Researchers Generate iPS Cells to Further Treatments for Lung Diseases

Researchers Generate iPS Cells to Further Treatments for Lung Diseases
Friday, 29 October 2010

A team of researchers from Boston University's Center for Regenerative Medicine and the Pulmonary Center have generated 100 new lines of human induced pluripotent stem cells (iPSC) from individuals with lung diseases, including cystic fibrosis and emphysema. The new stem cell lines could possibly lead to new treatments for these debilitating diseases. The findings, which appear in the current issue of Stem Cells, demonstrate the first time lung disease-specific iPSC have been created in a lab.

iPSCs are derived by reprogramming adult cells into a primitive stem cell state. This process results in the creation of cells that are similar to embryonic stem cells in terms of their capability to differentiate into different types of cells, including endoderm cells that can give rise to liver and lung tissue.

"iPSCs solve many major hurdles currently impacting embryonic stem cell research," said Darrell Kotton, the study's lead author and associate professor of medicine and pathology and laboratory medicine at Boston University's School of Medicine (BUSM).

iPSCs do not require embryos, and the process used to cultivate iPSCs is easier than the techniques used to obtain embryonic stem cells. iPSCs are genetically identical to the patient's cells and potentially can be transplanted back without rejection.

"In a laboratory dish, these cells have the ability to multiply indefinitely so that researchers have more time to investigate the diseased cell and correct its genes," said Kotton.

The study involved patients with different forms of lung disease – cystic fibrosis, alpha-1 antitrypsin deficiency-related emphysema, scleroderma (SSc) and sickle cell disease. The patients underwent skin biopsies and donated tissue samples, which the research team used to cultivate adult stem cells. Using a Boston University-patented vector in the form of a virus, named the Stem Cell Cassette (STEMCCA), the researchers were able to reprogram the skin cells into the primitive pluripotent stem cells known as iPSCs.

"The STEMCCA vector is proving invaluable for reprogramming cells from a variety of species, and this is the first report of the 'humanized' version of our vector for use in reprogramming human cells," said Gustavo Mostoslavsky, a co-author of the study and assistant professor of medicine at BUSM. Together Kotton and Mostoslavsky co-direct the new Boston University Center for Regenerative Medicine (CReM).

To test the differentiation power of the iPSCs, the team showed that the stem cells multiplied and could be differentiated into endoderm tissue, the natural precursor cells of the lung, the primary organ destroyed by the diseases cystic fibrosis and emphysema.

"We hope to build a bank of stem cells that could be used to help treat the two most common forms of inherited lung disease, cystic fibrosis and alpha-1 antitrypsin deficiency," said Kotton.

The next step, he said, is to correct the genetic mutations responsible for causing cystic fibrosis, emphysema and other lung diseases.

Source: Boston University Medical Center
Contact: Gina DiGravio

Reference:
Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette
Aba Somers, Jyh-Chang Jean, Cesar A. Sommer, Amel Omari, Christopher C. Ford, Jason A. Mills, Lei Ying, Andreia Gianotti Sommer, Jenny M. Jean, Brenden W. Smith, Robert Lafyatis, Marie-France Demierre, Daniel J. Weiss, Deborah L. French, Paul Gadue, George J. Murphy, Gustavo Mostoslavsky and Darrell N. Kotton
Stem Cells Vol. 28, Issue 10, pp. 1728–1740, October 2010, DOI: 10.1002/stem.495
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ZenMaster

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Gene Therapy Corrects Sickle Cell Disease in Laboratory

New St. Jude treatment alleviates long-term anaemia and organ damage in mice and paves the way for human applications Friday, 05 December 2008 Using a harmless virus to insert a corrective gene into mouse blood cells, scientists St. Jude Children's Research Hospital have alleviated sickle cell disease pathology. In their studies, the researchers found that the treated mice showed essentially no difference from normal mice. Although the scientists caution that applying the gene therapy to humans presents significant technical obstacles, they believe that the new therapy will become an important treatment for the disease. Sickle cell disease, which affects millions of people worldwide, arises because of a tiny genetic defect in the gene for beta-globin, a protein component of haemoglobin. This defect causes haemoglobin-containing red blood cells to tend to deform, clump and break apart. The resulting clogged blood vessels can lead to cognitive dysfunction by causing small strokes in the brain and cause damage to kidneys, liver, spleen and lungs. The only permanent cure for the disease is a bone marrow transplant to give recipients blood-forming cells that will form normal beta-globin. However, such transplants are rare because of the lack of compatible donors. Researchers have long known that symptoms of the disease could be alleviated by persistence in the blood of an immature foetal form of haemoglobin in red blood cells. This immature haemoglobin, which usually disappears after birth, does not contain beta-globin, but another form called gamma-globin. St. Jude researchers had found that treating patients with the drug hydroxyurea encourages the formation of foetal haemoglobin and alleviates disease symptoms. "While this is a very useful treatment for the disease, our studies indicated that it might be possible to cure the disorder if we could use gene transfer to permanently increase foetal haemoglobin levels," said Derek Persons, M.D., Ph.D., assistant member in the St. Jude Department of Hematology. He and his colleagues developed a technique to insert the gene for gamma-globin into blood-forming cells using a harmless viral carrier. The researchers extracted the blood-forming cells, performed the viral gene insertion in a culture dish and then re-introduced the altered blood-forming cells into the body. The hope was that those cells would permanently generate red blood cells containing foetal haemoglobin, alleviating the disease. In the experiments, reported in the advanced, online issue of the journal Molecular Therapy, the researchers used a strain of mouse with basically the same genetic defect and symptoms as humans with sickle cell disease. The scientists introduced the gene for gamma-globin into the mice's blood-forming cells and then introduced those altered cells into the mice. The investigators found that months after they introduced the altered blood-forming cells, the mice continued to produce gamma-globin in their red blood cells. "When we examined the treated mice, we could detect little, if any, disease using our methods," said Persons, the paper's senior author. "The mice showed no anaemia, and their organ function was essentially normal." The researchers also transplanted the altered blood-forming cells from the original treated mice into a second generation of sickle cell mice to show that the gamma-globin gene had incorporated itself permanently into the blood-forming cells. Five months after that transplantation, the second generation of mice also showed production of foetal haemoglobin and correction of their disease. "We are very encouraged by our results," Persons said. "They demonstrate for the first time that it is possible to correct sickle cell disease with genetic therapy to produce foetal haemoglobin. We think that increased foetal haemoglobin expression in patients will be well tolerated and the immune system would not reject the haemoglobin, in comparison to other approaches." While Persons believes that the mouse experiments will lead to treatments in humans, he cautioned that technical barriers still need to be overcome. "It is far easier to achieve high levels of gene insertion into mouse cells than into human cells," he said. "In our mouse experiments, we routinely saw one or two copies of the gamma-globin gene inserted into each cell. However, in humans this insertion rate is at least a hundred-fold less." Persons' laboratory is currently working with other animal and human cells to develop methods to achieve a high enough gene insertion rate to make the gene therapy clinically useful. About St. Jude Children's Research Hospital St. Jude Children's Research Hospital is internationally recognized for its pioneering work in finding cures and saving children with cancer and other catastrophic diseases. Founded by late entertainer Danny Thomas and based in Memphis, Tenn., St. Jude freely shares its discoveries with scientific and medical communities around the world. No family ever pays for treatments not covered by insurance, and families without insurance are never asked to pay. St. Jude is financially supported by ALSAC, its fundraising organization. ......... ZenMaster

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Genetically Modified Human Embryo Stirs Controversy

Scientists create first GM human embryo 
Tuesday, 13 May 2008 

Researchers at Cornell University in New York have made a breakthrough in genetics by creating the first genetically modified (GM) human embryo. The GM embryo was produced to study how early cells in the embryo develop, but the scientists destroyed it just after five days. Led by Nikica Zaninovic, researchers at Cornell University used a virus to add a gene, a green fluorescent protein, to an embryo left over from assisted reproduction. It is believed to be the first documented genetic modification of a human embryo. 

Zaninovic's achievement was announced at the American Society for Reproductive Medicine annual meeting in 2007, but was only publicized recently when the United Kingdom's reproductive technology regulators reviewed the research. One of the authors of the study said to AP that the work was focused on stem cells. He noted that the researchers used an abnormal embryo that could never have developed into a baby anyway. 

"None of us wants to make designer babies," said Dr. Zev Rosenwaks, director of the Center for Reproductive Medicine and Infertility at New York-Presbyterian/Weill Cornell Medical Center. Dr. Rosenwaks said the research had been approved by a review board at his medical center and been privately financed, so it did not violate federal restrictions on research involving human embryos. 

 Doctors already put foreign genes into people as part of gene therapy to treat diseases. But those genetic changes generally cannot be passed on to future generations because they are made to only certain types of cells in the body, like blood cells or muscle cells. Genetic changes made to an embryo would theoretically be heritable if the embryo became a baby. So far, this has been a no-go area for scientists and medical professionals. 

 The breakthrough has brought with it major concerns. The British regulator, the Human Fertilisation and Embryology Authority (HFEA), has even cautioned that such controversial experiments may lead to "large ethical and public interest issues". However, the HFEA has said that it is preparing for scientists to apply for licences to create GM embryos. 

A paper, published by the authority, states: “The bill has taken away all inhibitions on genetically altering human embryos for research. The Science and Clinical Advances Group [of the HFEA] thought there were large ethical and public interest issues and that these should be referred for debate.” 

 The House of Commons in Britain is about to consider legislation permitting this and other controversial reproductive technologies, such as the creation of chimeras – human-animal hybrid embryos. The first voting on this Bill took place yesterday in the British Parliament. There the MPs voted to allow, with a great majority, the plan to update the human embryology laws to continue to their next Parliamentary stage. The research raises a number of difficult ethical questions. 

Though adding a fluorescent protein was merely a proof-of-principle step, modified embryos could be used to research human diseases. Scientists say embryos wouldn't be allowed to develop for more than a few weeks, much less implanted in a woman and brought to term. If the embryos were allowed to develop, genetic modifications – which would be permanent and passed to future generations – might prevent disease. 

Modifications might also be used for other reasons – physical appearance, intellectual prowess and personality changes – though the necessary science remains hypothetical at this point. Developing such techniques would necessarily involve at this stage trial-and-error and risk-taking with human life.

Let's have that debate:
What do you think CellNEWS readers? 

• Should genetically modified human embryos be used in research, or reproduction? Both? Neither? 
• What would be the advantages or disadvantages? 
• Would it OK to produce ‘designer babies’ in the future, when the technique is perfected? 

Reference: 

Genetic Modification Of Preimplantation Embryos and Embryonic Stem Cells (ESC) by Recombinant Lentiviral Vectors: Efficient and Stable Method for Creating Transgenic Embryos and ESC 

N. Zaninovic, J. Hao, J. Pareja, D. James, S. Rafii, Z. Rosenwaks. 

ASRM 2007 Annual Meeting, Poster session. 

Other Online Resources: 

HFEA 

Center for Genetics and Society 

Human Genetics Alert 

Genetics and Public Policy Center 

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ZenMaster

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For more on stem cells and cloning, go to CellNEWS 
at http://cellnews-blog.blogspot.com/

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Reprogrammed adult cells treat sickle-cell anaemia in mice

Proof of principle for therapeutic use of induced pluripotent stem cells Friday, 07 December 2007 Mice with a human sickle-cell anaemia disease trait have been treated successfully in a process that begins by directly reprogramming their own cells to an embryonic-stem-cell-like state, without the use of eggs. This is the first proof-of-principle of therapeutic application in mice of directly reprogrammed “induced pluripotent stem” (iPS) cells, which recently have been derived in mice as well as humans. The research, reported in Science Express online on December 6, was carried out in the laboratory of Whitehead Member Rudolf Jaenisch. The iPS cells were derived using modifications of the approach originally discovered in 2006 by the Shinya Yamanaka laboratory at Kyoto University. The scientists studied a therapeutic application of iPS cells with the sickle-cell anaemia model mouse developed by the laboratory of Tim Townes of the University of Alabama at Birmingham. Sickle-cell anaemia is a disease of the blood marrow caused by a defect in a single gene. The mouse model had been designed to include relevant human genes involved in blood production, including the defective version of that gene. To create the iPS cells, the scientists started with cells from the skin of the diseased mice, explains lead author Jacob Hanna, a postdoctoral researcher in the Jaenisch lab. These cells were modified by a standard lab technique employing retroviruses customized to insert genes into the cell’s DNA. The inserted genes were Oct4, Sox2, Lif4 and c-Myc, known to act together as master regulators to keep cells in an embryonic-stem-cell-like state. iPS cells were selected based on their morphology and then verified to express gene markers specific to embryonic stem cells. To decrease or eliminate possible cancer in the treated mice, the c-Myc gene was removed by genetic manipulation from the iPS cells. Next, the researchers followed a well-established protocol for differentiating embryonic stem cells into precursors of bone marrow adult stem cells, which can be transplanted into mice to generate normal blood cells. The scientists created such precursor cells from the iPS cells, replaced the defective blood-production gene in the precursor cells with a normal gene, and injected the resulting cells back into the diseased mice. The blood of treated mice was tested with standard analyses employed for human patients. The analyses showed that the disease was corrected, with measurements of blood and kidney functions similar to those of normal mice. “This demonstrates that iPS cells have the same potential for therapy as embryonic stem cells, without the ethical and practical issues raised in creating embryonic stem cells,” says Jaenisch. While iPS cells offer tremendous promise for regenerative medicine, scientists caution that major challenges must be overcome before medical applications can be considered. First among these is to find a better delivery system, since retroviruses bring other changes to the genome that are far too random to let loose in humans. “We need a delivery system that doesn’t integrate itself into the genome,” says Hanna. “Retroviruses can disrupt genes that should not be disrupted or activate genes that should not be activated.” Potential alternatives include other forms of viruses, synthesized versions of the proteins created by the four master regulator genes that are modified to enter the cell nucleus, and small molecules, Hanna says. Despite the rapid progress being made with iPS cells, Jaenisch emphasizes that this field is very young, and that it’s critical to continue full research on embryonic stem cells as well. “We wouldn’t have known anything about iPS cells if we hadn’t worked with embryonic stem cells,” says Jaenisch. “For the foreseeable future, there will remain a continued need for embryonic stem cells as the crucial assessment tool for measuring the therapeutic potential of iPS cells.” Reference: Treatment of Sickle-Cell Anaemia Mouse Model with iPS Cells Generated from Autologous Skin Science Express online, December 6, 2007 ......... ZenMaster

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