Memories of the Way They Used to Be

Human iPS Cells Retain Some Gene Expression of Donor Cells Friday, 18 September 2009 A team of researchers from the University of California, San Diego School of Medicine and the Salk Institute for Biological Studies in La Jolla have developed a safe strategy for reprogramming cells to a pluripotent state without use of viral vectors or genomic insertions. Their studies reveal that these induced pluripotent stem cells (iPSCs) are very similar to human embryonic stem cells, yet maintain a “transcriptional signature.” In essence, these cells retain some memory of the donor cells they once were. The study, led by UCSD Stem Cell Program researcher Alysson R. Muotri, PhD, assistant professor in the Departments of Pediatrics at UCSD and Rady Children’s Hospital and UCSD’s Department of Cellular and Molecular Medicine, will be published online in PLoS ONE on September 17. “Working with neural stem cells, we discovered that a single factor can be used to re-program a human cell into a pluripotent state, one with the ability to differentiate into any type of cell in the body” said Muotri. Traditionally, a combination of four factors was used to create iPSCs, in a technology using viral vectors – viruses with the potential to affect the transcriptional profile of cells, sometimes inducing cell death or tumours. In addition, while both mouse and human iPSCs have been shown to be similar to embryonic stem cells in terms of cell behaviour, gene expression and their potential to differentiate into different types of cells, researchers had not achieved a comprehensive analysis to compare iPSCs and embryonic stem cells. “One reason is that previous methodologies used to derive iPSCs weren’t ‘footprint free,’” Muotri explained. “Viruses could integrate into the genome of the cell, possibly affecting or disrupting genes.” "In order to take full advantage of reprogramming, it is essential to develop methods to induce pluripotency in the absence of permanent changes in the genome," added Fred H. Gage, PhD, a professor in the Laboratory for Genetics at the Salk Institute and the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Diseases. By creating iPSCs from human neural stem cells without the use of viruses, the scientists learned something new. While the genetic transcriptional profile of the new iPSCs was closer to that of embryonic stem cells than to human neural stem cells, the iPSCs still carried a transcriptional “signature” of the original neural cell. “While most of the original genetic memory was erased when the cells were reprogrammed, some were retained,” said Muotri. He added that, in the past, it wasn’t known if this was caused by the use of viral vectors. “By using a footprint-free methodology, we have shown a safe way to generate human iPSCs for clinical purposes and basic research. We’ve also raised an interesting question about what, if any, effect the ‘memory retention’ of these cells might have.” The research was supported by start-up funds from the UCSD Stem Cell Research Program, and by grants from the California Institute of Regenerative Medicine and The Lookout Fund Foundation. Reference: Transcriptional Signature and Memory Retention of Human-Induced Pluripotent Stem Cells Maria C. N. Marchetto, Gene W. Yeo, Osamu Kainohana, Martin Marsala, Fred H. Gage, Alysson R. Muotri PLoS ONE 4(9): e7076. doi:10.1371/journal.pone.0007076 ......... ZenMaster

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Rare Genetic Disease Successfully Reversed using Stem Cell Transplantation

Scripps Research scientists correct gene defect in mice that causes lethal symptoms in children Thursday, 17 September 2009 A recent study by Scripps Research Institute scientists offers good news for families of children afflicted with the rare genetic disorder, cystinosis. In research that holds out hope for one day developing a potential therapy to treat the fatal disorder, the study shows that the genetic defect in mice can be corrected with stem cell transplantation. "After meeting the children who suffer from this disease, like an 18-year-old who has already had three kidney transplants, and the families who are desperately searching for help, our team is committed to moving toward a cure for cystinosis, a lysosomal storage disorder," says principal investigator Stephanie Cherqui, assistant professor in the Department of Molecular and Experimental Medicine. "This study is an important step toward that goal." In the study, which is published in the September 17, 2009 print edition of the journal Blood, the Scripps Research team used bone marrow stem cell transplantation to address symptoms of cystinosis in a mouse model. The procedure virtually halted the cystine accumulation responsible for the disease and the cascade of cell death that follows. Cystine is a by-product of the break down of cellular components the body no longer needs in the cell's "housekeeping" organelles, called lysosomes. Normally, cystine is shunted out of cells, but in cystinosis a gene defect of the lysosomal cystine transporter causes it to build up, forming crystals that are especially damaging to the kidneys and eyes. A Rare But Devastating Disease While cystinosis is rare — affecting an estimated 500 people in the United States and 2,000 worldwide — it is devastating. Three types of cystinosis have been described based on the age at diagnosis and the amount of cystine in cells: infantile onset, adolescent onset, and adult onset. Children as young as six months can begin to suffer renal dysfunction, which grows progressively worse with time. Other symptoms include diabetes, muscular disease, neurological dysfunction, and retinopathy. Infantile onset is the most common, as well as the most severe, form of the disease. The only available drug to treat cystinosis, cysteamine, while slowing the progression of kidney degradation, does not prevent it, and end-stage kidney failure is inevitable. "Cysteamine must be given every six hours, so children have to be woken up each night to take this drug, which has unpleasant side effects, and many others to treat various symptoms," Cherqui says. "So although there is treatment, it is difficult treatment that does not cure the disease." "Surprised and Encouraged" In the new study, the researchers found that transplanted bone marrow stem cells carrying the normal lysosomal cystine transporter gene abundantly engrafted into every tissue of the experimental mice. This led to an average drop in cystine levels of about 80 percent in every organ. In addition to preventing kidney dysfunction, there was less deposition of cystine crystals in the cornea, less bone demineralization, and an improvement in motor function. "The results really surprised and encouraged us," says Cherqui, who as a doctoral student in France in 1998 helped discover the gene involved in cystinosis. "Because the defect is present in every cell of the body, we did not expect a bone marrow stem cell transplant to be so widespread and effective." Cherqui, who generated the mouse model in 2000 that is currently used to study cystinosis, says that adult bone marrow stem cell therapy is particularly well suited as a potential treatment for cystinosis because these cells target all types of tissues. In addition, stem cells reside in the bone marrow for the duration of a patient's life, becoming active as needed, a particular benefit for a progressive disease like cystinosis. The work of Cherqui and her colleagues may have wider applications for other genetic diseases, providing proof of principle that adult stem cell transplants may be successful in humans for genetic diseases with systemic defects, especially those of a progressive nature. Cherqui expects to spend the next several years analyzing the safety of genetically modified autologous (obtained from the same individual) bone marrow transplants in the cystinosis mouse and other models before moving on to human clinical trials. This work was funded by the Cystinosis Research Foundation. Reference: Successful treatment of the murine model of cystinosis using bone marrow cell transplantation Kimberly Syres, Frank Harrison, Matthew Tadlock, James V. Jester, Jennifer Simpson, Subhojit Roy, Daniel R. Salomon, and Stephanie Cherqui Blood 2009 114: 2542-2552, DOI 10.1182/blood-2009-03-213934 ......... ZenMaster

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How microRNAs Drive Tumour Progression

How microRNAs Drive Tumour Progression Thursday, 17 September 2009 UCSF researchers have identified collections of tiny molecules known as microRNAs that affect distinct processes critical for the progression of cancer. The findings, they say, expand researchers' understanding of the important regulatory function of microRNAs in tumour biology and point to new directions for future study and potential treatments. The researchers refer to these microRNA collections as signatures, and their study results are reported in the September 15 issue of "Genes & Development.'' The study was led by the laboratory of Douglas Hanahan, PhD, an American Cancer Society Research Professor in the Department of Biochemistry and Biophysics at UCSF. Approximately five percent of all known human genes encode, or produce, microRNAs, yet scientists are only now — nearly a decade after their discovery — beginning to unlock the mystery of their functions. MicroRNAs are snippets of single-stranded RNAs that prevent a gene's code from being translated from messenger RNA into proteins, which are essential for cell growth and development. Produced in the nucleus and released into the cytoplasm, they home in on messenger RNAs that possess a stretch that is complementary to their genetic sequence. When they locate them, they latch on, preventing the messenger RNA from being processed by the protein-making machines known as ribosomes. As such, microRNAs are able to ratchet down a cell's production of a given protein. Over the last several years, several groups have identified hundreds of microRNAs that are deregulated between normal tissue and tumours, however researchers only understand what a handful of these powerful regulators are doing to drive tumour formation. "Virtually all cancers acquire approximately six distinct capabilities en route to tumour formation," said lead author Peter Olson, PhD, a postdoctoral fellow in the Diabetes Center and Helen Diller Family Comprehensive Cancer Center at UCSF. "When a cancer researcher observes a gene or microRNA go awry, it can be challenging to understand how that microRNA impacts tumourigenesis." To home in on the question, the authors turned to a mouse model of pancreatic neuroendocrine tumours in which lesions go through discrete stages before culminating in invasive and metastatic carcinomas. In the three-year microRNA study, they found that cells in the mouse model developed and functioned normally but started to replicate uncontrollably at five weeks. Several weeks later, some pancreatic islets had become angiogenic (forming new blood vessels) — a step in the journey from a dormant state to a malignant state — though had not yet formed a tumour. By 10 weeks, a subset of angiogenic lesions had progressed to the tumour stage, and by week 16, a small percentage of mice had developed liver metastasis. "This represents the spectrum of stages that we think are important for all tumours, including human disease," said Olson. By measuring the expression level of all known microRNA in pre-tumour stages, tumours and metastases, the authors were able to associate deregulated microRNAs with processes such as hyper-proliferation, angiogenesis and metastasis. Focusing on the metastatic signature, researchers found — in one of the most striking observations of the project — that tumours bore a startlingly divergent microRNA expression pattern compared to primary tumours. Moreover, a subset of primary tumours showed more similarity to metastases than to other primary tumours. "If you can identify tumours that have an increased propensity to metastasize, then it would have a very important clinical application," said Olson. "A lively debate in metastatic research has centred around whether primary tumour cells must suffer an additional mutation that endows that cell with a metastatic capability, or whether certain mutational combinations that are responsible for primary tumour formation also significantly increase the propensity of that cell to metastasize. These data provide evidence for the latter.'' Reference: MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer Peter Olson, Jun Lu, Hao Zhang, Anny Shai, Matthew G. Chun, Yucheng Wang, Steven K. Libutti, Eric K. Nakakura, Todd R. Golub, and Douglas Hanahan Genes Dev. September 15, 2009 23: 2152-2165; doi:10.1101/gad.1820109 ......... ZenMaster

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