The Niche

Below is a summary of a couple Cell Stem Cell papers that offer map-fragments to one of stem cells Holy Grails: culturing the cells that give rise to blood. This could lead to more broadly applicable alternatives to treatments that now use cord blood or bone marrow transplants. This will become a formal highlight next week.

Also, an article published yesterday in Nature Medicine shows how embryonic stem cells can be used to evauluate mutations implicated in breast cancer.

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Developing a way to reliably produce hematopoietic stem cells (HSCs) is a bloody tough problem. Unlike most tissues, cells of the hematopoietic system emerge from several embryonic sites and then circulate through the body. This mobility has perplexed researchers who hope that mimicking the in vivo environment will help them culture HSCs. Now though, two British research teams report complementary techniques for isolating HSCs in Cell Stem Cell. These could form the lifeblood of techniques creating easier alternatives to bone marrow transplantation.

Alexander Medvinsky and his colleagues at the University of Edinburgh went straight to the heart of HSC development — the aorta-gonad-mesonephros (AGM) region, where the first multipotent HSCs are thought to arise in the embryo. By dissociating and reconstructing the mouse AGM's three-dimensional structure, they developed a new method to expand and track the development of HSCs, and tracked down a population of cells containing markers (VE-cadherin and CD45) generally found on separate types of cells1.

"Medvinsky characterized a cell that's wearing two hats: It's an endothelial cell and a blood cell," says M. William Lensch, of Children's Hospital Boston and the Harvard Medical School. "When you see a cell like this, it lends credibility that they develop directly from the vasculature," rather than from elsewhere in the developing embryo.

Medvinsky’s population of cells contained one marker associated with the inner lining of blood vessels and another specific to blood cells themselves, and he thought that these cells might constitute “pre-HSC” progenitors capable of acquiring stem cell function. Indeed, when he injected the VE-cadherin+CD45+ cells in irradiated mice, they rapidly developed into a large pool of definitive HSCs that that restored hematopoiesis. Since these cells mature within the AGM microenvironment, this constitutes an active niche that drives the specification of fully mature HSCs, says Medvinsky.

Rather than trying to isolate HSCs directly, a team led by Majlinda Lako of Newcastle University developed a relatively efficient way of coaxing hematopoietic differentiation from human embryonic stem cells (hESCs). They co-cultured hESCs with AGM-derived stromal cell lines, and found that hematopoietic activity increased at least 31-fold compared to previous co-culturing methods2. They then injected the induced-hESCs into the femurs of immunocompromised mice, and found substantially greater engraftment efficiencies than previously reported — up to 16% for cells co-cultured with the best cell line. Finally, Lako’s group screened around 40 signaling molecules for positive enhancers of hematopoietic differentiation, and flagged the transcription factors TGF-β1 and TGF-β3 as the most efficient inducers of hematopoiesis.

Together, the studies show that nascent cells must mature within the proper context to become definitive HSCs, regardless of whether you start with pre-HSCs or hESCs, says Hanna Mikkola, of the University of California, Los Angeles. “The message from both papers is you really need to have the correct embryonic environment for functional maturation in culture.”

The question of how that maturation occurs remains unanswered. The TGF family members identified by Lako are probably involved, Mikkola says, but she doubts these factors tell the whole story. Lako agrees. As a follow-up, Lako's group is currently sifting through a library of other candidate factors, including calcium signaling molecules and insulin-like growth factors, for other key regulators of HSC development.

Lako’s results are impressive, says Medvinsky. But he thinks that co-culturing hESCs with his VE-cadherin+CD45+ cells could be even more successful. “With our system we might be able to produce a better outcome.” Lako, however, suspects her stromal cell lines may already contain some of Medvinsky’s “niche” factors. “It’s very likely that we’re using the same signals to induce our human ES cells,” she says. In either case, both authors recognize that more work will be needed to nail down the molecular cues before fully transplantable HSCs can be cultured. "Because you can do this all in a lab dish now, you have the ability to really focus on what the molecules are," notes Lensch. These two studies now inject new blood into achieving that goal.

References
1. Taoudi, S. et al. Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs. Cell Stem Cell 3: 99–108 (July 2008).

2. Ledran, M.H. et al. Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches. Cell Stem Cell 3: 85–98 (July 2008).

Author affiliation
Elie Dolgin is a Canadian science writer currently residing in Milwaukee, Wisconsin.

Posted by Monya Baker on July 07, 2008
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The California scientists most likely to receive state grants for making new cell lines were those who proposed comparing embryonic stem cell lines and induced pluripotent stem (iPS) cell lines. Overall, thirty-two percent of all grant applications (16 of 50) were funded. Four of the five grants that proposed comparisons got funds. The unfunded grant application crossed into less favored categories, as it also proposed making lines from parthenotes and through nuclear transfer. None of the grant applications that sought to make cell lines using human oocytes were funded. Two proposed cloning through nuclear transfer, one proposed stimulating unfertilized eggs to divide into parthenotes, and one application proposed using both methods.

Success rates for grants proposing the derivation of only ES or only iPS cells were each 33%, but there were twice as many grants for iPS cells. That’s astounding considering that the grant program was announced in October 2007, a month before the first publications that human cells could be successfully reprogrammed.

Four proposals to make pluripotent lines using cells derived from the placenta, testes, or amniotic fluid were rejected. But a proposal to make spermatagonial stem cells, ES cells, and iPS cells was funded and highly praised, with reviewers particularly keen to see a comparison of iPS and spermatagonial stem cells from the same individual.

Continue reading "What got funded: statistics on California’s new stem cell line grants" »

Posted by Monya Baker on July 02, 2008
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An edited version will appear on the site on later this month.
Differentiated cells can be reset to an embryonic-stem-cell-like state, but doing so requires using retroviruses to insert a suite of genes into a culture of cells. Because the viruses insert their cargo at random into the genome, infected cell populations from the same individual are genetically different, and it’s hard to know whether differences between the resulting stem cell lines are due to this genetic variation, the epigenetic state of the original cells, or chance events. Worse, the current use of retroviruses renders the cells unusable for clinical applications.

Now researchers led by Rudolf Jaenisch at the Whitehead Institute in Cambridge Massachusetts show a convenient way to generate genetically identical cell populations that can be converted to induced pluripotent stem (iPS) cells by adding a drug . What’s more, they show that cells from multiple organs can be successfully reprogrammed.

In previous work, Jaenisch and other laboratories had reprogrammed cells using genes that turn on in the presence of a drug called doxycycline. This allowed them to study how long transgenes need to stay active for reprogramming to occur. To prove that the cells with drug-inducible reprogramming genes were pluripotent, researchers mixed the cells with mouse embryos to create chimeric mice.

In this paper, the researchers show that cells from multiple organs within these chimeric mice can be efficiently reprogrammed with the addition of doxycycline: reprogrammed cells include neural progenitors, mesenchymal stem cells, and keratinocytes as well as cells taken from muscle, intestinal epithelium, the adrenal gland, and the hematopoietic lineage.

iPS cells generated from different tissues taken from the same mouse are genetically identical, allowing researchers to examine the effects of cell types and retroviral insertion sites. For example, the researchers were able to reprogram intestinal epithelium derived from one iPS cell line, but not another, suggesting that reprogramming requirements vary between cell types. In particular, the expression of transgenes seemed to vary with both cell type and site of insertion in the genome.

The reprogramming rate for `secondary iPS cells’ was 4 to 8 fold higher than for the production of primary iPS cells, presumably because cells in the mice already had the favorable number of proviruses inserted at appropriate sites of the genomes. Still the overall reprogramming rate is low, only between 2% and 4%.

The researchers believe this could be because the drug-inducible transgenes may be more or less responsive to doxycycline even within genetically identical cells and also because reprogramming depends on stochastic events, several of which are required for complete reprogramming.

A source of genetically identical cells will help researchers home in on these and other variables and greatly simplify the search for methods to create clinically acceptable reprogrammed cells.

Wernig, M. et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat. Biotechnol. doi:10.1038/nbt1483 (Advance online publication July 1, 2008)

Posted by Monya Baker on July 01, 2008
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Alexey Bersenev just sent in his detailed take on the ISSCR meeting, in which he also compiled a list of other blogs on the meeting. Thanks!

Just to keep the Nature comments on ISSCR in one place, here’s another list.
My speed-writing on impressions from ISSCR.
Nature’s Brendan Maher posted on Doug Melton’s talk on reprogramming in situ. Who needs pluripotency? Let’s go straight to the cells we need!

How do you know a reprogrammed cell is reprogrammed?
Scientists consider minimum standards for induced pluripotent stem cells
My blog post on the same topic links to articles on iPS cell advances.

Stem cell society condemns unproven treatments
The ISSCR is drawing up guidelines for clinical practice. How will patients and practitioners respond?

Read our feature Stem cell researchers face down stem cell tourism

Also, just weeks before the ISSCR meeting, stem cell researchers gathered for their version of summer camp, Cold Spring Harbor Laboratory Symposium, where the ratio of content learned
Read my impressions.

Posted by Monya Baker on June 30, 2008
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Here's a highlight summarizing three new papers.

Neural stem cells continuously form new neurons in the adult brain. But after injury, these stem cells seem able to become other cell types, such as glial cells that support neurons. This flexibility helps the brain heal naturally, but it poses a problem because researchers developing treatments for neurological diseases want to be able to direct cells to desired fates. In some diseases only new neurons are needed; for others the non-neuronal cells are essential. In a series of recent papers, two La Jolla, California, research groups provide potential solutions to these brain-bending problems, showing that overexpression of single genes can reliably direct neural differentiation.

Continue reading "Mind control: new work on differentiating neural stem cells" »

Posted by Monya Baker on June 30, 2008
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Ronin takes a place beside, but not with, Oct4, Nanog, and Sox2

(This is out in Cell today. The research highlight will appear on our site on July 10)
Embryonic stem cells exist in a state of suspended differentiation; they are able to become any cell type but resist taking on any particular fate. A trio of transcription factors is widely recognized as essential to this pluripotent state: Oct4, Nanog and Sox2. These proteins interact closely with each other and often bind the same or nearby sites on DNA. Now, work by Thomas Zwaka of the Baylor College of Medicine in Houston, Texas, and colleagues reveals another protein that works outside this pluripotency network1. He dubbed it Ronin, the word for a samurai warrior without a master.

Zwaka came upon Ronin serendipitously. His previous work had shown that caspase-3, an enzyme that regulates cell death, also affects pluripotency by cleaving Nanog. Zwaka suspected that this enzyme might have additional roles to play in pluripotency, so his lab carried out a laborious large-scale screen called a yeast two-hybrid assay to figure out what other proteins the caspase interacted with. This screen pulled out the protein that is now called Ronin, and Zwaka’s team set about conducting a series of experiments to find out what it does.

Ronin is expressed only in oocytes, early embryos and some regions of the brain where cells proliferate. Neither embryos nor embryonic stem (ES) cells lacking Ronin can survive. If Ronin was overexpressed, mouse stem cells failed to differentiate even under conditions (the removal of a key growth factor) that caused all stem cells with normal Ronin expression to do so.

To really know that overexpression of Ronin can maintain ES cells without growth factors, the cells should be grown over several passages, says Qi-Long Ying, a stem cell scientist at the University of Southern California, in Los Angeles, who recently reported a cocktail of small molecules that allow mouse ES cells to proliferate without the growth factors typically required2. “I don’t know that they tested for prolonged culture.” Nonetheless, he says, Ronin seems to be a new essential factor for maintaining both pluripotency and early embryonic development. “It’s very important and also very interesting.”

Ronin seems to act through chromatin regulation. It has a zinc-finger DNA-binding motif called THAP that is often found in proteins that modify chromatin; Ronin also binds another protein called host cell factor-1 (HCF-1) that, along with other proteins, helps to modify histones, the structures within cells that package DNA into chromatin and help regulate gene expression. Ronin also seems highly conserved across species: the gene that encodes it is quite similar in humans and zebrafish, and there is even an apparent homologue in sea urchins.

It makes sense that important functions like self-renewal are regulated by a network and not a handful of factors, says Sadhan Majumder at the M.D. Anderson Cancer Center, in Houston, Texas, who has identified components of pluripotency networks3. “I think scientists are focusing on a few molecules that have so far been discovered, but I am sure that the network is controlled by many,” he says. Any of these network components could shift a cell’s equilibrium between self-renewal and differentiation.

Zwaka says that the next steps are to search for a Ronin regulatory network and other Ronin-related proteins. He’s also testing to see if Ronin can reprogram cells to pluripotency in the absence of other known reprogramming factors.

Related articles
Off with differentiation

Repressing microRNAs for pluripotency

References
1. Dejosez, M. et al. Ronin is essential for embryogenesis and the pluripotency of mouse embryonic stem cells. Cell 133, 1162–1174 (2008). | Article |
2. Ying, Q.-L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008). | Article |
3. Singh, S. et al. REST/NRSF maintains self-renewal and pluripotency of embryonic stem cells. Nature 453, 223–227 (2008). | Article |

Posted by Monya Baker on June 26, 2008
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