Scientists have known for decades that we possess stem cells that replenish our bodies’ tissues. Blood-forming (hematopoietic) stem cells, for example, can spawn red cells, white cells and all the other types of blood cells. Most intriguing of all are human embryonic stem cells, capable of developing into any of the body’s more than 100 different types of tissue—a characteristic known as pluripotency.
Researchers suspected that if human embryonic stem cells could be isolated, they could be used to renew or repair all sorts of human tissues. But even the simple act of obtaining human stem cells for scientific study proved difficult.
A breakthrough came in 1998, when James Thomson of the University of Wisconsin discovered how to isolate stem cells from early human embryos and culture them in laboratory dishes. Ideally, these human embryonic stem cells could then be made to develop into any tissue type desired. But obtaining human embryonic stem cells meant sacrificing the embryo, triggering opposition to their use. In 2001, President George W. Bush limited federal funding for such research to 60 human embryonic stem cell lines then in existence.
The field was reinvigorated by Shinya Yamanaka, M.D., a Japanese researcher. In 2006, Dr. Yamanaka found that inserting four particular genes into adult mouse skin cells caused those cells to go backward developmentally and turn into cells closely resembling embryonic stem cells. He then showed that these engineered cells, dubbed “induced pluripotent stem cells (iPSCs),” could—like embryonic stem cells—be coaxed to differentiate into many cell types. A year later, he duplicated the experiment using adult human skin cells. His discovery that mature, fully differentiated cells could be reprogrammed to become pluripotent would earn him a share of the 2012 Nobel Prize in Physiology or Medicine.
“Thanks to Dr. Yamanaka’s breakthrough, researchers finally have a source of embryonic-like human stem cells that is free of ethical constraints,” says Paul S. Frenette, M.D., professor of medicine (hematology) and of cell biology, and chair and director of the Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research at Einstein. “While it’s too early to assess the full impact of this technology, iPSCs have great potential in everything from disease modeling to drug testing to regenerative medicine.”
Einstein investigators are currently using iPSCs to study autism, schizophrenia, cataracts, liver disease and blood disease. To encourage further iPSC research, the Gottesman Institute has established a Pluripotent Stem Cell Unit, which creates iPSCs for the Einstein research community and provides training in maintaining and differentiating iPSCs.
Let There Be Blood Cells
Since World War II, just about every aspect of healthcare, from surgery to radiology to record keeping, has undergone sweeping change. Blood banking is a notable exception. Blood today is collected, typed, screened and stored much as it was in the late 1940s, when a nationwide system of blood banks was first organized.
While this system works relatively well, it has significant flaws, says Eric E. Bouhassira, Ph.D., professor of cell biology and of medicine (hematology), the Ingeborg and Ira Leon Rennert Professor of Stem Cell Biology and Regenerative Medicine and director of the Pluripotent Stem Cell Unit.
With its brief shelf life, blood can’t be stockpiled, resulting in local shortages. And while all units of donated blood are screened for a variety of pathogens, nothing can be done to prevent the transmission of new ones, which is what happened with HIV in the 1980s. In addition, some people with sickle cell anemia and other conditions requiring chronic transfusions develop sensitivities to antigens in blood, making it difficult to find suitable blood matches.
Genetically modified stem cells offer perhaps the best hope for curing thalassemia.
Dr. Bouhassira is trying to use iPS to produce red blood cells (RBCs) on an industrial scale—a seemingly far-fetched idea that may not be so far off. In a 2011 study published in PloS One, he showed that various types of adult human cells could be reprogrammed into iPSCs, which could then be made to produce large quantities of fetal-like red blood cells. Unfortunately, fetal RBCs have a form of hemoglobin (the oxygen-carrying protein in RBCs) that differs from the kind in mature RBCs, and they would not sustain an adult’s oxygen needs.
“Our next challenge,” says Dr. Bouhassira, “is to induce iPSCs to differentiate far enough along the blood-forming pathway that we can create RBCs that possess adult hemoglobin.”
In addition to their freedom from ethical problems, iPSCs offer another key advantage over human embryonic stem cells: replacement tissues derived from iPSCs are unlikely to provoke an immune response resulting in tissue rejection. New nerve cells for a patient with Parkinson’s disease, for example, should be a good match for that patient, since they come from iPSCs derived from the patient’s own skin cells rather than from an embryo with a different genetic makeup. Dr. Bouhassira is taking advantage of this trait in work aimed at transforming iPSCs into cures for genetic blood disorders such as thalassemia.
People with thalassemia make an abnormal form of hemoglobin that causes mild to severe anemia, depending on the underlying genetic flaw. Thalassemia is typically treated with repeated blood transfusions. But over time, such transfusions can cause elevated blood levels of iron, which must be removed with costly chelation therapy.
Selected cases of thalassemia can be cured with bone marrow transplantation, in which the patient receives high doses of drugs or radiation to destroy the diseased hematopoietic (blood-forming) stem cells, followed by a marrow infusion from a compatible donor. But the risky procedure is generally reserved for patients with severe disease who have well-matched donors—typically siblings—available.
Genetically modified stem cells offer perhaps the best hope for curing thalassemia. In one approach, doctors harvest a patient’s hematopoietic stem cells, use viral vectors to insert normal copies of the affected gene into them and then return the cells to the patient. But using viral vectors risks inducing cancer-causing mutations in the stem cells.
Dr. Bouhassira is developing a potentially safer stem cell cure based on iPSCs, which can be genetically modified without viruses. The idea here is to convert the patient’s skin cells into iPSCs and then modify the iPSCs with a gene-insertion technique using zinc finger nucleases—synthetic proteins that carry little or no risk of causing cancer. Scientists would then induce the corrected iPSCs to develop into RBCs, which would be transfused back into the patient.
In a study published last year in Blood, Dr. Bouhassira showed that this technique could potentially correct the genetic flaws responsible for alpha thalassemia major, the most severe form of the disease. But as with the effort to form RBCs for transplantation, the genetically corrected iPSCs must progress beyond the fetal RBC stage and develop into adult RBCs before this therapy can be brought to clinical trials.
The ideal way to study disease at the molecular level is to analyze cells from the affected tissues of patients—not a problem for, say, dermatologists or hematologists, who can readily obtain skin or blood cells. But neuroscientists lack such access. The brain, encased in its bony vault, is well protected from insult, injury and prying hands. So those who study brain diseases have had to make do with tissue samples obtained at autopsy.
“This has been extremely limiting,” says Herbert Lachman, M.D., professor of psychiatry and behavioral sciences and of medicine (hematology). “Diseases such as schizophrenia may begin as early as embryogenesis. But with autopsy specimens, you’re typically looking at cells from adults, many decades after the disease first developed. In addition, the cells may be from someone who abused drugs or alcohol or had taken psychotropic medications, which can make it difficult to distinguish the primary disease from secondary influences.”
Dr. Lachman has embraced iPSC technology because he realizes it could provide him with live nerve cells (neurons) from living patients.
“It was a steep learning curve—iPSC technology is extremely complex, and I made a lot of rookie mistakes,” he admits. But his efforts are paying off. Three years down the line, he has mastered the fine art of transforming skin cells into iPSCs and then tweaking iPSCs into neurons, creating a bounty of research opportunities.
In a study funded by the National Institute of Mental Health, Dr. Lachman is comparing iPSC-derived neurons from patients with schizophrenia to neurons from healthy controls. He’s particularly interested in whether neurons from the two groups differ in their microRNAs—snippets of RNA that regulate gene expression.
MicroRNAs are known to play a key role in brain development and in forming synapses (connections between neurons), says Dr. Lachman, who is also an associate professor in the Dominick P. Purpura Department of Neuroscience and the department of genetics. And evidence from a genetic disease called velo-cardio-facial syndrome (VCFS) points to a role for microRNAs in schizophrenia.
VCFS is caused by a 22q11 microdeletion (the absence of a small portion of chromosome 22). Approximately one-third of VCFS patients suffer from schizophrenia. The specific gene defect responsible for these cases of VCFS has not been unequivocally identified, but one promising candidate is DGCR8, which codes for a protein involved in microRNA production.
Mice genetically engineered to have just one copy of DGCR8, instead of the usual two, exhibit changes in behavior, in neuronal branching (a measure of brain-cell connectivity) and in the expression of microRNAs in the hippocampus and cortex.
“Our goal is to find out which microRNAs are abnormally regulated in schizophrenia,” says Dr. Lachman, also an attending physician at Montefiore. In theory, those microRNAs could then be targeted with medications.
Dr. Lachman also wants to know whether microRNA expression is altered in patients with schizophrenia who do not have a 22q11.2 deletion. “If so, this would suggest that a single aberrant molecular pathway could account for the problems that characterize this disease, even if schizophrenia itself can originate from many different gene abnormalities,” says Dr. Lachman. “That would certainly simplify the search for a single treatment that would help most people with schizophrenia.”
In other iPSC research, Dr. Lachman is employing iPSCs to grow “mini-brains” in laboratory culture. No Frankenstein worries here: These creations are not brains in the traditional sense but rather small, in vitro three-dimensional aggregates of radial glial cells (neuron precursors) and maturing neurons. The mini-brains are intended to mimic neuronal structures that form in the developing forebrain.
“From autopsy samples, we know that a good fraction of patients with schizophrenia have abnormalities in their synaptic architecture,” he says. “Using these models, we can start looking at this architecture and see how it might be influenced by 22q11 deletions.” He notes that mini-brains could also be used to evaluate new medications for schizophrenia and other diseases.
Our goal is to find out which microRNAs are abnormally regulated in schizophrenia.
A third iPS cell project involves work that Dr. Lachman is doing with Brett S. Abrahams, Ph.D., assistant professor of genetics, to study neuron abnormalities in autism spectrum disorders (ASD).
As in his schizophrenia research, he is using iPSC technology to derive neurons from healthy children and compare them with neurons from children with ASD. The researchers are looking for variations in a portion of chromosome 15 known as 15q11.2. Deletions and duplications within this small area can increase the risk for autism and other behavioral disorders. Some individuals with these variations have no neurodevelopmental issues, while others are severely affected.
By looking at the molecular differences between neurons of affected individuals and healthy controls, Drs. Lachman and Abrahams hope to find precisely how variations at 15q11.2 increase the risk for autism and, ultimately, to develop ways of counteracting the effects of those gene defects.
Despite the great potential for creating disease models using iPSCs, Dr. Lachman cautions that it will take some time before therapies based on this technology reach patients. “Research into gene therapy began several decades ago, and we’re just starting to see results,” he says. “The same will probably hold true for iPSCs.”
Dr. Frenette is similarly cautious: “We don’t know whether iPSCs are exactly the same as stem cells that form naturally. Another issue is that the specialized cells derived from iPSCs have not always progressed to full maturity. Plus, when you have iPSCs that have not fully differentiated, there’s the small but real risk that they could conceivably cause cancer. In other words, we still have a lot of work to do.”
Top of page, cross-section of an aggregate of neurons, referred to as a “minibrain.” It originated from skin fibroblasts of a patient with a chromosome 22q11.2 deletion and schizophrenia. The fibroblasts were reprogrammed into iPSCs that closely resemble human embryonic stem cells.
Dr. Herb Lachman and lab technician Erika Pedrosa coaxed the iPSCs to develop into the “minibrain,” which models early brain development. Embedded in the sea of neurons (stained green) are circular structures resembling neural tubes (stained blue) that consist of radial glial cells organized around a central lumen. A protein encoded by a 22q11.2-linked gene (RANBP1) is stained orange.
Image credit: Erika Pedrosa, M.S.