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

How do you produce your iPS cells?

From AlleleForum: First off, thank you for choosing Allele Biotech for your iPSC experiment needs. Now onto your questions

You asked Q1: How many human fibroblast cells you normally to start for transfection. I understand you use 12-well plate? How many days you wait till the cells grow confluent? If the cells never grow confluent, should I still transfer them to feeder plate? Is it critical for the cells to reach confluent, if it is, could you suggest the reasons to me

We usually plate at 70% or about 10e4-10e5 cells and transduce the cells for 2-3 days. It should become confluent in 2-3 days. There is no need for the cells to become confluent before splitting onto feeder cells. Please note for primary cells, do not wait for the cells to get too confluent because contact inhibition may induce growth senescence before cells are reprogrammed.

Q2: How many cells you plate on the feeder plate, let’s say it is 6-well plate, and how many clones would normally pup out from each well?

From one well of a 12 well plate, you can plate 1/5 onto a well of a 6 well feeder cell plate. From there, you should get plenty of colonies.

Q3: At the time when you need to cut the Loxp sites, what passage number you do, do you have to dispense the iPS into single cell? Do you have a detailed protocol for that? Other than virus, do you have any other means to do the job, like plasmid?

Never dispense iPSC into single cells. They do not grow back well if split into single cells. iPSC colonies should be passaged in patches of cells. To excise loxP, the suggested timing is after 12-14 days when the cells are reprogrammed into iPSC colonies. Just transduce the iPSC colonies with Cre virus.

Q4: Is it true, that the 4-in-1 is more powerful than individual ones? Do you have the construct(4-in-one) for sale?

The 4-in-1 is somewhat more effective than 4 individual ones. For license issues, we do not distribute the construct to customers because we only offer packaging service. Similar type of plasmid DNAs may be accessible from other sources.

If you have any other questions or concerns, please let us know. Thanks again.

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Tags: 4-in-1, feeder cells, iPS, iPS colonies, iPS medium, iPS protocol, lentivirus, loxP Cre, splitting iPS cells

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

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.

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

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.

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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