stem cells

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 individualised 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 Dr Tobias Deuse, the Julien I.E. Hoffman, Endowed Chair in Cardiac Surgery at UCSF and lead author of the study.

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, Dr Sonja Schrepfer, 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,” Dr Deuse said. “Most approaches to individualised iPSC therapies have been abandoned because of this.”

Dr Deuse and Prof 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,” Dr 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, PhD – 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 – Prof 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 humanised 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 humanised 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,” Dr Deuse said. “Our technique can benefit a wider range of people with production costs that are far lower than any individualised approach. We only need to manufacture our cells one time and we’re left with a product that can be applied universally.”

The study was published in the journal Nature Biotechnology.

measles

Researchers have developed a method to use the measles virus vector to reprogram pluripotent stem cells.

Induced pluripotent stem cells begin as differentiated cells which are then reprogrammed to pluripotent stem cells through exposure to a complex set of genetic cocktails . Using the measles virus vector the team of researchers trimmed this process from a multi-vector four reprogramming factor process, down to a single vector process.

“If we’re going to successfully use reprogrammed stem cells to treat patients in the clinic, we need to ensure that they are safe and effective, that is, not prone to the risk of mutation and potential tumors,” said Dr Patricia Devaux, Mayo Clinic molecular scientist and senior author of the article.

“The measles virus vector has long been used safely at Mayo for treating cancer, so it is very safe. Now that we’ve combined a multiple-vectors process into one, it’s efficient as well.”

Four reprogramming proteins, OCT4, SOX2, KLF4 and cMYC, were introduced individually to the cells to induce change resulting in the desired outcome. This would lead to partially reprogrammed cells, because not all of the cells received the four factors that are required for reprogramming. The researchers in this study combined those factors within the measles virus vector so the process occurred in one step, and so all targeted cells have the potential to reprogram.

The team mentioned how the measles virus was attenuated, and so all dangerous aspects of the virus have been removed, as they are in a vaccine. The virus then becomes a vector or carrier for other genetic material. The measles virus vaccine strain is often used today because it is safe, fast and targetable.

A clinically applicable reprogramming system free from genomic modifications should go a long way to making widespread use of induced pluripotent stem cell therapies feasible, the team stated. These are therapies in which an individual’s own cells are reprogrammed can then be use to work in a particular diseased organ, thus avoiding risk of cell rejection, and can be seen through recent breakthrough immunotherapies for cancer. 

The study was published in the journal Gene Therapy.

mouse

Functional B1-cells that have been derived from mouse embryonic stem cells have been found to be capable of long-term engraftment as they secrete natural antibodies after transplantation.

Scientists were interested in B1-cells generated from pluripotent stem cells because they could be tested as a therapeutic for a broad range of immunological disorders.

“It is still challenging to produce transplantable immune cells from mouse embryonic stem cells, so obtaining transplantable functional B1-cells from mouse embryonic stem cells is a significant advance in the field,” said senior study author Dr Momoko Yoshimoto of the Center for Stem Cell & Regenerative Medicine at the McGovern Medical School at UTHealth in Houston. “The take-home message is that a portion of immune cells may be replaced by cell therapies utilising pluripotent stem cells in the future.”

Hematopoietic stem cells in the adult bone marrow – the soft, sponge-like tissue in the center of most bones – provide various blood cells throughout life. Hematopoietic stem cell transplants are now routinely used to treat patients with cancers and other disorders of the blood and immune systems. But with current in vitro methods, it is challenging to produce hematopoietic stem cells that recapitulate the properties of cells in living organisms without gene manipulation.

In particular, bone marrow transplantation may fail to reconstitute some immune cells called B1-cells, which produce immunoglobulin M (IgM) antibodies – the first type of antibody the immune system makes to fight a new infection. In addition to patients who receive stem cell transplants, IgM deficiency also occurs in individuals with some cancers, autoimmune diseases, allergic diseases, and gastrointestinal diseases, increasing the risk for life-threatening infections.

In the new study, Dr Yoshimoto and her colleagues demonstrated that functional, transplantable B1-cells can be generated from mouse embryonic stem cells without gene modifications. The researchers overcame previous barriers preventing this feat by using high-quality cell lines to support B-cell development. After being transplanted into recipient mice, stem cell-derived B progenitors matured into B1-cells that were maintained for more than 6 months and secreted natural IgM antibodies.

“Producing functional B1 progenitors in vitro from mouse embryonic stem cells is an important step to develop a cell therapy to provide natural IgM and innate B1-cells that may not be provided by bone marrow transplantation,” Dr Yoshimoto said.

In future studies, the researchers will attempt to generate B-cells from human induced pluripotent stem cells, which may be used for cell therapy to treat patients with immunological disorders. “This is just a first step in a long process to translate our findings to humans,” Dr Yoshimoto said.

The study was published in the journal Stem Cell Reports.

insulin

Scientists are developing ways that stem cells could be used to treat diabetes. These undifferentiated cells could be transformed into cells that produce insulin, the hormone that controls blood sugar.

But there’s a major challenge: the amount of insulin produced by theses cells is difficult to control.

By tweaking the recipe for coaxing human stem cells into insulin-secreting beta cells, a team of researchers at Washington University School of Medicine in St. Louis has shown that the resulting cells are more responsive to fluctuating glucose levels in the blood.

When they transplanted the beta cells into mice that could not make insulin, the new cells began secreting insulin within a few days, and they continued to control blood sugar in the animals for months.

“We’ve been able to overcome a major weakness in the way these cells previously had been developed. The new insulin-producing cells react more quickly and appropriately when they encounter glucose,” said principal investigator Dr Jeffrey R. Millman, an Assistant Professor of Medicine and of Biomedical Engineering. “The cells behave much more like beta cells in people who don’t have diabetes.”

The researchers now believe it may be time to evaluate whether the same stem-cell approach could produce insulin and effectively control blood sugar in people.

Prof Millman was a part of a research team at Harvard that, in 2014, converted skin cells into stem cells and, in 2016, did the same thing with skin cells from a patient with diabetes. Each time, the stem cells were then treated with various growth factors to coax them into insulin-secreting beta cells. The beta cells, however, didn’t work as well as the researchers had hoped.

“Previously, the beta cells we manufactured could secrete insulin in response to glucose, but they were more like fire hydrants, either making a lot of insulin or none at all,” he said. “The new cells are more sensitive and secrete insulin that better corresponds to the glucose levels.”

For this study, Prof Millman’s laboratory still grew beta cells from human stem cells, but they made numerous changes to the “recipe” for producing insulin-producing beta cells, treating the cells with different factors at different times as they grew and developed to help the cells mature and function more effectively.

After that process was complete, the researchers transplanted the beta cells into diabetic mice with suppressed immune systems so that they wouldn’t reject the human cells. Those transplanted cells produced insulin at levels that effectively controlled blood sugar in the mice, functionally curing their diabetes for several months, which, for most of the mice in the study, was about the length of their lives.

As laboratory researcher rather than a clinician, Prof Millman said he can’t predict exactly when such cells may be ready for human trials but believes there are at least two ways that stem cell-derived beta cells could be tested in human patients.

“The first would be to encapsulate the cells in something like a gel – with pores small enough to prevent immune cells from getting in but large enough to allow insulin to get out,” he said. “Another idea would be to use gene-editing tools to alter the genes of beta cells in ways that would allow them to ‘hide’ from the immune system after implantation.”

Prof Millman said that if stem cell-derived beta cells are proven safe and effective for people with diabetes, his method of manufacturing the cells quickly could be ramped up to an industrial scale. In his laboratory alone, his team is able to grow and develop more than a billion beta cells in just a few weeks.

The study was published in the journal Stem Cell Reports.