Activation of Dormant Viruses May Cause ALS


Inactive viruses that litter the human genome may become reactivated and contribute to the development of motor neuron disease, according to new research published today in the journal Science Translational Medicine.

Human endogenous retroviruses (HERVs) are the flotsam and jetsam of ancient viruses that integrated into our chromosomes long ago as the results of retrovirus infections that occurred over several million years of our history.  These HERV sequences account for about 8% of human DNA and the vast majority of them have acquired multiple genetic mutations that made rendered them innocuous.  Therefore, HERVs are sometimes referred to as “junk” DNA, although some of these sequences have been shown to have function (for example, see Dupressoir A, Lavialle C, Heidmann T. Placenta. 2012 Sep;33(9):663-7).

In 2011, Avindra Nath, the intramural clinical director of the National Institute of Neurological Disorders and Stroke, and his colleagues reported that proteins synthesized by one such HERV known as HERV-K are found in very high concentrations in the brains of patients who died of amyotrophic lateral sclerosis (ALS), which is a progressive and fatal neurodegenerative disease that destroys those motor neurons that control speech, movement, swallowing and breathing, which leads to death between three to five years after the symptoms first appear.

In their new study, Nath’s research group investigated the toxicity of viral proteins to nerve cells. They examined samples of nervous tissue from 11 patients who had died of ALS, 10 Alzheimer’s patients, and 16 people who showed no signs of neurological disease as controls.  They used RNA sequencing to confirm that transcripts of three HERV-K genes are present in tissue samples from the ALS patients but not in those from the Alzheimer’s patients or control patients.  In their next set of experiments, Nath and his coworkers showed that the proteins encoded by these viral genes localized to motor neurons in the brains and front halves of the spinal cords of ALS patients.  This is significant, since the ventral or font portions of the spinal cord contains the cell bodies of motor neurons that send their axonal fibers to the body’s skeletal muscles where they synapse with those muscles.  Thus the presence of the viral proteins strongly correlates with the tendency of these cells to die.

To definitively test the toxicity of these viral proteins to neurons, Nath and others transfected either the entire viral genome, or just the viral env gene, which encodes the virus’s coat protein, into cultured human neurons.  Once integrated into the genomes of the cultured cells, the viral genes were fully activated and used the cell’s molecular machinery to synthesize their respective proteins.  Expression of these viral genes killed off significant numbers of cells and caused them to retract their neural fibers.  Furthermore expression of only the env gene in these cultured neurons was sufficient to kill them.

To test their hypothesis in a living animal, Nath and others generated a strain of genetically engineered mice whose neurons express high levels of the HERV-K env gene.  Behavioral tests showed that these HERV-K env+ animals developed motor function abnormalities; they had difficulty walking and balancing compared to healthy mice.  These symptoms progressed rapidly between 3 and 6 months of age, and half of the animals had died before or shortly after reaching 10 months of age.

Closer examination revealed that neurons in the motor cortex had degenerated.  They also showed a decrease in the length, branching and complexity of dendrites, and a reduction in the number of dendritic spines (small, finger-like extensions that receive chemical signals from other cells).

All of these data strongly suggest that reactivation of dormant HERV-K contributes to neurodegeneration in the brain and spinal cord.  The absence of this virus in the brains of Alzheimer’s patients supports the conclusion that reactivation of it causes degeneration, rather than being a consequence of it, and further suggests that it is specific to ALS.

ALS is associated with genetic mutations in more than 50 different genes.  However, as is the case for Alzheimer’s, these inherited forms of the disease, which account for just 10-15% of cases. But this study only examined patients with sporadic, or non-inherited, ALS, the cause of which have been much harder to pin down.

Further genetic analyses may identify DNA sequence variations, in the HERV-K genes themselves, and others that interact with them, which might make the virus more prone to reactivation.  More work will need to be done to determine exactly how the reactivated virus genes contribute to the disease.

Meanwhile, Nath and his colleagues are collaborating with researchers at Johns Hopkins University to determine if anti-retroviral drugs might alleviate disease symptoms in subsets of ALS patients.

See Li, W., et al. (2015). Human endogenous retrovirus-K contributes to motor neuron disease. Sci. Trans. Med., 7: 307ra153.

Human Stem Cell Gene Therapy Appears Safe and Effective


Two recent studies in the journal Science have reported the outcome of virally-mediated gene correction in hematopoietic stem cells (HSCs) to treat human patients. These studies may usher in a new era of safe and effective gene therapy. These exciting new clinical findings both come from the laboratory of Luigi Naldini at the San Raffaele Scientific Institute, Milan, Italy. The first experiment examined the treatment of metachromatic leukodystrophy (MLD), which is caused by mutations in the arylsulfatase A (ARSA) gene, and the second, investigated treatments for Wiskott-Aldrich syndrome (WAS), which is caused by mutations in the gene that encodes WASP.

MLD is one of several diseases that affects the lysosome; a structure in cells that acts as the garbage disposal of the cell. So called “lysosomal storage diseases” result from the inability of cells to degrade molecules that come to the lysosome for degradation. Without the ability to degrade these molecules, they build up to toxic levels and produce progressive motor and cognitive impairment and death within a few years of the onset of symptoms.

To treat MLD, workers in Naldini’s laboratory isolated blood-making stem cells from the bone marrow of three pre-symptomatic MLD patients (MLD01, 02 and 03). These stem cells were infected with genetically engineered viruses that encoded the human ARSA gene. After expanding these stem cells in culture, they were re-introduced into the MLD patients after those same patients had their resident bone marrow wiped out. The expression of the ARSA gene in the reconstituted bone marrow was greater than 10 fold the levels measured in healthy controls and there were no signs of blood cancers or other maladies. One month after the transplant, the implanted cells showed very high-level and stable engraftment. Between 45%-80% of cells isolated and grown from bone marrow samples harbored the fixed ARSA gene. AS expected, the levels of the ARSA protein rose to above-normal levels in therapeutically relevant blood cells and above normal levels of ARSA protein were isolated from hematopoietic cells after one month and cerebrospinal fluid (CSF) one to two years after transfusion. This is remarkable when you consider that one year before, no ARSA was seen. This shows that the implanted cells and their progeny properly homed to the right places in the body. The patient evaluations at time points beyond the expected age of disease onset was even more exciting, since these treat patients showed normal, continuous motor and cognitive development compared to their siblings who had MLD, but were untreated. The sibling of the patient designated “MLD01” was wheelchair-bound and unable to support their head and trunk at 39 months, but excitingly, after treatment, patient MLD01 was able to stand, walk and run at 39 months of age and showed signs of continuous motor and cognitive development. Lastly, and perhaps most importantly, there was no evidence of implanted cells becoming cancerous, even though they underwent self-renewal, like all good stem cells. This is the first report of an MLD patient at 39 months displaying such positive clinical features.

The second study treated WAS, which is an inherited disease that affects the immune system and leads to infections, abnormal platelets, scaly skin (eczema), blood tumors, and autoimmunity. In this second study, blood-making stem cells were collected from three patients infected with genetically engineered viruses that expressed the WASP gene. These stem cells were then reinfused intravenously (~11 million cells ) three days after collection. Blood tests and bone marrow biopsies showed evidence of robust engraftment of gene-corrected cells in bone marrow and peripheral blood up to 30 months later. WASP expression increased with time in most blood cells. Although serious adverse infectious events occurred in two patients, overall clinical improvement resulted in reduced disease severities in all patients. None of the three patients demonstrated signs of blood cancers and the platelet counts rose, but, unfortunately, not to normal levels. Again, no evidence for adverse effects were observed.

Simply put, these authors have presented a strategy for ex vivo gene correction in HSCs for inherited disorders which works and appears safe in comparison to previous strategies. Long-term analyses will undoubtedly need to be intensely scrutinized, but this research surely represents a huge step forward in the safe treatment of these and similar genetic disorders.

Physical Cues Push Mature Cells into Induced Pluripotent Stem Cells


Bioengineers from the laboratory of Song Li at UC Berkeley have used physical cues to help push mature cells to de-differentiate into embryonic-like cells known as induced pluripotent stem cells.

Essentially, Li and his coworkers grew skin fibroblasts from human skin and mouse ears on surfaces with parallel grooves 10 micrometers apart and 3 micrometers high, in a special culture medium. This procedure increased the efficiency of reprogramming of these mature cells four-fold when compared to cells grown on a flat surface. Growing cells in scaffolds of nanofilbers aligned in parallel had similar effects.

Li’s study could significantly advance the protocols for making induced pluripotent stem cells (iPSCs). Normally iPSCs are made by genetically engineering adult cells so that they overexpress four different genes: Oct-4, Sox-2, Klf-4, and c-Myc. To put these genes into the cells, genetically modified viruses are used, or plasmids (small circles of DNA). Initially, Shinya Yamanaka, the scientist who invented iPSCs, and his co-workers used retroviruses that contained these four genes. When fibroblasts were infected with these souped-up retroviruses, the viruses inserted their viral DNA into the genomes of the host cells and expressed these genes.

retrovirus_life_cycle

Shinya Yamanaka won the Nobel Prize for this work in Physiology or Medicine in 2012 for this work. Unfortunately, retroviruses and can cause insertional mutations when they integrate into the genome (Zheng W., et al., Gene. 2013 Apr 25;519(1):142-9), and for this reason they are not the preferred way of making iPSCs. There are other viral vectors that do not integrate into the genome of the host cell (e.g., Sendai virus; see Chen IP, et al., Cell Reprogram. 2013 Dec;15(6):503-13). There are also techniques that use plasmids, which encode the four genes but do not integrate into the genome of the host cell. Finally, synthetic messenger RNAs that encode these four genes have also been used to make iPSCs (Tavernier G,, et al., Biomaterials. 2012 Jan;33(2):412-7).

The use of physical cues to make iPSCs may replace the need for gene overexpression, just as the use of particular chemicals can replace the need for particular genes (Zhu, S. et al. Cell Stem Cell 7, 651–655 (2010); Li, Y. et al. Cell Res. 21, 196–204 (2011)). If physical cues can replace the need for the overexpression of particular genes, then this discovery could revolutionize iPSC derivation; especially since the overexpression of particular genes in mature cells tends to cause genome instability in cells (Doris Steinemann, Gudrun Göhring, and Brigitte Schlegelberger. Am J Stem Cells. 2013; 2(1): 39–51).

“Our study demonstrates for the first time that the physical features of biomaterials can replace some of these biochemical factors and regulate the memory of a cell’s identity,” said study principal investigator Song Li, UC Berkeley, Professor of bioengineering. “We show that biophysical signals can be converted into intracellular chemical signals that coax cells to change.”

a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p<0.05 (two-tailed, unpaired t-test) compared with the control flat surface. Error bars represent one standard deviation. g, Immunostaining of a stable iPSC line expanded from colonies generated on 10 μm grooves. These cells express mESC-specific markers Oct4, Sox2, Nanog and SSEA-1 (scale bar, 100 μm). h, The expanded iPSCs in g were transplanted into SCID mice to demonstrate the formation of teratomas in vivo (scale bar, 50 μm).
a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p

To boost the efficiency of mature cell reprogramming, scientists also use a chemical called valproic acid, which dramatically affects global DNA structure and expression.

“The concern with current methods is the low efficiency at which cells actually reprogram and the unpredictable long-term effects of certain imposed genetic or chemical manipulations,” said the lead author of this study Timothy Downing. “For instance, valproic acid is a potent chemical that drastically alters the cell’s epigenetic state and can cause unintended changes inside the cell. Given this, many people have been looking at different ways to improve various aspects of the reprogramming process.”

This new study confirms and extends previous studies that showed that mechanical and physical cues can influence cell fate. Li’s group showed that physical and mechanical cues can not only affect cell fate, but also the epigenetic state and cell reprogramming.

a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p<0.05 (two-tailed, unpaired t-test) compared with the control surface with random nanofibres. e, Fibroblasts were micropatterned into single-cell islands of 2,000 μm2 area with a CSI value of 1 (round) or 0.1 (elongated). After 24 h, cells were immunostained for AcH3, H3K4me2 or H3K4me3 (in green). Phalloidin staining (red) identifies the cell cytoskeleton for cell shape accuracy. The white arrowhead indicates the location of the nucleus (scale bars, 20 μm). f, Quantification of fluorescence intensity in e (n = 34, 20 and 34 for AcH3, H3K4me2 and H3K4me3, respectively). *p<0.05 (two-tailed, unpaired t-test) compared with the circular micropatterned cells (CSI = 1). Error bars represent one standard deviation.
a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p

“Cells elongate, or example, as they migrate throughout the body,” said Downing, who is a research associate in Li’s lab. “In the case of topography, where we control the elongation of a cell by controlling the physical microenvironment, we are able to more closely mimic what a cell would experience in its native physiological environment. In this regard, these physical cues are less invasive and artificial to the cell and therefore less likely to cause unintended side effects.”

Li and his colleagues are studying whether growing cells on grooved surfaces eventually replace valproic acid and even replace other chemical compounds in the reprogramming process.

“We are also studying whether biophysical factors could help reprogram cells into specific cell types, such as neurons,” said Jennifer Soto, a UC Berkeley graduate student in bioengineering who was also a co-author on this paper.

Timothy Downing, et al., Nature Materials 12, 1154–1162 (2013).  

Tests to Improve Stem Cell Safety


Stem cell scientists from the Commonwealth Scientific and Industrial Research Organisation or CSIRO (the Australian version of the NIH) have developed a test to identify unsafe pluripotent stem cells that can potentially cause tumors. This test is one of the first tests specifically designed for human induced pluripotent stem cells or iPSCs.

The development of this test marks a significant breakthrough in improving the quality of iPSCs and identifying unwanted stem cells that can form tumors. The test also directly assesses the stability of iPSCs when they are grown in the lab.

Andrew Laslett and his team have spent the last five years working on this research project and perfecting their test.

Laslett explained: “The test we have developed allows us to easily identify unsafe iPSC cells. Ensuring the safety of these cell lines is paramount and we hope this test will become a routine screen as part of developing safe and effective iPS-based cell therapies.”

Laslett’s research focused on comparing different types of iPS cells with human embryonic stem cells. Induced pluripotent stem cells are, at this time, the most commonly used type of pluripotent stem cell in research.

Laslett’s method has established that iPSCs made in certain ways are inherently less stable and riskier than those made by alternative means. For example, the classical way of making iPSCs, with genetically engineered retroviruses that insert their genes into the chromosomes of the cells they infect, can cause insertional mutations and are inherently more likely to cause tumors. In comparison, iPSCs made with viruses that do not integrate into the host cell’s DNA (that is, with genetically engineered adenoviruses), or made with plasmid DNA, mRNA or modified proteins, do not form tumors.

Laslett hopes the study and the new test method will help to raise the awareness and the importance of stem cell safety. He also predicts that tests like his will promote a kind of quality control over the production of iPSC lines.

“It is widely accepted that iPS cells made using viruses should not be used for human treatment, but they can also be used in research to understand diseases and identify new drugs. Having the assurance of safe and stable cells in all situations should be a priority,” said Laslett.

This test utilizes laser technology that activates fluorescent dyes attached to antibodies that are bound to specific cell surface proteins.  If the cell has the cell surface protein bound by the antibody, the cell and its surface proteins fluoresce, and it is sent into the positive test tube.  If it does not fluoresce, it is sent to the negative test tube.  This technique is called fluorescence activated cell sorting or FACS.  In order to identify proteins found the surfaces of iPSCs, Laslett’s team used dye-conjugated antibodies that bound to surface proteins TG30 (CD9) and GCTM-2.  The presence of these specific cell-surface proteins provides a means to separate cells into safe and unsafe cell lines.  Very early-stage differentiated stem cells that expressed TG30 (CD9) and GCTM-2 on their cell surfaces tend to dedifferentiate into pluripotent cells after differentiation and cause tumors, whereas those very early-stage differentiation stem cell lines that do not express TG30 (CD9) and GCTM-2 on their cell surfaces do not cause tumors.  After separation of the stem cell lines by FACS, the iPSC lines were further monitored as they grew in culture.  Unsafe iPS cell lines that form tumors usual clump together to make recognizable clusters of cells.  However, the safe iPS cell lines do no such thing. This test can also be applied to somatic cell nuclear transfer human embryonic stem cells.

Professor Martin Pera, the Program Leader of Stem Cells, Australia said, “Although cell transplantation therapies based on iPS cells are being fast tracked for testing in humans, there is still much debate in the scientific community over the potential hazards of this new technology.”

Induced Pluripotent Stem Cells


Embryonic stem cells might provide the means to heal a variety of physical ailments. However the problem with embryonic stem cells is not necessarily in their use, but in their derivation. In order to make embryonic stem cell lines, human embryos are destroyed.

The following video shows Alice Chen from Doug Melton’s laboratory at Harvard University destroying embryos to make embryonic stem cells:  http://www.jove.com/index/details.stp?ID=574.

Now that federal funding is available to not only work with existing embryonic stem cell lines but to MAKE new lines, there is nothing to stop researchers from thawing and (I’m sorry to be so blunt) killing human embryos. Can we have our “cake and eat it too?” Can we have the benefits of embryonic stem cells and not destroy embryos? Perhaps we can.

In 2001, Masako Tada reported the fusion of embryonic stem cells with a connective tissue cell called a fibroblast. This fusion reprograms the fibroblasts so that they behave like embryonic stem cells (Current Biology 11, no. 9 (2001): 1553–8). This suggests that something within embryonic stem cells can redirect the machinery of somatic cells to become more like that of embryonic stem cells. In 2006 Kazutoshi Takahashi and Shinya Yamanaka were able to generate embryonic stem cell lines by introducing four specific genes into mouse skin fibroblasts. These “induced pluripotent stem cells” (iPSCs) shared many of the properties of embryonic stem cells derived from embryos, but when transplanted into mouse embryos, they were not able to participate in the formation of an adult mouse (Cell 126, no. 4 (2006): 663–76). This experiment showed that it is possible to convert adult cells into something that resembles an embryonic stem cell. Could we push adult cells further? In 2007, three different research groups used retroviruses to transfer four different genes (Oct3/4, Sox2, c-Myc and Klf4) into mouse skin fibroblasts and completely transformed them into cells that had all the features and behaviors of embryonic stem cells (Cell Stem Cell 1, no. 1 (2007): 55–70; Nature 448 (2007): 313–7; Nature 448 (2007): 318–24.).

These experiments drew a great deal of excitement, but there were several safety concerns that had to be addressed before iPSCs could be used in human clinical trials.  Scientists used engineered retroviruses to introduce genes into adult cells in order to reprogram them into iPSCs (Current Topics in Microbiology and Immunology 261 (2002): 31-52).  Retroviruses insert a DNA copy of their genome into the chromosomes of the host cell they have infected.  If that viral DNA inserts into a gene, it can disrupt it and cause a mutation.  This can have dire consequences (see Folia Biologia 46 (2000): 226-32; Science 302 (2003): 415-9).  Fortunately this is not an intractable problem.  The conversion of adult cells into iPSCs only requires the transient expression of the inserted genes.  Secondly, scientists have created retroviruses that self-inactivate after their initial insertion (Journal of Virology 72 (1998): 8150-7; Virology 261, (1999).  One laboratory has also discovered a way to make iPSCs with a virus that does not insert into host cell chromosomes (Science 322 (2008): 945-9).  Other researchers have designed ingenious ways to move the necessary genes into adult cells without using viruses (Science 322 (2008): 949-53).  Both procedures avoid the dangers associated with the use of retroviruses.

A second concern involves the genes used to convert re-program adult cells into iPSCs.  One of these genes, c-Myc, is found in multiple copies in human and animal tumors.  Thus increasing the number of copies of the c-Myc gene might predispose such cells to form tumors (Recent Patents on Anticancer Drug Discovery 1 (2006): 305-26; Seminars in Cancer Biology 16 (2006): 318-30). Indeed, the increased ability of iPSCs made by Yamanaka to cause tumors in laboratory animals underscore this concern (Hepatology 46, no 3 (2009): 1049-9).  Several groups, however, have succeeded in making iPSCs from adult cells without the use of the c-Myc gene (Science 321 (2008): 699­-702; Nature Biotechnology 26 (2008): 101-6; Science 318 (2007): 1917–20), although the conversion is much less efficient.  Additionally, several groups have established that particular chemicals, in combination with the addition of a subset of the four genes originally used, can effectively transform particular cells into iPSCs (Cell Stem Cell 2 (2008): 525-8).   Thus the larger safety concerns facing iPSCs have been largely solved.

Finally, patient-specific iPSCs have been made in several labs, even though they have not been used in clinical trials to date.  Here is a short list of some of the diseases for which patient-specific iPSCs have been made:

Amylotrophic Lateral SclerosisScience 321 (2008): 1218­21.

Spinal Muscular AtrophyNature 457 (2009): 277­81.

Parkinson’ DiseaseCell 136, no. 5 (2009): 964­77.

Adenosine deaminase deficiency-related severe combined immunodeficiency – Cell 134, no. 5 (2008): 877­86.

Shwachman-Bodian-Diamond syndrome – Cell 134, no. 5 (2008): 877­86.

Gaucher disease – Cell 134, no. 5 (2008): 877­86.

Duchenne and Becker muscular dystrophy – Cell 134, no. 5 (2008): 877­86.

Huntington disease – Cell 134, no. 5 (2008): 877­86.

Juvenile-onset type 1 diabetes mellitus – Cell 134, no. 5 (2008): 877­86.

Down syndrome – Cell 134, no. 5 (2008): 877­86.

Lesch-Nyhan syndromeCell 134, no. 5 (2008): 877­86.

Thus iPSCs represent an exciting, embryo-free alternative to embryonic stem cells that provide essentially all of the opportunities for regenerative medicine without destroying embryos.