The Niche

The standard technique for creating make differentiated cells behave like embryonic stem cells uses viruses to insert the genes cMyc, Klf4, Oct4, and Sox2 into cells, but adding these genes to cells makes them less predictable and more likely to form tumors. Researchers have been able to reprogram neural stem cell using only Oct4, but these cells are not readily available from patient biopsies and so researchers are searching for alternate techniques. New work published in Cell Stem Cell shows that a small druglike molecule can effectively replace two of the four genes typically used to generate induced pluriptotent stem cells.
To begin their hunt for compounds that could help reprogram cells, researchers led by Kevin Eggan and Lee Rubin of the Harvard Stem Cell Institute used cultures of mouse skin cells engineered to express green fluorescent protein as a marker of pluripotency. They first screened for small molecules that allowed mouse cells to be reprogrammed without adding the gene for Sox2. When three such molecules were identified, the researchers tried again and found that one of the molecules could reprogram cells even in the absence of cMyc, a tumour-promoting gene that, while not required for reprogramming, greatly boosts reprogramming rates.
To make sure the cells were really reprogrammed, the researchers performed a series of tests, including mixing them with mouse embryos and demonstrating that they could contribute to every type of tissue in chimeric mice. They named the identified molecule RepSox for its ability to replace Sox2 and also after the Red Sox, the local baseball team. Previous studies had identified this molecule as inhibiting a pathway known as TGF-beta signaling. Careful work showed that RepSox did not work by activating the Sox2 gene in fibroblasts, as might be expected. Instead, the molecule functions in partially reprogrammed cells that accumulate in the absence of Sox2, apparently by inducing and stabilizing Nanog expression. Thus, the researchers write, the discovery of RepSox is important not only for replacing one of the reprogramming factors but for illuminating a new strategy to identifying such molecules. “There need not always be a discrete, one-to-one mapping between the functions of the reprogramming factors and their chemical replacements.”
Robert Blelloch, who studies reprogramming at the University of California San Francisco, praised the team’s strategy of only screening compounds whose mechanisms are at least partly understood. “They find a small molecule that replaces a factor, but they take it further and use it to understand the biology.”

See also: Induced pluripotent stem cells: down to one factor

Posted by Monya Baker on October 08, 2009
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Differentiated human cells have been reprogrammed to an embryonic-like state with the addition of only one gene, rather than the standard four [1]. This should advance techniques for the efficient production of high-quality patient-specific stem cells.

The ability to make induced pluripotent stem (iPS) cells using cells from specific patients could enable unprecedented new ways to study disease and also ease the development of cell therapies. However, such applications have been stymied in part because making induced pluripotent stem cells efficiently requires the introduction of pluripotency genes, which are typically inserted at random sites throughout the genome. This unwanted source of variation stymies rigorous comparisons between cells, and could make them behave in unpredictable, dangerous ways if used for cell therapies. Several techniques to make cells without permanent insertion of the genes have been reported, including some that do not use genetic material at all.
See: Human iPS cells with no genetic integration
Virus free pluripotency for human cells
Integration-free iPS cells
Reprogramming to pluripotency without genetic engineering
Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins

However, researchers are eager for additional, ‘gentler’ ways to reprogram cells, and one possibility would be starting with cells that are more prone to reprogramming. Evidence in mice suggests that the tissue of origin affects how often and how well differentiated cells reprogram.
See: Cell origin and variation in induced pluripotent stem cell lines
Stomach and liver cells reprogrammed

Scholer and colleagues reasoned that neural cells would be a good candidate, since these cells already express high levels of three of the four standard pluripotency factors (Sox2, Klf4 and c-myc). The team had previously shown that this strategy worked in mice. The researchers used viruses to insert copies of the fourth pluripotency factor, Oct4, into the cells. This produced reprogrammed cells that passed all standard tests of pluripotency.

The current study reprogrammed neural stem cells from human fetal tissue. While adult tissues tend to be more difficult to reprogram, and brain biopsies are difficult to obtain, Scholer and colleagues say they are already working out practical solutions. More-accessble cells, such as those found in dental pulp, might also be good candidates.

1. Kim, J.B. Direct reprogramming of human neural stem cells by OCT4. Nature advance online publication, doi:10.1038/nature08436 (28 August 2009)

Posted by Monya Baker on August 28, 2009
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A thorough analysis finds tissue-specific stem cells make chemically and functionally different bone than embryonic ones

Many sorts of cells are able to form superficial bone-like nodules in culture, but how these nodules compare to native bone has been unclear. New work reveals that embryonic stem cells form a bone-like material quite different from that formed by adult-derived cells1. This finding has implications for osteogenic engineering, which could be used in the over two million bone-replacement procedures that take place every year.

Molly Stevens’s team at Imperial College London compared the nodules formed by neonatal osteoblasts from mice with those differentiated from both mouse mesenchymal stem cells (MSCs) and mouse embryonic stem (ES) cells. Although all of the nodules became calcified, this did not necessarily mean that they were forming similar bone-like material.

Continue reading "Building bones from stem cells" »

Posted by Monya Baker on August 10, 2009
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This piece, by Elie Dolgin, builds on the more-general article now up on Nature News.

The tumor suppressor gene p53 is usually thought of as a master regulator that helps stave off cancer, but it's also a major barrier to cellular reprogramming. Blocking the p53 pathway vastly improves the efficiency of transforming differentiated cells into induced pluripotent stem (iPS) cells and with fewer genes than the commonly used reprogramming recipes, new research shows. The findings, which should make it easier to derive patient-specific stem cells from any tissue, provide a bridge between tumour formation and cellular reprogramming that could force a rethink about cancer development.

Continue reading "p53, guardian of the genome, also blocks reprogramming" »

Posted by Monya Baker on August 10, 2009
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Two groups of researchers have at last completed a stringent test to show that induced pluripotent stem cells have the same developmental potential as embryonic stem cells: inserted into a special embryo, they can contribute to all the cells in a new mouse, litters of which have now been produced. (See the Nature news story)

GoogleNews was saturated this morning with stories of how to regenerate the heart:

Continue reading "Round-up of regenerative medicine stories and a big, squeaking accomplishment" »

Posted by Monya Baker on July 23, 2009
Categories: Clinical trials, Heart, Regenerative medicine, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

This account is by Teisha Rowland, a student at UC Santa Barbara who uses hESCs and iPSCs. She runs a blog called allthingsstemcell.com

Physiological differences have not been reported between embryonic stem cells and induced pluripotent stem cells, but new work shows consistent gene expression differences between them. One of the biggest questions in the field has been how similar these cells really are. If there are differences, are those due to suboptimal techniques for making iPS cells, or the fact that iPS cells don’t come from an embryo?
At ISSCR in Barcelona, Kathrin Plath of UCLA reported that there are some distinct gene and miRNA expression differences between them. She compared four iPSC lines made using different methods and found that 15 genes that are consistently expressed in these lines have significantly different expression levels in ESCs. In particular, basic cellular processes are down-regulated in iPSCs compared to ESCs, while regulation of genes involved in differentiation is up-regulated. Plath suggests that this may be because the fibroblasts from which the iPSCs are made aren’t sufficiently reprogrammed. Interestingly, late-passage iPSC gene and miRNA expression more closely resemble the ESC profile than the early-passage iPSC does, in Plath’s analysis. Plath hypothesizes that this may be caused by selecting iPS cells that most resemble ESCs over many passages. Ultimately, Plath suggests that iPSCs should be thought of as a different type of pluripotent stem cell, distinct from ESCs.
See also Plath’s recent publication in Cell Stem Cell
Session and write-up info
Speaker: Kathrin Plath
Talk title: Mechanisms of Transcription Factor-Induced Reprogramming
(Thursday, Concurrent Session I, Track A)

Note from Niche editor This post comes as a response to my solicitation in June calling for people to submit their accounts of ISSCR 2009. I’d asked people to describe what most interested them and to disclose any conflicts of interest. I’m very grateful for these volunteers’ help making more information available.

Posted by Monya Baker on July 15, 2009
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Posted for David Cyranoski; Cross-posted from The Great Beyond

Researchers in China have made pluripotent stem cells from a pig. The cells could be useful for making humanized pig organs for transplant to humans, pig models of human disease useful for testing drugs, and for improving pig farming productivity and nutritional value.

Lei Xiao, head of the research group at the Shanghai Institutes for Biological Sciences where the research was done, admits that none of these will happen for the next several years. But his creation in pigs of induced pluripotent stem (iPS)-cells which share with embryonic stem cells the ability to differentiate into any cell type in the body-is still a huge accomplishment. (Paper).

The isolation and culture of embryonic stem (ES) cells from mice in 1981 revolutionized the use of mice as a developmental and biomedical research model. But it is a difficult process. It took 17 years to culture human iPS
cells. Even now there are ES cells for only four mammals: mice, humans, monkeys, and rats. Pig ES cells, despite many attempts, still do not exist.

A new paper in Cell Stem Cell describes how scientists reprogrammed human cells to pluripotency without using any DNA at all. Instead, reprogramming proteins were engineered so that they could enter the nucleus. These proteins were produced in cultures of mammalian cells and secreted into the culture media. When fibroblasts derived from newborns were exposed to those cell extracts, the cells reprogrammed to teratoma-producing induced pluripotent stem (iPS) cells, a big first for human cells.

Nature’s David Cyranoski covered the story for Nature News, and he generously provided some outtakes of quotes from his reporting, although even these are condensed from what he provided. His original notes were over ten pages!

Those quoted include several well-known experts:

The scientists who led the most-recent work: Kwang-Soo Kim of CHA Stem Cell Institute in Seoul, South Korea, and Harvard Medical School in Cambridge, Massachusetts; Robert Lanza, chief scientific officer of Advanced Cell Technology in Santa Monica, California.

The scientists who, among other work, reported protein-only reprogramming in mouse cells in April: Sheng Ding of The Scripps Research Institute in La Jolla, California; Hans Schöler of the Max Planck Institute for Molecular Biomedicine in Münster, Germany.

Outside experts: James Thomson of the University of Wisconsin–Madison, the first to derive human embryonic stem cells and human iPS cells; Shinya Yamanaka of Kyoto University, Japan, and the Gladstone Institute in San Francisco, the first to reprogram both mouse and human cells.

Below, they all discuss the significance of the results and hurdles ahead, the differences between the human and mouse techniques and the need to compare different cells.

Continue reading "Gene-free reprogramming in human cells" »

Posted by Monya Baker on May 28, 2009
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In a testament to the pace of the field, on the very day two features on induced pluripotent stem cells publish, so does a new paper showing how to reprogram without DNA, perhaps the most obvious milestone in the reprogramming race.. (See the previous blog post)

Here are related stories on Nature Reports. The links are divided into four sections. The first lists overarching stories, including my two features and lovely Q&As with Tom Graf and James Thomson. The others key off the specific research publications noted. They are grouped according to 1) reprogramming techniques 2) understanding pluripotency 3) embryonic stem cell behavior in culture.

Such lists are always incomplete. Send comments to theniche[at]nature.com

Related Nature Reports features, Q&As and commentaries

What does reprogramming do?
Researchers grapple with the factors that make cells pluripotent

Stem cells: fast and furious (Subscription required)
The field of induced pluripotent stem cells has gone from standing start to headlong rush in less than three years.

Continue reading "Induced pluripotent stem cells, reading list" »

Posted by Monya Baker on April 23, 2009
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Researchers make iPS cells without inserting DNA

For the first time, researchers have reprogrammed cells to pluripotency without using DNA. Ever since Shinya Yamanaka of Kyoto University in Japan showed that cultured skin cells could be made to behave like embryonic stem cells by inserting additional pluripotency genes, researchers have been trying to find ways to avoid genetic engineering as a reprogramming strategy. The additional genes make the cells less predictable, more variable and more prone to undergo unwanted proliferation. Even if DNA is not inserted into the cells, researchers worry that undetected integration could occur and could change the behaviour of those cells, limiting their use in cell therapy, drug screening and disease modelling.

An obvious alternative would be to add the proteins for the gene products instead of the genes themselves, but for that to happen, proteins would have to be made not only to enter cells but also to enter the nucleus, a particularly difficult task. Furthermore, the proteins would have to persist at high enough levels in the nucleus for the duration of the reprogramming process.

Now, researchers led by Sheng Ding of The Scripps Research Institute in La Jolla, California, have found a way to overcome this barrier in mouse fibroblasts. Besides Scripps scientists, the team included Hans Schöler of the Max Planck Institute for Molecular Biomedicine in Münster, Germany, who has published with Ding before, as well as scientists from two California-based companies — Proteomtech, in Costa Mesa, and LD Biopharma, in San Diego1.

The secret to getting the proteins across the cell and nuclear membrane turns out to be adding a sort of transportation tag to each of the four proteins (c-Myc, Klf4, Oct4 and Sox2) that is typically used to reprogram cells. The tag consisted of 11 linked copies of the amino acid arginine, a highly polar species that helped the proteins pass through membranes. In addition, the researchers added valproic acid, which has been shown to boost reprogramming rates both for induced pluripotent stem cells and in somatic cell nuclear transfer. The proteins were added 4 times over 6 days at 36-hour intervals. Researchers observed the cells over 30 passages and found that they were “morphologically indistinguishable” from embryonic stem cells and expressed similar markers. Though the researchers have not yet completed the step showing that the cells can form viable sperm and eggs, the cells did pass a related test. When the cells were mixed in with normal mouse embryos and allowed to develop in a surrogate mother, the reprogrammed cells contributed to the germ layers in 13.5-day-old embryos.

Though the work has not yet been reported in human cells, and other groups will need to replicate the results, Ding predicts that his and similar techniques will replace those requiring DNA, partially because it does not require the preparation of viruses and plasmids. “Whenever you use a genetic method, even if you claim there's nothing left [of the added DNA], it's still not as convenient as using chemically defined methods.”

Nonetheless, Ding and other researchers agree that further studies are essential to assessing induced pluripotent stem cells made by various methods. “At the end of the day, what you want to do is just make normal cells and reduce the risk of things like mutation,” says James Thomson of the University of Wisconsin–Madison, who recently published a technique to reprogram cells without requiring any genetic integration.2 There will eventually be many ways of making the cells, he says. “Evaluating the cells — that’s going to be the hard part.”

Nonetheless, techniques like this are essential to such evaluation. “Once the cells are vector free,” says Thomson, “they can be characterized by a lot of labs.”

Related articles
What does reprogramming do?

Small molecules boost reprogramming rates

References
1. Zhou, H. et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell doi:10.1016/j.stem.2009.04.005 (published online 23 April 2009).
2. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science doi:10.1126/science.1172482 (published online 26 March 2009).

Further reading
iPS reading list

Author affiliation
Monya Baker is editor of Nature Reports Stem Cells.

Posted by Monya Baker on April 23, 2009
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For the first time, human skin cells have been reprogrammed to pluripotency without requiring genetic elements to insert themselves into the reprogrammed cells. Though so-called induced pluripotent stem cells promise to be as powerful as embryonic stem cells in their ability to differentiate into all cell types, standard techniques use viruses to insert multiple copies of reprogramming genes into the cells; this makes the cells less predictable, and it creates a higher risk of a cancerous growth. As a result, many laboratories have been racing to publish techniques to reprogram cells without permanent genetic modification.

The latest paper, by Junying Yu and James Thomson at the University of Wisconsin–Madison, and colleagues, uses a plasmid that does not integrate into chromosomes to reprogram the cells[1].

Continue reading "Human iPS cells without genetic integration: Six reprogramming factors, a plasmid, and a holy grail" »

Posted by Monya Baker on March 26, 2009
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The bogey of making cells that behave like embryonic stem cells has been genetic engineering: so far, reprogramming human cells has required permanent genetic modification, a fact that raises worries of increased cancer risk and unpredictability.
Three prominent papers this week describe reprogramming cells without permanent genetic baggage. They use genetic material that gets into cells, reprograms them, and snips themselves out.

Work published Sunday in Nature dispensed with viruses and instead engineered a transposon called piggyBac to take itself in and out of the cells. That work was led by Andreas Nagy, of Mount Sinai Hospital in Toronto, Canada, and Keisuke Kaji, of the University of Edinburgh, UK. (See Virus free pluripotency for human cells ) On Thursday, a paper by MIT’s Rudolf Jaenisch published in Cell took the cells much further (See Test tube disease models one step closer.) They reprogrammed cells from five patients with Parkinson’s disease and then showed that these reprogrammed cells could be differentiated into neurons. They use an engineered virus that snips out the integrated reprogramming genes once the cells have transformed. Though it leaves behind some remnants of the virus, it removes potentially dangerous genes. Perhaps more importantly, the iPS cells from which the reprogramming genes have been removed behave more like embryonic stem cells.

As Nature’s Erika Check Hayden reports: “While 48 genes were expressed differently between the factor-free iPS cells and the embryonic stem cells, 271 genes differed between the factor-free iPS cells and the iPS cells that retained the factors.”

Though the work is exciting, the race is still on for new techniques to derive iPS cells. The holy grail is to transform the cells without using DNA at all, presumably by adding small molecules and proteins that cause adult cells to reactivate their own, silenced version of pluripotency genes.

Though the cells appear to be very, very similar in their undifferentiated state, and mouse iPS cells can contribute to all tissues in a chimeric mouse, researchers warn that the mechanism by which cells are reprogrammed is not fully understood. The next steps are to study cells differentiated from both ES and iPS cells to see which would be best for cell therapies and drug screening.

Integration-free iPS cells describes other DNA-based techniques to reprogram cells without permant genetic modification.

Cells reprogrammed using only one gene describes a recent paper using proteins and small molecules to reprogram cells and includes links to several related research highlights.

For lay coverage of the self-excising reprogramming factors, see the LA Times , which covers all three papers, the New York Times, which covers the Jaenisch paper and has a nice description of the Jaenisch construct, and the Washington Post, which covers the transposon papers and how techniques to make pluripotent stem cells without embryos might impact political battles over the funding of ES cell work.

Posted by Monya Baker on March 05, 2009
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There's a paper just out in Cell today moving researchers closer to reprogramming without adding oncogenes. Sure, it starts with a cell type that's not readily accessed in humans, but it does indicate that the cell type could matter. Also of interest should be a paper that was published in Cell last week, which compared where the four Yamanaka factors are binding in fully reprogrammed cells, partially reprogrammed cells, and fibroblasts. (See how the four factors reprogram)

Cells that behave like embryonic stem cells can be made from cultured skin, liver, and stomach cells. All techniques so far require the addition of at least two pluripotency genes, which renders the cells much less attractive for cell therapy and drug screening. Now, researchers led by Hans Schöler of the Max Planck Institute for Molecular Biomedicine in Münster, Germany show that cells can be reprogrammed to pluripotency using just one of the standard four genes.1 “With only one “switch,” the gene Oct4, we have turned adult somatic cells into stem cells that are very similar to embryonic stem cells,” he says.

Schöler’s team began not with fibroblasts, the cultured skin cells most frequently used to make so-called induced pluripotent stem cells (iPS cells), but with mouse neural stem cells, which naturally express three of the four standard transcription factors. They were able to induce pluripotency by adding the gene for the missing factor, Oct4. Oct4, which is officially called Pou5F1, is expressed in embryonic stem cells and germ cells, and has long been considered a key regulator of pluripotency. The team had previously been able to reprogram neural stem cells using two of the four factors. The trick to using just one was waiting longer for cells to reprogram. Reprogramming generally takes about three weeks, but Schöler and his colleagues cultured the Oct4 infected cells for four to five weeks. The resulting cells passed several tests of pluripotency, including germline transmission in chimeric mice. The reprogramming efficiency was similar to that of reprogramming mouse embryonic fibroblasts with all four factors, about 0.014%.

Practical implications may be a ways off. Unlike skin cells, brain cells cannot be obtained readily from a human biopsy. However, Schöler says these cells present a good model for reprogramming not only because they can be transformed readily but also because they can be grown easily in pure cultures, so researchers can be certain what type of cells are being reprogrammed.

“The study sets the basis to understand, at a mechanistic level, whether Oct4 alone, in the absence of other oncogenes, could be used to reprogram different adult stem cells,” says Juan Carlos Izpisua Belmonte of the Salk Institute in La Jolla, California, whose work has shown that cells from plucked human hair reprogram much more swiftly and efficiently than fibroblasts. If such an approach could be made to work with more easily obtained cell types, the therapeutic implications would be “extraordinary,” he says. In the meantime, understanding what cell types are most susceptible to reprogramming “will surely help at unveiling the nuts and bolts of the process.”

Other techniques will also be helpful. Schöler and other researchers and other researchers previously showed that fetal neural stem cells could be reprogrammed without requiring the insertion of the Oct4 gene, though doing so required insertion of the other pluripotency genes plus a small molecule that inhibits an enzyme known as G9a histone methyltransferase. However, Schöler says that since this molecule turns on many genes, it requires the addition of the other three factors to focus the “crucial action” of Oct4.

[[Author Affiliation]] Monya Baker is editor of Nature Reports Stem Cells

1. Kim, J.B. et al. Oct4-induced pluripotency in adult neural stem cells. Cell 136, 411–419 (2009)

Related articles

Easing out the viruses in induced pluripotency

Small molecules boost reprogramming rates

Integration-free iPS cells

Embryonic-like stem cells from a single human hair

Posted by Monya Baker on February 05, 2009
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Consider this:
Mouse somatic cells were reprogrammed in 2006, 25 years after mouse embryonic stem cells were created.
Human somatic cells were reprogrammed in 2007, 9 years after human embryonic stem cells.
Rat somatic cells were actually reprogrammed a few weeks before rat embryonic stem cells were created.
(See Real rat embryonic stem cells )

Science declared reprogramming to be the breakthrough of the year and, while I’m certainly biased, it does make sense.

A subscribers-only review article by Doug Melton (whose in vivo reprogramming paper made a big splash this year) and John Gurdon (whose cloning of frogs in the 1960s anticipated current breakthroughs) chronicles the reprogramming field. They invoke the notion of “fleeting access” to explain why reprogramming rates are so low, particularly in specialized cells. The complexes of gene-inactivating proteins that cling to DNA sporadically dissociate from DNA allowing very short intervals during which reprogramming proteins can get to work. The concept explains why some cells are easier to reprogrma than others; most genes in embryonic cells and a subset of active genes in specialized cells will be more accessible. The actual reprogramming molecules differ depending on the technique (nuclear transfer into an oocyte, lineage switching, inducing pluripotency), but they conclude, however, the concept of fleeting access should appy in all cases.

In a news article describing reprogramming as the breakthrough of the year, Gretchen Vogel does a nice job surveying the year for non-specialists, but the format doesn’t let her list citations. (You can read Vogel's article for free if you register)

Here are some relevant (free) articles from Nature Reports Stem Cells. The first stems from the Melton paper which reprogrammed pancreatic cells in vivo.

Smash the (Cell) State!
A new quest for short cuts between specialized states could lay bare the machinery governing cell fate

Embryonic Stem Cells 2.0
Scientists' enthusiasm grows for induced pluripotent cells

Thomas Graf: Cellular identity and transdifferentiation

In the quest to switch one cell type to another, how far can tweaking transcription factors go?

Selected research highlights and meeting notes

Continue reading "A 2008 reprogramming round-up" »

Posted by Monya Baker on December 31, 2008
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Here’s a research highlight that will appear soon on Nature Reports Stem Cells. This version has the addition of outside comment. It came in too late to be incorporated into the highlight, but I'm putting it here because I think it's interesting.
Multitasking methyltransferase: G9a silences gene expression two ways
As embryonic stem cells differentiate, the pluripotency gene known as Oct4 goes on lockdown. In fact, the guards to gene expression are doublelocked:the gene-encoding DNA strands are wound up into a structure called heterochromatin, in which the DNA is complexed with histones and other proteins in such a way that it is inaccessible to the transcriptional machinery. Furthermore, gene-expression machinery is kept at bay by chemical modifications to the DNA that signals the start of a gene. New work published in Nature Structural and Molecular Biology1 shows not only that both of these modifications are regulated by a single master protein, the histone methyltransferase G9a, but that this enzyme apparently brings about the inactivation of many early embryonic genes.
Led by Howard Cedar and Yehudit Bergman of Hadassah Hebrew University in Jerusalem, Israel, a team of researchers examined mouse embryonic stem (ES) cells as they began to differentiate. They used microarrays to compare cells at different stages of differentiation as well as cells that could and could not express G9a. This identified a number of genes besides Oct4 that were newly methylated during differentiation; in other words, as cells lost pluripotency, a set of genes was silenced by way of chemical modification to certain regions of DNA, and this methylation also seems to be under the control of G9a.
G9a helps convert chromatin into heterochromatin, in which gene expression is blocked.\Interestingly, DNA methylation and heterochromatinization seem to be independent of each other. The researchers blocked the heterochromatinization activity of G9a by introducing a mutation into the protein that prevents it from methylating a lysine 9 residue on histone protein H3 . This mutation did not, however, change patterns of gene methylation.
G9a seems to regulate DNA methylation by recruiting two well known DNA methyltransferases (Dnmt3a and Dnmt3b) to relevant sites in the genome, sites where G9a is helping to create heterochromatin. The authors speculate that other histone methyltransferases may also promote DNA methylation as well as heterochromatinization.
G9a directs both processes as mouse embryos transition between pre- and post-implantation stages. For a cell to be reprogrammed back to a pluripotent state, these states must be reversed, and while heterochromatin can be remodelled during cell division, DNA methylation patterns are often faithfully reproduced, and this, the authors believe, constitutes the main barrier to reprogramming. "G9a seems to serve as a master regulator involved in turning off pluripotency," says Cedar. His future work will be aimed at understanding how these processes reverse themselves when somatic cell genomes are reprogrammed to pluripotency.
1. Epsztejn-Litman, S. et al. De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes. Nature Struct. Mol. Biol., advance online publication, doi:10.1038/msmb.1476 (26 October 2008). | Article |

When I write research highlights, I try to find other scientists to put that work in perspective. For the highlight below, I contacted the corresponding author of an earlier paper on the role of microRNAs and methylation in embryonic stem cells, which seemed to suggest another mechanism for silencing Oct4 as ES cells differentiate. I received a reply too late to work it into the highlight, so I am putting the responses to my four questions below. This is a joint response from Lasse Sinkkonen in Friedrich Miescher Institute for Biomedical Research in Basel and Petr Svoboda from Institute of Molecular Genetics in Prague.

1) How does this finding from Cedar and Bergman fit in with your work, particularly in terms of the methylation of Oct4?

Our work was actually inspired by their work because we saw these data
at Bergman's talk while ago and we decided to test if Dicer-/-ES cells
behave like G9a and DNMT3a/b-/-. Their work is in no contradiction to
our work as we show that miRNAs regulate DNMT3a/b, which are required
for G9a-induced methylation. We only saw a small effect of miRNAs on G9a mRNA levels (~1.5-fold) and since H3K9methylation of Oct-3 promoter
occurs without problems, we believe that miRNAs mainly affect
methylation downstream of G9a and independently of its DNMT3 binding.

2) How surprising/important is this most recent finding?

It is very interesting because it was known for a long time that there is a connection between histone modifications and DNA methylation. G9a work is important because it provides a mechanistical link between histone methylation and DNA methylation by dual function histone
methyltransferase. And it is surprising to see that if the histone
methylation activity of G9a is abolished, G9a can still repress its
target gene via DNA methylation.

3) What additional studies should be done before the paper's conclusions can be accepted?

There are three studies from October (two in EMBO J), which corroborate
the model, so I don't have any major problem with accepting NCB
conclusions.

4) What new questions/ applications does this finding open up?

DNA methylation has been perceived by many as a security lock on
transcriptionally silenced genes, which appears after histone
modifications. The latest results raise a question whether DNA
methylation could have a more active role in gene silencing and could be more of a parallel silencing system than a follow up guardian.

Posted by Monya Baker on November 04, 2008
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Ever since human cells were reprogrammed to behave like embryonic stem cells, a large group of scientists have said confidently that the feat can be accomplished without modifying the genome. Just this week, there has been a flurry of papers showing advances. Perhaps getting the most press are reports that biopsies from testes can be reprogrammed to pluripotency without any genetic modification at all.

Most recently comes a report from Harvard’s Doug Melton in Nature Biotechnology. (Here’s the Reuters report ). The first descriptions of the reprogramming technique required multiple copies of four separate genes to be permanently inserted into cells without using retrovirus. Melton shows that a common chemical, valproic acid, can be used in place of two of the four genes when reprogramming cultured human skin cells. The two that were replaced were Klf4 and c-Myc, both associated with tumorigenesis. The researchers kept Oct4 and Sox2, known pluripotency genes.

This work follows closely on work previously reported in the mouse.
BTW: Other researchers have reprogrammed without Sox2, so no single factor seems essential for reprogramming. It’s just a matter of finding one of the right combinations.

Integration-free iPS cells

Last week, Shinya Yamanaka of Kyoto University described in Science that mouse cells could be transformed to pluripotency apparently without using viruses and, as far as he could tell, without permanently changing cells’ genome. The week before, Harvard’s Konrad Hochedlinger reported that his team had reprogrammed cells using a virus that does not insert itself into chromosomes. My research highlight on that goes live on Thursday, but I’ll paste a sneak preview below.

A major impediment to clinical application of a technique for creating embryonic-like stem cells without using embryos has been removed, at least in principle.

Specialized cells can be reset to an unspecialized state capable of becoming any cell type in the body. Though induced pluripotent stem (iPS) cells made from individual patients could be incredibly valuable for drug screening or cell therapies, the established process to create the cells requires using retroviruses to insert several extra copies of genes into each cell. This renders the cells less predictable and more prone to forming tumours, and may make them unacceptable for human transplantation.

Two recent papers in Science show that, at least for certain cell types in mice, viral integration is not necessary; one technique does away with viruses altogether. A team led by Konrad Hochedlinger at Harvard Medical School in Boston used a type of virus that does not insert itself into the genome to deliver the genes necessary for converting cells to pluripotency. Hochedlinger’s team focused on converting adult liver cells, which have previously been shown to require fewer sites of viral integration and are more easily infected by the adenovirus vector the team was using. This approach generated cells that passed stringent tests of pluripotency1. If transplanted into mouse embryos, the cells go on to produce a wide range of tissue types within newly born mice, including sperm. Interestingly, though, about a quarter of the cell lines had twice the usual number of chromosomes, an abnormality that is not observed with other techniques used to produce iPS cells.

After using adenoviruses to figure out the best way to combine genes, Shinya Yamanaka at Kyoto University in Japan then tried a technique that did not use a virus. They transfected mouse cells cultured from embryonic tissue (mouse embryonic fibroblasts) with plasmids over several days. The team also generated cell lines that expressed markers of pluripotency and formed sperm in chimeric mice, but the researchers started with specialized embryonic cells instead of adult cells2.

Kathrin Plath, at the University of California, Los Angeles has created mouse and human iPS cells. She says the starting cell type might be very important when using non-integrating viral vectors, but she’s confident that the plasmid technique Yamanaka used on embryonic fibroblasts can be made to work using fibroblasts derived from skin biopsies from adult mice.

She also is not particularly worried that the yields of iPS cells are so much lower with this method than those seen when using retroviruses. “The efficiencies are quite low, but at the end if you get them, you don’t care.”

Besides efficiency, there are hurdles between this work in mice and being able to reprogram human cells from easily accessible tissue samples like a skin biopsy. The rates at which cells convert to pluripotency are far lower than the rates obtained using integrating viruses, and neither team can be absolutely certain that no fragments of the introduced DNA integrated into the cells’ genomes. Nonetheless, the work indicates that permanent genetic modification is not necessary for creating iPS cells. Though the cells still need to be more thoroughly compared to ES cells, iPS cells’ potential use for therapy and research seems greater than ever.

References
1. Stadtfield, M. et al. Induced pluripotent stem cell generated without viral integration. Science doi:10.1126/science.1162494 (published online 25 September 2008).

2. Okita, K. Generation of mouse induced pluripotent stem cells without viral vectors. Science doi:10.1126/science.1164270 (published online 9 October 2008).

Posted by Monya Baker on October 14, 2008
Categories: Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

Credit crunch hurts California stem-cell facility
When the California Institute of Regenerative Medicine granted $271 million for building new laboratories, it had a few conditions. One was that research institutions had to come up with matching funds (see yesterday’s post on funds to Stanford). But one of the awardees, the Buck Institute for Age Research, has blamed the credit crunch for a stall in securing its share of the funds, according to an article in GenomeWeb. According to the article, nine of the grant recipients report that their building projects are moving ahead, and CIRM says it is too early to assess whether it should extend the deadline by which buildings must be completed.

Falsified data
Years before news that human skin cell could be reprogrammed to a state as powerful as embryonic stem cells, enthusiasm centered on potentially powerful cells in the bone marrow termed MAPCs (multipotent adult progenitor cells). Excitement dimmed when other researchers were unable to replicate the results. (This stands in sharp contrast to the reprogramming work, which has been repeated in multiple labs in multiple countries.) Now, a panel at the University of Minnesota reports that data was falsified in several figures. The investigation cleared the lead investigator in the lab, Catherine Verfaillie who is still retains a part-time U of M position but is now at the Catholic University in Leuven, Belgium, and the blame falls to a graduate student in the lab.

The story was first reported in New Scientist, which had previously brough attention to discrepancies.

The University has asked that an article published in Blood be retracted and notes discrepancies but not falsification in another article in the Journal of Clinical Investigation. Other peer-reviewed articles are not mentioned in the materials made available to the media. (Nature issued a correction on related work in June last year, though authors say conclusions are still valid. See Flawed data in multipotent cell study and Stem-cell paper corrected.)

Here is an excerpt from the U of M statement:
In four of seven figures in the Blood paper, the panel concluded that aspects of the figures were altered in such a way that the manipulation misrepresented experimental data and sufficiently
altered the original research record to constitute falsification under federal regulations and University policy. Manipulations identified by the panel included: elimination of bands on blots, altered orientation of bands, introduction of lanes not included in the original figure, and covering objects or image density in certain lanes.

Posted by Monya Baker on October 07, 2008
Categories: Funding and Resources, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

The scientist that helped clone Dolly the sheep has moved away from cloning and toward making embryonic-like stem cells without eggs. The shift is described in an article and interview in Scientific American. Wilmut (and others) think that iPS cells might one day replace ES cells for clinical applications and drug-testing applications, but no one thinks that day is now here. Bits of the SciAm articles are floating around the blogosphere, but these (willfully?) strip away some of the nuances, so it’s worth reading the full articles from the source. I also think that the article conflated and neglected a few ideas, which I’ll outline below.

Continue reading "Ian Wilmut's move from cloning: getting practical with iPS" »

Posted by Monya Baker on July 23, 2008
Categories: Ethics, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

There are a couple cool stem cell papers in this month’s Cell.

Using a screen of chromatin regulating proteins in embryonic stem cells, UCSF’s Barbara Panning discovers something surprising. (See below)

Also see another cool article by Amy Wagers at Harvard, where her team was able to identify skeletal stem cells from look-alike cells and then show that these stem cells could rescue the phenotype of a mouse model of muscular dystrophy. It was written up in the Washington Post. and ScienceNews.

Packaging DNA for pluripotency
An RNA interference screen reveals a surprising player

Continue reading "Cell papers: purified skeletal muscles help mice; packaging DNA for pluripotency" »

Posted by Monya Baker on July 10, 2008
Categories: Regenerative medicine, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

Once again, there are more great papers out there than I can write about. Below are two that will show up on the site in a few days. (Nature Reports web production schedule requires a week). Also check out Tom Zwaka's paper that finds another, powerful control over Nanog; Sheng Ding shows that small molecules can substitute for two of the four Yamanaka factors, inching closer to reprogramming without viruses; in a high-throughput screen, Lorenz Studer shows us how known drugs affect human embryonic stem cells, a technique that might reveal unwanted side effects. (Those are all in the most recent Cell Stem Cell; see our Q&A with Sheng Ding on the potential of small molecules.)

See below for these papers along with links to less specialized articles.
A metasignalling network makes muscles age (Irina Conboy on skeletal muscle)
Two networks of pluripotency (Chia-Lin Wei and Huck-Hui Ng map transcription factor binding sites to find 'stemness hotspots')

Continue reading "Old stem cells made young; more maps of pluripotency" »

Posted by Monya Baker on June 17, 2008
Categories: Regenerative medicine, Reprogramming/Pluripotency | Permalink | Comments (2) | TrackBacks (0)

The same day results of a new approach to growing stem cells in culture were published in Nature, Stem Cell Sciences in Australia announced that it was launching a new product based on that work. The culture system will grow mouse embryonic stem cells without feeder cells or serum. And yes, they've patented it. That was announced just a few days before the paper published.

The paper from Austin Smith’s lab shows that it’s not necessary to actively trigger self-renewal to grow stem cells, but inhibiting differentiation seems to be sufficient. That’s interesting because while activating self-renewal seems to rely on complex biological components, often secreted from feeder cells, inhibiting pathways can often be accomplished more easily and using cheaper small molecules.

(Smith is one of the scientists that found self-renewal factor Nanog for humans and LIF for mice; so it’s sort of full circle that now his lab discovers how to get around these renewal factors.) I interviewed Qi-Long Ying, the lead author on this work for a Making the Paper. I’ve also recently interviewed Sheng Ding on how to use small molecules to facilitate self renewal and differentiation.

Here’s a snip from the press release:
“Professor Smith’s research is a major step forward in embryonic stem cell research and elucidates some of the early mechanisms involved in self-renewal and differentiation,” noted Dr Tim Allsopp, Chief Scientific Officer of Stem Cell Sciences. “We have now leveraged this significant advance into our novel media product Culticell iSTEM, which we believe will help provide researchers with a more pure starting point for embryonic stem cell research.”

Posted by Monya Baker on May 23, 2008
Categories: Reprogramming/Pluripotency | Permalink | Comments (1) | TrackBacks (0)

“Now we have the technology that can make a cloned child” reads the headline of the most-read article in the Independent right now. But the article does not actually break any news, nor does it use the common method of cloning; rather it discusses a well-understood implication of that recent reprogramming breakthroughs might yield yet another weird way of making a baby.

If a technician wanted to do this, here’s how it would work: First, cells would be gathered from an existing human, probably through a skin biopsy. Second, these cells would be reprogrammed to an embryonic like state. (Current techniques to do this require engineered viruses to insert copies of genes into the reprogrammed cells. This makes the cells’ behavior less predictable and more prone to form tumours, but many scientists believe that new reprogramming techniques will soon be available that don’t require genetic modification.) Next, the reprogrammed cells would be merged with an early stage embryo, created by sperm fusing with egg in a laboratory dish. The “chimeric” embryo would be cultured for a few days and then implanted into a woman. If a baby was born, he or she would contain cells from two genetic individuals: the embryo and the human who supplied the cells. The baby would have three parents: two who gave the gametes for the embryo, one who gave the cells from a biopsy. (Such an individual would not be a clone. However, it is feasible that the chimeric embryo could be manipulated such that the original embryo only forms placenta and the reprogrammed cells form the body. This has been accomplished with mixtures mouse embryonic stem cells and mouse embryos, but not with mixtures of reprogrammed mouse cells and mouse embryos. )

The results of some quick internet research suggests that using human iPS cells this way would not be allowed: In the UK, creating or using embryos outside the body requires a special license from the government, so I’d guess that permission would need to happen proactively. The US lacks legislation on reproductive cloning, though some individual states ban it. Australia distinguishes between research embryos (created through technical manipulation or by mixing genes from three or more people) and reproductive embryos (created through fusion of sperm and egg) and allows only reproductive embryos to used to create an embryo. A document dated to 2004 from Japan banned, among other things, the creation of chimeric human-human embryos for research.

Continue reading "Cloning by reprogramming?" »

Posted by Monya Baker on April 15, 2008
Categories: Cloning, Ethics, Reprogramming/Pluripotency | Permalink | Comments (2) | TrackBacks (0)

Skin cells taken from patients with some eight different diseases have been reprogrammed to an embryonic-like state. These could be invaluable for studying disease and testing drugs.

Here’s the statement from the UK’s Science Media Centre, which announced the result:
‘Dr Willy Lensch from the Children's Hospital in Boston and colleagues in his laboratory have generated stem cell lines from iPS cells with the genetic characteristics of more than six different diseases, including Huntingdon’s disease, Down’s syndrome and a type of muscular dystrophy. These can be used to study how these diseases affect fundamental development. They also can be used for surrogate testing for drug development, accelerating the development of therapies for devastating diseases.’ The announcement has been reported by the BBC. UPDATE: When I asked folks at Children's Hospital about this, I was told that the work wasn't ready for coverage; it had simply been mentioned at a seminar, and the fact that the UK press picked it up was surprising.

Reprogramming human cells was first reported in November, using cell cultures that could be bought commercially. Converting cells from a fresh patient biopsy was reported the following month by the lab led by George Daley and where Willy Lensch works as a senior scientist. By now, multiple labs have independently reported reprogramming cells, demonstrating that the technique is reliable and reproducible.

There are a variety of steps that will need to happen before the cells will start yielding information that will be useful for clinical applications. These are discussed in a commentary by the California Institute of Regenerative Medicine and a feature article written after mouse cells were fully reprogrammed.

The cells will need to be differentiated into the cell types that are affected in the various diseases. According to the BBC, a team at Nottingham University is already using reprogrammed cells to study heart conditions. Human cells differentiate very slowly compared to mouse cells. Turning embryonic stem cells into apparent photoreceptors, for example, took close to a year.

The first step in telling if cells are differentiating is checking out the molecules they display on their surfaces. Then comes the much more arduous task of looking at cells’ shape and function. (If it’s a nerve cell, does it release neurotransmitters? If it’s a heart cell, does it beat?) Even then scientists worry whether the cells in a dish behave like the ones in the body.

Other obstacles are getting enough of the cells and purifying the differentiated cells away from other cells growing in the dish that have not transformed fully.

Finally, drugs that are known to treat particular diseases will be tested on the differentiated cells. Results from these cell-based tests will be compared to established tests, most likely tests carried out on mice and rats.

Developing cells to become therapies (transplanting them to perk up or replace diseased hearts, brains, or other organs) will require considerably more work than developing cells to test therapies. One worry is that techniques to reprogram cells change them genetically, and clinical work in gene therapy resulted in patients’ deaths, making researchers leery of trying again.

Posted by Monya Baker on April 07, 2008
Categories: Reprogramming/Pluripotency | Permalink | Comments (1) | TrackBacks (0)

I’ve gotten a couple of emails about an editorial Nature recently ran urging scientists in the iPS field not to rush. It starts by relating an anonymous attack against Shinya Yamanaka for a minor problem. That’s supposed to get folks’ attention, but it is absolutely not the point of the article, which is to urge caution to everyone who is in and rushing into a very hot, very young field that is also politically charged.

The article is not questioning Shinya Yamanaka as a scientist. (It’s common for mistakes to slip through, and there are mechanisms to correct that.) The editorial is about what happens (confusion) and can sometimes happen (fraud) in hot, new fields, and this is going to be even worse for stem cell scientists because the field is politically charged as well. Shinya Yamanaka has already dealt with the accusations in a way that seems to have satisfied Science, and so delving any more into them would actually elevate the accusations of an anonymous emailer, giving the accusations more attention than they deserve.

The idea for the editorial started after PrimeGen decided to publish its findings on viral-free reprogramming by press release. Here was an accomplishment that the whole community was waiting for, but no one could assess it, and so Nature felt that we needed to say something about how people need to be more patient in a hot field. And then a few days later, the anonymous email got sent to many journalists and journal editors, and it seemed a call for caution was even more necessary.

So again, the editorial is urging caution in a hot, politically charged field. It is not about one of the field’s best-loved and most-respected scientists.

I’ll blog again as I get more feedback and hear more thoughts, but I wanted to get this up quickly. In the meantime, I want to say that much thought went into this editorial. You might be interested in how I think some decisions are made. (I don’t have first-hand knowledge of much of this, but I think I can guess.) Also, I should emphasize that stuff I've written above is just me; I haven't yet weighed in on the collective wisdom of NPG.

Continue reading "Recent editorial is meant to urge caution, not attack a scientist" »

Posted by Monya Baker on March 27, 2008
Categories: Community, Reprogramming/Pluripotency | Permalink | Comments (1) | TrackBacks (0)

Here's an accounting of how an interesting Nature paper, published online this Sunday, came to be.

Sadhan Majumder (pictured right) sought a better understanding of childhood brain cancer. He ended up finding a new regulator of self-renewal in embryonic stem cells along with a previously unknown mechanism of how this state is maintained.

While working on one of the most malignant childhood brain tumours, medulloblastoma, Majumder and his group thought much of the blame for the cancer might lie on a protein called REST (which has the burdensome full name of repressor element-1 silencing transcription factor/neuron-restrictive silencing factor). REST was originally believed to repress the final stages of neuronal differentiation. “We were working on REST in neurogenesis like everyone else,” Majumder recalls, “and we found that in medulloblastoma, abnormal expression of REST maintains the proliferative state of neural stem or progenitor cells and blocks their differentiation. Basically, it maintains the ‘stemness’ of these cells.” In fact, if REST is introduced artificially into neural stem cells, he says, they will not differentiate. “That told us that it may have a role in self-renewal,” says Majumder. “Then this paper from Gail Mandel came out that showed REST was expressed in embryonic stem cells.” He laughs as he described how he decided on his next experiments. “I have a tremendous grasp of the obvious.”

Continue reading "A new protein in the pluripotency circuit represses microRNA" »

Posted by Monya Baker on March 23, 2008
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Forbes has just reported a company’s announcement that it can reprogram adult human cells to an embryonic-stem-cell-like state without using viruses. All reported successes so far use viruses to introduce new genes into the cells, a technique that most believe make them unsuitable for clinical use. At a stem-cell conference in New York, PrimeGen, based in Irvine-Calif, said that it got the technique to work by attaching “carbon-based delivery vehicles” to the proteins that the genes encode and putting the proteins inside the cells. Unusually, it announced its results outside a peeri-reviewed journal without disclosing many of the details leading to its conclusions. That means that the results are likely to be met with high skepticism from the scientific community.

There are a lot of researchers trying to reprogram cells by introducing the proteins directly. Problems that they have encountered include the fact that some proteins enter and persist in cells better than others, and it’s hard to get enough proteins in for a long enough time and in the proper ratios, which are still not understood. Research in mouse cells indicate that the proteins from the viruses need to persist for a week or more in order for reprogramming to occur.

I haven’t read anything on this specifically but the Forbes article, but here are my thoughts based on my understanding of the researchers who have successfully reprogrammed human skin cells (Kyoto University’s Shinya Yamanaka, the University of Wisconsin’s James Thomson, Harvard’s George Daley, and the University of California, Los Angeles's Kathin Plath) and mouse skin cells (Yamanaka, Plath, Harvard’s Konrad Hochedlinger, MIT’s Rudy Jaenisch).

One problem is that it’s a lot easier to get the cells to just divide really fast than to reprogram, and these can initially resemble reprogrammed cells. The PrimeGen researchers don’t describe the techniques used to conclude that the cells as reaching the embryonic-like state. It implies that the cells have not been tested to see if they can make teratomas, weird tumors that make cells characteristic of the three main types of tissue and the most rigorous test available for human cells. PrimeGen does say that perhaps cells don’t need to be fully reprogrammed to be clinically useful.

According to the Forbes article, PrimeGen says it is collaborating with James Thomson, the scientists who first created human embryonic stem cells and leader of one of the teams that first reprogrammed differentiated cells. Thomson told Forbes he knows little about the company and denies he’s a collaborator.

The researchers say that they have reprogrammed testicular, skin, and retinal cells. There’s some evidence that testicular cells (depending on the actual type) are amenable over time in culture to becoming highly flexible cells, though less flexible than embryonic stem cells. There are several kinds of skin cells. Shinya Yamanaka recently showed that epithelial cells (cells that cover or line organs, and include some skin cells) can be reprogrammed using fewer copies of the viral genes than cultured skin cells (fibroblasts) need, which would make some cell types more amenable to being reprogrammed with proteins than others. Also, Daley found that one problem in reprogramming is that viral genes are silenced too quickly, thus inserting proteins directly might pose some advantages.

PrimeGen said that it made its announcement in order to attract investors. If a company has technology this hot, one would expect a company to approach investors with non-disclosure contracts in hand, not announce it so openly. Also, they say they need partners to expand the cells, but embryonic stem cells should be able to expand indefinitely. Also, the partners aren’t going to want to expand the cells until they know what they are. Embryonic stem cells are hard to grow, so possibly, researchers who could create the cells would have trouble growing them.

The Forbes article quotes several scientists saying they cannot evaluate the work until they see results in more detail. PrimeGen says it will soon be publishing its results in a peer-reviewed journal.

Posted by Monya Baker on February 27, 2008
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The former head of President Bush’s council on bioethics, now says there shouldn’t be a ban against cloning human embryos for research. Instead, there should be a five-year moratorium against the process. Writing in the Weekly Standard, Leon Kass decries the fact that the US Congress did not pass a law blocking all forms of human cloning, and then says that this stricter form of the law is unnecessary now that researchers can turn to alternate ways of reprogramming.

Instead, he argues for a law that would ban “all attempts to conceive a child save by the union of egg and sperm (both taken from adults).” That’s because the new reprogramming techniques mean that a skin cell could generate egg and sperm cells, whether taken from a man or a woman (or a boy or a girl, for that matter).

Embryos created for the purposes of research would not be outlawed, but instead banned for four or five years as researchers are given more funds to perfect the reprogramming techniques. He does not rebut, because he does not raise, the argument that stopping work the creation of embryos for research through somatic cell nuclear transfer will delay efforts to prefect reprogramming techniques.

Kass writes “Cloning for the purpose of biomedical research has lost its chief scientific raison d'être” (i.e. making a pluripotent cell line genetically matched to a patient.) That’s because it will probably be much easier to reprogram whole cells from adult biopsies than it will be to pull out an adult cell’s nucleus, plop it into a donated egg, grow that “reconstituted embryo” to a blastocyst and make embryonic stem cells.

Kass is probably right, but he fails to mention two caveats.

First, while many scientists are hopeful that so-called induced pluripotent stem cells will really behave like embryonic stem cells, they still aren’t sure. Possibly, a reprogrammed skin cell could be coaxed into a pancreas cell or a heart cell, transplanted, and then “remember” that it started out as a skin cell. Also, no one wants to use the current technique (using viruses to insert genes at random places in the cells’ chromosomes) to make cells that would actually get put into people. Those are serious problems, but most scientists think they can be overcome.

Second, and more important, many scientists think that to understand how reprogramming works with viruses, they have to understand how reprogramming works in an egg. Most people think that requires transferring adult nuclei into eggs or early embryos, and trying to figure out what happens.

Just a little quibble: Kass says that recent success by Stemagen in cloning a human blastocyst depended on the technique that Shoukhrat Mitalipov’s team in Oregon used to clone monkey blastocysts to make embryonic stem cells . Actually, Stemagen did not use this technique but credits its success not with a new technique but with a supply of high quality eggs.

Posted by Monya Baker on February 21, 2008
Categories: Cloning, Ethics, Policy, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

The possibility of reprogramming adult cells to behave like embryonic stem cells without overexpressing cancer genes or relying on hard-to-control viruses has gotten a bit closer. Publishing in Science, Shinya Yamanaka and colleagues from Kyoto University, in Japan (see From skin cells to stem cells), show that the reprogramming techniques he previously demonstrated on cultured mouse skin cells also work on two other mouse cell types: those that line the stomach and those from the liver1.

Because far fewer than 1 in 100 treated cells are successfully reprogrammed, several stem cell scientists had raised concerns that reprogramming does not work on fully differentiated cells but rather on rare stem cells residing undetected within the culture. That would make the reprogrammed cells less interesting scientifically and, potentially, therapeutically. To address this concern, Yamanaka used a genetic marking system that permanently labeled liver cells once they differentiated enough to express albumin, and he found that these cells could be reprogrammed to so-called induced pluripotent stem (iPS) cells that can contribute to all cell types in an adult mouse.

“The old question in cloning was exactly the same: was Dolly derived from a fully differentiated cell?” said Rudolf Jaenisch of the Whitehead Institute, in Cambridge, Massachusetts, who showed that mice can be cloned from terminally differentiated cells such as neurons. Yamanaka provides “good evidence” that reprogramming works in differentiated cells, said Jaenisch, but that conclusion assumes both a reliable labeling system and that only mature cells express the albumin gene. Yamanaka himself stopped short of calling the initial cells fully differentiated: “Our data showed that lineage-committed albumin-producing cells can be reprogrammed.”

Perhaps more interesting, Yamanaka also shows that the epithelial cells lining the stomach can generate iPS cells using a less rigorous screening system than that used with cultured skin cells or fibroblasts. Reprogramming currently uses viruses to insert several copies of three or more pluripotency genes into cells, one of which is particularly implicated in cancer. Tumours develop in about a third of mice created using iPS cells derived from fibroblasts; no tumours were found in mice created from iPS cells derived from stomach and liver cells. Though viruses were less efficient at infecting the stomach-lining cells with the necessary genes, the cells that were transformed contained fewer copies of the transgenes compared with fibroblasts, perhaps because epithelial cells are more similar to embryonic stem cells than fibroblasts are. (These mice were more likely to die in utero, but live-born mice appeared healthy.)

Yamanaka found that the transgenes do not need to be inserted into specific sites within the genome for liver and stomach cells to be reprogrammed. “This is encouraging to those of us who are seeking a nonviral means of generating iPS cells,” said George Daley of Children’s Hospital Boston, who recently compared the efficiencies of reprogramming human fibroblasts from different sources.

References
1. Aoi, T. et al. Generation of pluripotent stem cells from adult mouse liver and stomach cell. Science published online 14 February 2008; doi:10.1126/science.1154884

Posted by Monya Baker on February 14, 2008
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An independent laboratory has been able to reprogram differentiated human skin cells to an embryonic-like state using the method originally reported by Shinya Yamanaka of Kyoto University.1,2 Also late last year, two other laboratories announced reprogramming differentiated human cells via slightly different methods.3,4 The latest work, by Kathrin Plath of the University of California, Los Angeles, indicates that the technique is broadly feasible and introduces a potentially easier method to pluck reprogrammed cells from culture.5

Notably, all the groups that have reported success with this method have experience working with embryonic stem cells. At least two have derived human embryonic stem cells, and the genes that must be introduced to reprogram cells were identified through embryonic stem cells.

Meanwhile, the California Institute of Regenerative Medicine announced that it has received 50 applications for up to 16 grants totaling $25 million for creating new human pluripotent cell lines either from embryos from fertility clinics or from other sources of cells. In an executive order and his state of the union address, President Bush has directed the NIH to direct more funds to methods to create pluripotent stem cell lines without destroying embryos, but no dedicated funding programs have yet been announced.

The scientist leading the work just published in the Proceedings of the National Academy of Sciences has received a $1.5 million NIH grant set aside for “exceptionally innovative investigators.” Only 41 such grants were awarded, less than one of 50 applicants including biomedical researchers across the nation. She also received a $2.2 million grant from CIRM to study how reprogramming works in mice. Two other scientists who have successfully reprogrammed human cells (Yamanaka and James Thomson) have part-time appointments in California that will allow them to access some funds from the CIRM Medicine, but Plath is the first full-time faculty California to lead such an effort.

Plath’s team infected cultured skin cells originally collected from circumcisions and infected them with retroviruses carrying the four genes originally identified in reprogramming work. After about two weeks they began to see colonies proliferating. They looked clearly different from the cultured skin cells, but they did not go on to become induced pluripotent cells, and analysis showed that they had not taken in copies of all four genes. Colonies of cells destined to become reprogrammed lines showed up about 21 days after infection; they clustered together as human embryonic stem cells do, and they also displayed a variety of cell-surface markers characteristic of ES cells. A week later, Plath and researchers hand-selected colonies staining positive for one particular cell-surface marker. All cells analyzed from these colonies contained copies of all four genes.

Of the 30 colonies isolated, cells from seven were closely analyzed. As expected, all of these cells were expressing endogenous genes associated with pluripotency, and the viral genes were silenced. Though the cells express markers of the three main types of tissues forming the body, the team has not yet tested the functionality of the cells by differentiating them into teratomas or other cell types.

1. Takahashi K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell advance online publication 20 November 2007. doi: 10.1016/j.cell.2007.11.019 | Article |
2. Takahashi K. & Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–76 (2006) | Article | PubMed | ISI | ChemPort |
3. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science advance online publication 20 November 2007. doi: 10.1126/science.1151526 | Article |
4. Park, I. H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature advance online publication, doi:doi: 10.1038/nature06534 (23 December 2007).
5. Lowry, W.E. et al. Generation of human-induced pluripotent cells from dermal fibroblasts. Advance online publication www.pnas.org_cgi_doi_10.1073_pnas.0711983105 11 February 2008

Posted by Monya Baker on February 10, 2008
Categories: Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

I’ve been reading the coverage on making embryonic-like stem cells without embryos in the religious press, and two quotes going through my mind, both sarcastic. One is “Shocked! Shocked!” (from Casablanca) and the other is “Oh, Lord! Make me pure, but not just yet.” (from St. Augustine).

Continue reading "Inconsistent Christian views on reprogramming" »

Posted by Monya Baker on January 22, 2008
Categories: Ethics, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

George Daley, author of a paper published online today in Nature says that a simple skin biopsy can yield stem cells specific to an individual patient, and may mean that a bank of genetically matched stem cell lines is possible. Further, any group that knows how to keep human or mouse embryonic stem cells alive will probably be able to make and maintain induced pluripotent stem (iPS) cells.

Here's an advance peak of an article that will appear on our site in January 2008.

In January 2007, George Daley of Harvard University published proof of principle that pluripotent stem cells could be created so that they would not cause an immune response when differentiated for cell transplantation1. His forthcoming publication in January 2008 shows much the same thing, but through an entirely different technique. The first paper used unfertilized mouse eggs; the more recent one uses a skin biopsy from a human volunteer2. It is the first to demonstrate such complete reprogramming without starting from embryos or cell cultures available from commercial vendors.

These bookends highlight the major stem cell advance of this year: multiple laboratories have now shown that adult human skin cells can be reprogrammed to an embryonic stem-cell-like state.

Daley’s lab began work shortly after Kyoto University’s Shinya Yamanaka announced the four genes that could transform cultured mouse skins cells, or fibroblasts, into an embryonic-like state3. This summer, Yamanaka and two other groups proved that the mouse fibroblasts could be made truly pluripotent by showing that the cells could become sperm and eggs4-6. (See Skin cell to stem cell)

Meanwhile, labs across the world were racing to reprogram human cells. This November, Yamanaka and James Thomson of the University of Wisconsin-Madison became the first labs to announce that they had done so7,8. “Our paper was already submitted when the others were published,” Daley says. “It was frustrating, but the point is that this is a robust technology that lots of people can reduce to practice.” Indeed, he says, any group that knows how to keep human or mouse embryonic stem cells alive will probably be able to make and maintain induced pluripotent stem (iPS) cells.

Continue reading "Personalizing pluripotency" »

Posted by Monya Baker on December 23, 2007
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Since the recent announcement of successful reprogramming, editorials carrying statements such as “[r]arely has a president - so vilified for a moral stance - been so thoroughly vindicated” have been springing up across the United States. Now the fightback seems to be gearing up.

Key to their argument is the fact that ‘reprogrammed’ cells – where instead of obtaining stem cells from an embryo ‘induced pluripotent stem cells’ are created from adult human skin – are not yet safe for clinical use.

“For doing basic research on human cells, IPS as a method has won - it's huge. But for the ultimate goal of getting cells into a patient, it's a lot less clear. These cells may never be useful for direct therapy,” says George Q. Daley, a stem cell researcher at Children’s Hospital Boston, in the Boston Globe.

Douglas A. Melton, codirector of the Harvard Stem Cell Institute, is even firmer, saying: “It will never be approved [by the FDA] to put these cells in a patient.”

See also our Q&A on the topic with the head of the NIH Stem Cell Task Force and what scientists had to say

Posted by Monya Baker on December 21, 2007
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This will appear as a regular, archived article on Nature Reports Stem Cells eventually. However, our production cycle will be even slower over the holidays, and I wanted to put this up as soon as possible. --Monya

Nature Reports: Did the induced pluripotent stem (iPS) cell breakthrough happen faster than you thought?

Landis: Yes.

Nature Reports: What do you think of the public response to this breakthrough?

Landis: It’s kind of very sad. Instead of focusing on the scientific potential—what you can learn in terms of reprogramming and the epigenetics of the cells—people seem to have focused on “We don’t need embryonic stem cells” or “Oh yes we do need embryonic stem cells”. It’s as if the science has been consumed by the political argument.

Nature Reports: What still needs to be assessed with induced pluripotent stem cells?

Landis: There are a zillion questions. The assumption on the part of a large part of the public that this does away with the need for embryonic stem cells is premature.

I find it hard to believe that you’d get back to the same starting point that a pristine embryonic stem cell would represent. You don’t know what the undifferentiated state actually is and you don’t know how they [the cells] are going to respond to differentiation.

If you’re taking a fibroblast that’s obviously gone through several developmental stages to get to its differentiated state and then you’re getting it to go back to its undifferentiated state, I would be surprised if it took the same pathway backwards.

[Regarding pluripotent stem cells as disease models] An interesting catch could be that the mutations that give rise to the disease could interfere with the ability to reprogram. Everyone has just assumed that they won’t, but I don’t think we have any data on that.

Nature Reports: How can researchers compare human iPS cells to embryonic stem cells?

Landis: Given that they’ve had the mouse embryonic stem cells and mouse iPS cells for some time and have not yet completed the epigenetic comparison, I think it will take a lot to do the human.

Nature Reports: But comparisons can’t be funded for the newer human embryonic stem cell lines.

Landis: You would be constrained to the identified lines that are available for funding. Obviously it would be better to have more lines. Jamie Thomson[who led one of the groups making the reprogramming breakthrough and was the first to generate human embryonic stem cells] has pointed out that one of the major disadvantages of the limited number of lines is that they come from a pretty narrow genetic repertoire.

Nature Reports: Scientists have called for comparisons between iPS and hES cells, but there is some ambiguity about what kinds of these studies the NIH could fund. For example, can people use data or RNA or techniques from newer embryonic stem cell lines that aren’t eligible for NIH funding?

Landis: That’s kind of outside my paygrade, that kind of regulation. Apparently Harvard has a very good policy that’s written up that outlines what Harvard feels are the appropriate safeguards to make sure that you don’t violate the NIH policy.

Nature Reports: What’s going to happen now in terms of what science is being done and who’s doing it?

Landis: [The buzz makes it sound] like it’s really easy and that anyone who’s cultured cells should be able to make their own pluripotent stem cells. In talking to people on the phone, it sounds like it’s much more complicated than that. Jamie Thompson said that it took him four years.

There will be new grant applications to take advantage of this scientific advance, whether or not they will be outstanding grant applications is unclear. Also, with the advance of SCNT [somatic cell nuclear transfer] in primates, I expect we’ll get more grant applications based on that.

Since this is a new area, and not many investigators have the expertise to make pluripotent stem cell lines, the issue won’t be that there are too many [grant applications] that are outstanding but that there won’t be enough that are outstanding.

Nature Reports: How will grants be chosen?

Landis: One of the most contentious issues at NIH is how much money is assigned by what the review says is the scientific merit of the grant versus how much money is assigned based on programmatic considerations.

If 50 grants come in and none of them are deemed outstanding, the institutes can then say ‘none of them make the payline, but this [research] is absolutely critical.’

Nature Reports: How do you feel about NIH’s leadership role in global science?

Landis: Do we want therapeutic advances using human embryonic research to come out of Singapore, China, Britain? That’s a piece of the tension that exists.

I don’t think that the NIH can do anything except talk about the fact that the science does not support the President’s policy and at the same time to implement the President’s policy.

Posted by Monya Baker on December 17, 2007
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Last month, we posted an article that asked how one could declare human cells pluripotent, when the most robust tests are neither ethical nor feasible. Here are some of our favorite responses. If you've got more to say, please add your own comments.

Peter Andrews, Sheffield University

I rather think the discussion is becoming like the Middle Ages' discussions about how many angels can stand on the head of a pin. Does it matter? Sometimes in science it helps to have terms that are not so precisely defined - indeed the term 'gene' is an example. In fact it can mean a variety of subtly different things - which in fact makes it generally useful. When people wanted more precise terms, new ones were invented, like the 'cistron' based upon a very specific assay.

The same may be true of pluripotency. To me it means what it says - the ability of a cell to be capable of generating many cell types by differentiation. When we come to ES (and related EC) cells, we can actually find a very broad range of capacities - ranging from cells that have completely lost their ability to differentiatiate (nullipotent) to those with a very broad range – ultimately all somatic cell types. But we know very little about the molecular basis of pluripotency and what controls the range of cells into which a stem cell can differentiate. On the face of it at the moment I think we have little or no way of identifying which ES cells can generate a whole mouse in the tetraploid assay and which cannot even form the germ line in chimeras. In the face of this type of uncertainty, I would advocate retaining 'pluripotency' as a somewhat vague, term meaning ability to differentiate into a lot of cell types, and then as the need arises invent new terms with precise definitions based on specific assays - very much as the concept of the gene and its associated terminologies evolved.

Shinya Yamanaka, Kyoto University

This is an important, but difficult question. First of all, we don't know whether human ES cells are really ES cells or not. Because the lack of chimera experiments, we will not be able to answer this question. This means we lack a positive control. I have been telling my students that one of the worst experiments you can do is one without positive and negative controls.

In human ES cell field, all the scientists are forced to perform bad experiments without positive control. The best we can do is to describe how the cells are similar to human ES cells. This includes not only teratoma formation, but also surface marker, gene expression, DNA methylation, telomerase activity. You are right that some iPS cells can make teratomas, but do not give rise to germline transmission. However, these cells have different gene expression and DNA methylation.

I don't think it is governments to make definition of pluripotency. It should be scientific community.

Paul Tesar, Laboratory of Molecular Biology, National Institutes of Health, NINDS

Since I’m associated with NIH, I won’t comment on the recent nomenclature alteration.

I do, however, think that the definition of pluripotency sits at the heart of modern biology. Currently it is more of a semantic argument but I think further study will clarify the issue. Existing methodologies such as blastocyst injection and teratoma formation are inclusive but not exclusive when defining pluripotency. Additionally, they require secondary characteristics that are not necessarily involved in pluripotency. For example, cells that do not incorporate into the ICM, maybe because of cell adhesion or cell cycle differences, can not be examined by blastocyst injection. This does not mean that they are not pluripotent. Likewise, cells that do not rapidly proliferate when transplanted to an ectopic site will not form a teratoma. Can quiescent cells be pluripotent? Does growth or cell adhesion have to be linked to pluripotency? I think, thus far, pluripotent cells have satisfied one or the other of these basic assays but it is becoming harder to pinpoint the defining characteristic of pluripotency.
It sounds a bit outlandish but one could imagine something like a ‘pluripotency score’ which could be computed from a variety of cellular characteristics. It is difficult to define what exactly would need to be input, but in a current sense one could imagine looking across the genome at a large number of histone and DNA modifications. The ‘pluripotency score’ would basically be the probability that the chromatin is immediately capable of changing to form a panoply of differentiated tissues. SCNT and iPS cells have shown us that most, if not all, cells are capable of being pluripotent, but only after reprogramming. A much deeper understanding of multiple aspects of cell biology are necessary for something like a ‘pluripotency score’ to be a reliable and predictive measure, but at least it provides a framework to move forward instead of walking away or simply arguing semantics.

William Gunn, Tulane University
I would like to share what the consensus view is trending towards in my field, multipotent stromal cells(aka mesenchymal stem cells, MSCs).

I think the subtlety that is most often missed when talking about differentiation capacity is that differentiation is a cell-intrinsic process, but it's only assayed at the level of a whole culture. In other words, you're assaying a heterogeneous population of cells for phenotypes that different subpopulations possess to various degrees.

Further, these populations interact through cell-cell contacts and paracrine signaling, forming microenvironments which change constituency over time. Outside of ESCs and HSCs, it's an open question whether there's really one cell in a stem cell culture that could make all the various tissues, or if the pluripotency we see is a result of a mixture of progenitors of the various types that we just haven't learned how to distinguish yet. When exactly these progenitors may have become committed to a lineage isn't known.

The heterogeneity and dynamic nature of pluripotent cells is what has been confounding the studies which try to pin down markers of pluripotency or "stemness", and I'm not sure we'll get a satisfying answer until we develop the tools to study these cells on the single-cell level.

Evidence supporting this can be found in the work of Kuznetsov et al,
back in 1997: http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=9286749&ordinalpos=41&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

Posted by Monya Baker on December 13, 2007
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Over the past few days, I’ve seen a series of press releases from stem cell companies. They’ve taken the excitement generated by recent breakthroughs to draw people’s attention to their existence. And why not latch on to the general sense of euphoria? Two big advances happened within a week of each other, and both were ones that the stem-cell community had been waiting for.

First, came the announcement that embryonic stem cells could be made from cloned monkey embryos, a feat that many had deemed impossible. Nature Reports Stem Cells had a feature describing what made the advance possible as well as exclusive information on what the anonymous peer reviewers had to say about the advance. Well before the accomplishment was printed, the Niche had posted expert opinion on whether cloning papers needed additional layers of scrutiny, and Nature had decided on independent verification for cloning papers.

Next, came the announcement that human skin cells could be reprogrammed to pluripotency. Back when the breakthrough was published for mice this summer, Nature Reports Stem Cells covered what would need to happen to generate useful cells through direct reprogramming. A month earlier, we’d explored how pluripotency could be defined for human cells since the most rigorous tests are neither feasible nor ethical for humans. We also ran a profile of Shinya Yamanaka, who found the suite of genes and slogged through the screens showing that differentiated cells could be reset to a state similar to that found in the embryo. A highlight of the papers showing how human cells can be reprogrammed with only three genes.

More Nature articles are listed here.

Posted by Monya Baker on December 04, 2007
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The excitement from the reprogramming and cloning breakthroughs from earlier this month is fading, and people are looking to future paths and profits. Monkey cloner Shoukhrat Mitalipov has teamed up with a start-up company in San Diego, though it’s not exactly clear what it will be doing. Reprogramming cells without eggs or embryos will require less money, skill, and hard-to-procure material, so expect both academics and entrepreneurs to jump into the space. I’ve already seen one stem cell company touting the advance in a press release.
The intellectual property field may be more open as well. One of the teams that reprogrammed human skin cells was led by James Thomson of the University of Wisconsin, who also led the first team to generate human embryonic stem cells from leftover embryos provided by an IVF clinic. His patents covering human embryonic stem cells are controlled by WARF (Wisconsin Alumni Research Foundation) and have raised howls of protest from the community. Thomson says the intellectual property surrounding reprogramming techniques will "be complicated." When I asked WARF what that meant, I was told that the patent situation is complex because two groups made the discovery at the same time and the science is moving very rapidly. Another complicating factor is that the two groups used different techniques to reprogram cells, and whispers of forthcoming techniques are growing into shouts.
That doesn't yet mean patient advocates should be dancing in the streets. To keep us levelheaded, Newsweek’s Sharon Begley has an article that’s informative and easy to read. Also, while several prolife blogs are hoping the end is nigh for embryonic stem cells, the scientists leading the egg-free reprogramming breakthrough are making a strong case that studies of embryonic stem cells hold the keys for using these so-called induced pluripotent cells. See an editorial in Nature and this article from AFP.
For what it’s worth: Of the articles that appeared at the time of announcement, I particularly enjoyed the articles from Bloomberg , Nature, and Science.

Posted by Monya Baker on November 29, 2007
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That’s a paraphrase of what James Thomson at the University of Wisconsin-Madison told reporters at a press conference this morning when he announced that he’d induced human skin cells to take on the trappings of embryonic stem cells. His work is published online today in Science. Tying (or narrowly beating) Thomson is Kyoto University’s Shinya Yamanaka who reports his accomplishment in Cell. A news article from Nature is available here.
Thomson said that other researchers would be hard-pressed to distinguish his cells from human embryonic stem cells (ESCs) but repeated several times that whether these cells differ from ESCs in important ways remains to be seen. It does seem, however, that highly flexible cells could be made without collecting eggs from women and without destroying an early stage embryo.

Moreover, these pluripotent cells could be made from patients with known diseases. If the root causes of disease were genetic that could be a better way to study disease. It could also mean that replacement tissues for, say, diabetes patients using genetically identical cells. Thomson predicted that this research could lead to researchers testing drugs in ‘panels” of cell lines to figure out if toxicity and efficacy varied across genotypes.

Both Yamanaka and Thomson used a suite of four genes to transform cultures of skin cells. Both quartets included OCT3 and SOX2, well known markers of pluripotency. For the other two, Yamanaka used the KLF4 and c-Myc, which he’d shown earlier in mice. Thomson used NANOG (identified a few years ago as a master switch of pluripotency) and LIN28, implicated in processing mRNA. (According to a news article by Science.)

Besides these groups, there are many, many whispers of others about to publish similar accomplishments. Some report overcoming a remaining drawback: the transformed cells contain multiple copies of genes inserted into the genome by engineered viruses. “Nobody thinks we’re going to have those vectors even a year from now,” Thomson told reporters who had called in.

But he said, the major barriers still exist. The manipulations move cells back to what he called “a ground state” but for therapies and drug screening, researchers need a differentiated state. That was something he said was coming.

Synergies with other research

Thomson said that the time, cost, and expertise needed to make embryonic stem cells would likely push researchers to prefer genetically reprogrammed cells. Induced pluirpotent cells made by Yamanaka and Thomson come directly from cultured skin cells. Embryonic stem cells are made by scooping out cells from within an embryo and culturing them. Both types of cells can form teratomas and be differentiated into other cell types. Embryonic stem cells can also be made from cloned embryos, in which the nucleus of a differentiated cell is placed in an oocyte that is then activated to divide to form an embryo.

That feat was never been accomplished in humans (earlier reports were fraudlent). Nature did report it this week.

Thomson said that it would be useful to reprogram cells from the same monkey whose nucleus was used to make the embryonic stem cell lines. Then, cells generated from oocyte-assisted reprogramming and genetically engineered reprogramming could be compared directly.

Much of the speculation about what would need to happen to make the technique useful was reported when Yamanaka and other groups reported the accomplishment in mice. Here is a link to that article .

Posted by Monya Baker on November 20, 2007
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The Nobel prize has been awarded to three scientists who created the techniques for “knockout mice”. Here’s one article.

The award announcement acknowledged the powerful cells that made the technique possible. The prize went to Sir Martin Evans at Cardiff University, Oliver Smithies, from the University of North Carolina, and Mario Capecchi, from the University of Utah for "principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

Knockout (and knock-in) mice are one of genetics’ power apps. Scientists create mice with nonfunctioning (or, in some cases, differently functioning) versions of specific genes. In one recent example, scientists knocked out one of many mouse genes that allow neurons to communicate and ended up, surprisingly, with a potential animal model for obsessive compulsive disorder. That’s just one of thousands of experiments using these engineered mice. The technique and its iterations are so routine that it’s hard to imagine biology without it.

Knockout mice are, both directly and indirectly, responsible for the breakthroughs showing that mouse skin cells can be reprogrammed to a state almost exactly like embryonic stem cells. Also, Shinya Yamanaka, the scientist who discovered which genes to insert to cause reprogramming, read about knockout mice well over a decade ago. He was doing classic pharmacology on dogs, but decided to strike out on a completely different path because the ability to pick any gene and delete its function was so intriguing.

When Yamanka attempted to reprogram differentiated cells, he started by genetically modifying mice so that their cells could signal (by growing green) when they had been reprogrammed.

This year’s Nobel Prize in Medicine lauds a tool and technique used by scientists in many disciplines across the world. It also illustrates a powerful platform that already owes its existence to embryonic stem cells. Cell therapies aside, many scientists who hope to study a disease in a dish believe there are more to come.

Posted by Monya Baker on October 08, 2007
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Today, the Human Fertilisation and Embryology Authority (HFEA) in the UK said that scientists could combine human chromosomes with animal eggs and try to make embryonic stem cells. It’s easier to collect unfertilized eggs from, say, cows than it is to collect them from women.

Interested scientists will learn in November if they’ll be licensed to make the attempts, which must be carried out under certain guidelines, but an article this month in Nature Cell Biology reminds us that even if the government says `yes’, some laws of science might say ‘no’.

In chimera-embryos (properly called `cybrid-embryos’ in this context), the chromosomes will be human, but at least some of the mitochondria will not.

Continue reading "Britain gives go-ahead on chimeras. Will science now block the way?" »

Posted by Monya Baker on September 06, 2007
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Horst-Dietrich Elvers, Burkhard Jandrig, and Christof Tannert write:
The Nature News story “Simple switch turns cells embryonic” (Nature 447, 618-619; 2007) presents the results of three independent research teams showing that normal skin cells can be reprogrammed to an embryonic state in mice. If this can be successfully adapted to human cells, the creation of human germ cells out of these pluripotent cells should be possible (as was indicated already by Huebner et al. Science 300, 1251-1256, 2003). Now, the road seems to be prepared to create human tissues for therapeutic purposes without using or destructing human embryos. This is, doubtless, an important progress for the whole field of regenerative medicine and avoids many morally questionable decisions, which so far have led to an international mix of regulatory frameworks. Therefore it is not surprising that excitement is overall huge at the moment.
The published results seem to indicate that the ethical problems of human embryo research are solved now.

Continue reading "Reprogramming breakthrough does not displace ethical debate" »

Posted by Monya Baker on July 09, 2007
Categories: Correspondence, Reprogramming/Pluripotency | Permalink | Comments (2) | TrackBacks (0)

In June, widely publicized work from three labs showed that specialized cells could be reprogrammed after transfection with four genes. In this correspondence, James Trosko suggests an alternative explanation, that the reprogrammed cells identified by groups led by Shinya Yamanaka, Rudolf Jaenisch, and Konrad Hochedlinger and Kathrin Plath could in fact be skin stem cells reprogrammed to an embryonic state.

Another thread discusses how reprogramming work alters perceptions of whether dedifferentiation is active or passive, and adds insight from in silico modeling. http://blogs.nature.com/reports/theniche/2007/07/reprogramming_insights_in_sili.html

Below is the email correspondence between the scientists:

Continue reading "Do induced pluripotent stem cells arise from skin stem cells?" »

Posted by Monya Baker on July 09, 2007
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Eric Werner writes:
The recent results of dedifferentiating adult mouse fibroblast cells into stem cells brings into focus the fundamental question of how differentiation and development are controlled. (Cyranoski, D., Nature 447, 618-9; 2007). (Okita, K., et al. Nature doi 10.1038/nature05934, 2007). The fact that just four regulatory genes inserted into cells using viral vectors, can transform normal, differentiated cells into pluripotent stem cells indicates that for some cell types, at least, the process of dedifferentiation is more a process of activation rather than deactivation (Reik, W., Nature 447, 425-32; 2007). Indeed, this falls in line with in silico studies where stem cells, and, more generally, multicellular differentiation and development are modeled on computers.

Continue reading "Reprogramming insights: in silico modeling suggests active dedifferentiation" »

Posted by Monya Baker on July 09, 2007
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By Natalie DeWitt and Monya Baker

Monkey embryonic stem cells have, for the first time, been created through somatic cell nuclear transfer (SCNT). All attempts to make human embryonic stem cells through nuclear transfer so far have failed, but Jamie Thomson got the recipe for human embryonic stem cells by first doing so in monkeys, so researchers will likely be going to Shoukhrat Mitalipov of Oregon National Primate Research Center for advice. Mitalipov made his announcement Monday at the International Society for Stem Cell Research in Cairns, Australia, in a special add-on presentation . This finding represents a proof of principle that therapeutic cloning to create patient-specific ES cell lines could work in primates.

Continue reading "Oregon scientist reports first ES cells from cloned primate embryos" »

Posted by Natalie DeWitt on June 18, 2007
Categories: Cloning, Regenerative medicine, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (0)

Posted by Natalie DeWitt for Monya Baker

South Korean scientist Woo Suk Hwang actually did achieve an important first, just not the one he claimed. I was at the meeting where Hwang said, falsely, that he’d created the first human embryonic stem cell through cloning. It felt like a rock concert, except attendees held up recorders instead of lighters.

It turns out that Hwang might have gotten some rock-star status just by sticking to the truth. The human embryonic stem cells he made came from a parthenote, or an activated, unfertilized egg, and he really did do it first. George Daley, a stem cell scientist from Children’s Hospital, Boston, announced this fact to an absolutely packed crowd in an exhibit hall at the International Society for Stem Cell Research in Cairns, Australia. That Hwang's line came from a parthenote had been suspected, but this line of evidence hadn't been presented before.

(Last year, Tiziana Brevini and Fulvio Gandolfi of the University of Milan announced that they had derived two stem cell lines from 104 eggs that had been donated to fertility clinics. The news story is here: http://www.nature.com/nature/journal/v441/n7097/full/4411038a.html)

Over a year and a half ago, everyone assumed that cloning human embryonic stem cells had been reduced to practice. Now, Hwang is a symbol for the biggest scientific fraud so far this century.

Daley described how embryonic stem cells derived from parthenotes could generate transplant tissue less subject to immune rejection, and I think about how when I bump in from stem cell scientists from South Korea, they tend to bring up Hwang in the first few sentences. They have done nothing wrong, but they still seem embarrassed. Had Hwang simply stuck to his real achievement, they would be proud.

(In a subsequent post, I’ll describe Daley’s work comparing how embryonic stem cells made through cloning differ from their parthenote-derived equivalents.)

Posted by Natalie DeWitt on June 18, 2007
Categories: Cloning, Regenerative medicine, Reprogramming/Pluripotency | Permalink | Comments (0) | TrackBacks (1)