MEF

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.

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Thursday, August 19th, 2010 iPSCs and other stem cells No Comments

How to Generate Conditional Knockout Mice with Cre

The bacterial Cre recombinase targets a specific DNA sequence called loxP and deletes a segment of DNA flanked by loxP sequences. This system is often used in the generation of knockout and conditional knockout animals.

The knockout of specific genes leading to embryonic lethal phenotype will not yield adult animals. Cre-lox recombination provides a means to knockout the specific genes in adult mice, or to introduce a knockout phenotype in specific tissues (conditional knockout) using tissue-specific promoter driven Cre or an inducible Cre.

The cutting by Cre at the loxP sites and rejoining by ligase is an efficient process. During this process, inverted loxP sites will result in an inversion, whereas direct repeat will cause a deletion. Cre/lox recombination is a one-way reaction so there is no need for continued Cre expression. Therefore, Cre can be introduced by adenovirus or lenti/retrovirus. Here is an example of using adnovirus-Cre in one lab: for MEF, on a 70% confluent P10 cm plate (probably 2-2.5 million cells), use 6ul of 1.1×10^12 adenovirus-Cre, which will give 80% infection; or use 10ul of 1.1×10^12 adenovirus-Cre to get 90% infection, with GFP as marker and analyzed by FACS.

Adenovirus could post a toxicity problem when used at very high titers to reach high percentage of transduction. An alternative is to use only lentivirus-Cre, at only about 1-2 ul and still obtain >80% infection. However, a silencing event needs to occur before the expression of Cre from lentivirus is shut off. The timing and degree of silencing is not controlled in such experiments. Continued expression of Cre should not influence most experiments.

To be certain that the Cre enzyme can be successfully delivered into the nucleus for conditional knockout to occur, the bacterial Cre gene needs to be engineered to contain a nuclear localization (nl) signal of eukaryotic cells. The function of the nuclear-localized Cre (nlCre) can be tested using a loxP-nuclear localized lacZ (nlacZ) reporter cell line, which can be used to monitor the function of the nlCre recombinase.

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Wednesday, March 3rd, 2010 Viruses and cells No Comments

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.

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Wednesday, October 7th, 2009 iPSCs and other stem cells No Comments