Reprogramming Neurons into New Cells

Researchers from Harvard’s Department of Stem Cell and Regenerative Biology have succeeded in reprogramming one type of neuron into a different type of neurons in a living animals.  Such an experiment has never been done before.  These researchers, Paola Arlotta and Caroline Rouaux said that their work “tells you that maybe the brain is not as immutable as we always thought, because at least during an early window of time one can reprogram the identity of one neuronal class into another”  Arlotta, an associate professor in Harvard’s Department of Stem Cell and Regenerative Biology (SCRB).

Direct lineage reprogramming of differentiated cells within the body was first proven by the SCRB co-chair and Harvard Stem Cell Institute  (HSCI) co-director Doug Melton and colleagues five years ago.  Workers in Melton’s lab succeeded in reprogramming exocrine pancreatic cells directly into insulin-producing beta cells.  Now Arlotta and Rouaux now have shown that neurons can change too.  Their work has been published in the journal Nature Cell Biology

In their experiments, Arlotta and Rouaux targeted a group of neurons known as callosal projection neurons.  Collosal projection neurons connect the two hemispheres of the brain.  After specific treatments, the collosal projections neurons in this study were converted into corticofugal projection neurons.  The significance of corticofugal projection neurons are not lost on Arlotta and Rouaux because they are a type of corticospinal motor neuron, which is one of two populations of neurons destroyed in Amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease.

To achieve such reprogramming of neuronal identity, the researchers inserted a gene for a transcription factor known as Fezf2 into the collosal neurons.  Fexf2 plays a central role in the development of corticospinal neurons in the embryo.  The collosal neurons retracted their connects to the other hemisphere and made connections with neurons in the lower layers of the cerebral cortex.

Luci Bruijn, a neuroscientist who was not directly involved in this work noted, “This discovery tells us again that the brain is a somehow flexible system and gives us more evidence that reprogramming neurons to take on new identities and, perhaps, that new functions are possible. For those working to treat neurodegenerative diseases, that is reassuring.”

This work did not take take place in a culture dish in a laboratory.  Instead it was done in the brains of living mice.  The mice were young, so it is still not certain if such reprogramming could occur in older animals or even humans.  If such reprogramming is possible, the implications for the treatment of neurodegenerative diseases could be enormous.

“Neurodegenerative diseases typically affect a specific population of neurons, leaving many others untouched. For example, in ALS it is corticospinal motor neurons in the brain and motor neurons in the spinal cord, among the many neurons of the nervous system, that selectively die,” Arlotta said. “What if one could take neurons that are spared in a given disease and turn them directly into the neurons that die off? In ALS, if you could generate even a small percentage of corticospinal motor neurons, it would likely be sufficient to recover basic functioning.”

Bruijn said of this work, “Understanding the constraints and possibilities of nervous system development allows us to consider new experiments and new strategies for therapy development. The most immediate importance of this finding is likely to be in the laboratory, where it will help us understand more about how the nervous system may respond when neurons are injured as they are in ALS.”

Human Ovaries Harbor Egg-Making Stem Cell Population

We have read it before, countless times, that women are born with a particular number of eggs and after they die during ovulation or are ovulated, the women is out of eggs and goes through menopause. She does not have the ability to make any more eggs.

Well, another dogma falls by the wayside. As it turns out, egg-making stem cells exist in the woman’s ovaries. An article published in Nature Medicine by Jonathan Tilly and his colleagues who work in a laboratory at Massachusetts General Hospital in Boston, confirms earlier work by Tilly in 2004 that found ovarian stem cells exist in mouse ovaries (Joshua Johnson, et al., Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428, 145-150 (11 March 2004) | doi:10.1038/nature02316), and 2009 publication by a laboratory in Shanghai, China, that confirmed Tilly’s controversial publication (Kang Zou, et al., Production of offspring from a germline stem cell line derived from neonatal ovaries. Nature Cell Biology 11 (2009): 631 – 636).

Despite the rigor of earlier experiments by Tilly’s group and Ji Wu’s, many ovarian experts remained quite skeptical that such a stem cell population existed in humans. This present publications, however, seems to seal the deal. According the Tilly, “This is unequivocal proof that not only was the mouse biology correct, but what we proposed eight years ago was also correct — that there was a human population of stem cells in young adult tissue.” says Tilly.

By listening to their critics, Tilly and is group developed techniques to address the concerns to those skeptical of their findings. They a protocol by which they could isolate and identify mouse ovarian stem cells. This new methods use “fluorescence-activated cell sorting” or FACS. FACS requires the attachment of a fluorescent tag onto the surface of those cells you wish to isolate. Tilly and his group used antibodies linked to a fluorescent dye that could bind tightly to a surface protein called “Ddx4.” Ddx4 is found on the surfaces of ovarian stem cells, but is quickly lost once the stem cells differentiate into egg cells. Treating ovarian cells with the fluorescently-labeled antibodies essentially “painted” them a glowing color. Then the cells were given to a cell sorter than placed cells into one container or another, based on whether or not their surfaces glowed. The cell sorter also distinguishes between whole cells and dead or damaged ones that might fluoresce by accident. In this way, Tilly’s lab invested a protocol for isolating and identifying ovarian stem cells that was highly selective and sensitive.

This new protocol confirmed that Ddx-4-expressing stem cells were present in mouse ovaries. The group did not stop there. They asked if human ovaries had the same stem cell population. They turned to Yasushi Takai, who is a former research fellow in Tilly’s lab, but now works as a reproductive biologist at Saitama Medical University in Japan. Takai provided Tilly with frozen, whole ovaries that had been removed from young women during sexual-reassignment procedures. Tilly says, “It was 9 November when we did the first human FACS sort and I knew immediately that it had worked. I cannot even put into words the excitement — and, to some degree, the relief — I felt.”

From the FACS experiment, Tilly’s group isolated human oogonial stem cells (OSCs). When cultured in the laboratory these OSCs spontaneously produced normal immature oocytes. How did the OSCs do this? To get an inside view of OSC differentiation, Tilly’s team labeled OSCs with green fluorescent protein in order to trace them. Then they injected the green-labeled OSCs into bits of cultured human ovarian tissue, and then transplanted the whole thing under the skin of mice. One to two weeks after transplantation, they found that the OSCs had formed green-glowing cells that greatly resembled ovaries and expressed two ovary-specific genes.

Tilly sounded a cautious note: “There’s no confirmation that we have baby-making eggs yet, but every other indication is that these cells are the real deal — bona fide oocyte precursor cells.” To do this, Tilly must show that the OSC-derived oocytes can be fertilized and form an early embryo. Such work must be done with private funding, since federal funding cannot legally be used for any research that will result in the destruction of a human embryo, regardless of the source of the embryo. Another strategy might be to procure a license from the UK Human Fertilisation and Embryology Authority to do the work with collaborators in the United Kingdom.

Tilly’s experiments have actually converted one scientist who was rather skeptical of his results. Evelyn Telfer, a reproductive biologist at the University of Edinburgh, UK, did not believe Tilly’s initial results. Now, however, she has become a believer. Telfer testified, “I’ve visited [Tilly’s] lab, seen these cells and how they behave. They’re convincing and impressive.” Telfer, has studied in vitro maturation of human eggs, and she wants to work with Tilly to try to grow the OSC-derived eggs to the point at which they are ready for fertilization.

Telfer noted that even though OSCs can form egg-like cells in culture, there is presently no evidence that they can do so in the ovary or that they actually do form new eggs in the ovary. However, the ability to convert OSCs into eggs in vitro might make them usable for in vitro fertilization (IVF), and this achievement would change assisted reproduction forever.

However, Tilly admonishes, “That’s a huge ‘if’.” However, it could means that women who under cancer treatments and experience early menopause could have OSCs removed before treatment and for later fertility use. In fact, according to Tilly, follow-up experiments have shown that OSCs actually exist in the ovaries of women well into their 40s. Even giving women another five years would cover most women affected by IVF.