Scientists successfully expand bone marrow-derived stem cells in culture

Scientists successfully expand bone marrow-derived stem cells in culture

  8:13:00 PM    Science Lover

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All stem cells-regardless of their source-share the remarkable capability to replenish themselves by undergoing self-renewal. Yet, so far, efforts to grow and expand scarce hematopoietic (or blood-forming) stem cells in culture for therapeutic applications have been met with limited success.

An image of fully functional hematopoietic
stem cells (or blood-forming) that are successfully

proliferating amongst other bone marrow-derived

cells in a culture dish. Credit: Dr. John Perry,
Stowers Institute forMedical Research

Now, researchers at the Stowers Institute for Medical Research teased apart the molecular mechanisms enabling stem cell renewal in hematopoietic stem cells isolated from mice and successfully applied their insight to expand cultured hematopoietic stem cells a hundredfold.

Their findings, which will be published in the Sept. 15, 2011, edition of Genes & Development, demonstrate that self-renewal requires three complementary events: proliferation, active suppression of differentiation and programmed cell death during proliferation.

"The previous efforts so far to grow and expand scarce hematopoietic stem cells in culture for therapeutic applications have been met with limited success", says Stowers investigator Linheng Li, Ph.D., who led the study. "Being able to tap into stem cell's inherent potential for self-renewal could turn limited sources of hematopoietic stem cells such as umbilical cord blood into more widely available resources for hematopoietic stem cells," he adds while cautioning that their findings have yet to be replicated in human cells.

The transplantation of human hematopoietic stem cells isolated from bone marrow is used in the treatment of anemia, immune deficiencies and other diseases, including cancer. However, since bone marrow transplants require a suitable donor-recipient tissue match, the number of potential donors is limited.

Hematopoietic stem cells isolated from umbilical cord blood could be a good alternative source: Readily available and immunologically immature, they allow the donor-recipient match to be less than perfect without the risk of immune rejection of the transplant. Unfortunately, their therapeutic use is limited since umbilical cord blood contains only a small number of stem cells.

Although self-renewal is typically considered a single trait of stem cells, Li and his team wondered whether it could be pulled apart into three distinct requirements: proliferation, maintenance of the undifferentiated state, and the suppression of programmed cell death or apoptosis. "The default state of stem cells is to differentiate into a specialized cell types," explains postdoctoral researcher and first author John Perry, Ph.D. "Differentiation must be blocked in order for stem cells to undergo self-renewal."




Proliferation of stem cells in an undifferentiated state, however, calls tumor suppressor genes into action. These genes help prevent cancer by inducing a process of cell death known as apoptosis. "Consequently, self-renewal of adult stem cells must also include a third event, the active suppression of apoptosis," says Perry.

To test their hypothesis, Perry and his colleagues isolated hematopoietic stem cells from mice and analyzed two key genetic pathways—the Wnt/β-catenin and PI3K/Akt pathways. Wnt proteins had been identified as "self-renewal factors," while PI3K/Akt activation had been shown to induce proliferation and promote survival by inhibiting apoptosis.

Surprisingly, activation of the Wnt/β-catenin pathway alone blocked differentiation but eventually resulted in cell death, while activation of the PI3K/Akt pathway alone increased differentiation but facilitated cell survival. Only when both pathways were activated, did the pool of hematopoietic stem cells start expanding. "This demonstrated both pathways had to cooperate to promote self-renewal," says Perry.

Although altering both pathways drives self-renewal of hematopoietic stem cells, it also permanently blocks their ability to mature into fully functional blood cells. To sidestep the differentiation block and generate normal, functioning hematopoietic stem cells usable for therapy, the Stowers scientists used small molecules to reversibly activate both the Wnt/β-catenin and PI3K/Akt pathways in culture.

"We were able to expand the most primitive hematopoietic stem cells, which, when transplanted back into mice gave rise to all blood cell types throughout three, sequential transplantation experiments," says Li. "If similar results can be achieved using human hematopoietic stem cells from sources such as umbilical cord blood, this work is expected to have substantial clinical impact."

Provided by Stowers Institute for Medical Research

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Nerves, Muscle Regenerated With Sodium Ions

Nerves, Muscle Regenerated With Sodium Ions

  12:35:00 AM    Science Lover

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Sodium gets a bad rap for contributing to hypertension and cardiovascular disease. Now biologists at Tufts University's School of Arts and Sciences have discovered that sodium also plays a key role in initiating a regenerative response after severe injury. The Tufts scientists have found a way to regenerate injured spinal cord and muscle by using small molecule drugs to trigger an influx of sodium ions into injured cells.

Tufts biologists have regenerated spinal cord and 
muscle by using a "pharmaceutical cocktail" to 
trigger an influx of sodium ions into injured cells. The 
treatment method is most directly applicable to spinal
cord repair and limb loss, which are significant problems
worldwide, but the proof-of-principle may apply to many 
complex organs and tissues. The Tufts team found that a 
localized increase in sodium ions was necessary for young 
tadpoles to regenerate their tails -- complex appendages
containing spinal cord, muscle and other tissue. Like 
human beings, who regenerate fingertips only as children, 
tadpoles lose the ability to regenerate their tail with 
age. The Tufts biologists showed that such "refractory" 
tadpoles whose tails had been removed could be induced 
to make a perfect new tail by only an hour of treatment. 
The tadpole on the left, which received the "cocktail" to 
trigger an influx of sodium ions, grew a perfectly formed tail. 
The control tadpole on the right did not regenerate. This 
approach breaks new ground in biomedicine because it 
requires no gene therapy; can be effectively administered
for some time after an injury has occurred; and is bioelectric, 
rather than chemically based. (Credit: Ai-Sun Tseng and 
Michael Levin-Tufts University)

The approach breaks new ground in the field of biomedicine because it requires no gene therapy; can be administered after an injury has occurred and even after the wound has healed over; and is bioelectric, rather than chemically based.

In a paper appearing as the cover story of the September 29, 2010, issue of the Journal of Neuroscience, the Tufts team reported that a localized increase in sodium ions was necessary for young Xenopus laevis tadpoles to regenerate their tails - complex appendages containing spinal cord, muscle and other tissue.

Like human beings, who regenerate fingertips only as children, these tadpoles lose the ability to regenerate their tail with age. Most remarkably, it was shown that such "refractory" tadpoles whose tails had been removed could be induced to make a perfect new tail by only an hour of treatment with a specific drug cocktail.

The findings have tremendous implications for treating wounds sustained in war as well as accidental injuries. The treatment method used is most directly applicable to spinal cord repair and limb loss, which are highly significant medical problems world-wide. It also demonstrates a proof-of-principle that may be applicable to many complex organs and tissues.

"We have significantly extended the effective treatment window, demonstrating that even after scar-like wound covering begins to form, control of physiological signals can still induce regeneration. Artificially causing an influx of sodium for just one hour can overcome a variety of problems, such as the decline in regenerative ability that comes with age and the effect of regeneration-blocking drugs," said Tufts Professor of Biology Michael Levin, Ph.D., corresponding author on the paper and director of the Center for Regenerative and Developmental Biology at Tufts. Co-authors were Research Associate Ai-Sun Tseng, Postdoctoral Associate Wendy S. Beane, Research Associate Joan M. Lemire, and Alessio Masi, a former post-doctoral associate in Levin's laboratory.

The transport of ions in and out of cells is regulated by electronic security doors, or gates, that let in specific ions under certain circumstances. A role for sodium current in tissue regeneration had been proposed in the past, but this is the first time the molecular-genetic basis of the ion flow has been identified, and a specific drug-based treatment demonstrated. Until now, advances in this model system had involved administering therapies before the injury was sustained.

"This is a novel, biomedically-relevant approach to inducing regeneration of a complex appendage," noted Levin.

The Tufts research established a novel role in regeneration for the sodium channel Nav1.2, a crucial component of nerve and cardiac function. It showed that local, early increase in intracellular sodium is required for initiating regeneration following Xenopus tail amputation, while molecular and pharmacological inhibition of sodium transport causes regenerative failure. The new treatment induced regeneration only of correctly-sized and patterned tail structures and did not generate ectopic or other abnormal growth.

"The ability to restore regeneration using a temporally-controllable pharmacological approach not requiring gene therapy is extremely exciting," said the researchers.

Of critical importance, they said, was the discovery that the tail could be induced to regenerate as late as 18 hours after amputation, revealing that tissues normally fated for regenerative failure still maintain their intrinsic characteristics and can be programmed to reactivate regeneration.

Amphibians such as frogs can restore organs lost during development, including the lens and tail. The frog tail is a good model for human regeneration because it repairs injury in the same way that people do: each tissue makes more of itself. (In contrast, regeneration in some other animals occurs through transdifferentiation (one cell type turns into another cell type) or adult stem cell differentiation. Furthermore, though small, the Xenopus larval tail is complex, with muscle, spinal cord, peripheral nerves and vasculature cells.

The National Institutes of Health, National Highway Traffic Safety Administration, Department of Defense and Defense Advanced Research Projects Agency funded the work.

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