Jefferson Scientists Find New Way To Convert Adult Human Stem Cells To Dopamine Neurons
Researchers at Jefferson Medical College have found a new way to coax bone marrow stem cells into becoming dopamine-producing neurons. If the method proves reliable, the work may ultimately lead to new therapies for neurological diseases such as Parkinson's disease, which is marked by a loss of dopamine-making cells in the brain.
Developmental biologist Lorraine Iacovitti, Ph.D., associate director of the Farber Institute for Neurosciences at Thomas Jefferson University in Philadelphia and her co-workers had previously shown that by using a potion of growth factors and other nutrients in the laboratory, they were able to convert adult human bone marrow stem cells into adult brain cells. Human adult bone marrow stem cells – also known as pluripotent stem cells – normally give rise to human bone, muscle, cartilage and fat cells.
While nearly all cells looked like neurons with axonal processes, they invariably reverted back to their original undifferentiated state in two to three days.
Dr. Iacovitti and her co-workers instead attempted to grow the cells in a different way. Rather than an attached monolayer of skin-like cells, they grew the bone marrow cells in suspension as neurospheres – groups of cells early in development – akin to the way neural stem cells are grown.
They found that the newly differentiated cells didn't merely look like dopamine neurons, but expressed traits of neurons and related cells called astrocytes and oligodendrocytes – cells derived from neural stem cells. What's more, the neurons produced tyrosine hydroxylase, an enzyme needed to make dopamine.
She reports her team's findings October 25, 2004 at the annual meeting of the Society for Neuroscience in San Diego.
The Jefferson scientists also found a second enzyme involved in dopamine production, and an important molecule called the dopamine transporter.
Interestingly, Dr. Iacovitti notes, some of the cellular markers that would be expected to be expressed by new bone marrow cells were present in bone marrow stem cells grown in the original monolayers, though they were fewer in number.
"The markers don't disappear," explains Dr. Iacovitti, who is also professor of neurology at Jefferson Medical College of Thomas Jefferson University. "The cells seem to have markers of both bone marrow cells and dopamine neurons all the time. They don't forsake what they normally would be."
While she can't say for sure whether or not the stem cells grown with the new method have markers of both bone marrow stem cells and dopamine neurons, the new dopamine neurons did not revert back to stem cells.
"There are limitations to differentiating adult stem cells the way we want them – to get them to permanently give up being what they were meant to be and become neurons," she says. "Maybe this is a way to grow these stem cells to get them to truly become dopamine neurons instead of just looking like neurons.
"If we can now appropriately direct the differentiation of bone marrow stem cells, these cells could provide an abundant source of adult human neurons for use in the treatment of neurodegenerative diseases," she says.
Friday, December 7, 2007
Adult Cells, Reprogrammed To Embryonic Stem Cell Like State, Treat Sickle-cell Anemia In Mice
Adult Cells, Reprogrammed To Embryonic Stem Cell Like State, Treat Sickle-cell Anemia In Mice
Mice with a human sickle-cell anemia 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 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.
Scientists studied a therapeutic application of IPS cells with the sickle-cell anemia model mouse developed by the laboratory of Tim Townes of the University of Alabama at Birmingham. Sickle-cell anemia 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."
*The research article "Treatment of Sickle-Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin" was published online in Science Express on December 6, 2007.
Mice with a human sickle-cell anemia 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 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.
Scientists studied a therapeutic application of IPS cells with the sickle-cell anemia model mouse developed by the laboratory of Tim Townes of the University of Alabama at Birmingham. Sickle-cell anemia 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."
*The research article "Treatment of Sickle-Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin" was published online in Science Express on December 6, 2007.
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