The Niche

Round-up of recent publications

Here’s a round-up of recent publications that I thought were interesting. I’ve had some written up as research highlights which will go out in our newsletter. For others, I’ll point you to articles in the popular press.

(Update 2/22/08, on NPR’s ScienceFriday, Geoerge Daley just gave a clear explanation of his Science paper along with an interesting backstory I hadn’t appreciated when I read the paper. The audio should become available through NPR. )

Also, Cell just came out with a big collection of review and overview articles on stem cells. For those reading after Feb 28, it’s the Feb 22, 2008 issue.

We’ll start with a list….

MicroRNA, embryonic stem cells, and Lin-28, oh my! — in a Science paper, Daley and other Harvard folk show what Lin-28 might be doing in the reprogramming mix (Scroll down to 1)

Embryonic stem cells yield pancreatic progenitors that stall diabetes in mice—a Nature Biotechnology paper from Novocell (Scroll down to 2)

Different embryonic stem cell lines prefer different destinies—in a Nature Biotechnology paper, Harvard’s Melton watches his lines differentiate and finds some like the heart, others brain (Scroll down to 3)

Sorting the starting steps in self-renewal—in a couple Cell Stem Cell articles, Harvard’s Hochedlinger and MIT’s Jaenisch monitor how transgenes switch on cells’ own self-renewal machinery, information useful to get away from hard-to-control viruses (Scroll down to 4)

Reprogramming is not just for skin cells—in Science, Kyoto University’s Yamanaka shows that some cells seem easier to reprogram than fibroblasts, and the transformed cells were originally differentiated (Scroll down to 5)

Transplanted human embryonic stem cells help rats with strokes, no tumours either—in PLOS, Stanford’s Steinberg differentiates ES cells to neural stem cells, injects them in 10 rats, an finds they walk better

Read it in Scientific American

Or in the Guardian

Precancerous stem cells make blood vessels different than healthy cells do—in PLOS , Ohio State University’s Gao explains why some cancer treatments that try to starve tumours of their blood supply don’t work so well

Read about it in Medical News Today


1) More reprogramming tips

A gene used to reprogram differentiated cells blocks microRNA processing

Researchers are rushing to create a how-to manual for reprogramming differentiated cells to an embryonic-stem-cell-like state, and new work shows that processing gene-regulating RNAs known as microRNA will be an important part.

So far, four sets of researchers have reported reprogramming human skin cells to behave like embryonic stem cells. The trick is to add various genes into cells using viruses. Mostly, scientists stuck to the ‘Yamanaka’s quartet’, the genes that Shinya Yamanaka of Kyoto University used originally to reprogram mouse cells, but James Thomson of the University of Wisconsin tweaked the technique, swapping in other genes known to be expressed in undifferentiated embryonic stem cells. Their reprogramming recipe included a gene called Lin-28, known to bind RNA in the cytoplasm.

In a paper published 21 February in ScienceExpress, researchers from Harvard University pinned down what this mysterious protein is doing. Srinivas Viswanathan, George Daley, and Richard Gregory observed that a type of unprocessed microRNA disappears in differentiated cells, but not in embryonic stem cells, embryonal carcinoma cells, and certain tumours. When they identified the proteins binding to this type of microRNA, they mostly found proteins that were part of common RNA-processing machinery, but they also found Lin-28. Lin-28 has been implicated in the development of worms, chickens, and mice, so this protein seemed like a good candidate to explore. In a series of experiments that boosted and decreased levels of functioning Lin-28 in different cell types, the researchers found that certain unprocessed microRNAs (pri-let-7) built up in cells with more Lin-28. In the absence of Lin-28, the microRNAs appeared in its processed form (mature let-7).

A version of Lin-28 is activated in some liver cancers, and the researchers speculate that processing this type microRNA helps “silence the self-renewal machinery” thus promoting a differentiated state. In fact, independent researchers have reported that breast cancer stem cells have lower levels of let-7.) Further, blocking this processing helps cells move from a specialized identity to a less differentiated one, a process that happens both when cancer begins and when cells are reprogrammed in culture.

2) Perfect Pancreatic Cells?

Pancreatic endoderm derived from embryonic stem cells forms glucose responsive endocrine cells in vivo

By Simone Alves

This week, California-based Novocell, Inc. showed cell replacement therapy for Type I diabetes mellitus seems to work, at least in mice. The Holy Grail for researchers working to treat diabetes this way is a reliable source of cells which produce sufficient quantities of insulin in response to glucose; starting with embryonic stem cells, a team led by Emmanual Baetge have apparently just done so1.

Type I diabetes occurs when insulin-producing β-cells are destroyed by the immune system. Treating patients by transplanting a pancreas or pancreatic cells is reasonably successful, but the tissues are hard to get, and the transplants require a difficult immunosuppression routine. Nonetheless, the potential of transplanting functional cells has fuelled attempts to derive insulin-producing cells in vitro using human embryonic stem cells (hESC). Though β-cells have previously been produced in vitro, they have not been unable to respond to insulin by secreting high amount of glucose in vitro2 or in vivo3.

Previous work has shown that, when transplanted into diabetic mice, immature fetal pancreatic tissue can differentiate and correct hyperglycaemia. Baetge’s team decided that instead of trying to fully differentiate the cells in vitro using the protocol they’d previously developed, they would try using cells at an earlier stage, when they resembled fetal pancreatic tissue, and see if they could continue to differentiate in vivo. These ‘pancreatic endoderm’ cells were grafted into immunocompromised mice. Only 30 days after engraftment, they produced human insulin in response to glucose, and the effect became more robust over time. The grafts expressed endocrine hormones independently of nearby tissue and their structure resembled the mature pancreatic islets where β-cells are usually found. Perhaps the most convincing of the technique’s potential, the engrafted mice still did not get diabetes after researchers used a cytotoxin that specifically destroys endogenous mouse β-cells. It is still unknown how long the grafted cells will survive; work in heart and brain diseases show that grafted cells tend to die over time.

Still, this is exciting news and has already generated a flurry of media attention. Researchers have finally coaxed hESCs into giving rise to a mature pancreatic cell population that can respond to glucose stimulation, and produce insulin in a regulated fashion. The authors feel this is a promising step in using hESC to develop a renewable source of insulin producing islets for diabetes cell replacement therapies.

References:

1. Kroon, E., et al. Pancreatic endoderm derived from himan embryonic astem cells generates glucose responsive insulin secreting cells in vivo. Nat Biotechnol. AOP 20 Feb 2008, doi10.1038/nbt/1393

2. D’Amour, K.A. et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24, 1392–1401 (2006).

3. Jiang, J. et al. Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells 25, 1940–1953 (2007).

Further reading:

Stainier, D. No stem cell is an Island (Yet), New England Journal of Medicine, Vol. 354, No. 5, pp. 521-523, February 2, 2006.

Simone Alves is an intern with Nature Publishing Group in London

3) Underlying Differences

Various embryonic stem cell lines trend toward particular cell types

By Simone Alves

Embryonic stem cell lines seem similar in culture, but researchers have long suspected that some lines are better at forming some cell types than others. Now, that suspicion, based largely on anecdotal evidence, has been confirmed systematically. A team led by Douglas Melton at the Harvard Stem Cell Institute compared markers expressed by 17 human embryonic stem cell lines while undifferentiated, after spontaneous differentiation, and after being induced toward cardiac and pancreatic cell types.

When undifferentiated, key pluripotency genes such as OCT4 and NANOG were similarly expressed in all 17 cell lines over several time points. The researchers examined the spontaneously differentiating cells for markers representing eight organs and the three germ layers, the broad categories of ectoderm, mesoderm, and endoderm into which all non-sex cells can be sorted. Expression levels of these markers could vary as much as 3,000-fold between lines.

For example, after spontaneous differentiation, HUES 6 expressed neuronal and other ectoderm genes, but particularly low levels of endodermal and pancreatic markers SOX17 and PDX1. Another line, HUES8, expressed particularly high levels of these markers. The group then induced the embryonic stem cell lines specifically to express SOX17 and PDX1 by treating them with activin A, and HUES 8 expressed both markers at higher levels than did HUES 6. Similarly, HUES 3 expressed particularly high levels of heart-muscle markers while HUES1 expressed particularly low levels during spontaneous differentiation. When induced toward cardiomyocytes, HUES 3 generated a larger number of cardiomyocytes that actually contracted. The differences depended on the cell lines and not on how long the cells had been grown in culture.

The work shows that indeed, there are differing proclivities in differentiation. Melton’s group found that these proclivities could not be explained by cell type, karyotype, sex chromosome ratio or derivation protocol. Indeed, all the lines were derived in the Melton lab. They suggest instead that genetic variation and the epigenetic status of the cell lines play a role and suggest that deriving additional cell lines may be useful for creating specific cell lineages.

Reference:

Osafune, K., et al; Marked differences in differentiation propensity among human embryonic stem cell lines; Nat Biotechnol, AOP 17 Feb 2008; doi: 10.1038/nbt1383

Simone Alves is an Intern at Nature Publishing Group in London.

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4) Sorting the steps to self renewal

Multiple labs have now shown that adult cells can be reprogrammed into an embryonic-like state. A suite of virally introduced genes are necessary to kick-start the reprogramming process, but gradually the cell’s own endogenous pluripotency genes become active, and the viral genes are silenced. Differentiated cells take weeks to become pluripotent, and some estimate that fewer than one in five hundred cells that take in copies of each gene are transformed. Two papers in Cell Stem Cell dissect events in the reprogramming process. This insight could help researchers attempt new, reprogramming approaches that do not use viruses.

The research teams, led by Rudolf Jaenisch at the Massachusetts Institute of Technology and Konrad Hochedlinger at Massachusetts General Hospital started with cultured mouse skin cells (fibroblasts) and added viruses carrying the four genes originally established for inducing pluripotency (Oct4, Sox2, Klf4 and c-Myc). Both teams had reprogrammed fibroblasts with this method before. This time, however, they used versions of genes that could only turn on in the presence of the small molecule doxycycline. That way, they could tell how long reprogramming cells needed the transgenes to stay active.

Hochedlinger’s team looked for the initiation of several events necessary for pluripotency: downregulation of genes normally active in fibroblasts, reactivation of the X chromosome, plus the upregulation of proteins characteristic of embryonic stem cells like telomerase, Sox2, Oct4, and stage-specific embryonic antigen-1 (SSEA-1). They found that the fibroblast markers shut down quickly, in as little as two or three days. Cells first started expressing the gene for SSEA-1 at around 5 days. Other events happened later. At each step, the researchers could sort cells according to pluripotency markers (or absence of differentiation markers) and get a higher percentage of cells that would go on to produce induced pluripotent stem cells. “This now allows to zoom into these intermediate cell populations and ask what’s going on at the molecular level,” says Hochedlinger.

Jaenisch’s team first showed that endogenous pluripotency genes must become active in a particular sequence, a finding that is consistent with other studies. For this, they used cell-sorting techniques, plus mouse cell lines engineered so that green fluorescent protein would be produced when one of two endogenous pluripotency genes (Nanog or Oct4) was expressed. The first gene to activate was that encoding alkaline phosphatase, at around three days; the next was the SSEA-1, at around nine days. Nanog and endogenous Oct4 were detectable by cell-sorting machines at around 16 days. Even 21 days after culture, transgene activity greatly boosted reprogramming rates, though the rates were still low.Though the cells looked morphologically different as early as three days after infection, they always reverted back to fibroblasts if doxycycline was removed from the culture within two weeks of delivery of the transgenes.

Hochedlinger’s team also showed that the transgenes were necessary for about ten days, after which cells’ endogenous machinery sustained pluripotency. To study the kinetics of transgene silencing, Hochedlinger infected cells with a retrovirus coding for red fluorescent protein and found that silencing began to occur as early as three days after infection. In colonies of cells examined 13 days after transfection, the researchers found that cells expressing red fluorescent protein (meaning the retrovirus had not been silenced) never expressed a green fluorescent protein tied to expression of pluripotency gene Sox2, and vice versa. This suggests that full reactivation of the pluripotency genes requires silencing of the viruses.

Jaenisch’s team created versions of the transgenes that could not be silenced and found that although transformed cells took on properties that are characteristic of embryonic stem (ES) cells, they did not differentiate when injected into mice, unlike ES cells or other induced pluripotent stem cells.

Next steps include exploring roles and relationships of different transgenes and trying to figure out why reprogramming cells through this method seems to take so much more time than reprogramming through nuclear transfer. “Many sequential events have to happen. We have to re-establish the core circuitry of pluripotency,” says Jaenisch.

References

1. Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2,151–159 (2008).

2. Stadtfeld et al., Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse, Cell Stem Cell doi:10.1016/j.stem.2008.02.001 (Advance online publication 14 February 2008)

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5) Stomach and liver cells reprogrammed

Cells need fewer extra gene copies to become pluripotent, and the resulting mice don’t get tumours

The possibility of reprogramming adult cells to behave like embryonic stem cells (ES cells) without overexpressing potential cancer-causing genes or relying on hard-to-control viruses has just got a bit closer. Publishing in Science, Shinya Yamanaka and colleagues from Kyoto University, in Japan, show that the reprogramming techniques they previously demonstrated on cultured mouse skin cells (see From skin cell to stem cell) also work on two other mouse cell types: liver cells and the epithelial cells lining the stomach1. Not only did these cells seem easier to reprogram, mice generated from the reprogrammed cells did not develop cancer.

Several labs have now reprogrammed fibroblasts, and because far fewer than 1 in 100 treated cells are successfully transformed, several stem-cell researchers 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 in his new study, Yamanaka used a genetic marking system that permanently labels liver cells once they differentiate enough to express albumin, and he found that these cells could be reprogrammed to so-called induced pluripotent stem cells (iPS cells) that can contribute to all cell types in an adult mouse. Yamanaka and colleagues also showed that the epithelial cells lining the stomach can generate iPS cells, and doing so requires a less rigorous screening system than that used with cultured skin cells, or fibroblasts.

“The old question in cloning was exactly the same: was Dolly derived from a fully differentiated cell?” says Rudolf Jaenisch of the Whitehead Institute, 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, says Jaenisch, but that conclusion assumes both a reliable labeling system and that only mature cells express the albumin gene. Yamanaka himself stops short of calling the initial cells fully differentiated in an email: “Our data showed that lineage-committed albumin-producing cells can be reprogrammed,” he wrote in an email.

Whereas tumours develop in about a third of mice created using iPS cells derived from fibroblasts, no tumours were found in the mice created from iPS cells derived from stomach and liver cells. These mice were more likely to die in utero than those generated from fibroblasts, but the live-born mice appeared healthy. Compared with fibroblasts, viruses were less efficient at infecting the stomach-lining cells with the necessary genes, but the transformed cells contained fewer copies of the transgene compared with fibroblasts, perhaps because epithelial cells are more similar to ES cells than fibroblasts are.

Yamanaka also 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 non-viral means of generating iPS cells,” says George Daley of Children’s Hospital, Boston, who has recently compared the efficiencies of reprogramming human fibroblasts from different sources2.

References

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

2. Park IH et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 451, 141-6. (2008)

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