FAQ About Feeder Cells for Stem Cells –Part One

The cost of preparing feeder cells for induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) is mainly due to 1. serum and media, 2. labor for growing and treating cells, and 3. expenses for freezing media and vials. Ready-to-use feeder cells saves one important labor-intensive step of iPSC generation, it should be an important help for iPSC and stem cell researchers. We know that most of our colleagues are tired of preparing fresh early passages of MEFs and treating them with expensive mitomycin C or finding an irradiator to pre-treat the MEFs. A lot of iPSC researchers lost iPS stem cells due to the lack of patience in handling MEF feeders. The offering of Allele’s feeder cell product line is really an easy solution and convenience to iPSC researchers.

Question 1: There are companies offering drug-resistant feeder cells such as MEF cells expressing neo-, puro-, or hygromycin-resistance genes. Is it important to have such drug-resistance genes when choosing feeder cells?

Adding drug resistant markers to these cells should not be necessary because iPSCs grown on feeder cells are usually not cultured in antibiotics-containing medium. The feeder cells will not be selected by drug resistance nor will they contaminate iPS cells since they can not propagate after irradiation. However, for those who do need to use drug selection for any reason, we will provide drug-resistant feeder cells upon request.

Question 2: There are publications showing the use of cells lines as feeder cells instead of primary fibroblasts, e.g. SL10, MRC-5, STO. Are there any advantages of using these cell lines?

Not really. Handling primary cells requires certain amount of experience and may be tedious; using cell lines, on the other hand, would be easier for preparing feeder cells. We provide feeder cells from immortalized early passage human foreskin fibroblasts at prices often lower than those from cell lines.

Question 3: Should I choose fluorescent protein expressing feeder cells for easy separation from iPSCs?

You do not need to include fluorescent protein in feeder cells, as feeder cells are quite different in morphology from iPS cells or ES cells. In fact, many labs use iPS factors that are co-expressed with fluorescent markers, in which cases feeder cell expressed fluorescent proteins will confuse the readout.

Question 4: What are the main advantages of using bFGF-expressing feeder cells?

Our bFGF-feeder cells not only eliminate the needs for added recombinant bFGF to stem cell cultures, but also form very nice cell lawn to serve iPSC colony formation because of their strictly controlled passage and growth conditions. We have used these cells without coating dishes with gelatin and obtained nice iPSC colonies.

Preview: Next Part of FAQ on Feeder Cells: choosing mouse or human fibroblasts, selecting iPSC colonies…

Announcement: An audience-orientated User Forum will be added to Allele Biotech webpages so that people can freely discuss or review products and technologies. A distilled version of discussions will be presented in a related but separate FAQ section, which will also include all Allele eNewsletters sent to our contacts about every quarter. Look for the links on www.allelebiotech.com in coming weeks.

Tags: bFGF, biology FAQ, biology forum, biotech enewsletter, ES cells, ESC, ESCs, feeder cells, human fibroblasts, iPS cells, iPSC, iPSCs, irradiation, MEF, MEFs, mitomycin C, stem cell culture