The Niche

Stem cells in NPG this week

Our feature, A closer look at the single cell , profiles innovations in imaging from individual laboratories as well as commercial applications in gene expression.

I decided to commission it while reporting a story on cancer stem cells: researcher after researcher told me that the heterogeneity of cells within a tumour made it both essential, and impossible, to figure out what individual cells are doing. It does not include excellent genetic lineage experiments but rather focuses on studying cells in vitro.

Besides our feature, there’s a write-up of a recent conference bringing together scientists and industry.

Also, how did researchers get differentiated cells to beat when no one else had? Here’s our highlight of that Nature paper from Gladstone’s Benoit Bruneau.

And this highlight for researchers who want to start reprogramming human cells but lack the patience or expertise to pick the right colonies, check out this reprogramming tool published in Nature Methods by James Ellis of the University of Toronto.

Scroll below for a sneak peaks of three highlights.

A pair of papers in PNAS and Cell Stem Cell show ways to stabilize pluripotency and so make embryonic stem cells from non-permissive embryos. From MIT’s Rudolf Jaenisch and Scripp’s Phil Schultz

A Cell paper finds a microRNA for differentiation. It represses three major pluripotency genes and is repressed by one of them. From Ken Kosik of the University of California, Santa Barbara

A Nature Medicine paper finds early embryos have many more chromosomal abnormalities than anticiptated. What does that mean for embryonic stem cells? (This topic is covered for embryos in a News and Views)

Elsewhere in NPG this week:

Gene therapy researcher, James Wilson, warns stem-cell scientists not to repeat his field’s mistakes

South Korea re-enters human stem-cell research

Neuroscientists claim growing pains with a commercial media

With OCT4, less is more : the first characterization of a post-translational regulation event occurring on OCT4 in a hES cell system

A panel of experts weigh in on a recent Nature Cell Biology that adult mice can generate new oocytes

A couple Nature Biotechnology papers compare the methylome in a variety of cells, including fibroblrasts, embryonic stem cells, and induced pluripotent stem cells. Read more

Here are peeks of the highlights that will go live next week.


MicroRNA a major repressor of pluripotency

How pluripotency is controlled is one of biology’s major puzzles. New research led by Kenneth Kosik of the University of California, Santa Barbara reveals not only a new piece in the puzzle but also how some major pieces fit together.

A set of transcription factors — Klf4, Oct4 and Sox2 — are sufficient to reprogram specialized cells to pluripotency. As pluripotent cells take on a differentiated fate, these transcription factors disappear. Kosik found that one microRNA (miRNA) is able to repress all three of these proteins.

Although these small RNA molecules can silence gene expression in all sorts of cells, their role in pluripotent stem cells is unclear. Kosik screened for miRNAs whose expression increased as embryonic stems cells differentiated.

One of these was miRNA-145, which exists in frogs, fish and mammals and is also enriched in many germline tissues. Computational tools predicted that it would hit mRNA transcripts for Klf4, Oct4 and Sox2.

To make sure that miRNA-145 interacted with the pluripotency proteins, the researchers attached the untranslated regions of Klf4, Oct4 and Sox2 onto the gene for luciferase and found that miRNA-145 repressed luciferase expression in several types of cells, whereas other miRNA constructs did not. Many more experiments, involving mutating the untranslated regions and upregulating and downregulating miRNA-145, all provided evidence that miRNA-145 inhibits these core pluripotency factors.

“[The work] is exciting for many reasons,” says Hannele Ruohola-Baker, who studies stem cells and miRNAs at the University of Washington in Seattle. This knowledge could be exploited not only to understand what keeps cells pluripotent or prompts them to differentiate, but also to make cell therapy safer by providing a mechanism to eliminate unwanted stem cells.

Right now, though, she says the research is at an exploratory stage, with researchers just starting to uncover the miRNAs that control stem cell states. “Let-7, miRNA-21 and now miRNA-145 have all been shown to be miRNAs that are not expressed in stem cells and, at varying levels of certainty, have been shown to make cells differentiate,” she says. “Who knows, maybe this one [miRNA-145] is the most powerful so far.”

And miRNA-145 seems to have a particularly interesting interaction with stem cells’ pluripotency machinery. Further study of the genome identified an Oct4 binding site near the part of the genome coding for miRNA-145. Using a luciferase reporter tied to the miRNA-145 region, the researchers found that higher levels of Nanog did not affect the miRNA’s expression, but higher levels of Oct4 did. Thus, miRNA-145 represses three powerful pluripotency transcription factors and is itself repressed by one of them — Oct4.

Kosik says that the identification of miRNA-145 and this interesting mechanism opens up many additional questions. “What we need to do now is understand how the double-negative feedback loop is creating a homeostatic state or an altered state. To do that we need a lot more information.”

For instance, he says, the number of copies of the various components could help set the balance between the differentiated state and the pluripotent state. Understanding that kind of stoichiometry will be difficult but could be essential to manipulating miRNAs.

There is a precedent for this sort of feedback loop in murine embryonic stem cells, says Isidore Rigoutsos, manager of the bioinformatics and pattern discovery group at the IBM Thomas J. Watson Research Center in Yorktown Heights, New York. He and others have shown that miRNA-296 targets Nanog and that Nanog and Oct4 regulate miRNA-296 (refs 2,3).

Still, Kosik’s work is “particularly exciting,” Rigoutsos says. “It shows that the details of a feedback loop may change fairly drastically from organism to organism even if one confines oneself to studying specific genes.” Understanding such differences could be crucial to manipulating cell states for applications from disease modeling to drug discovery to cell therapy.

Related articles

Versatile microRNAs, two-faced transcription factors and embryonic stem cells

MicroRNAs boost reprogramming, boot out cMyc

A gene used to reprogram differentiated cells blocks microRNA processing

Efficient ES cell differentiation: the right tweak at the right time

Haifan Lin: peeling back layers of stem cell control

MicroRNAs help embryonic stem cells grow fast

MicroRNA reins in tumor-initiating cells

Repressing microRNAs for pluripotency

References

1. Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J. A. & Kosik, K. S. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell doi:10.1016/j.cell.2009.02.038 (published online 30 April 2009).

2. Tay, Y., Zhang, J., Thomson, A., Lim, B. & Rigoutsos, I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 455, 1124–1128 (2008).

3. Loh, Y-H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Genet. 38, 431–440 (2006).

Single-cell genetic analysis on embryos generated by IVF finds surprisingly high rates of rearrangements

By Alicia Chung

The gain or loss of chromosomal segments causes many problems in cell function, with malignant growth being the most worrying. Screening for such abnormalities is standard practice in fertility clinics for embryos generated by in vitro fertilization, and it has revealed that these embryos, sometimes donated for the generation of human embryonic stem cell lines, often possess chromosome imbalances. In a recent study published in Nature Medicine, Joris Vermeesch and colleagues at the Center for Human Genetics at the Catholic University Leuven, in Belgium, used high-resolution single-cell genetic analysis to show that such abnormalities are also found in embryos generated by in vitro fertilization from fertile young women (less than35 years old) and are likely to be an inherent feature of early human development1.

Current screening for chromosome abnormalities on embryos generated by in vitro fertilization does not cover the entire genome. Using new array-based approaches, Vermeesch’s group performed a genome-wide scan of chromosome copy number variation and single-nucleotide polymorphisms at 25,000 positions on a single-cell level. Their study of 23 pre-implantation embryos from 9 fertile couples found that chromosome instability was much higher than anticipated. Previous studies using fluorescence in situ hybridization on 10 chromosomes had shown aneuploidies occurring at a rate of around 50%, says Vermeesch, a rate considered the upper limit for embryos from fertile young women. “Since we have for the first time looked at all chromosomes, we find 90% of embryos with abnormal cells.” Additionally, rearrangements such as segmental imbalances, thus far only observed in tumours, were observed in 70% of the 23 embryos tested by Vermeesch’s group. “Because of the genome wideview,” he says, “we see much more.”

The reason why chromosomes are unstable during early human embryogenesis is unclear, but the findings have implications not only for fertility research but also for the derivation of embryonic stem cell (ESC) lines. Vermeesch believes chromosomal imbalances may be present before a line is derived. On the other hand, says Martin Pera, director for the Center for Regenerative Medicine and Stem Cell Research at the University of Southern California in Los Angeles, normal diploid cells do seem to have selective advantages over abnormal cells during both in vivo fertilization and human embryonic stem cell (hESC) line establishment, so genetically normal hESC lines could be established from embryos, even if some of their cells contain genetic abnormalities. However, he says, abnormalities in ESCs often arise during culture. According to Pera, these genetic changes tend to be recurrent, resembling those found in germ cell tumours of the testis, and can confer a growth advantage to pluripotent cells.

It’s possible that hESC lines created from very early-stage embryos might be more inclined toward unstable karyotypes, says Steven Stice, director of the Regenerative Bioscience Center at the University of Georgia. But he thinks techniques to monitor ESC lines for abnormalities, inherent or acquired, will overcome such issues. The implications of Vermeesch’s study are not likely to apply to the selection of embryos from which to derive cell lines but rather to better assessment of lines once they have been derived.

Related articles

How to assess a stem cell genome

Robust embryonic stem cells may harbor precancerous surprises

Stem cell lines accrue cancer mutations

From one, many genetically different embryonic stem cells

References

1. Vanneste, E. et al. Chromosome instability is common in human cleavage-stage embryos. Nature Med. advance online publication, doi:10.1038/nm.1924 (26 April 2009).

New types of embryonic stem cells generated by stabilizing pluripotency

Embryos from nonobese diabetic mice don’t yield stable embryonic stem cells, which makes the mice unsuitable for several sorts of experiments. New research reveals not only how to generate these stem cells but also how to toggle between different states of pluripotency.

Rudolf Jaenisch of the Massachusetts Institute of Technology in Cambridge says the work, reported in Cell Stem Cell, grew out of several strands of research1. In 2007, scientists led by Ron McKay at the National Institutes of Health in Bethesda, Maryland, and Roger Pedersen at the University of Cambridge, UK, had found that mouse embryos could generate a type of embryonic stem (ES) cell that very much resembled human ES cells in terms of gene expression and requirement of growth factors. Because these stem cells came from the epiblast, or the outer layer of early ball-shaped embryos, these cells are called epiSCs2,3. Unlike mouse ES cells, which are generated from the inner cell mass of ball-shaped embryos, epiSCs do not, if mixed with a mouse embryo, contribute widely to tissues in the resulting mouse pups.

Another strand of the research comes from work published this year by Austin Smith at the University of Cambridge, which showed that epiSCs could be converted into regular ES cells with the insertion of the gene for pluripotency factor Klf4 (ref. 4). If the gene was removed, the cells reverted back to epiSCs.

That got Jaenisch wondering if the epiSCs that can be generated from nonobese diabetic mice could also be converted into typical ES cells. His lab began to explore the issue by trying to reprogram fibroblasts from these mice, using the four standard reprogramming genes: cMyc, Klf4, Oct4 ) and Sox2. The cells reprogrammed, but only if genes for either cMyc or Klf4 remained active.

Then came the pursuit of small molecules that could replace these pluripotency factors. Researchers led by Peter Schultz of The Scripps Research Institute in La Jolla, California, developed a high-throughput screen that could test greater numbers and diversity of molecules than previous screens5. (The assay, which relies on a gene for the glowing luciferase protein tied to the Nanog promoter, can be performed in standard 96-well plates.)

This identified a promiscuous kinase inhibitor called kenpaullone that was capable of replacing Klf4. Other small molecule combinations that had previously been shown to prevent ES cell differentiation or allow derivation of rat ES cells could also work to generate stable ES cells from the nonobese diabetic mice. If these small molecules were withdrawn, however, the cells became unstable.

Jaenisch says his work shows both that a cells’ genetic background controls how cells become pluripotent and that external factors, like culture conditions, can compensate for nonpermissive backgrounds. That, in turn, could lead to the generation of ES cells from more species, a research tool that could not only help livestock science but also create better ways to study human disease. Interestingly, work published last month by researchers at the University of Washington in Seattle also showed the ability of human ES cells to apparently toggle between different pluripotent states, depending on the concentration of a histone deacetylase inhibitor called butyrate, which affects the way cells control whether genes are active or silent6.

But Jaenisch says he’s not sure whether this work will lead to techniques to convert human ES cells to a more mouse-like state. “Mouse cells are much easier to work with than human cells, but I’m not sure that I can transfer them so easily.”

Related articles

Off with differentiation

Real potential in rat embryonic stem cells

References

1. Hanna, J. et al. Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell doi:10.1016/j.stem.2009.04.015 (published online 7 May 2009)).

2. Brons, I.G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007). | Article | PubMed | ISI | ChemPort |

3. Tesar, P.G. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007). | Article | PubMed | ISI | ChemPort

4. Guo, G. et al. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063–1069 (2009).

5. Lyssiotis, C. A. et al. Reprogramming of murine fibroblasts to iPS cells with chemical complementation of Klf4. Proc. Natl. Acad. Sci. USA (in the press).

6. Ware, C. B. et al. Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells. Cell Stem Cell 4, 359–369 (2009).

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