Wired.com, August 5, 2010  —  By temporarily turning off a pair of genes identified in research on limb-regrowing newts, researchers turned back the biological clock on mouse muscle cells, allowing them to divide anew and form fresh tissue.

“It’s regeneration a la newt,” said Stanford University cell biologist Helen Blau, who performed the feat in an April 5 Cell Stem Cell study.

In most animals, including ourselves, cells stop dividing when they’ve attained their mature, tissue-specific form. Chop off a limb or carve up an organ, and it doesn’t grow back. A few creatures, however, including newts and axolotl salamanders, break those rules. They can regrow new limbs, even organs.

That’s made them the focus of regenerative medicine researchers, who until recently suspected that regenerators had an extra-large supply of stem cells. In biology’s version of alchemy, these cells can take multiple forms — or, in the case of embryonic stem cells, any form.

But newts and salamanders don’t rely on stem cells, at least not exclusively. Instead, their standard, adult-issue tissue cells, supposedly incapable of dividing again, revert to a slightly more immature form, and start dividing again.

In the case of new muscle, “they still know they’re muscle cells. They retain their identity. They make more copies of themselves, and then specialize again,” said Blau. “It’s an alternative to stem cells.”

Blau’s team experimented with two genes: A tumor suppressor called Rb that was flagged in earlier regeneration studies, and another tumor suppressor called Ink4A that they found in the genomes of higher-order vertebrates, but not in newts and salamanders.

Some researchers have been able to make cells regenerate after turning off Rb, but others have failed. Blau’s team wondered if both Rb and Ink4A needed to be tweaked. They speculated that Ink4A’s protection against cancer came at the expense of regeneration.

In laboratory cultures of muscle cells, they shut down both genes. The cells divided, and kept on dividing. They grew out of control, as in cancer. The researchers tried again, but used a short-lived protein switch to inactivate the genes. That did the trick: The cells divided until the protein wore off, then stopped.

When put into damaged muscles in a mouse, the engineered cells “made really nice muscle fibers,” said Blau.

The findings echo research from the lab of Wistar Institute cell biologist Ellen Heber-Katz, who in March described how turning off the p19 gene, which is regulated by Ink4A, led to limited regeneration in mouse ears.

Katz cautioned that much remains to be determined about regeneration, and that it likely requires a complex, varying and as-yet-unknown mixture of cell types and stages. “There are enormous numbers of possibilities,” she said.

Blau echoed her restraint. But even if full-blown regeneration is unrealistic, a temporary burst of cell regrowth would still be beneficial, she said.

“Doing what newts do, making a hand or an arm, is incredibly complex. But I foresee this being useful in a different way. You could put cells in a culture dish, allow them to make more copies of themselves, then put them back in the tissue,” she said. “Alternatively, You could get the cells to make copies of themselves in the tissue itself, ie. get localized growth and repair. I’m thinking of the heart after a heart attack, or the pancreas in early diabetes.”

Blau plans to study other, non-muscle cell types, including nerve cells.

“This might serve as a really interesting alternative to stem cells,” she said.

Image: In the left column, a cross-section of torn mouse muscle tissue, without additional imaging; in the middle and right columns, with different types of fluorescent imaging. In the top row, the Ink4a and Rb genes have been permanently inactivated in injected cells; as indicated by the scattered green dots of their nuclei, these are not gathering to form new muscle fibers. In the bottom row, the genes were only only temporarily turned off in injected cells; these are clustering and forming new fibers, so the green dots are larger./Cell Stem Cell.

Read More http://www.wired.com/wiredscience/2010/08/mouse-regeneration/#ixzz0vke8lLAy

By Bryan Nelson, Mother Nature Network

The turritopsis nutricula species of jellyfish may be the only animal in the world to have truly discovered the fountain of youth.

Since it is capable of cycling from a mature adult stage to an immature polyp stage and back again, there may be no natural limit to its life span. Scientists say the hydrozoan jellyfish is the only known animal that can repeatedly turn back the hands of time and revert to its polyp state (its first stage of life).

The key lies in a process called transdifferentiation, where one type of cell is transformed into another type of cell. Some animals can undergo limited transdifferentiation and regenerate organs, such as salamanders, which can regrow limbs. Turritopsi nutricula, on the other hand, can regenerate its entire body over and over again. Researchers are studying the jellyfish to discover how it is able to reverse its aging process.

Because they are able to bypass death, the number of individuals is spiking. They’re now found in oceans around the globe rather than just in their native Caribbean waters.  “We are looking at a worldwide silent invasion,” says Dr. Maria Miglietta of the Smithsonian Tropical Marine Institute.

Bryan Nelson is a regular contributor to Mother Nature Network, where a version of this post originally appeared.

 


Jellyfish Turritopsis Can Change the Direction of Its Life Cycle


July/August 2010

Biologist Teisha Rowland Adds more to the research on Turritopsis

To be forever young. That has been the elusive dream for people since the dawn of humanity, sparking such myths as the “Fountain of Youth.” Yet for one tiny jellyfish, that dream is merely the way of life.

Completely grown-up, adult jellyfish belonging to the genus Turritopsis possess an amazing ability: They can reverse their life cycle and turn back into their “newborn” form. This genus belongs to a group called the hydrozoans, which includes some small, predatory jellyfish, hydra, and the Portuguese Man o’ War, among others.

While most hydrozoans die after reproducing, Turritopsis has other plans. In fact, they’re the only animals in the world that have been found to be able to reverse their life cycle like this.

To truly appreciate this remarkable feat, it’s important to understand the basic, “normal” life cycle of the Turritopsis jellyfish. Turritopsis’s life cycle is made up of two basic stages: an immature polyp and a sexually mature medusa (also commonly referred to as the jellyfish). When the Turritopsis jellyfish create a fertilized egg, it settles to the ocean floor, in shallow or deep waters. The eggs can hatch within a few days. A single egg turns into a polyp, a cup-shaped, sessile (rooted in place), immature form that has tentacles on its raised mouth to help it catch food, and a large central cavity to then digest its food. Many polyps cluster together to form interconnected polyp colonies.

So how do the polyps turn into the adult medusae? Fascinatingly, individual polyps actually form a bud that breaks off and turns into an adult medusa. This is done asexually, without any of the polyps exchanging genetic material. The medusa looks like the classic jellyfish form we’re so familiar with: solitary, somewhat umbrella-shaped, and adorned with many tentacles.

That’s the normal, tried-and-true life cycle for many jellyfish, but Turritopsis nutricula and a related species, Turritopsis dohrnii, can do this and much more.

T. nutricula is quite small, about only one-fifth of an inch in diameter, with transparent walls and a vibrantly red-colored large stomach. It can have an astonishing number of tentacles, around 80 to 90.

But its appearance isn’t what’s gained it so much attention; it’s what it does. Under certain conditions, T. nutricula can successful transform to its juvenile stage 100 percent of the times tested in laboratories.

What does this mean in terms of the seemingly “bizarre” jellyfish lifestyle? The sexually mature, adult medusa can actually transform into a ball of tissue, like a cyst. It does this by somehow reabsorbing the external parts of its body. The cyst attaches to the ocean floor and, within a few days, depending on the temperature, grows into a colony of immature cylindrical polyps, and the cycle starts all over again; new medusae bud off of the polyps, forming genetically identical jellyfish.

The cysts can even wait for months before turning into polyps, if temperatures are very cool. Consequently, they’re virtually immortal. But this reversion doesn’t happen all the time: It only happens when the jellyfish are starved, physically injured, or under other stresses, and most likely many jellyfish die before undergoing this transformation.

So how exactly does the Turritopsis get rid of its wrinkles and gray hairs? It’s still a big mystery, but current thought suggests that the process of transdifferentiation is involved. Transdifferentiation is when an adult, mature cell turns into a completely different kind of cell. In the Turritopsis nutricula medusa, it’s thought, certain adult tissue layers transform into different, specific tissue layers found in the new cyst. While this is somewhat understood on a general tissue level, on the cellular level there is much that remains to be elucidated. Additionally, there is some debate over whether stem cells are necessary for the process of transdifferentiation, and how it works in Turritopsis is even more unclear.

Transdifferentiation occurs in other animals as well, though usually only in regrowing parts of the animal; Turritopsis is probably the only known case of an entire organism undergoing transdifferentiation (though there are other contenders). A long-standing classic example of transdifferentiation is the salamander: Cut off a leg and it regrows a new one, presumably from the mature cells on the leg stump. However, this was recently challenged by research done by Elly Tanaka (at the Max Planck Institute of Molecular Cell Biology and Genetics and the Center for Regenerative Therapies at the University of Technology, both in Dresden, Germany); it now appears that salamanders use several different kinds of limited stem cells (not mature cells) to regenerate their severed limbs. Clearly this is a rapidly evolving field as our understanding of these amazing abilities grows.

As we learn more about how animals such as Turritopsis can revert all of their cells to a much earlier stage in life, it may help us better understand human diseases and just the basic definition of what it is to be a cell. Transdifferentiation occurs in humans, but mostly in diseases such as cancer. But this is just one of the many hints, including the existence of Turritopsis, that the cells in our adult bodies are not necessarily fixed in their ways. Given the right conditions, they can change their very identities.

And the tiny Turritopsis is greatly profiting from its innate knowledge. Turritopsis have actually been leading a “silent invasion” of the world’s oceans. Research done by Dr. Maria Pia Miglietta (Pennsylvania State University) revealed that, just in recent times, the animals’ numbers have been significantly increasing. They have also been rapidly spreading around the world, most likely from water released from the ballasts of ships in ports. While they may only be one-fifth of an inch in diameter and lack a central nervous system, these jellyfish sure seem to have a lot of things figured out.

For more on transdifferentiation and Turritopsis’s ability to change from an adult to a juvenile, see Discover magazine’s blog post “The Curious Case of the Immortal Jellyfish,” Stefano Piraino’s article on “Reversing the Life Cycle: Medusae Transforming into Polyps…,” Maria Pia Miglietta’s article on “A Silent Invasion,” Shifaan Thowfeequ’s article on “Transdifferentiation in Developmental Biology, Disease, and in Therapy,” Hongbao Ma’s article on “Turritopsis nutricula,” and Wikipedia’s article on “Turritopsis nutricula,”

Biology Bytes author Teisha Rowland is a science writer, blogger at All Things Stem Cell, and graduate student in molecular, cellular, and developmental biology at UCSB, where she studies stem cells. Send any ideas for future columns to her at science@independent.com.

More by biologist, Teisha Rowland

In vitro growth of human embryonic stem cells. In this image, the red lines are microtubules of the cytoskeleton. The blue circles are the cell nucleus, with Oct4 shown in green. Oct4 is a stem cell marker because only human embryonic stem cells have this transcription factor, which binds to specific genes and upregulates them. This transcription factor seems to control the genes that are required to keep a stem cell reproducing, rather than differentiating into different kinds of cells. Feeder-free conditions used in the HSCCF ensure that these images show hESCs uncontaminated by mouse material. Photo: Samantha Zeitlin, Ph.D., University of California, San Diego.

  

 

 

AllThingsStemCell.com, by Teisha Rowland  —  A goal of regenerative medicine has been to be able to take any cell from a person’s body and turn it in to any other cell type that may be desired (such as insulin-producing beta-cells for treating diabetes, or creating neurons to treat a neurodegenerative disease). This would eliminate several donor-compatibility problems, and potentially eliminate the need for a donor (who isn’t the patient) altogether. In 2007, human induced pluripotent stem cells (iPSCs) were created and this goal seemed a bit closer (Yu et al., 2007; Takahashi et al., 2007). iPSCs are cells that can be take from adult tissue and “reprogrammed” into embryonic stem cell (ESC)-like cells. Because iPSCs are pluripotent, these cells can then differentiate into (or become) any cell type (for more information, see the All Things Stem Cell article on “Induced Pluripotent Stem Cells: A New Stem Cell Line with a Long History”).

But is it possible to get rid of the iPSC-middle man? Is it possible to take any cell in the adult body and directly reprogram it, skipping the iPSC state, into the final desired cell type? There have been several studies over the last few decades that show this is quite possible, though it still has a ways to go before it can be regularly used in the clinic.

Reprogramming of cells to a different cell type is usually done by either somatic cell nuclear transfer (SCNT) or by using transcription factors. This post will focus on work done with transcription factors (for more information on using SCNT, see the “Induced Pluripotent Stem Cells…” post). Transcription factors are expressed (or made) at different levels in different cell types, and control what genes are expressed in every cell, making sure, for example, that a liver cell remains a liver cell and does not become a neuron. A famous example of how transcription factor expression can be used to alter a cell’s identity is the creation of iPSCs, where adult cells were forced to express transcription factors normally expressed in ESCs, which made the adult cells express genes specific to ESCs, and consequently become nearly identical to ESCs.

There are many degrees of direct reprogramming that have been reported over the last few decades. Several progenitor cells, cells that appear to be committed to their fate but not yet fully differentiated, have been shown to be capable of dedifferentiating into a different cell type; this process is called transdetermination. However, in a few cases it has been shown that a fully differentiated cell can actually become a different cell type; this process is called transdifferentiation (Graf and Enver, 2009). Over the last few decades, much progress has been made in direct reprogramming with muscle, blood, the pancreas, and neurons.

Muscle

In the 1980s, the first reprogramming experiments using transcription factors took place. In 1987, a group reported using MyoD to make fibroblasts become muscle cells (Davis et al., 1987). Fibroblasts are cells important for wound healing (they secrete essential extracellular matrix proteins) and are common in connective tissues. The specific fibroblasts used were embryonic mouse fibroblasts. Because they were embryonic, this process is called transdetermination; the embryonic fibroblasts could probably differentiate more easily than adult fibroblasts (Graf and Enver, 2009). To convert the fibroblasts into muscle cells, the researchers transfected the fibroblasts with the cDNA of MyoD, forcing the cells to express MyoD (Davis et al., 1987). MyoD is normally only expressed in skeletal muscle, and it was later found to be a transcription factor involved in the differentiation of muscle cells and also a very early marker of muscle cell fate commitment.

Because of its success with the fibroblasts, MyoD was subsequently used in many other reprogramming studies to see what other cells it could make into muscle. It was found that while MyoD could indeed convert many different cell types into muscle, including fibroblasts in the dermal layer of skin, immature chondrocytes (cells in cartilage), smooth muscle, and retinal cells (Choi et al., 1990), MyoD could not turn any cell type into muscle; it was found incapable of making muscle out of hepatocytes (cells in the liver) (Schäfer et al., 1990).

Blood

In the 1990s, another key direct reprogramming factor was discovered, specifically involved in hematopoiesis. Hematopoiesis is the process by which the different types of blood cells are generated in the body (the term literally means “to make blood”). (For information on hematopoietic stem cells, see the All Things Stem Cell article “Hematopoietic Stem Cells: A Long History in Brief”). The central hematopoiesis-regulating factor discovered was the transcription factor GATA-1.

In 1995, a group reported that when GATA-1 was added to or removed from avian monocyte precursors, it could turn them into erythrocytes, megakaryocytes, and eosinophils (Kulessa et al., 1995). To understand the significance of these findings an inspection of hematopoiesis is required (see Figure). During hematopoiesis, hematopoietic stem cells (HSCs) (also called hemocytoblasts) give rise to all the different types of blood cells. Specifically, HSCs can first differentiate into either a common myeloid progenitor cell or a common lymphoid progenitor cell; either progenitor then further differentiates into specific blood cell types.

Direct Reprogramming in the Hematopoietic System. Several different transcription factors have been found that can directly reprogram one type of blood cell into another. Changing the expression levels of GATA-1 in monocytes (red) can make them differentiate into eosinophils, erythrocytes, or megakaryocytes. Making B-cells (B lymphocytes) express C/EBP transcription factors (blue) can cause them to differentiate into macrophages. Lastly, C/EBPs can also inhibit the function of the transcription factor Pax5; when Pax5 is deleted in B-cells they differentiate into T-cells (T lymphocytes), though they first dedifferentiate into a common lymphoid progenitor.

The common myeloid progenitors can directly become megakaryocytes or erythrocytes. (Megakaryocytes reside in the bone marrow and generate platelets (thrombocytes), which are necessary for blood clotting. Erythrocytes, or red blood cells, are the most common blood cell and deliver oxygen to the body through the blood system.) Common myeloid progenitors can also become monocytes and eosinophils, but to do this it must first become a myeloblast. (Monocytes are white blood cells that create macrophages, while eosinophils are white blood cells that combat infections.)

With this understanding of hematopoiesis, the importance of the 1995 report (Kulessa et al., 1995) becomes clearer. Their findings showed that when high levels of GATA-1 were expressed in monocyte precursors (cells that have not yet fully differentiated into monocytes), these cells could dedifferentiate into cells that occurred an earlier point in hematopoiesis differentiation, the erythrocytes and megakaryocytes. This makes sense with GATA-1’s normal role in hematopoiesis; GATA-1 is an important transcription factor for erythrocyte and megakaryocyte differentiation. GATA-1 is expressed in hematopoietic progenitors, but becomes downregulated in monocytes during differentiation. Interestingly, when lower levels of GATA-1 were expressed, the monocytes became eosinophils; these lower levels are normally present in eosinophils (Kulessa et al., 1995).

While all of the cells in this study were descendants of common myeloid progenitors, it was shown in 2004 that descendants of the other hematopoietic branch, those derived from common lymphoid progenitors, could also be coaxed into becoming a descendant of common myeloid progenitors (Xie et al., 2004). Common lymphoid progenitors can normally become B-cells, also called B lymphocytes (white blood cells that make antibodies against invaders). In 2004, it was reported that B-cells could be reprogrammed into macrophages by making the B-cells express C/EBP transcription factors (C/EBP stands for CCAAT-enhancer-binding proteins). C/EBPs are necessary for cells to normally differentiate from monocytes into macrophages. Interestingly, B-cell progenitors much more efficiently became macrophages than fully differentiated B-cells, again emphasizing the key role that the differentiation state plays in the ability to reprogram a cell. This 2004 report was also significant in that it was the first report showing that fully differentiated cells could be reprogrammed using transcription factors; the first report of transdifferentiation.

While C/EBPs work to enforce a macrophage fate, they also actively work to prevent the B-cell fate. C/EBPs inhibit Pax5 (paired box gene 5), which is a transcription factor that reinforces the B-cell’s commitment (Nutt et al., 1999; Xie et al., 2004). The function of Pax5 has been investigated through ablation studies; when the Pax5 gene is deleted, B-cells become dedifferentiated, turning into common lymphoid progenitor-like cells, which can then be differentiated into T-cells (lymphocytes) (Cobaleda et al., 2007). However, this is not quite direct reprogramming, as it requires the lymphoid progenitor cell state. (T-cells can also be reprogrammed using C/EBPs; its expression can induce T-cells to undergo macrophage differentiation (Laiosa et al., 2006).)

Most recently, reprogramming in the hematopoietic system also taught researchers an important reprogramming lesson: the order in which the cells are exposed to the transcription factors affects reprogramming, probably in a way similar to in vivo (Graf and Enver, 2009).

Pancreas

While the hematopoietic system appears to have some rather flexible cells differentiation-wise, it was some time before such reprogramming abilities were proven in other cellular systems. In 2008, the ability to reprogram one type of pancreatic cell, exocrine cells, into a functionally different type, beta-cells, was reported (Zhou et al., 2008). Exocrine cells are highly specialized pancreatic cells which produce digestive enzymes for the small intestine. Beta-cells (http://en.wikipedia.org/wiki/Beta_cell) reside in the islets of Langerhans, inside the pancreas, where they produce insulin, a hormone that regulates blood glucose levels. Insulin stimulates multiple organs to take glucose in their cells from the blood stream. Diabetes can develop due to high blood glucose levels, caused by the body not producing enough insulin or not responding to insulin it produces. Because diabetes can be caused by a lack of insulin production, the ability to create beta-cells is quite appealing.

From the start, the group set out to find the key transcription factors that could reprogram exocrine cells into beta-cells. They screened over 1,100 transcription factors and found around 20 were only expressed in mature beta cells, and 9 of these caused an abnormal developmental phenotype when mutated, indicating their functional importance in the development of the pancreas. These 9 were used for the initial reprogramming screens in mice, using adenoviral vectors to infect only the pancreatic exocrine cells. The studies were done in mice, and not in culture, to let the natural environment aid in survival and maturation of the cells and allow for direct comparisons of the reprogrammed cells to the native beta-cells. Ultimately, the combination of transcription factors that worked best was Ngn3 (Neurogenin3), Pdx1, and Mafa. Expressing these factors resulted in exocrine cells becoming beta-like-cells that had the same size, shape, structure, and protein expression as native beta-cells, and could also produce insulin (Zhou et al., 2008). However, while exocrine cells and beta cells are functionally quite different, both are derived from the pancreatic endoderm; it still remained to be seen whether more developmentally removed cells could be reprogrammed into each other.

Fibroblasts and Neurons

The most recent breakthrough on direct reprogramming of cells reported the ability to convert fibroblasts into neurons (Vierbuchen et al., 2010). Specifically, the researchers used mouse embryonic fibroblasts and postnatal fibroblasts and, using three transcription factors known to be important in specifying the neural-lineage fates, made the cells into functional neurons in vitro. The researchers first tested 19 candidate transcription factors, chosen for their expression in neural cells or their ability to reprogram cells to pluripotency. Infecting the fibroblasts using lentiviral vectors, the researchers screened for the ability of the candidates to induce a neuronal phenotype, and indeed found some that became neuronal-like. The researchers narrowed down the candidates to a smaller group to see what was necessary for the neuronal-like phenotype, and discovered three transcription factors to be key: Ascl1, Brn2, and Myt1l. While Ascl1 alone could induce immature neuronal features, the other two were required for mature neuron-like cells. The resultant neurons expressed neuron-specific proteins and functioned like neurons (they could generate action potentials and form functional synapses).

Future Steps

While direct reprogramming of adult cells into other cell types is clearly possible, the process by which it happens remains largely not understood. Much research needs to be done to understand the vital molecular mechanisms at play, as well as what occurs at the cellular level. Specifically, it is unclear whether, during direct reprogramming experiments, a cell turns into a progenitor briefly and then differentiates into the final cell type, or the cell actually differentiates directly to the final cell type (Graf and Enver, 2009).

Direct reprogramming efforts in the future may incorporate many factors in addition to transcription factors to be most effective. Studies are already testing the effects of altering expression of microRNAs and factors involved in chromatin remodeling, along with effective chemicals, on cell identity and differentiation; in the future, these approaches will most likely be used along with changing the expression of key transcription factors to find the most effective combinations (Graf and Enver, 2009). Additionally, many studies to date have been in mice and mouse cells; these must be repeated with human cells before they can be used clinically in humans. The resultant cells will be important not only for creating patient-specific cells for cellular therapies and regenerative medicine, but also for studying cell differentiation, plasticity during development, and cell identity problems that occur during diseases such as cancer.