In experiments in mice, Johns Hopkins Medicine researchers say they have developed a way to successfully transplant certain protective brain cells without the need for lifelong anti-rejection drugs.
A report on the research, published Sept. 16 in the journal Brain, details the new approach, which selectively circumvents the immune response against foreign cells, allowing transplanted cells to survive, thrive and protect brain tissue long after stopping immune-suppressing drugs.
The ability to successfully transplant healthy cells into the brain without the need for conventional anti-rejection drugs could advance the search for therapies that help children born with a rare but devastating class of genetic diseases in which myelin, the protective coating around neurons that helps them send messages, does not form normally.
Approximately 1 of every 100,000 children born in the U.S. will have one of these diseases, such as Pelizaeus-Merzbacher disease.
This disorder is characterized by infants missing developmental milestones such as sitting and walking, having involuntary muscle spasms, and potentially experiencing partial paralysis of the arms and legs, all caused by a genetic mutation in the genes that form myelin.
“Because these conditions are initiated by a mutation causing dysfunction in one type of cell, they present a good target for cell therapies, which involve transplanting healthy cells or cells engineered to not have a condition to take over for the diseased, damaged or missing cells,” says Piotr Walczak, M.D., Ph.D., associate professor of radiology and radiological science at the Johns Hopkins University School of Medicine.
A major obstacle to our ability to replace these defective cells is the mammalian immune system.
The immune system works by rapidly identifying ‘self’ or ‘nonself’ tissues, and mounting attacks to destroy nonself or “foreign” invaders.
While beneficial when targeting bacteria or viruses, it is a major hurdle for transplanted organs, tissue or cells, which are also flagged for destruction.
Traditional anti-rejection drugs that broadly and unspecifically tamp down the immune system altogether frequently work to fend off tissue rejection, but leave patients vulnerable to infection and other side effects. Patients need to remain on these drugs indefinitely.
In a bid to stop the immune response without the side effects, the Johns Hopkins Medicine team sought ways to manipulate T cells, the system’s elite infection-fighting force that attacks foreign invaders.
Specifically, Walczak and his team focused on the series of so-called “costimulatory signals” that T cells must encounter in order to begin an attack.
“These signals are in place to help ensure these immune system cells do not go rogue, attacking the body’s own healthy tissues,” says Gerald Brandacher, M.D., professor of plastic and reconstructive surgery and scientific director of the Vascularized Composite Allotransplantation Research Laboratory at the Johns Hopkins University School of Medicine and co-author of this study.
The idea, he says, was to exploit the natural tendencies of these costimulatory signals as a means of training the immune system to eventually accept transplanted cells as “self” permanently.
To do that, the investigators used two antibodies, CTLA4-Ig and anti-CD154, which keep T cells from beginning an attack when encountering foreign particles by binding to the T cell surface, essentially blocking the ‘go’ signal.
This combination has previously been used successfully to block rejection of solid organ transplants in animals, but had not yet been tested for cell transplants to repair myelin in the brain, says Walczak.
In a key set of experiments, Walczak and his team injected mouse brains with the protective glial cells that produce the myelin sheath that surrounds neurons.
These specific cells were genetically engineered to glow so the researchers could keep tabs on them.
The researchers then transplanted the glial cells into three types of mice: mice genetically engineered to not form the glial cells that create the myelin sheath, normal mice and mice bred to be unable to mount an immune response.
Then the researchers used the antibodies to block an immune response, stopping treatment after six days.
Each day, the researchers used a specialized camera that could detect the glowing cells and capture pictures of the mouse brains, looking for the relative presence or absence of the transplanted glial cells.
Cells transplanted into control mice that did not receive the antibody treatment immediately began to die off, and their glow was no longer detected by the camera by day 21.
The mice that received the antibody treatment maintained significant levels of transplanted glial cells for over 203 days, showing they were not killed by the mouse’s T cells even in the absence of treatment.
“The fact that any glow remained showed us that cells had survived transplantation, even long after stopping the treatment,” says Shen Li, M.D., lead author of the study.
“We interpret this result as a success in selectively blocking the immune system’s T cells from killing the transplanted cells.”
The next step was to see whether the transplanted glial cells survived well enough to do what glial cells normally do in the brain — create the myelin sheath.
To do this, the researchers looked for key structural differences between mouse brains with thriving glial cells and those without, using MRI images.
In the images, the researchers saw that the cells in the treated animals were indeed populating the appropriate parts of the brain.
GRPs confocol in brain. The image is credited to Johns Hopkins Medicine.
Their results confirmed that the transplanted cells were able to thrive and assume their normal function of protecting neurons in the brain.
Walczak cautioned that these results are preliminary.
They were able to deliver these cells and allow them to thrive in a localized portion of the mouse brain.
In the future, they hope to combine their findings with studies on cell delivery methods to the brain to help repair the brain more globally.
Other researchers involved in this study include Byoung Chol Oh, Chengyan Chu, Antje Arnold, Anna Jablonska, Georg Furtmüller, Huamin Qin and Miroslaw Janowski of The Johns Hopkins University; Shen Li of the Dalian Municipal Central Hospital and The Johns Hopkins University; Johannes Boltze of the University of Warwick; and Tim Magnus and Peter Ludewig of the University of Hamburg.
Funding: This research was funded by the National Institute on Neurological Disorders and Stroke (R01NS091110, R01NS091100, R01NS102675, 2017-MSCRFD-3942).
The authors report no competing interests.
UC San Francisco scientists have used the CRISPR-Cas9 gene-editing system to create the first pluripotent stem cells that are functionally “invisible” to the immune system, a feat of biological engineering that, in laboratory studies, prevented rejection of stem cell transplants.
Because these “universal” stem cells can be manufactured more efficiently than stem cells tailor-made for each patient – the individualized approach that dominated earlier efforts – they bring the promise of regenerative medicine a step closer to reality.
“Scientists often tout the therapeutic potential of pluripotent stem cells, which can mature into any adult tissue, but the immune system has been a major impediment to safe and effective stem cell therapies,” said Tobias Deuse, MD, the Julien I.E. Hoffman, MD, Endowed Chair in Cardiac Surgery at UCSF and lead author of the new study, published Feb. 18 in the journal Nature Biotechnology.
The immune system is unforgiving.
It’s programmed to eradicate anything it perceives as alien, which protects the body against infectious agents and other invaders that could wreak havoc if given free rein.
But this also means that transplanted organs, tissues or cells are seen as a potentially dangerous foreign incursion, which invariably provokes a vigorous immune response leading to transplant rejection.
When this occurs, donor and recipient are said to be – in medical parlance – “histocompatibility mismatched.”
“We can administer drugs that suppress immune activity and make rejection less likely. Unfortunately, these immunosuppressants leave patients more susceptible to infection and cancer,” explained Professor of Surgery Sonja Schrepfer, MD, Ph.D., the study’s senior author and director of the UCSF Transplant and Stem Cell Immunobiology (TSI) Lab at the time of the study.
In the realm of stem cell transplants, scientists once thought the rejection problem was solved by induced pluripotent stem cells (iPSCs), which are created from fully-mature cells – like skin or fat cells – that are reprogrammed in ways that allow them to develop into any of the myriad cells that comprise the body’s tissues and organs. If cells derived from iPSCs were transplanted into the same patient who donated the original cells, the thinking went, the body would see the transplanted cells as “self,” and would not mount an immune attack.
But in practice, clinical use of iPSCs has proven difficult. For reasons not yet understood, many patients’ cells prove unreceptive to reprogramming. Plus, it’s expensive and time-consuming to produce iPSCs for every patient who would benefit from stem cell therapy.
“There are many issues with iPSC technology, but the biggest hurdles are quality control and reproducibility. We don’t know what makes some cells amenable to reprogramming, but most scientists agree it can’t yet be reliably done,” Deuse said. “Most approaches to individualized iPSC therapies have been abandoned because of this.”
Deuse and Schrepfer wondered whether it might be possible to sidestep these challenges by creating “universal” iPSCs that could be used in any patient who needed them.
In their new paper, they describe how after the activity of just three genes was altered, iPSCs were able to avoid rejection after being transplanted into histocompatibility-mismatched recipients with fully functional immune systems.
“This is the first time anyone has engineered cells that can be universally transplanted and can survive in immunocompetent recipients without eliciting an immune response,” Deuse said.
The researchers first used CRISPR to delete two genes that are essential for the proper functioning of a family of proteins known as major histocompatibility complex (MHC) class I and II. MHC proteins sit on the surface of almost all cells and display molecular signals that help the immune system distinguish an interloper from a native. Cells that are missing MHC genes don’t present these signals, so they don’t register as foreign. However, cells that are missing MHC proteins become targets of immune cells known as natural killer (NK) cells.
Working with Professor Lewis Lanier, Ph.D.—study co-author, chair of UCSF’s Department of Microbiology and Immunology, and an expert in the signals that activate and inhibit NK cell activity—Schrepfer’s team found that CD47, a cell surface protein that acts as a “do not eat me” signal against immune cells called macrophages, also has a strong inhibitory effect on NK cells.
Believing that CD47 might hold the key to completely shutting down rejection, the researchers loaded the CD47 gene into a virus, which delivered extra copies of the gene into mouse and human stem cells in which the MHC proteins had been knocked out.
CD47 indeed proved to be the missing piece of the puzzle. When the researchers transplanted their triple-engineered mouse stem cells into mismatched mice with normal immune systems, they observed no rejection.
They then transplanted similarly engineered human stem cells into so-called humanized mice—mice whose immune systems have been replaced with components of the human immune system to mimic human immunity—and once again observed no rejection.
Additionally, the researchers derived various types of human heart cells from these triple-engineered stem cells, which they again transplanted into humanized mice.
The stem cell-derived cardiac cells were able to achieve long-term survival and even began forming rudimentary blood vessels and heart muscle, raising the possibility that triple-engineered stem cells may one day be used to repair failing hearts.
“Our technique solves the problem of rejection of stem cells and stem cell-derived tissues, and represents a major advance for the stem cell therapy field,” Deuse said. “Our technique can benefit a wider range of people with production costs that are far lower than any individualized approach. We only need to manufacture our cells one time and we’re left with a product that can be applied universally.”
Source:
Johns Hopkins Medicine
Media Contacts:
Rachel Butch – Johns Hopkins Medicine
Image Source:
The image is credited to Johns Hopkins Medicine.
Original Research: Open access
“Induction of immunological tolerance to myelinogenic glial-restricted progenitor allografts “. Piotr Walczak et al.
Brain doi:10.1093/brain/awz275.
