[ZucchiniFlower]

Received this email today:

Marius Wernig and Alexander Meissner from the Jaenisch lab are tentatively scheduled to appear tonight on the NBC Nightly News at 6:30 pm (Channel 7 in the greater Boston area). Due to the unpredictability of network news, the segment could be rescheduled at the last minute for another night.

Wernig and Meissner will discuss today's groundbreaking Nature paper that describes taking mouse fibroblasts (a kind of cell that gives rise to connective tissue) and, through genetic manipulation, turning them into cells that resemble embryonic stem cells in every way that can be tested.

Although this work has been done in mice, scientists are optimistic that these findings will translate to human cells and one day enable researchers to create embryonic stem cells without utilizing an egg or embryo.

[ZucchiniFlower]

Nature
Published online: 6 June 2007; | doi:10.1038/447618a
Simple switch turns cells embryonic
Technique removes need for eggs or embryos.

David Cyranoski

Research reported this week by three different groups shows that normal skin cells can be reprogrammed to an embryonic state in mice1, 2, 3. The race is now on to apply the surprisingly straightforward procedure to human cells.

If researchers succeed, it will make it relatively easy to produce cells that seem indistinguishable from embryonic stem cells, and that are genetically matched to individual patients. There are limits to how useful and safe these would be for therapeutic use in the near term, but they should quickly prove a boon in the lab.



The birth of this chimaeric mouse suggests that the cells used to generate it behave like embryonic stem cells.
S. OGDEN


"It would change the way we see things quite dramatically," says Alan Trounson of Monash University in Victoria, Australia. Trounson wasn't involved in the new work but says he plans to start using the technique "tomorrow". "I can think of a dozen experiments right now — and they're all good ones," he says.

In theory, embryonic stem cells can propagate themselves indefinitely and are able to become any type of cell in the body. But so far, the only way to obtain embryonic stem cells involves destroying an embryo, and to get a genetic match for a patient would mean, in effect, cloning that person — all of which raise difficult ethical questions.

As well as having potential ethical difficulties, the 'cloning' procedure is technically difficult. It involves obtaining unfertilized eggs, replacing their genetic material with that from an adult cell and then forcing the cell to divide to create an early-stage embryo, from which the stem cells can be harvested. Those barriers may have now been broken down.

"Neither eggs nor embryos are necessary. I've never worked with either," says Shinya Yamanaka of Kyoto University, who has pioneered the new technique.

Last year, Yamanaka introduced a system that uses mouse fibroblasts, a common cell type that can easily be harvested from skin, instead of eggs4. Four genes, which code for four specific proteins known as transcription factors, are transferred into the cells using retroviruses. The proteins trigger the expression of other genes that lead the cells to become pluripotent, meaning that they could potentially become any of the body's cells. Yamanaka calls them induced pluripotent stem cells (iPS cells). "It's easy. There's no trick, no magic," says Yamanaka.

It's unbelievable, just amazing. It's like Dolly. It's that type of accomplishment.

The results were met with amazement, along with a good dose of scepticism. Four factors seemed too simple. And although the cells had some characteristics of embryonic cells — they formed colonies, could propagate continuously and could form cancerous growths called teratomas — they lacked others. Introduction of iPS cells into a developing embryo, for example, did not produce a 'chimaera' — a mouse carrying a mix of DNA from both the original embryo and the iPS cells throughout its body. "I was not comfortable with the term 'pluripotent' last year," says Hans Schöler, a stem-cell specialist at the Max Planck Institute for Molecular Biomedicine in Münster who is not involved with any of the three articles.

This week, Yamanaka presents a second generation of iPS cells1, which pass all these tests. In addition, a group led by Rudolf Jaenisch2 at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, and a collaborative effort3 between Konrad Hochedlinger of the Harvard Stem Cell Institute and Kathrin Plath of the University of California, Los Angeles, used the same four factors and got strikingly similar results.

"It's a relief as some people questioned our results, especially after the Hwang scandal," says Yamanaka, referring to the irreproducible cloning work of Woo Suk Hwang, which turned out to be fraudulent. Schöler agrees: "Now we can be confident that this is something worth building on."

The improvement over last year's results was simple. The four transcription factors used by Yamanaka reprogramme cells inconsistently and inefficiently, so that less than 0.1% of the million cells in a simple skin biopsy will be fully reprogrammed. The difficulty is isolating those in which reprogramming has been successful. Researchers do this by inserting a gene for antibiotic resistance that is activated only when proteins characteristic of stem cells are expressed. The cells can then be doused with antibiotics, killing off the failures.

The protein Yamanaka used as a marker for stem cells last year was not terribly good at identifying reprogrammed cells. This time, all three groups used two other protein markers — Nanog and Oct4 — to great effect. All three groups were able to produce chimaeric mice using iPS cells isolated in this way; and the mice passed iPS DNA on to their offspring.

Jaenisch also used a special embryo to produce fetuses whose cells were derived entirely from iPS cells. "Only the best embryonic stem cells can do this," he says.

"It's unbelievable, just amazing," says Schöler, who heard Jaenisch present his results at a meeting on 31 May in Bavaria. "For me it's like Dolly [the first cloned mammal]. It's that type of accomplishment."

The method is inviting. Whereas cloning with humans was limited by the number of available eggs and by a tricky technique that takes some six months to master, Yamanaka's method can use the most basic cells and can be accomplished with simple lab techniques.

But applying the method to human cells has yet to be successful. "We are working very hard — day and night," says Yamanaka. It will probably require more transcription factors, he adds.

If it works, researchers could produce iPS cells from patients with conditions such as Parkinson's disease or diabetes and observe the molecular changes in the cells as they develop. This 'disease in a dish' would offer the chance to see how different environmental factors contribute to the condition, and to test the ability of drugs to check disease progression.


But the iPS cells aren't perfect, and could not be used safely to make genetically matched cells for transplant in, for example, spinal-cord injuries. Yamanaka found that one of the factors seems to contribute to cancer in 20% of his chimaeric mice. He thinks this can be fixed, but the retroviruses used may themselves also cause mutations and cancer. "This is really dangerous. We would never transplant these into a patient," says Jaenisch. In his view, research into embryonic stem cells made by cloning remains "absolutely essential".

If the past year is anything to judge by, change will come quickly. "I'm not sure if it will be us, or Jaenisch, or someone else, but I expect some big success with humans in the next year," says Yamanaka.

Additional reporting by Heidi Ledford
For more on alternative stem-cell work, see 'Stem cells: Recycling the abnormal'; and see http://www.nature.com/stemcells

Article brought to you by: Nature

References

1. Okita, K., Ichisaka, T. & Yamanaka, S. Nature doi:10.1038/nature05934 (2007).
2. Wernig, M. et al. Nature doi:10.1038/nature05944 (2007).
3. Maherali, N. et al. Cell Stem Cell doi:10.1016/j.stem.2007.05.014 (2007).
4. Takahashi, K. & Yamanaka, S. Cell 126, 663–676 (2006). | Article | PubMed | ChemPort |

http://www.nature.com/news/2007/0706...l/447618a.html

[ZucchiniFlower]

Nature 447, 649-650 (7 June 2007) | doi:10.1038/447649a; Published online 6 June 2007

full article:

http://www.nature.com/nature/journal...ture05879.html

Stem cells: Recycling the abnormal

Alan Colman1 & Justine Burley2

Abstract

Using human eggs in the quest to make donor-specific embryonic stem cells is controversial. A method developed in mice, if applicable to humans, could eliminate the need to obtain eggs for this purpose.

On page 679 of this issue, Egli et al.1 describe a promising method for generating embryonic stem-cell (ESC) lineages using the technique of somatic-cell nuclear transfer (SCNT). Conventional SCNT involves replacement of the nuclear genetic material of an unfertilized egg (oocyte), with that of a somatic (non-germ) cell. After 'fertilization', which is induced by chemical or electrical triggers, the embryo undergoes several rounds of cell division and, after implantation into a foster mother, may develop to term. So far, this technique has been used successfully to clone 12 species. It also has been used in mice to generate ESCs from a 3.5-day-old mouse embryo2 — a blastocyst.

Since Dolly the Sheep was cloned by SCNT more than ten years ago3, it has been hoped that this technique would serve to create patient-matched ESCs for therapy, and human-disease-specific ESC lines for use in basic research and drug development. However, in contrast to SCNT in mice, the use of this technique in humans has been thwarted by technical difficulties, as well as logistical and ethical concerns about obtaining oocytes. Now, Egli and colleagues1 describe a different approach to produce donor/disease-specific ESC lines that may well revolutionize the field of human stem-cell research, and that removes one of the main ethical objections to such work. The crux of their contribution is the use of fertilized eggs, instead of oocytes, as SCNT recipients.

Historically, fertilized mouse eggs at the one-cell stage — the zygote — have been successfully used as recipients of nuclear genetic material4, but only when the donor cells were also zygotes and not from later developmental stages5. Possible reasons for this limitation include loss of essential non-DNA factors with the removed genetic material6, and inadequate time for the reprogramming of the donor's genetic material in its new environment5.

Egli et al. reasoned that the loss of the crucial factors could be minimized or eliminated if nuclear transfer is conducted when both the recipient zygote and the donor cell are temporarily arrested at the mitotic cell division. To test this, they used the drug nocodazole to arrest mitosis in mouse zygotes at the stage when chromosomes condense. Replacing nocodazole with another inhibitor allowed chromosome alignment along the mitotic spindle, but prevented further cell-cycle progression. The spindle could then be seen using optical devices, and removed mechanically.

Donor zygotes and two- and eight-celled embryos were also arrested with nocodazole. The condensed chromosomes were then identified, removed from individual cells, and injected into the cytoplasm of treated recipient zygotes. Removal of the inhibitors allowed development to resume, and the resulting blastocysts were returned to foster mothers. Donors from all three stages of development led to some live births.

Next, the authors used mouse ESCs as donors. The resultant blastocysts were either returned to foster mothers — leading to nine live births — or were used to make new ESC lines. By injecting these SCNT-derived ESCs into normal host blastocysts they showed that these cells had the full range of developmental potencies expected from bona fide mouse ESCs.

Finally, adult tail-tip cells were used as recipients to make donor-specific SCNT-derived ESCs. Previously, mitotic, embryonic7 and somatic cells8 have all been used as donors in nuclear transfer experiments. But Egli et al. are the first to use a mitotic cytoplasm as a recipient. Using cells at this stage of the cell cycle as recipients may expedite reprogramming of donor chromosomes, because at other stages reprogramming factors are probably sequestered within cells' nuclei9.

In terms of efficiency, the method reported by Egli et al.1 is not better than previous ones. So why all the excitement? After all, the new method seems to be ethically inferior, as in generating SCNT-derived ESCs, two, rather than one, developing embryos are disrupted — the original zygote and the SCNT-derived embryo. The answer can be found in the results of their last experiment.

The researchers generated an embryo containing three sets of chromosomes — in which two sperm cells fertilized a single oocyte. Such embryos never develop normally. Nevertheless, replacement of these three sets of chromosomes with one set from an ESC led to a normally developing embryo, which could potentially be used to generate a new ESC line (Fig. 1). This finding could have a profound effect on developing a viable and tractable method of SCNT in humans.
Figure 1: Somatic-cell nuclear transfer using abnormal embryos.
Figure 1 : Somatic-cell nuclear transfer using abnormal embryos. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

Egli et al.1 generated abnormal mouse zygotes, in which the egg was fertilized with two sperm cells (n indicate pronuclei). Such zygotes are often useless by-products of human in vitro fertilization procedures. However, using inhibitors, the authors allowed the abnormal mouse zygote to progress through the cell cycle up to the point during mitosis at which its chromosomes aligned on the mitotic spindle. They then mechanically removed the spindle and replaced it with the condensed chromosomal content of a donor embryonic stem cell (ESC), also arrested at mitosis. Removal of the inhibitors allowed development to resume, and a blastocyst formed. This is a promising technical feat, as the authors also found that blastocysts formed in this way but using ESCs or adult tail-tip cells as donors and normal zygotes as recipients led to live offspring or, alternatively, new ESCs.
High resolution image and legend (70K)

The failure of SCNT in humans and monkeys has been attributed by some10 to fundamental differences between primate and non-primate unfertilized eggs in the way their spindles form during cell division. However, even if this difficulty could be surmounted, obtaining freshly ovulated human oocytes would remain of logistical and ethical concern; unfortunately, in contrast to recent success in mice11, aged, unfertilized oocytes — a by-product of normal in vitro fertilization (IVF) procedures — have been inadequate for SCNT in humans12. However, if the technique developed by Egli and colleagues could be used successfully in humans, all of these problems would be circumvented.

It is estimated that 3–5% of fertilized human zygotes contain supernumerary sets of chromosomes13. Such zygotes are always excluded from clinical use in IVF centres because they cannot develop, and are therefore disposed of. The possibility of recycling non-viable zygotes to produce ESC lines obviates the need for oocyte donation. So those who have been troubled by this ethical aspect of human SCNT stem-cell research will be very encouraged by the results of Egli and his colleagues1.


References

1. Egli, D., Rosains, J., Birkhoff, G. & Eggan, K. Nature 447, 679–685 (2007). | Article |
2. Munsie, M. et al. Curr. Biol. 10, 989–992 (2000). | Article | PubMed | ISI | ChemPort |
3. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. & Campbell, K. H. Nature 385, 810–813 (1997). | Article | PubMed | ISI | ChemPort |
4. McGrath, J. & Solter, D. Science 220, 1300–1302 (1983). | PubMed | ISI | ChemPort |
5. McGrath, J. & Solter, D. Science 226, 1317–1319 (1994).
6. Polejaeva, I. A. et al. Nature 407, 86–90 (2000). | Article | PubMed | ISI | ChemPort |
7. Kwon, O. Y. & Kono, T. Proc. Natl Acad. Sci. USA 93, 13010–13013 (1996). | Article | PubMed | ChemPort |
8. Ono, Y., Shimozawa, N., Ito, M. & Kono, T. Biol. Reprod. 64, 44–50 (2001). | Article | PubMed | ISI | ChemPort |
9. Do, J. T. & Scholer, H. R. Stem Cells 22, 941–949 (2004). | Article | PubMed | ISI | ChemPort |
10. Simerly, C. et al. Science 300, 297 (2003). | Article | PubMed | ISI |
11. Wakayama, S. et al. Curr. Biol. 17, R120–R121 (2007).
12. Hall, V. J. et al. Hum. Reprod. 22, 52–62 (2007).
13. Aoki, V. W. et al. J. Exp. Clin. Assist. Reprod. 2, 3 (2005).

[Thelma]

Millions and millions of dollars being spent in the quest for the answer to satisfy a religious supposition surrounding their view of what life really is.

why???????????????????????????????????????????????

Do they want a cure for these diseases or not???????????????

Is it really a quest for a cure or for a large profit based platform for more lmonies for useless research endeavors???????????????????

Cure first with what can do work land then find that which can be used for generic treatments for the religious few.

I am so sick of all the hype that is going on on all sides.

[ZucchiniFlower]

It may be better to use a patient's own cells to create cells with the same properties as embryonic stem cells. Considering the number a patients who need the benefit, it would probably be best to use their own bodies to help cure themselves.

Public release date: 6-Jun-2007


Contact: Erin Doonan
edoonan@cell.com
617-397-2802
Cell Press
A twist of fate -- Reprogrammed fibroblasts resemble embryonic stem cells

Stem cell biology takes another exciting leap forward as scientists report that normal tissue cells can be reprogrammed to exhibit many of the properties that are characteristic of embryonic stem cells, including the ability to give rise to multiple cell types and contribute to the germline. These findings, published in the inaugural issue of the journal Cell Stem Cell, published by Cell Press, provide strong support for the rationale that it may be possible to generate stem cells with nearly unlimited potential directly from a patient’s own cells, an idea that has significant implications for regenerative therapeutics.

Although transplantation of stem cells generated from human embryos is considered to be a promising option for replacement of damaged or diseased tissues, there are serious difficulties and concerns associated with this methodology. Controversial ethical issues are associated with the use of human embryos, and tissue rejection remains a major concern with stem cell transplants, just as it is for organ transplants. One way to avoid these potential problems is to find a way to reliably reprogram an individual’s differentiated tissue cells into cells that behave like embryonic stem cells and can give rise to any fetal or adult cell type.

Recent research discovered that expression of four transcription factors can induce a pluripotent state in adult fibroblasts. Dr. Konrad Hochedlinger from the Massachusetts General Hospital Center for Regenerative Medicine and the Harvard Stem Cell Institute, Dr. Kathrin Plath from the Institute for Stem Cell Biology and Medicine at UCLA, and their colleagues improved this approach and combined it with an efficient selection process, which allowed them to generate induced pluripotent cells from fibroblasts that were, based on the assays used, indistinguishable from ES cells. For example, genome-wide analysis revealed that the induced stem cells were highly similar to embryonic stem cells with regards to global DNA methylation and histone methylation patterns. In addition, female-induced stem cells showed reactivation of the X chromosome that was silenced in differentiated cells and exhibited random X inactivation upon differentiation.

“Our results demonstrate that the ectopic expression of four transcription factors is sufficient to globally reset the epigenetic landscape of fibroblasts into that of pluripotent cells that are remarkably similar to embryonic stem cells,” explains Dr. Hochedlinger. Dr. Plath adds “The fact that our induced pluripotent cells are epigenetically similar to ES cells suggests that epigenetic abnormalities will not pose a problem for the potential therapeutic applications of induced pluripotent cells.” The researchers went on to show that the induced pluripotent cells could differentiate into numerous cell types, including blood cells in culture and oocytes in animals. Importantly, two related papers being published in the journal Nature demonstrate that similar induced pluripotent cells can also give rise to fertilized embryos and viable offspring, respectively. Future studies are needed to examine whether direct reprogramming of human cells follows these promising patterns observed in mice.

These studies and other recent advances in strategies aimed at generating patient-specific stem cell lines are discussed in the review article by Dr. Shinya Yamanaka in the current issue of Cell Stem Cell.
###

Journalists please note: The related papers in the journal Nature will go live at the same time and follow the same embargo, 1:00pm Eastern Time US on Wednesday, 6 June 2007. For further information please contact Ruth Francis, Senior Press Officer at Nature: r.francis@nature.com or visit Nature's press site at http://press.nature.com/press.

The researchers include Nimet Maherali of Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine and Harvard Stem Cell Institute in Boston, MA and Harvard University in Cambridge, MA; Rupa Sridharan, Wei Xie, Robin Yachechko, Jason Tchieu, and Kathrin Plath of UCLA School of Medicine in Los Angeles, CA; Jochen Utikal, Sarah Eminli, Katrin Arnold, Matthias Stadtfeld, and Konrad Hochedlinger of Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine and Harvard Stem Cell Institute in Boston, MA; Rudolf Jaenisch of the Whitehead Institute at Massachusetts Institute of Technology in Cambridge, MA.



Maherali et al.: “Directly Reprogrammed Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribution.” Publishing in Cell Stem Cell 1, 55–70, July 2007. DOI 10.1016/j.stem.2007.05.014

FULL ARTICLE:

http://www.cellstemcell.com/content/...34590907000203

FROM THE ARTICLE:

"Successful reprogramming of somatic cells by nuclear transfer or cell fusion is thought to require faithful remodeling of epigenetic modifications such as DNA methylation, histone modifications, and reactivation of a silent X chromosome in female cells (Rideout et al., 2001). Aberrant epigenetic reprogramming is assumed to be the principal reason for the developmental failure and abnormalities seen in animals cloned by nuclear transfer. Thus, the question of epigenetic reprogramming is of particular relevance for the potential therapeutic applications of iPS cells, as epigenetic aberrations can result in pathological conditions such as cancer (Gaudet et al., 2003).

Here, we have generated iPS cells from fibroblasts by using novel selection approaches and assessed their epigenetic status at a gene-specific, chromosome-wide, and genome-wide level. Our results demonstrate that the ectopic expression of four transcription factors is sufficient to globally reset the epigenetic state of fibroblasts into that of pluripotent cells that are remarkably similar to ES cells."