Australian Researchers Make A Kidney in the Laboratory With Stem Cells


Stem cell researchers from the University of Queensland in Australia have successfully grown a kidney in the laboratory with stem cells. This new breakthrough will almost certainly open the door to improved treatments for patients with kidney disease, and bodes well for the future of organ bioengineering.

Mini-kidney in dish. (Source: University of Queensland)
Mini-kidney in dish. (Source: University of Queensland)

The principal investigator of this research project, Professor Melissa Little, from University of Queensland’s Institute for Molecular Bioscience (IMB), said that new treatments for kidney disease were urgently needed.

“One in three Australians is at risk of developing chronic kidney disease and the only therapies currently available are kidney transplant and dialysis,” Little said. “Only one in four patients will receive a donated organ, and dialysis is an ongoing and restrictive treatment regime. We need to improve outcomes for patients with this debilitating condition, which costs Australia $1.8 billion a year.”

Little’s research team designed a new step-wise protocol to coax embryonic stem cells to gradually form all the required kidney-specific cell types and to induce them to “self-organize” into a mini-kidney in a dish.  The embryonic stem cell line HES3 was used in this work, which derived by Reubinoff and others in the laboratory of Alan Trounson in 2000.

“During self-organization, different types of cells arrange themselves with respect to each other to create the complex structures that exist within an organ, in this case, the kidney,” Little said. “The fact that such stem cell populations can undergo self-organization in the laboratory bodes well for the future of tissue bioengineering to replace damaged and diseased organs and tissues. It may also act as a powerful tool to identify drug candidates that may be harmful to the kidney before these reach clinical trial.”

Despite the success of this research, Little cautioned that she and other kidney researchers had a great deal of work to do to before this protocol might be ready for human trials. Regardless, it is a very exciting step forward.

The Queensland Minister for Science and Innovation Ian Walker congratulated Little and her co-workers for their advances, and added that biomedical research was crucial in ensuring a healthier future for Queenslanders.

“The work by the IMB research team is an important milestone in developing improved treatments for chronic kidney disease and will ensure those with the condition can continue to live fulfilling and productive lives,” Walker said.

Little’s research team included Dr. Minoru Takasato, Pei Er, Melissa Becroft, Dr. Jessica Vanslambrouck, from IMB, and her collaorators, Professors Andrew Elefanty and Ed Stanley, from the Murdoch Children’s Research Institute and Monash University.

The research is published in the scientific journal Nature Cell Biology and supported by the Queensland Government, the Australian Research Council, as part of the Stem Cells Australia Strategic Research Initiative, and the National Health and Medical Research Council of Australia.

A More Efficient Way to Make Human Induced Pluripotent Stem Cells


Stem cell researchers at the University of California, San Diego have designed a simple, reproducible, RNA-based method of generating human induced pluripotent stem cells (iPSCs). This new technique broad applications for the successful production of iPSCs for use in therapies and human stem cell studies.

Human iPSCs are made from adult cells by genetically engineering adult cells to overexpress four different genes (Oct4, Klf4, Sox2, and c-Myc). This overexpression drives the cells to de-differentiate into pluripotent stem cells that have many of the same characteristics as embryonic stem cells, which are made from embryos. However, because iPSCs are made from the patient’s own cells, the chances that the immune system of the patient will reject the implanted cells is low.

The problem comes with the overexpression of these four genes. Initially, retroviruses have been used to reprogram the adult cells. Unfortunately, retroviruses plop their DNA right into the genome of the host cell, and this change is permanent. If these genes get stuck in the middle of another gene, then that cell has suffered a mutation. Secondly, if these genes are stuck near another highly-expressed gene, then they too might be highly expressed, thus driving the cells to divide uncontrollably.

Several studies have shown that in order to reprogram these cells, these four genes only need to be overexpressed transiently. Therefore, laboratories have developed ways of reprogramming adult cells that do not use retroviruses. Plasmid-based systems have been used, adenovirus and Sendai virus-based systems, which do not integrate into the genome of the host cell, have also been used, and even RNA has been used (see Federico González, Stéphanie Boué & Juan Carlos Izpisúa Belmonte, Nature Reviews Genetics 12, 231-242).

The UC San Diego team led by Steven Dowdy has used Venezuelan equine virus (VEE) that they engineered to express the reprogramming genes required to make iPSCs from adult cells. Because this virus does not integrate into the host genome, and expresses RNA in the host cell only transiently, it seems to be a safe and effective way to make buckets of messenger RNA over a short period of time.

The results were impressive. The use of this souped-up VEE produced good-quality iPSCs very efficiently. Furthermore, it worked on old and young human cells, which is important, since those patients who will need regenerative medicine are more likely to be young patients than old patients. Also, changing the reprogramming factors is rather easy to do as well.

Stem Cell Gene Therapy for Sickle Cell Disease Moves Toward Clinical Trials


UCLA stem cell researchers and “gene jockeys” have successfully proven the efficacy of using genetically engineered hematopoietic (blood cell-making) stem cells from a patient’s own bone marrow to treat sickle-cell disease (SCD).

In a study led by Donald Kohn, professor of pediatrics and microbiology at the UCLA Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, an “anti-sickling” gene was introduced into the hematopoietic stem cells (HSCs) from patients with SCD. Because the HSCs divide continuously throughout the life of the individual, all the blood cells they make will possess the anti-sickling gene and will therefore no sickle. This breakthrough gene therapy technique is scheduled to begin clinical trials by early 2014.

SCD results from a specific mutation in the beta-globin gene. Beta-globin is one of the two proteins that makes the multisubunit protein hemoglobin. Hemoglobin is a four-subunit protein that ferries oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs. It is tightly packed into each red blood cell.

The structure of hemoglobin, each subunit is in a different color.
The structure of hemoglobin, each subunit is in a different color.

Hemoglobin has a very high affinity for oxygen when oxygen concentrations are high, but a low affinity for oxygen when oxygen concentrations are low. Therefore, hemoglobin does a very good job of binding oxygen when it is in the lungs, where oxygen is plentiful, and a very good job of releasing oxygen in the tissues, where oxygen is not nearly as plentiful. This adaptive ability displayed by hemoglobin is the result of cooperativity between the four polypeptide chains that compose hemoglobin. Two of these polypeptide chains are alpha-globin proteins and the other two a beta globin proteins. Hemoglobin acts as though it is an alpha-beta dimer, or as though it is composed of two copies of an alpha-globin.beta-globin pair. The interactions between these polypeptide chains and the movement of the hemoglobin subunits relative to each other creates the biochemical properties of hemoglobin that are so remarkable.

A mutation in the beta-globin gene that substitutes a valine residue where there should be a glutamic acid residue (position number 6), creates a surface on the outside of the beta-globin subunit that does not like water, and when oxygen concentrations drops, the mutant hemoglobin molecule changes shape and this new water-hating surface becomes a site for protein polymerization.

Sickle cell hemoglobin

The mutant hemoglobin molecules for long, stiff chains that deform the red blood cells into a quarter moon-shaped structure that clogs capillaries.

Sickle cell RBCs

 

This new therapy seeks to correct the genetic mutation by inserting into the genome of the HSC that makes the abnormal red blood cells a gene for beta-globin that encodes a normal version of beta globin rather than a version of it that causes sickle-cell disease.  By introducing those engineered HSCs back into the bone marrow of the SCD patient, the engineered HSCs will make normal red blood cells that do not undergo sickling under conditions of low oxygen concentration.

Dr. Kohn noted that the results from his research group “demonstrate that our technique of lentiviral transduction is capable of efficient transfer and consistent expression of an effective anti-sickling beta-globnin gene in human SCD bone marrow progenitor cells, which improved the physiologic parameters of the resulting red blood cells.” Dr. Kohn’s statement may lead the reader to believe that this was done in a human patient, but that is not the case.  All this work was done in culture and in laboratory animals.

Kohn and his co-workers showed that in laboratory experiments, genetically engineered HSCs from SCD patients produced new non-sickled blood cells at a rate that would effectively allow SCD patients to show significant clinical improvement.  These new red blood cells also survived longer than those made by the nonengineered SCD HSCs.  The in vitro success of this technique has convinced the US Food and Drug Administration to grant Kohn the right to conduct clinical trials in SCD patients by early next year.

SCD affects more than 90,000 patients in the US, but it most affects people of sub-saharan African descent.  As stated before, the mutation that causes SCD produces red blood cells that are stiff, long, and get stuck in the tiny blood vessels known as capillaries that feed organs.  SCD causes multi-organ dysfunction and failure and can lead to death.

Sickle_cell_01

Treatment of SCD include bone marrow transplants, but immunological rejection of such transplants remains a perennial problem.   The success rate of bone marrow transplants is low and it is typically restricted to those patients with very severe disease who are on the verge of dying.

If Kohn’s clinical trials are successful, this stem cell-based treatment will hopefully become the gold standard for treatment of patients with SCD.  One potential problem with this technique is the use of lentiviral vectors to introduce a new gene into the HSCs.  Because lentiviruses are retroviruses, they insert their DNA into the genome of the host cells.  Such insertions can produce mutations, and it will be incumbent on Kohn and his colleagues to carefully screen each transformed HSC line to ensure that the insertion is not problematic and that the transformed cells are not sick or potentially tumorous.  However, such a vector is necessary in order to ensure permanent residence of the newly-introduced gene.

Even with these caveats, Kohn’s SCD treatment should go forward, and we wish all the best to Dr. Kohn, his team, and to the patients treated in this trial.

Stem Cell Transplants Restore Fertility in Monkeys


Injections of banked sperm-making stem cells can restore fertility to male non-human primates. This work comes from stem cell researchers at the University of Pittsburgh School of Medicine and the Magee-Womens Research Institute and was published in the journal Cell Stem Cell.

This is a remarkable finding because some men or boys must undergo cancer treatment before they have their families. Since cancer drugs destroy dividing cells and do not discriminate between normal cells and cancer cells, the stem cells that make sperm tend to take a serious beating during chemotherapy. The cancer might be destroyed, but the patient will be rendered sterile.

The senior investigator for this work, Kyle Orwig, associate professor in the Department of Obstetrics, Gynecology and Reproductive Medicine at the University of Pittsburgh School of Medicine said: “Men can bank sperm before they have cancer treatment if they hope to have biological children later in their lives,” he says. “But that is not an option for young boys who haven’t gone through puberty, can’t provide a sperm sample, and are many years away from thinking about having babies.”

Young boys that have yet to experience puberty do not yet make any sperm, but they have a modicum of spermatogonial stem cells in their testes that are waiting in the wings to produce sperm during puberty. During puberty, neurons in a part of the brain called the hypothalamus release a 10-amino acid peptide called “gonadotropin releasing hormone” (GnRH). Because these neurons release their GnRH into blood vessels that feed the pituitary gland, just below the hypothalamus, it flows directly to the pituitary.

GnRH stimulates the anterior lobe of the pituitary to release two trophic hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are collectively known as gonadotropins. FSH initiates sperm cell production in the tubules within the testes and LH stimulates the synthesis of the steroid hormone testosterone, which is necessary for sperm maturation.

Orwig and his colleagues wanted to determine if it is possible to restore fertility by freezing and banking these spermatogonial stem cells and then reintroducing them back into the testes after the completion of chemotherapy. Orwig and others took biopsies from the testes of young, adult male macaque monkeys that had yet to experience puberty. This tissue was cryopreserved in small samples. Then the monkeys were treated with chemotherapy agents that are known to reduce fertility.

A few months after chemotherapy treatment, Orwig and his colleagues transplanted each monkey’s own spermatogonial stem cells into their own testes by means of ultrasound-guided surgery. Nine of twelve adult animals showed restoration of sperm production and three of five very young animals that had not yet experienced puberty demonstrated an ability to make functional sperm after they reached maturity.

In a second experiment, spermatogonial stem cells from unrelated monkeys were transplanted into infertile animals. These transplanted cells generated sperm that had the DNA fingerprint of the donor. Because the testes contain a barrier to the immune system that prevents access of the sperm to the immune system, the implanted tissue could survive without being attacked by the immune system. This is a problem is males who have immune responses to sperm. For example, men who have had a vasectomy or make homosexuals have immune responses to human sperm. Laboratory tests showed that sperm from transplant recipients successfully fertilized 81 eggs that lead to embryos that developed normally. Donor parentage was confirmed in these embryos.

“This study demonstrates that spermatogonial stem cells from higher primates can be frozen and thawed without losing their activity, and that they can be transplanted to produce functional sperm that are able to fertilize eggs and give rise to early embryos,” Orwig says.

Several centers in the U.S. and elsewhere are already banking testicular tissue for young male cancer patients. This is in future anticipation that new stem cell-based therapies will be developed that will help them achieve pregnancy and have their own biological children. Thus this proof-of-principle experiment has generated no small degree of excitement for clinicians and patients who have compromised fertility.

According to Orwig, “These patients and their families are the pioneers that inspire our research and help drive the development of new medical breakthroughs.” He continued: “Many questions remain to be answered,” Orwig notes. “Should we re-introduce the spermatogonial cells as soon as treatment is over, or wait until the patient is considered cured of his disease, or when he is ready to start a family? How do we eliminate the risk of cancer recurrence if we give back untreated cells that might include cancer cells? These are issues we still must work through, but this study does show us the concept is feasible.”