Harald Ott and his team at Massachusetts General Hospital recently regenerated the limb of a rat in the lab, by flushing the arm of all cells and then regrowing them with cells from a different rat.
The process takes place in a tightly controlled environment with set temperatures, humidity, pH, oxygen levels, and pressure.
The researchers at Massachusetts General have used human cells to regenerate the arm of a macaque monkey. The aim is to one day grow human arms for transplantation.
The main option for amputees today are prosthetic limbs or transplants, where available. The use of transplants requires recipients to take lifelong immunosuppressive drugs which increase the risk of other infections and potential development of cancers.
The process involves removing all cells from the using fluids and detergents. Cells are then regrown and replaced using human progenitor cells, which can develop into blood, nerve or muscle cells.
In the Massachusetts lab, a monkey arm is stripped down as far as its individual cells. All that’s left behind is a bare, frail scaffold.
But that’s not the end of the road for this arm. The scaffold is rebuilt with infusions of cells from another being — be it a monkey, or a human — which grow and transform the limb.
The aim is ultimately to restore the limb to its fully functional form. But this transformed limb will contain the blood, bones, muscles, cartilage — and more — not of the animal it once was, but instead, the animal providing these new cells.
The hope is to eventually use human cells to make limbs that can be transplanted in humans — and the technology is already being trialled in monkeys.
Replacing lost limbs
“There are no good options to replace lost limbs,” says Ott.
In the United States, it is estimated that approximately 185,000 amputations occur each year, and more than 2 million people are currently living with limb loss, according to the Amputee Coalition.
The current options for amputees are a diverse range of prosthetics — incorporating many new forms of technology to help them feel real — or transplants, when matching donors are available. But with these options come limitations in terms of movement and control. In the case of transplants, the limiting factor is the need for life-long immunosuppressive drugs to stop a recipient’s immune system from attacking their new limb. Suppressing immunity in this way opens up the risk of new infections and certain cancers.
Ott’s ambitious technique therefore has an ambitious goal — to one day provide amputees with fully functional limbs that can be transplanted as if they were their own.
The idea is to create limbs made up of cells from the amputee’s own body to produce an arm — or one day leg — tailored to them and therefore unlikely to be attacked by their immune system.
“If it works out you could regenerate … on demand,” says Ott.
Avoiding immune attack
To date, Ott has managed to use this technique to grow organs — including lungs and a beating heart — and in June 2015, successfully extended it further to regenerate the arm of a rat in his lab.
He has now scaled things up — to monkeys.
To do this, Ott is using what is known as progenitor cells, which have the ability to differentiate into a wide range of cell types — such as blood or muscle cells — within the body. They are similar to stem cells but slightly more specialized making them more easily pushed into creating the specific cells desired by the team.
In their trial on monkeys, the team are using the scaffold of a monkey’s arm but with progenitor cells obtained from humans and stimulating them into becoming blood cells and vessels.
“The aim is … the formation of a fully lined vascular system,” says Ott.
Building body parts
Regenerating a limb is far from easy, but the complicated process comes down to some key stages, beginning with finding a limb to regenerate and flushing out all the cells inside with saline and a sequence of detergents to remove the donor’s cells completely. It’s a process known as decellularization and can take up to two weeks to complete.
“This changes the nature of the tissue entirely,” says Ott referring to the scaffold left when all possible cells have been stripped.
Next comes the hardest part — to repopulate the limb with progenitor cells from another individual and guide them to generate specific cells such as blood vessels, or muscle — known as recellularization.
All stages need to happen inside a tightly controlled environment with set temperatures, humidity, pH, oxygen levels, and pressure. The regrowth of the arm takes place inside a bioreactor providing nutrients and stimulation for the limb to reform.
After working with organs, Ott is preparing to jump the hurdles ahead of him when creating limbs.
“The challenge is their composite nature … limbs contain muscles, bone, cartilage, blood vessels, tendons, ligaments and nerves — each of which has to be rebuilt and require a specific supporting structure called the matrix,” he says.
After a start with blood cells and vessels, next comes muscle and then connective tissue, bone, cartilage, fat cells and more, with the final challenge of nerve cells to make a working arm.
“What’s important is to eventually let that limb become functional again,” says Ott.
Ott’s success with a rat’s arm was the first bio-limb to ever be created. After weeks of tending to this miniscule limb, the result was an arm capable of producing blood and muscle cells and with the ability to contract when electrically stimulated in the lab.
The next challenge is to create nerve cells and enable them to integrate and work once attached to the muscle of the animal receiving the new limb. “The nerves need to not only grow, but re-connect to the muscle,” says Ott.
But the success of human hand transplants in achieving this connection gives Ott some hope. “We’ve learned from the transplant community,” he says.
“[We need] to show we can apply this process to limbs of human scale,” says Ott. In his current work with Macaque monkeys his team has so far been able to grow the cells of human vascular tissue and its lining.
And in terms of time, going bigger doesn’t necessarily mean taking longer. “It’s a larger field that you plant with more seeds,” says Ott. The formation of a new human limb should, in theory, take the same amount of time as the rat.
But there’s a long road ahead
“It’s a very good first step,” says Maximina Yun, a regenerative biology researcher at University College London. But she adds: “there are still a few challenges to overcome.”
Yun’s work on limb regeneration has focused on the biology of salamanders, which regrow their own limbs readily when needed. “They can regrow straight after amputation,” she says.
Through her expertise on the biology of salamanders, Yun hopes to one day apply this knowledge to humans. She stresses the challenges ahead when growing limbs for humans in terms of growing nerves in muscles, maintaining their movement and preventing immune rejection.
“We need a limb that doesn’t pose any risk to humans,” she says. Ott agrees and is hopeful he will be working in science long enough to see his work come to fruition.
“I will live to see the clinical application of this,” he says.
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