The Niche

Mini-research round-up

There’s some cool papers out this week.

Rudolf Jaenisch and Jacob Hanna and others at the Whitehead Institute has not only reprogrammed a fully differentiated cell, but has also generated reprogramming-ready mice. According to everything that’s been published so far, reprogramming specialized cells to an embryonic-like state meant transfecting them with viruses and hoping random chance went your way. Cells in these chimeric mice already contain copies of the transgenes necessary for reprogramming, and these versions of the genes become active when exposed to doxycyclin.

Mike Clarke and Bolaji Akala and others at Stanford use triple mutant mice to help explain a looming question in stem-cell biology is why haematopoietic stem cells (HSCs) self-renew but their progenitors cannot.


Reprogramming turns an end to a beginningLess than a year ago, researchers showed that cultured skin cells could be transformed into a state almost indistinguishable from embryonic-like stem cells using a quartet of inserted genes. Amidst the excitement, basic-science researchers wondered whether the reprogrammed cells were more ‘lucky’ (that is, they happened to get enough copies of the four genes) or ‘special’ (that is, they arose from rare stem cells pre-existing within the cultured skin cells, or fibroblasts). Now, evidence from Rudolf Jaenisch and colleagues at the Whitehead Institute in Massachusetts sets the winner’s cap on the lucky-cell hypothesis by demonstrating that terminally differentiated cells can indeed be reprogrammed1.

The paper, published this month in Cell, is not the first evidence for the lucky-cell hypothesis. Shinya Yamanaka, from Kyoto University in Japan, engineered cells to rearrange their genomes once they differentiated enough to make albumin and then showed that reprogrammed cells had the specific rearrangement2. But albumin-producing cells need not be fully differentiated, and that left room for doubt about whether a fully differentiated cell could adopt an embryonic-like state. To really settle the question, says Jaenisch, reprogramming must be accomplished in terminally differentiated cells in which the genetic rearrangement occurs without relying on an introduced gene.

As part of the process that generates a flexible immune system, B cells, the white blood cells that produce antibodies, naturally cut out a piece of their DNA in their final maturation step. The researchers thought they could use this quality as a marker to check whether the reprogrammed cells came from fully differentiated or immature cells. They found that both mature and immature B cells could be reprogrammed, and they verified that these so-called induced pluripotent cells were truly reprogrammed by using them to make chimeric mice.

Nonetheless, advocates of the special-cell hypothesis will find some solace in the paper. Though immature B cells could be reprogrammed using the same four genes already used to reprogram fibroblasts, the mature B cells required something more: researchers had to interrupt the gene expression typical of B cells, either with an additional factor (CCAAT/enhancer binding protein alpha) or by knockdown of the B cell transcription factor Pax5. After trying some 20 factors to convert B cells to a reprogrammable state, the researchers chose one that had been used to convert B cells to macrophages. “That worked almost immediately,” recalls Jaenisch.

In addition to addressing the basic science question of lucky cells versus special ones, the work has at least two sets of practical applications. One is to create better ways of studying autoimmune diseases by using reprogrammed mice generated from white blood cells that attack myelin or insulin-producing cells to simulate multiple sclerosis or diabetes pathology.

The other application will be to ease some of the difficulties of reprogramming experiments. The viruses typically used to reprogram cells don’t infect B cells very well, so the researchers created mice that had reprogramming-ready cells — cells that already carried several copies of each of the four genes. These genes could be turned on at will by exposing the cells to the small molecule doxycycline. Under this system, the reprogramming rate is about 1 in 30 cells, says Jaenisch, a surprisingly high rate. Determining reprogramming efficiency rates requires many assumptions, says Jaenisch, because there’s no way to directly measure how many cells are infected with the viruses. He hopes to use the reprogramming-ready cells to compare efficiency rates in other cell types.

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References

1. 1. Hanna, J. et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250–264 (2008). | Article |

2. 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

Cells that regenerate blood increase ten-fold in mutant mice

Haematopoietic stem cells make blood by first generating ‘multipotent progenitors’. Though they can yield all sorts of mature blood cells, the progenitors cannot sustain themselves, and so each progenitor can produce only a finite number of cells. A looming question in stem-cell biology is why haematopoietic stem cells (HSCs) self-renew but their progenitors cannot. Publishing in Nature, Michael Clarke and colleagues at Stanford University have now uncovered some of the molecular mechanisms that limit progenitors’ ability to expand indefinitely.

The researchers created triple mutant mice that lack proteins repressed by Bmi1, a protein necessary to sustain adult HSCs, and found that the frequency of cells able regenerate blood systems increased ten-fold. The ability to artificially create long-lived progenitors also hints at how cells could, naturally, become long-lived malignant cells in blood cancers.

The study focused on three proteins repressed by Bmi1: Trp53, plus p16Ink4a and p19Arf, two proteins produced by alternative reading frames of a single gene also called Cdkn2a. Deleting these loci individually could not completely rescue HSC function in mice that lacked Bmi1. However, when all three were deleted in genetically engineered mice, bone marrow from the triple mutant mice was particularly potent at reconstituting blood systems in normal mice whose natural blood-making capacity had been destroyed. The mice receiving transplants produced all the mature blood cell types, and all appeared functional.

However, when the researchers examined the bone marrow of triple mutant mice, they did not see many more HSCs, and several in vivo and in vitro tests showed that the progenitors formed a population distinct from HSCs.

To understand why these progenitor cells were so potent, the researchers compared triple mutant and wild type progenitor cells in culture. The triple mutant progenitors died less often, with rates of apoptosis were two to three-fold below that of wild type progenitors. They were also better able to form proliferating colonies. However, more-specified proliferating progenitors (myeloid progenitors and granulocyte-marcophage progenitors) were not able to reconstitute blood systems. This finding suggests that self-renewal constraints increase with differentiation, and that several mechanisms regulate expansion capability in vivo. Understanding how such regulation can be dismantled at the multipotent progenitor stage can help explain both how cancers get started and how blood systems sustain themselves healthily.

Akala, O.O. et al. Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/-multipotent progenitors. Nature, doi 10.1038/nature06869 advance online publication April 17 2008.

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