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An)] TJ ET 0.271 0.267 0.267 rg BT 152.513 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 157.934 663.247 Td /F1 9.8 Tf [(Daniel Montoro)] TJ ET 0.271 0.267 0.267 rg BT 224.038 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 229.459 663.247 Td /F1 9.8 Tf [(Lisa M. Ellerby)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Zhang N, An MC, Montoro D, Ellerby LM. Characterization of Human Huntingtons Disease Cell Model from Induced Pluripotent )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Stem Cells. PLOS Currents Huntington Disease. 2010 Oct 28 . Edition 1. doi: 10.1371/currents.RRN1193.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(exon of the gene Huntingtin \(Htt\). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(progresses. A useful advance in HD would be the generation of a human-derived HD model to use for drug screening and )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HD-iPS cells into )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(striatal fate required several steps. First, we generated nestin+/PAX6+/SOX1+/OCT4- neural stem cells \(NSCs\) from HD-iPS )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(cells using the method of embryoid body formation. HD-NSCs were then subjected to a differentiation condition combining )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(with ?-III tubulin, calbindin and GABA but not DARPP-32 \(dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000\) )] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons )] TJ ET BT 26.250 471.333 Td /F1 9.8 Tf [(resulted in striatal neurons expressing markers like DARPP-32. The HD-iPS cells derived striatal neurons and neuronal )] TJ ET BT 26.250 459.429 Td /F1 9.8 Tf [(precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. )] TJ ET BT 26.250 447.524 Td /F1 9.8 Tf [(Moreover, the HD-NSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs \(from )] TJ ET BT 26.250 435.619 Td /F1 9.8 Tf [(iPS or H9 NSCs\). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD )] TJ ET BT 26.250 423.714 Td /F1 9.8 Tf [(mechanisms and drug screening.)] TJ ET BT 26.250 387.112 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 367.158 Td /F1 9.8 Tf [(This work was funded by the Buck Institute for Age Research and NIH T32 training grant AG000266 \(MCA\).)] TJ ET BT 26.250 338.055 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(The Huntingtons disease \(HD\) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(the N-terminus of the huntingtin protein. Greater than 36-38 CAG repeats in huntingtin will cause HD and longer CAG repeat )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(lengths correlate with earlier onset of the disease )] TJ ET 0.267 0.267 0.267 rg BT 240.847 294.291 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 265.242 294.291 Td /F1 9.8 Tf [( . The most dramatic pathological change in HD brain is the massive )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(loss of medium spiny neurons \(MSNs\) in the striatum and loss of neurons in the cortex. The disease results in chorea, dementia )] TJ ET BT 26.250 270.482 Td /F1 9.8 Tf [(and eventually death. There are numerous mechanisms proposed for HD including proteolysis to generate toxic N-terminal )] TJ ET BT 26.250 258.577 Td /F1 9.8 Tf [(fragments, alterations in vesicular trafficking, mitochondrial function and transcriptional dysregulation )] TJ ET 0.267 0.267 0.267 rg BT 461.919 258.577 Td /F1 9.8 Tf [([3] [4])] TJ ET 0.271 0.267 0.267 rg BT 486.313 258.577 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(Currently there is no cure for HD. Treatments alleviate symptoms but do not prevent or delay disease progression )] TJ ET 0.267 0.267 0.267 rg BT 518.274 239.172 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 529.116 239.172 Td /F1 9.8 Tf [( . Studies )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(aimed at understanding the cause of MSN cell loss in HD and efforts to develop new therapeutics would benefit from the )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(generation of human medium spiny neurons carrying the genetic mutation for Htt. Recent technology to reprogram patient )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(specific skin fibroblasts to a pluripotent state offers this possibility )] TJ ET 0.267 0.267 0.267 rg BT 309.653 203.458 Td /F1 9.8 Tf [([6])] TJ ET 0.271 0.267 0.267 rg BT 320.495 203.458 Td /F1 9.8 Tf [( . Multiple high throughput screenings are also ongoing in )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(search for potential drug candidates using cell culture models derived from overexpression of human Htt or mouse knockin cells )] TJ ET 0.267 0.267 0.267 rg BT 26.250 179.649 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 37.092 179.649 Td /F1 9.8 Tf [( . Generation of a human and patient specific HD cell model would offer a number of advantages in our search for targets and )] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(therapeutics for HD including \(1\) accounting of genetic factors in each patients cell type, \(2\) generation of different cell types to )] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(understand selective vulnerability, \(3\) large supply human and patient specific primary cells, \(4\) ability to recapitulate HD )] TJ ET BT 26.250 143.934 Td /F1 9.8 Tf [(disease phenotype and \(5\) a possible cell therapy that avoids immune rejection.)] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(We have utilized a recently established HD-specific induced pluripotent stem cell \(iPSC\) line to generate a human HD cell )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(model with a CAG expansion mutation in the endogenous huntingtin gene. The HD-specific iPSC \(HD-iPSC\) line was originally )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(derived from a HD patient with a 72-repeat CAG tract by Park )] TJ ET BT 293.936 100.720 Td /F5 9.8 Tf [(et al)] TJ ET 0.267 0.267 0.267 rg BT 312.364 100.720 Td /F1 9.8 Tf [([8])] TJ ET 0.271 0.267 0.267 rg BT 323.206 100.720 Td /F1 9.8 Tf [( . Although mutant huntingtin is already expressed in HD-)] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(iPSCs \(unpublished data\), neuronal cells from the HD-iPSCs would more closely mimic the affected cells in HD. Here we show )] TJ ET BT 26.250 76.911 Td /F1 9.8 Tf [(that we can differentiate the HD specific neural stem cells \(HD-NSCs\) into neurons with striatal characteristics using a modified )] TJ ET BT 26.250 65.006 Td /F1 9.8 Tf [(protocol based on work of Aubry )] TJ ET BT 168.775 65.006 Td /F5 9.8 Tf [(et al)] TJ ET 0.267 0.267 0.267 rg BT 187.203 65.006 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 198.045 65.006 Td /F1 9.8 Tf [( . The HD-iPSC-derived neurons contain the same expanded CAG repeat number as )] TJ ET BT 26.250 53.101 Td /F1 9.8 Tf [(the original HD-iPSC line and the HD patient fibroblasts from which this HD-iPSC line was generated. One important feature of )] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(HD pathology is the elevation of caspase-3/7 activity. When we measure caspase-3/7 activity of the HD-NSCs and wild-type )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Characterization of Human Huntingtons Disease Cell Model )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(from Induced Pluripotent Stem Cells)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(October 28, 2010)] TJ ET 0.267 0.267 0.267 rg BT 26.250 663.247 Td /F1 9.8 Tf [(Ningzhe Zhang)] TJ ET 0.271 0.267 0.267 rg BT 92.365 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 97.786 663.247 Td /F1 9.8 Tf [(Mahru C. An)] TJ ET 0.271 0.267 0.267 rg BT 152.513 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 157.934 663.247 Td /F1 9.8 Tf [(Daniel Montoro)] TJ ET 0.271 0.267 0.267 rg BT 224.038 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 229.459 663.247 Td /F1 9.8 Tf [(Lisa M. Ellerby)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Zhang N, An MC, Montoro D, Ellerby LM. Characterization of Human Huntingtons Disease Cell Model from Induced Pluripotent )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Stem Cells. PLOS Currents Huntington Disease. 2010 Oct 28 . Edition 1. doi: 10.1371/currents.RRN1193.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(exon of the gene Huntingtin \(Htt\). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(progresses. A useful advance in HD would be the generation of a human-derived HD model to use for drug screening and )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HD-iPS cells into )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(striatal fate required several steps. First, we generated nestin+/PAX6+/SOX1+/OCT4- neural stem cells \(NSCs\) from HD-iPS )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(cells using the method of embryoid body formation. HD-NSCs were then subjected to a differentiation condition combining )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(with ?-III tubulin, calbindin and GABA but not DARPP-32 \(dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000\) )] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons )] TJ ET BT 26.250 471.333 Td /F1 9.8 Tf [(resulted in striatal neurons expressing markers like DARPP-32. The HD-iPS cells derived striatal neurons and neuronal )] TJ ET BT 26.250 459.429 Td /F1 9.8 Tf [(precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. )] TJ ET BT 26.250 447.524 Td /F1 9.8 Tf [(Moreover, the HD-NSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs \(from )] TJ ET BT 26.250 435.619 Td /F1 9.8 Tf [(iPS or H9 NSCs\). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD )] TJ ET BT 26.250 423.714 Td /F1 9.8 Tf [(mechanisms and drug screening.)] TJ ET BT 26.250 387.112 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 367.158 Td /F1 9.8 Tf [(This work was funded by the Buck Institute for Age Research and NIH T32 training grant AG000266 \(MCA\).)] TJ ET BT 26.250 338.055 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(The Huntingtons disease \(HD\) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(the N-terminus of the huntingtin protein. Greater than 36-38 CAG repeats in huntingtin will cause HD and longer CAG repeat )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(lengths correlate with earlier onset of the disease )] TJ ET 0.267 0.267 0.267 rg BT 240.847 294.291 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 265.242 294.291 Td /F1 9.8 Tf [( . The most dramatic pathological change in HD brain is the massive )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(loss of medium spiny neurons \(MSNs\) in the striatum and loss of neurons in the cortex. The disease results in chorea, dementia )] TJ ET BT 26.250 270.482 Td /F1 9.8 Tf [(and eventually death. There are numerous mechanisms proposed for HD including proteolysis to generate toxic N-terminal )] TJ ET BT 26.250 258.577 Td /F1 9.8 Tf [(fragments, alterations in vesicular trafficking, mitochondrial function and transcriptional dysregulation )] TJ ET 0.267 0.267 0.267 rg BT 461.919 258.577 Td /F1 9.8 Tf [([3] [4])] TJ ET 0.271 0.267 0.267 rg BT 486.313 258.577 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(Currently there is no cure for HD. Treatments alleviate symptoms but do not prevent or delay disease progression )] TJ ET 0.267 0.267 0.267 rg BT 518.274 239.172 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 529.116 239.172 Td /F1 9.8 Tf [( . Studies )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(aimed at understanding the cause of MSN cell loss in HD and efforts to develop new therapeutics would benefit from the )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(generation of human medium spiny neurons carrying the genetic mutation for Htt. Recent technology to reprogram patient )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(specific skin fibroblasts to a pluripotent state offers this possibility )] TJ ET 0.267 0.267 0.267 rg BT 309.653 203.458 Td /F1 9.8 Tf [([6])] TJ ET 0.271 0.267 0.267 rg BT 320.495 203.458 Td /F1 9.8 Tf [( . Multiple high throughput screenings are also ongoing in )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(search for potential drug candidates using cell culture models derived from overexpression of human Htt or mouse knockin cells )] TJ ET 0.267 0.267 0.267 rg BT 26.250 179.649 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 37.092 179.649 Td /F1 9.8 Tf [( . Generation of a human and patient specific HD cell model would offer a number of advantages in our search for targets and )] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(therapeutics for HD including \(1\) accounting of genetic factors in each patients cell type, \(2\) generation of different cell types to )] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(understand selective vulnerability, \(3\) large supply human and patient specific primary cells, \(4\) ability to recapitulate HD )] TJ ET BT 26.250 143.934 Td /F1 9.8 Tf [(disease phenotype and \(5\) a possible cell therapy that avoids immune rejection.)] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(We have utilized a recently established HD-specific induced pluripotent stem cell \(iPSC\) line to generate a human HD cell )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(model with a CAG expansion mutation in the endogenous huntingtin gene. The HD-specific iPSC \(HD-iPSC\) line was originally )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(derived from a HD patient with a 72-repeat CAG tract by Park )] TJ ET BT 293.936 100.720 Td /F5 9.8 Tf [(et al)] TJ ET 0.267 0.267 0.267 rg BT 312.364 100.720 Td /F1 9.8 Tf [([8])] TJ ET 0.271 0.267 0.267 rg BT 323.206 100.720 Td /F1 9.8 Tf [( . Although mutant huntingtin is already expressed in HD-)] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(iPSCs \(unpublished data\), neuronal cells from the HD-iPSCs would more closely mimic the affected cells in HD. Here we show )] TJ ET BT 26.250 76.911 Td /F1 9.8 Tf [(that we can differentiate the HD specific neural stem cells \(HD-NSCs\) into neurons with striatal characteristics using a modified )] TJ ET BT 26.250 65.006 Td /F1 9.8 Tf [(protocol based on work of Aubry )] TJ ET BT 168.775 65.006 Td /F5 9.8 Tf [(et al)] TJ ET 0.267 0.267 0.267 rg BT 187.203 65.006 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 198.045 65.006 Td /F1 9.8 Tf [( . The HD-iPSC-derived neurons contain the same expanded CAG repeat number as )] TJ ET BT 26.250 53.101 Td /F1 9.8 Tf [(the original HD-iPSC line and the HD patient fibroblasts from which this HD-iPSC line was generated. One important feature of )] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(HD pathology is the elevation of caspase-3/7 activity. When we measure caspase-3/7 activity of the HD-NSCs and wild-type )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Characterization of Human Huntingtons Disease Cell Model )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(from Induced Pluripotent Stem Cells)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(October 28, 2010)] TJ ET 0.267 0.267 0.267 rg BT 26.250 663.247 Td /F1 9.8 Tf [(Ningzhe Zhang)] TJ ET 0.271 0.267 0.267 rg BT 92.365 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 97.786 663.247 Td /F1 9.8 Tf [(Mahru C. An)] TJ ET 0.271 0.267 0.267 rg BT 152.513 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 157.934 663.247 Td /F1 9.8 Tf [(Daniel Montoro)] TJ ET 0.271 0.267 0.267 rg BT 224.038 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 229.459 663.247 Td /F1 9.8 Tf [(Lisa M. Ellerby)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Zhang N, An MC, Montoro D, Ellerby LM. Characterization of Human Huntingtons Disease Cell Model from Induced Pluripotent )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Stem Cells. PLOS Currents Huntington Disease. 2010 Oct 28 . Edition 1. doi: 10.1371/currents.RRN1193.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(exon of the gene Huntingtin \(Htt\). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(progresses. A useful advance in HD would be the generation of a human-derived HD model to use for drug screening and )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HD-iPS cells into )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(striatal fate required several steps. First, we generated nestin+/PAX6+/SOX1+/OCT4- neural stem cells \(NSCs\) from HD-iPS )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(cells using the method of embryoid body formation. HD-NSCs were then subjected to a differentiation condition combining )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained )] TJ ET BT 26.250 495.143 Td /F1 9.8 Tf [(with ?-III tubulin, calbindin and GABA but not DARPP-32 \(dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000\) )] TJ ET BT 26.250 483.238 Td /F1 9.8 Tf [(were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons )] TJ ET BT 26.250 471.333 Td /F1 9.8 Tf [(resulted in striatal neurons expressing markers like DARPP-32. The HD-iPS cells derived striatal neurons and neuronal )] TJ ET BT 26.250 459.429 Td /F1 9.8 Tf [(precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. )] TJ ET BT 26.250 447.524 Td /F1 9.8 Tf [(Moreover, the HD-NSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs \(from )] TJ ET BT 26.250 435.619 Td /F1 9.8 Tf [(iPS or H9 NSCs\). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD )] TJ ET BT 26.250 423.714 Td /F1 9.8 Tf [(mechanisms and drug screening.)] TJ ET BT 26.250 387.112 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 367.158 Td /F1 9.8 Tf [(This work was funded by the Buck Institute for Age Research and NIH T32 training grant AG000266 \(MCA\).)] TJ ET BT 26.250 338.055 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(The Huntingtons disease \(HD\) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(the N-terminus of the huntingtin protein. Greater than 36-38 CAG repeats in huntingtin will cause HD and longer CAG repeat )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(lengths correlate with earlier onset of the disease )] TJ ET 0.267 0.267 0.267 rg BT 240.847 294.291 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 265.242 294.291 Td /F1 9.8 Tf [( . The most dramatic pathological change in HD brain is the massive )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(loss of medium spiny neurons \(MSNs\) in the striatum and loss of neurons in the cortex. The disease results in chorea, dementia )] TJ ET BT 26.250 270.482 Td /F1 9.8 Tf [(and eventually death. There are numerous mechanisms proposed for HD including proteolysis to generate toxic N-terminal )] TJ ET BT 26.250 258.577 Td /F1 9.8 Tf [(fragments, alterations in vesicular trafficking, mitochondrial function and transcriptional dysregulation )] TJ ET 0.267 0.267 0.267 rg BT 461.919 258.577 Td /F1 9.8 Tf [([3] [4])] TJ ET 0.271 0.267 0.267 rg BT 486.313 258.577 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(Currently there is no cure for HD. Treatments alleviate symptoms but do not prevent or delay disease progression )] TJ ET 0.267 0.267 0.267 rg BT 518.274 239.172 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 529.116 239.172 Td /F1 9.8 Tf [( . Studies )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(aimed at understanding the cause of MSN cell loss in HD and efforts to develop new therapeutics would benefit from the )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(generation of human medium spiny neurons carrying the genetic mutation for Htt. Recent technology to reprogram patient )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(specific skin fibroblasts to a pluripotent state offers this possibility )] TJ ET 0.267 0.267 0.267 rg BT 309.653 203.458 Td /F1 9.8 Tf [([6])] TJ ET 0.271 0.267 0.267 rg BT 320.495 203.458 Td /F1 9.8 Tf [( . Multiple high throughput screenings are also ongoing in )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(search for potential drug candidates using cell culture models derived from overexpression of human Htt or mouse knockin cells )] TJ ET 0.267 0.267 0.267 rg BT 26.250 179.649 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 37.092 179.649 Td /F1 9.8 Tf [( . Generation of a human and patient specific HD cell model would offer a number of advantages in our search for targets and )] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(therapeutics for HD including \(1\) accounting of genetic factors in each patients cell type, \(2\) generation of different cell types to )] TJ ET BT 26.250 155.839 Td /F1 9.8 Tf [(understand selective vulnerability, \(3\) large supply human and patient specific primary cells, \(4\) ability to recapitulate HD )] TJ ET BT 26.250 143.934 Td /F1 9.8 Tf [(disease phenotype and \(5\) a possible cell therapy that avoids immune rejection.)] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(We have utilized a recently established HD-specific induced pluripotent stem cell \(iPSC\) line to generate a human HD cell )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(model with a CAG expansion mutation in the endogenous huntingtin gene. The HD-specific iPSC \(HD-iPSC\) line was originally )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(derived from a HD patient with a 72-repeat CAG tract by Park )] TJ ET BT 293.936 100.720 Td /F5 9.8 Tf [(et al)] TJ ET 0.267 0.267 0.267 rg BT 312.364 100.720 Td /F1 9.8 Tf [([8])] TJ ET 0.271 0.267 0.267 rg BT 323.206 100.720 Td /F1 9.8 Tf [( . Although mutant huntingtin is already expressed in HD-)] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(iPSCs \(unpublished data\), neuronal cells from the HD-iPSCs would more closely mimic the affected cells in HD. Here we show )] TJ ET BT 26.250 76.911 Td /F1 9.8 Tf [(that we can differentiate the HD specific neural stem cells \(HD-NSCs\) into neurons with striatal characteristics using a modified )] TJ ET BT 26.250 65.006 Td /F1 9.8 Tf [(protocol based on work of Aubry )] TJ ET BT 168.775 65.006 Td /F5 9.8 Tf [(et al)] TJ ET 0.267 0.267 0.267 rg BT 187.203 65.006 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 198.045 65.006 Td /F1 9.8 Tf [( . The HD-iPSC-derived neurons contain the same expanded CAG repeat number as )] TJ ET BT 26.250 53.101 Td /F1 9.8 Tf [(the original HD-iPSC line and the HD patient fibroblasts from which this HD-iPSC line was generated. One important feature of )] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(HD pathology is the elevation of caspase-3/7 activity. When we measure caspase-3/7 activity of the HD-NSCs and wild-type )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /Font /Subtype /Type1 /Name /F5 /BaseFont /Helvetica-Oblique /Encoding /WinAnsiEncoding >> endobj 13 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 14 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 13 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 4223>> stream xە8=,8C#h!hGv#0=j$q1uaNĥT*J&a_Zaa0 a0 a0 H^UUUUX!0 3SY|^G0 ÌÔ{]wa_>"OeYaG!8e}a6̖{4M&d:"qʲ\QUUMS<ϛNld2l6Z a<=2B(2vK7M\.GW0 rrWWavߖe9C?Eq8Gqy~~}_qdϽ(neYʒGjK4EQNjͷm{X?|7m۶mqP:Ȳp8Kmu]lNzuZ,NTeY"jlUUQW\,aRiVbȗLkHkEsjd.L6=Iו P]1t7Sc2uIuMM; K5>A$Ǒ$I׋nY 0_Hat;aq䃰U$nR ,zO7bT mhhԫܻu1F*qm'P}ܙ<ϕALO6Lw3nB.T4co{߽.lV $laԀIu!gwChxJ]Ne (+M"d-Eqc=P ! 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Our results indicate that the HD-NSCs might serve as a human HD cell model with )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(endogenous CAG expansion suitable for HD mechanistic studies and drug screenings.)] TJ ET BT 26.250 707.064 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 687.110 Td /F4 9.8 Tf [(1. HD-iPSCs maintain ES cell markers after extended culture)] TJ ET BT 26.250 667.705 Td /F1 9.8 Tf [(We cultured the HD-iPSCs and normal iPSCs \( from Dr. George Daley\) in the same conditions of conventional human ESCs )] TJ ET BT 26.250 655.800 Td /F1 9.8 Tf [(and immunostained these cells periodically to ensure expression of hESC markers. Previous work demonstrated that the HD-)] TJ ET BT 26.250 643.896 Td /F1 9.8 Tf [(iPSCs were pluripotent and capable of multilineage differentiation \(8\) . After extended culture of the HD-iPSCs on either MEF )] TJ ET BT 26.250 631.991 Td /F1 9.8 Tf [(feeder cells or Matrigel \(around 60 passages\) these cells still stained positive for classic hESC markers including OCT4, )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(NANOG, SOX2 and SSEA4 [Figure 1], indicating HD-iPSCs retained stem cell markers during passaging. We did note that the )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(normal-iPSCs and H9 ESCs when compared to HD-iPSCs had distinct levels of ERK activation in response to bFGF and the )] TJ ET BT 26.250 596.277 Td /F1 9.8 Tf [(level of ERK was significantly dampened in HD-iPSCs. This is consistent with previous reports in the literature of HD cell culture )] TJ ET BT 26.250 584.372 Td /F1 9.8 Tf [(models of altered ERK activation )] TJ ET 0.267 0.267 0.267 rg BT 170.394 584.372 Td /F1 9.8 Tf [([10])] TJ ET 0.271 0.267 0.267 rg BT 186.657 584.372 Td /F1 9.8 Tf [( [Figure 2].)] TJ ET 0.965 0.965 0.965 rg 26.250 364.864 555.000 209.627 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 574.491 m 581.250 574.491 l 581.250 573.741 l 26.250 573.741 l f 26.250 364.864 m 581.250 364.864 l 581.250 365.614 l 26.250 365.614 l f q 225.000 0 0 129.750 35.250 434.991 cm /I3 Do Q q 35.250 376.114 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 419.467 Td /F4 9.8 Tf [(Fig. 1: HD-iPS cells express classic ES cell markers.)] TJ ET BT 35.250 400.097 Td /F1 9.8 Tf [(HD-iPS cells were cultured in the same conditions as human ES cells and immunostained for classic ES cell markers )] TJ ET BT 35.250 386.361 Td /F1 9.8 Tf [(OCT4, NANOG, SOX2 and SSEA4.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 114.264 555.000 243.100 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 357.364 m 581.250 357.364 l 581.250 356.614 l 26.250 356.614 l f 26.250 114.264 m 581.250 114.264 l 581.250 115.014 l 26.250 115.014 l f q 225.000 0 0 135.750 35.250 211.864 cm /I4 Do Q q 35.250 125.514 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 196.340 Td /F4 9.8 Tf [(Fig. 2: HD-iPSCs have altered signaling pathways when compared to control iPS.)] TJ ET BT 35.250 176.970 Td /F1 9.8 Tf [(HD-iPSCs, normal iPSCs and H9 cells were treated with 0, 4 or 20 ng/ml bFGF 24h after previous feeding. 30 min after )] TJ ET BT 35.250 163.234 Td /F1 9.8 Tf [(bFGF treatment cells were harvested for protein lysates. Western blotting was performed to detect phospho-p44/42 )] TJ ET BT 35.250 149.497 Td /F1 9.8 Tf [(\(ERK1/2\) \(Thr202/Tyr204\) as well as total p44/42 \(ERK1/2\) in these cells. The two bands represent p44 and p42 \(either )] TJ ET BT 35.250 135.761 Td /F1 9.8 Tf [(phosphor form or total protein\) respectively.)] TJ ET Q BT 26.250 97.240 Td /F4 9.8 Tf [(2. HD-iPSCs can be differentiated into HD-NSCs)] TJ ET BT 26.250 77.835 Td /F1 9.8 Tf [(We differentiated HD-iPSCs into neural lineages with a previously established method that utilizes the formation of embryoid )] TJ ET BT 26.250 65.931 Td /F1 9.8 Tf [(body \(EB\) intermediates. Neural rosettes appeared after attachment of EBs onto poly-ornithine/laminin \(pO/L\) coated surfaces )] TJ ET BT 26.250 54.026 Td /F1 9.8 Tf [(in neural differentiation medium supplemented with bFGF. We manually isolated the rosette cells, disrupted rosettes into smaller )] TJ ET BT 26.250 42.121 Td /F1 9.8 Tf [(pieces mechanically and plated these rosette cells on pO/L coated surfaces in neural proliferation medium supplemented with )] TJ ET Q q 15.000 27.835 577.500 749.165 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(NSCs \(WT-NSCs from normal iPS or H9 ESCs\) 24 hours after withdrawal of growth factors, the HD-NSCs but not WT-NSCs )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(showed enhanced caspase activity. Our results indicate that the HD-NSCs might serve as a human HD cell model with )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(endogenous CAG expansion suitable for HD mechanistic studies and drug screenings.)] TJ ET BT 26.250 707.064 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 687.110 Td /F4 9.8 Tf [(1. HD-iPSCs maintain ES cell markers after extended culture)] TJ ET BT 26.250 667.705 Td /F1 9.8 Tf [(We cultured the HD-iPSCs and normal iPSCs \( from Dr. George Daley\) in the same conditions of conventional human ESCs )] TJ ET BT 26.250 655.800 Td /F1 9.8 Tf [(and immunostained these cells periodically to ensure expression of hESC markers. Previous work demonstrated that the HD-)] TJ ET BT 26.250 643.896 Td /F1 9.8 Tf [(iPSCs were pluripotent and capable of multilineage differentiation \(8\) . After extended culture of the HD-iPSCs on either MEF )] TJ ET BT 26.250 631.991 Td /F1 9.8 Tf [(feeder cells or Matrigel \(around 60 passages\) these cells still stained positive for classic hESC markers including OCT4, )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(NANOG, SOX2 and SSEA4 [Figure 1], indicating HD-iPSCs retained stem cell markers during passaging. We did note that the )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(normal-iPSCs and H9 ESCs when compared to HD-iPSCs had distinct levels of ERK activation in response to bFGF and the )] TJ ET BT 26.250 596.277 Td /F1 9.8 Tf [(level of ERK was significantly dampened in HD-iPSCs. This is consistent with previous reports in the literature of HD cell culture )] TJ ET BT 26.250 584.372 Td /F1 9.8 Tf [(models of altered ERK activation )] TJ ET 0.267 0.267 0.267 rg BT 170.394 584.372 Td /F1 9.8 Tf [([10])] TJ ET 0.271 0.267 0.267 rg BT 186.657 584.372 Td /F1 9.8 Tf [( [Figure 2].)] TJ ET 0.965 0.965 0.965 rg 26.250 364.864 555.000 209.627 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 574.491 m 581.250 574.491 l 581.250 573.741 l 26.250 573.741 l f 26.250 364.864 m 581.250 364.864 l 581.250 365.614 l 26.250 365.614 l f q 225.000 0 0 129.750 35.250 434.991 cm /I3 Do Q q 35.250 376.114 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 419.467 Td /F4 9.8 Tf [(Fig. 1: HD-iPS cells express classic ES cell markers.)] TJ ET BT 35.250 400.097 Td /F1 9.8 Tf [(HD-iPS cells were cultured in the same conditions as human ES cells and immunostained for classic ES cell markers )] TJ ET BT 35.250 386.361 Td /F1 9.8 Tf [(OCT4, NANOG, SOX2 and SSEA4.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 114.264 555.000 243.100 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 357.364 m 581.250 357.364 l 581.250 356.614 l 26.250 356.614 l f 26.250 114.264 m 581.250 114.264 l 581.250 115.014 l 26.250 115.014 l f q 225.000 0 0 135.750 35.250 211.864 cm /I4 Do Q q 35.250 125.514 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 196.340 Td /F4 9.8 Tf [(Fig. 2: HD-iPSCs have altered signaling pathways when compared to control iPS.)] TJ ET BT 35.250 176.970 Td /F1 9.8 Tf [(HD-iPSCs, normal iPSCs and H9 cells were treated with 0, 4 or 20 ng/ml bFGF 24h after previous feeding. 30 min after )] TJ ET BT 35.250 163.234 Td /F1 9.8 Tf [(bFGF treatment cells were harvested for protein lysates. Western blotting was performed to detect phospho-p44/42 )] TJ ET BT 35.250 149.497 Td /F1 9.8 Tf [(\(ERK1/2\) \(Thr202/Tyr204\) as well as total p44/42 \(ERK1/2\) in these cells. The two bands represent p44 and p42 \(either )] TJ ET BT 35.250 135.761 Td /F1 9.8 Tf [(phosphor form or total protein\) respectively.)] TJ ET Q BT 26.250 97.240 Td /F4 9.8 Tf [(2. HD-iPSCs can be differentiated into HD-NSCs)] TJ ET BT 26.250 77.835 Td /F1 9.8 Tf [(We differentiated HD-iPSCs into neural lineages with a previously established method that utilizes the formation of embryoid )] TJ ET BT 26.250 65.931 Td /F1 9.8 Tf [(body \(EB\) intermediates. Neural rosettes appeared after attachment of EBs onto poly-ornithine/laminin \(pO/L\) coated surfaces )] TJ ET BT 26.250 54.026 Td /F1 9.8 Tf [(in neural differentiation medium supplemented with bFGF. We manually isolated the rosette cells, disrupted rosettes into smaller )] TJ ET BT 26.250 42.121 Td /F1 9.8 Tf [(pieces mechanically and plated these rosette cells on pO/L coated surfaces in neural proliferation medium supplemented with )] TJ ET Q q 15.000 27.835 577.500 749.165 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(NSCs \(WT-NSCs from normal iPS or H9 ESCs\) 24 hours after withdrawal of growth factors, the HD-NSCs but not WT-NSCs )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(showed enhanced caspase activity. Our results indicate that the HD-NSCs might serve as a human HD cell model with )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(endogenous CAG expansion suitable for HD mechanistic studies and drug screenings.)] TJ ET BT 26.250 707.064 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 687.110 Td /F4 9.8 Tf [(1. HD-iPSCs maintain ES cell markers after extended culture)] TJ ET BT 26.250 667.705 Td /F1 9.8 Tf [(We cultured the HD-iPSCs and normal iPSCs \( from Dr. George Daley\) in the same conditions of conventional human ESCs )] TJ ET BT 26.250 655.800 Td /F1 9.8 Tf [(and immunostained these cells periodically to ensure expression of hESC markers. Previous work demonstrated that the HD-)] TJ ET BT 26.250 643.896 Td /F1 9.8 Tf [(iPSCs were pluripotent and capable of multilineage differentiation \(8\) . After extended culture of the HD-iPSCs on either MEF )] TJ ET BT 26.250 631.991 Td /F1 9.8 Tf [(feeder cells or Matrigel \(around 60 passages\) these cells still stained positive for classic hESC markers including OCT4, )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(NANOG, SOX2 and SSEA4 [Figure 1], indicating HD-iPSCs retained stem cell markers during passaging. We did note that the )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(normal-iPSCs and H9 ESCs when compared to HD-iPSCs had distinct levels of ERK activation in response to bFGF and the )] TJ ET BT 26.250 596.277 Td /F1 9.8 Tf [(level of ERK was significantly dampened in HD-iPSCs. This is consistent with previous reports in the literature of HD cell culture )] TJ ET BT 26.250 584.372 Td /F1 9.8 Tf [(models of altered ERK activation )] TJ ET 0.267 0.267 0.267 rg BT 170.394 584.372 Td /F1 9.8 Tf [([10])] TJ ET 0.271 0.267 0.267 rg BT 186.657 584.372 Td /F1 9.8 Tf [( [Figure 2].)] TJ ET 0.965 0.965 0.965 rg 26.250 364.864 555.000 209.627 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 574.491 m 581.250 574.491 l 581.250 573.741 l 26.250 573.741 l f 26.250 364.864 m 581.250 364.864 l 581.250 365.614 l 26.250 365.614 l f q 225.000 0 0 129.750 35.250 434.991 cm /I3 Do Q q 35.250 376.114 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 419.467 Td /F4 9.8 Tf [(Fig. 1: HD-iPS cells express classic ES cell markers.)] TJ ET BT 35.250 400.097 Td /F1 9.8 Tf [(HD-iPS cells were cultured in the same conditions as human ES cells and immunostained for classic ES cell markers )] TJ ET BT 35.250 386.361 Td /F1 9.8 Tf [(OCT4, NANOG, SOX2 and SSEA4.)] TJ ET Q 0.965 0.965 0.965 rg 26.250 114.264 555.000 243.100 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 357.364 m 581.250 357.364 l 581.250 356.614 l 26.250 356.614 l f 26.250 114.264 m 581.250 114.264 l 581.250 115.014 l 26.250 115.014 l f q 225.000 0 0 135.750 35.250 211.864 cm /I4 Do Q q 35.250 125.514 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 196.340 Td /F4 9.8 Tf [(Fig. 2: HD-iPSCs have altered signaling pathways when compared to control iPS.)] TJ ET BT 35.250 176.970 Td /F1 9.8 Tf [(HD-iPSCs, normal iPSCs and H9 cells were treated with 0, 4 or 20 ng/ml bFGF 24h after previous feeding. 30 min after )] TJ ET BT 35.250 163.234 Td /F1 9.8 Tf [(bFGF treatment cells were harvested for protein lysates. Western blotting was performed to detect phospho-p44/42 )] TJ ET BT 35.250 149.497 Td /F1 9.8 Tf [(\(ERK1/2\) \(Thr202/Tyr204\) as well as total p44/42 \(ERK1/2\) in these cells. The two bands represent p44 and p42 \(either )] TJ ET BT 35.250 135.761 Td /F1 9.8 Tf [(phosphor form or total protein\) respectively.)] TJ ET Q BT 26.250 97.240 Td /F4 9.8 Tf [(2. HD-iPSCs can be differentiated into HD-NSCs)] TJ ET BT 26.250 77.835 Td /F1 9.8 Tf [(We differentiated HD-iPSCs into neural lineages with a previously established method that utilizes the formation of embryoid )] TJ ET BT 26.250 65.931 Td /F1 9.8 Tf [(body \(EB\) intermediates. Neural rosettes appeared after attachment of EBs onto poly-ornithine/laminin \(pO/L\) coated surfaces )] TJ ET BT 26.250 54.026 Td /F1 9.8 Tf [(in neural differentiation medium supplemented with bFGF. 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After the first passage with 0.05% trypsin these monolayer cells were cultured routinely as NSCs. The yield for this step )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(was greater than 95%. Immunofluorescent staining showed that these cells expressed NSC markers Nestin, SOX1 and PAX6 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(but not the ESC marker OCT4 [Figure 3].)] TJ ET 0.965 0.965 0.965 rg 26.250 584.158 555.000 149.627 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 733.786 m 581.250 733.786 l 581.250 733.036 l 26.250 733.036 l f 26.250 584.158 m 581.250 584.158 l 581.250 584.908 l 26.250 584.908 l f q 225.000 0 0 69.750 35.250 654.286 cm /I5 Do Q q 35.250 595.408 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 638.762 Td /F4 9.8 Tf [(Fig. 3: HD-NSCs derived from HD-iPS cells.)] TJ ET BT 35.250 619.392 Td /F1 9.8 Tf [(The HD-NSCs derived from HD-iPS cells immunostained positive for NSC markers Nestin, SOX1 and PAX6 while )] TJ ET BT 35.250 605.656 Td /F1 9.8 Tf [(negatively for ES cell marker OCT4.)] TJ ET Q BT 26.250 567.135 Td /F4 9.8 Tf [(3. HD-NSCs can be differentiated into striatal neurons)] TJ ET BT 26.250 547.730 Td /F1 9.8 Tf [(Next we further developed conditions to produce striatal neurons from the HD-NSCs. We modified a previously published )] TJ ET BT 26.250 535.825 Td /F1 9.8 Tf [(protocol based on work of Aubry et al. )] TJ ET 0.267 0.267 0.267 rg BT 192.624 535.825 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 203.466 535.825 Td /F1 9.8 Tf [( by treating the HD-NSCs directly with SHH, DKK1 and BDNF. In addition, we included )] TJ ET BT 26.250 523.920 Td /F1 9.8 Tf [(the ROCK inhibitor Y27632 in the differentiation condition to promote cell survival. After 8-10 days in SHH, DKK1, BDNF and )] TJ ET BT 26.250 512.016 Td /F1 9.8 Tf [(Y27632 \(Stage 1\), cells were further differentiated with BDNF, cAMP, valproic acid and Y27632 for an additional 1-3 days )] TJ ET BT 26.250 500.111 Td /F1 9.8 Tf [(\(Stage 2\). At Stage 1, cells already expressed GABAergic neuron marker GABA, some even stained with striatal neuron marker )] TJ ET BT 26.250 488.206 Td /F1 9.8 Tf [(calbindin, but no MSN specific marker DARPP-32 positive cells were observed [Figure 4A]. At stage 2 some cells started to )] TJ ET BT 26.250 476.301 Td /F1 9.8 Tf [(express DARPP-32 [Figure 4B]. This differentiation experiment demonstrated that these HD-NSCs were capable of giving rise )] TJ ET BT 26.250 464.397 Td /F1 9.8 Tf [(to striatal neurons, which may also be used as an HD cell model. The yield of DARPP-32 neurons was approximately 10%. )] TJ ET BT 26.250 452.492 Td /F1 9.8 Tf [(Since we induced striatal differentiation stepwise, it is not easy to estimate overall percentage of striatal neurons generated from )] TJ ET BT 26.250 440.587 Td /F1 9.8 Tf [(original iPS cells. After EB attachment rosettes represented a small portion of total cells \(approximately 20%\). However these )] TJ ET BT 26.250 428.682 Td /F1 9.8 Tf [(rosettes cells are enriched NSCs \(greater than 95% based on Nestin staining\) and they can be passaged for over 20 passages )] TJ ET BT 26.250 416.778 Td /F1 9.8 Tf [(and still stain positive for NSC markers Nestin, Sox1 and Pax6. So the amount of NSCs was not a limiting factor. The yield from )] TJ ET BT 26.250 404.873 Td /F1 9.8 Tf [(NSCs to DARPP32 positive neurons was low. Higher purity DARPP32 neurons may require introducing a striatal specific )] TJ ET BT 26.250 392.968 Td /F1 9.8 Tf [(reporter and cell sorting.)] TJ ET 0.965 0.965 0.965 rg 26.250 159.724 555.000 223.364 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 383.087 m 581.250 383.087 l 581.250 382.337 l 26.250 382.337 l f 26.250 159.724 m 581.250 159.724 l 581.250 160.474 l 26.250 160.474 l f q 225.000 0 0 129.750 35.250 243.587 cm /I6 Do Q q 35.250 170.974 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 228.063 Td /F4 9.8 Tf [(Fig. 4: Striatal neurons derived from HD-iPS cells.)] TJ ET BT 35.250 208.693 Td /F1 9.8 Tf [(A \(Stage 1\) Immature striatal neurons express neuronal marker ?III-tubulin \(Tuj1\), GABAergic neuron marker GABA and )] TJ ET BT 35.250 194.957 Td /F1 9.8 Tf [(striatal marker calbindin but not medium spiny neuron \(MSN\) marker DARPP-32. B \(Stage 2\) Mature striatal neurons )] TJ ET BT 35.250 181.221 Td /F1 9.8 Tf [(express DARPP32 as well as well as ?III-tubulin, GABA and Calbindin.)] TJ ET Q BT 26.250 142.700 Td /F4 9.8 Tf [(4. The endogenous HD mutation persisted in all cell types)] TJ ET Q q 15.000 30.081 577.500 746.919 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(bFGF. After the first passage with 0.05% trypsin these monolayer cells were cultured routinely as NSCs. The yield for this step )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(was greater than 95%. Immunofluorescent staining showed that these cells expressed NSC markers Nestin, SOX1 and PAX6 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(but not the ESC marker OCT4 [Figure 3].)] TJ ET 0.965 0.965 0.965 rg 26.250 584.158 555.000 149.627 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 733.786 m 581.250 733.786 l 581.250 733.036 l 26.250 733.036 l f 26.250 584.158 m 581.250 584.158 l 581.250 584.908 l 26.250 584.908 l f q 225.000 0 0 69.750 35.250 654.286 cm /I5 Do Q q 35.250 595.408 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 638.762 Td /F4 9.8 Tf [(Fig. 3: HD-NSCs derived from HD-iPS cells.)] TJ ET BT 35.250 619.392 Td /F1 9.8 Tf [(The HD-NSCs derived from HD-iPS cells immunostained positive for NSC markers Nestin, SOX1 and PAX6 while )] TJ ET BT 35.250 605.656 Td /F1 9.8 Tf [(negatively for ES cell marker OCT4.)] TJ ET Q BT 26.250 567.135 Td /F4 9.8 Tf [(3. HD-NSCs can be differentiated into striatal neurons)] TJ ET BT 26.250 547.730 Td /F1 9.8 Tf [(Next we further developed conditions to produce striatal neurons from the HD-NSCs. We modified a previously published )] TJ ET BT 26.250 535.825 Td /F1 9.8 Tf [(protocol based on work of Aubry et al. )] TJ ET 0.267 0.267 0.267 rg BT 192.624 535.825 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 203.466 535.825 Td /F1 9.8 Tf [( by treating the HD-NSCs directly with SHH, DKK1 and BDNF. In addition, we included )] TJ ET BT 26.250 523.920 Td /F1 9.8 Tf [(the ROCK inhibitor Y27632 in the differentiation condition to promote cell survival. After 8-10 days in SHH, DKK1, BDNF and )] TJ ET BT 26.250 512.016 Td /F1 9.8 Tf [(Y27632 \(Stage 1\), cells were further differentiated with BDNF, cAMP, valproic acid and Y27632 for an additional 1-3 days )] TJ ET BT 26.250 500.111 Td /F1 9.8 Tf [(\(Stage 2\). At Stage 1, cells already expressed GABAergic neuron marker GABA, some even stained with striatal neuron marker )] TJ ET BT 26.250 488.206 Td /F1 9.8 Tf [(calbindin, but no MSN specific marker DARPP-32 positive cells were observed [Figure 4A]. At stage 2 some cells started to )] TJ ET BT 26.250 476.301 Td /F1 9.8 Tf [(express DARPP-32 [Figure 4B]. This differentiation experiment demonstrated that these HD-NSCs were capable of giving rise )] TJ ET BT 26.250 464.397 Td /F1 9.8 Tf [(to striatal neurons, which may also be used as an HD cell model. The yield of DARPP-32 neurons was approximately 10%. )] TJ ET BT 26.250 452.492 Td /F1 9.8 Tf [(Since we induced striatal differentiation stepwise, it is not easy to estimate overall percentage of striatal neurons generated from )] TJ ET BT 26.250 440.587 Td /F1 9.8 Tf [(original iPS cells. After EB attachment rosettes represented a small portion of total cells \(approximately 20%\). However these )] TJ ET BT 26.250 428.682 Td /F1 9.8 Tf [(rosettes cells are enriched NSCs \(greater than 95% based on Nestin staining\) and they can be passaged for over 20 passages )] TJ ET BT 26.250 416.778 Td /F1 9.8 Tf [(and still stain positive for NSC markers Nestin, Sox1 and Pax6. So the amount of NSCs was not a limiting factor. The yield from )] TJ ET BT 26.250 404.873 Td /F1 9.8 Tf [(NSCs to DARPP32 positive neurons was low. Higher purity DARPP32 neurons may require introducing a striatal specific )] TJ ET BT 26.250 392.968 Td /F1 9.8 Tf [(reporter and cell sorting.)] TJ ET 0.965 0.965 0.965 rg 26.250 159.724 555.000 223.364 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 383.087 m 581.250 383.087 l 581.250 382.337 l 26.250 382.337 l f 26.250 159.724 m 581.250 159.724 l 581.250 160.474 l 26.250 160.474 l f q 225.000 0 0 129.750 35.250 243.587 cm /I6 Do Q q 35.250 170.974 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 228.063 Td /F4 9.8 Tf [(Fig. 4: Striatal neurons derived from HD-iPS cells.)] TJ ET BT 35.250 208.693 Td /F1 9.8 Tf [(A \(Stage 1\) Immature striatal neurons express neuronal marker ?III-tubulin \(Tuj1\), GABAergic neuron marker GABA and )] TJ ET BT 35.250 194.957 Td /F1 9.8 Tf [(striatal marker calbindin but not medium spiny neuron \(MSN\) marker DARPP-32. B \(Stage 2\) Mature striatal neurons )] TJ ET BT 35.250 181.221 Td /F1 9.8 Tf [(express DARPP32 as well as well as ?III-tubulin, GABA and Calbindin.)] TJ ET Q BT 26.250 142.700 Td /F4 9.8 Tf [(4. The endogenous HD mutation persisted in all cell types)] TJ ET Q q 15.000 30.081 577.500 746.919 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(bFGF. After the first passage with 0.05% trypsin these monolayer cells were cultured routinely as NSCs. The yield for this step )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(was greater than 95%. Immunofluorescent staining showed that these cells expressed NSC markers Nestin, SOX1 and PAX6 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(but not the ESC marker OCT4 [Figure 3].)] TJ ET 0.965 0.965 0.965 rg 26.250 584.158 555.000 149.627 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 733.786 m 581.250 733.786 l 581.250 733.036 l 26.250 733.036 l f 26.250 584.158 m 581.250 584.158 l 581.250 584.908 l 26.250 584.908 l f q 225.000 0 0 69.750 35.250 654.286 cm /I5 Do Q q 35.250 595.408 537.000 52.877 re W n 0.271 0.267 0.267 rg BT 35.250 638.762 Td /F4 9.8 Tf [(Fig. 3: HD-NSCs derived from HD-iPS cells.)] TJ ET BT 35.250 619.392 Td /F1 9.8 Tf [(The HD-NSCs derived from HD-iPS cells immunostained positive for NSC markers Nestin, SOX1 and PAX6 while )] TJ ET BT 35.250 605.656 Td /F1 9.8 Tf [(negatively for ES cell marker OCT4.)] TJ ET Q BT 26.250 567.135 Td /F4 9.8 Tf [(3. HD-NSCs can be differentiated into striatal neurons)] TJ ET BT 26.250 547.730 Td /F1 9.8 Tf [(Next we further developed conditions to produce striatal neurons from the HD-NSCs. We modified a previously published )] TJ ET BT 26.250 535.825 Td /F1 9.8 Tf [(protocol based on work of Aubry et al. )] TJ ET 0.267 0.267 0.267 rg BT 192.624 535.825 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 203.466 535.825 Td /F1 9.8 Tf [( by treating the HD-NSCs directly with SHH, DKK1 and BDNF. In addition, we included )] TJ ET BT 26.250 523.920 Td /F1 9.8 Tf [(the ROCK inhibitor Y27632 in the differentiation condition to promote cell survival. After 8-10 days in SHH, DKK1, BDNF and )] TJ ET BT 26.250 512.016 Td /F1 9.8 Tf [(Y27632 \(Stage 1\), cells were further differentiated with BDNF, cAMP, valproic acid and Y27632 for an additional 1-3 days )] TJ ET BT 26.250 500.111 Td /F1 9.8 Tf [(\(Stage 2\). At Stage 1, cells already expressed GABAergic neuron marker GABA, some even stained with striatal neuron marker )] TJ ET BT 26.250 488.206 Td /F1 9.8 Tf [(calbindin, but no MSN specific marker DARPP-32 positive cells were observed [Figure 4A]. At stage 2 some cells started to )] TJ ET BT 26.250 476.301 Td /F1 9.8 Tf [(express DARPP-32 [Figure 4B]. This differentiation experiment demonstrated that these HD-NSCs were capable of giving rise )] TJ ET BT 26.250 464.397 Td /F1 9.8 Tf [(to striatal neurons, which may also be used as an HD cell model. The yield of DARPP-32 neurons was approximately 10%. )] TJ ET BT 26.250 452.492 Td /F1 9.8 Tf [(Since we induced striatal differentiation stepwise, it is not easy to estimate overall percentage of striatal neurons generated from )] TJ ET BT 26.250 440.587 Td /F1 9.8 Tf [(original iPS cells. After EB attachment rosettes represented a small portion of total cells \(approximately 20%\). However these )] TJ ET BT 26.250 428.682 Td /F1 9.8 Tf [(rosettes cells are enriched NSCs \(greater than 95% based on Nestin staining\) and they can be passaged for over 20 passages )] TJ ET BT 26.250 416.778 Td /F1 9.8 Tf [(and still stain positive for NSC markers Nestin, Sox1 and Pax6. So the amount of NSCs was not a limiting factor. The yield from )] TJ ET BT 26.250 404.873 Td /F1 9.8 Tf [(NSCs to DARPP32 positive neurons was low. Higher purity DARPP32 neurons may require introducing a striatal specific )] TJ ET BT 26.250 392.968 Td /F1 9.8 Tf [(reporter and cell sorting.)] TJ ET 0.965 0.965 0.965 rg 26.250 159.724 555.000 223.364 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 383.087 m 581.250 383.087 l 581.250 382.337 l 26.250 382.337 l f 26.250 159.724 m 581.250 159.724 l 581.250 160.474 l 26.250 160.474 l f q 225.000 0 0 129.750 35.250 243.587 cm /I6 Do Q q 35.250 170.974 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 228.063 Td /F4 9.8 Tf [(Fig. 4: Striatal neurons derived from HD-iPS cells.)] TJ ET BT 35.250 208.693 Td /F1 9.8 Tf [(A \(Stage 1\) Immature striatal neurons express neuronal marker ?III-tubulin \(Tuj1\), GABAergic neuron marker GABA and )] TJ ET BT 35.250 194.957 Td /F1 9.8 Tf [(striatal marker calbindin but not medium spiny neuron \(MSN\) marker DARPP-32. B \(Stage 2\) Mature striatal neurons )] TJ ET BT 35.250 181.221 Td /F1 9.8 Tf [(express DARPP32 as well as well as ?III-tubulin, GABA and Calbindin.)] TJ ET Q BT 26.250 142.700 Td /F4 9.8 Tf [(4. 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To make sure )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(our HD cell model contained the same length of CAG repeats as parental cells, we performed a PCR to amplify the CAG )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(trinucleotide tract. The PCR result showed that all the cells derived from HD-iPSCs \(line HD-iPS-4\), including HD-NSCs and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(striatal differentiated neurons, had 72 CAG repeats, same as the HD patient fibroblasts from which the HD-iPSCs were )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(generated [Figure 5]. In contrast normal iPS cells \(line F-iPS-5 from Dr. George Daley\) only contained alleles with normal CAG )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(repeats. The stability of the CAG trinucleotide repeats in our HD-NSCs and striatal differentiated neurons ensured the )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(consistency and reproducibility for the use of these cells as HD model. Note that iPS \(line HD-iPS-1\) was determined to have )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(normal repeat length \(data not shown\).)] TJ ET 0.965 0.965 0.965 rg 26.250 431.676 555.000 242.586 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 674.262 m 581.250 674.262 l 581.250 673.512 l 26.250 673.512 l f 26.250 431.676 m 581.250 431.676 l 581.250 432.426 l 26.250 432.426 l f q 225.000 0 0 121.500 35.250 543.012 cm /I7 Do Q q 35.250 442.926 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 527.488 Td /F4 9.8 Tf [(Fig. 5: All cells derived from HD-iPS cells contain the same CAG expansion mutation as the parental HD-fibroblasts.)] TJ ET BT 35.250 508.118 Td /F1 9.8 Tf [(Total DNA was extracted from normal iPS cells, HD-iPS cells, NSC and striatal neurons derived from HD-iPS cells, as well )] TJ ET BT 35.250 494.382 Td /F1 9.8 Tf [(as HD-fibroblasts from which the HD-iPS cells were generated. A PCR reaction amplifying the CAG trinucleotide tract )] TJ ET BT 35.250 480.646 Td /F1 9.8 Tf [(containing huntingtin sequence was performed with total DNA samples. cDNAs containing known number of CAG repeats )] TJ ET BT 35.250 466.909 Td /F1 9.8 Tf [(were also included. Unlike Normal iPS cells, HD-iPS cells and cells derived from HD-iPS cells have the exact number of )] TJ ET BT 35.250 453.173 Td /F1 9.8 Tf [(CAG repeat as the HD-Fibroblasts.)] TJ ET Q BT 26.250 414.652 Td /F4 9.8 Tf [(5. Enhanced caspase activity in growth factor deprived HD-NSCs when compared to WT-NSCs)] TJ ET BT 26.250 395.247 Td /F1 9.8 Tf [(One of the most important applications of a HD cell model is to screen for therapeutic compounds with cellular assays. One )] TJ ET BT 26.250 383.343 Td /F1 9.8 Tf [(such assay utilized in the field is the measurement of caspase activity. Caspase activity is elevated in HD cell culture models, )] TJ ET BT 26.250 371.438 Td /F1 9.8 Tf [(mouse models and postmortem tissue when compared to controls )] TJ ET 0.267 0.267 0.267 rg BT 313.446 371.438 Td /F1 9.8 Tf [([11])] TJ ET 0.271 0.267 0.267 rg BT 329.709 371.438 Td /F1 9.8 Tf [( . Because caspase cleavage of huntingtin is an )] TJ ET BT 26.250 359.533 Td /F1 9.8 Tf [(important step to generate toxic fragments, the activity of caspase reflects Htt-mediated cellular toxicity. To test if our human HD )] TJ ET BT 26.250 347.628 Td /F1 9.8 Tf [(iPSC derived model is suitable for assay based drug screenings, we measured caspase activity in HD-NSCs and WT-NSCs )] TJ ET BT 26.250 335.724 Td /F1 9.8 Tf [(\(NSCs derived from normal H9 ES cells )] TJ ET 0.267 0.267 0.267 rg BT 200.161 335.724 Td /F1 9.8 Tf [([11])] TJ ET 0.271 0.267 0.267 rg BT 216.424 335.724 Td /F1 9.8 Tf [( using the same EB method or from iPSC with normal HD repeat length\) after )] TJ ET BT 26.250 323.819 Td /F1 9.8 Tf [(growth factor withdrawal for 24h. In WT-NSCs, 24h of growth factor withdrawal did not affect caspase-3/7 activity significantly. )] TJ ET BT 26.250 311.914 Td /F1 9.8 Tf [(However, in HD-NSCs caspase-3/7 activity was stimulated under the same condition [Figure 6]. This result demonstrates the )] TJ ET BT 26.250 300.009 Td /F1 9.8 Tf [(potential use of this human HD cell model in drug screenings.)] TJ ET 0.965 0.965 0.965 rg 26.250 49.378 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 290.128 m 581.250 290.128 l 581.250 289.378 l 26.250 289.378 l f q 178.500 0 0 225.000 35.250 55.378 cm /I8 Do Q q 35.250 49.378 537.000 0.000 re W n Q Q q 15.000 49.378 577.500 727.622 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(The CAG expansion mutation of HD is sometimes unstable and may contract or expand spontaneously )] TJ ET 0.267 0.267 0.267 rg BT 472.781 767.476 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 497.175 767.476 Td /F1 9.8 Tf [( . To make sure )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(our HD cell model contained the same length of CAG repeats as parental cells, we performed a PCR to amplify the CAG )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(trinucleotide tract. The PCR result showed that all the cells derived from HD-iPSCs \(line HD-iPS-4\), including HD-NSCs and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(striatal differentiated neurons, had 72 CAG repeats, same as the HD patient fibroblasts from which the HD-iPSCs were )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(generated [Figure 5]. In contrast normal iPS cells \(line F-iPS-5 from Dr. George Daley\) only contained alleles with normal CAG )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(repeats. The stability of the CAG trinucleotide repeats in our HD-NSCs and striatal differentiated neurons ensured the )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(consistency and reproducibility for the use of these cells as HD model. Note that iPS \(line HD-iPS-1\) was determined to have )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(normal repeat length \(data not shown\).)] TJ ET 0.965 0.965 0.965 rg 26.250 431.676 555.000 242.586 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 674.262 m 581.250 674.262 l 581.250 673.512 l 26.250 673.512 l f 26.250 431.676 m 581.250 431.676 l 581.250 432.426 l 26.250 432.426 l f q 225.000 0 0 121.500 35.250 543.012 cm /I7 Do Q q 35.250 442.926 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 527.488 Td /F4 9.8 Tf [(Fig. 5: All cells derived from HD-iPS cells contain the same CAG expansion mutation as the parental HD-fibroblasts.)] TJ ET BT 35.250 508.118 Td /F1 9.8 Tf [(Total DNA was extracted from normal iPS cells, HD-iPS cells, NSC and striatal neurons derived from HD-iPS cells, as well )] TJ ET BT 35.250 494.382 Td /F1 9.8 Tf [(as HD-fibroblasts from which the HD-iPS cells were generated. A PCR reaction amplifying the CAG trinucleotide tract )] TJ ET BT 35.250 480.646 Td /F1 9.8 Tf [(containing huntingtin sequence was performed with total DNA samples. cDNAs containing known number of CAG repeats )] TJ ET BT 35.250 466.909 Td /F1 9.8 Tf [(were also included. Unlike Normal iPS cells, HD-iPS cells and cells derived from HD-iPS cells have the exact number of )] TJ ET BT 35.250 453.173 Td /F1 9.8 Tf [(CAG repeat as the HD-Fibroblasts.)] TJ ET Q BT 26.250 414.652 Td /F4 9.8 Tf [(5. Enhanced caspase activity in growth factor deprived HD-NSCs when compared to WT-NSCs)] TJ ET BT 26.250 395.247 Td /F1 9.8 Tf [(One of the most important applications of a HD cell model is to screen for therapeutic compounds with cellular assays. One )] TJ ET BT 26.250 383.343 Td /F1 9.8 Tf [(such assay utilized in the field is the measurement of caspase activity. Caspase activity is elevated in HD cell culture models, )] TJ ET BT 26.250 371.438 Td /F1 9.8 Tf [(mouse models and postmortem tissue when compared to controls )] TJ ET 0.267 0.267 0.267 rg BT 313.446 371.438 Td /F1 9.8 Tf [([11])] TJ ET 0.271 0.267 0.267 rg BT 329.709 371.438 Td /F1 9.8 Tf [( . Because caspase cleavage of huntingtin is an )] TJ ET BT 26.250 359.533 Td /F1 9.8 Tf [(important step to generate toxic fragments, the activity of caspase reflects Htt-mediated cellular toxicity. To test if our human HD )] TJ ET BT 26.250 347.628 Td /F1 9.8 Tf [(iPSC derived model is suitable for assay based drug screenings, we measured caspase activity in HD-NSCs and WT-NSCs )] TJ ET BT 26.250 335.724 Td /F1 9.8 Tf [(\(NSCs derived from normal H9 ES cells )] TJ ET 0.267 0.267 0.267 rg BT 200.161 335.724 Td /F1 9.8 Tf [([11])] TJ ET 0.271 0.267 0.267 rg BT 216.424 335.724 Td /F1 9.8 Tf [( using the same EB method or from iPSC with normal HD repeat length\) after )] TJ ET BT 26.250 323.819 Td /F1 9.8 Tf [(growth factor withdrawal for 24h. In WT-NSCs, 24h of growth factor withdrawal did not affect caspase-3/7 activity significantly. )] TJ ET BT 26.250 311.914 Td /F1 9.8 Tf [(However, in HD-NSCs caspase-3/7 activity was stimulated under the same condition [Figure 6]. This result demonstrates the )] TJ ET BT 26.250 300.009 Td /F1 9.8 Tf [(potential use of this human HD cell model in drug screenings.)] TJ ET 0.965 0.965 0.965 rg 26.250 49.378 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 290.128 m 581.250 290.128 l 581.250 289.378 l 26.250 289.378 l f q 178.500 0 0 225.000 35.250 55.378 cm /I8 Do Q q 35.250 49.378 537.000 0.000 re W n Q Q q 15.000 49.378 577.500 727.622 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(The CAG expansion mutation of HD is sometimes unstable and may contract or expand spontaneously )] TJ ET 0.267 0.267 0.267 rg BT 472.781 767.476 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 497.175 767.476 Td /F1 9.8 Tf [( . To make sure )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(our HD cell model contained the same length of CAG repeats as parental cells, we performed a PCR to amplify the CAG )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(trinucleotide tract. The PCR result showed that all the cells derived from HD-iPSCs \(line HD-iPS-4\), including HD-NSCs and )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(striatal differentiated neurons, had 72 CAG repeats, same as the HD patient fibroblasts from which the HD-iPSCs were )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(generated [Figure 5]. In contrast normal iPS cells \(line F-iPS-5 from Dr. George Daley\) only contained alleles with normal CAG )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(repeats. The stability of the CAG trinucleotide repeats in our HD-NSCs and striatal differentiated neurons ensured the )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(consistency and reproducibility for the use of these cells as HD model. Note that iPS \(line HD-iPS-1\) was determined to have )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(normal repeat length \(data not shown\).)] TJ ET 0.965 0.965 0.965 rg 26.250 431.676 555.000 242.586 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 674.262 m 581.250 674.262 l 581.250 673.512 l 26.250 673.512 l f 26.250 431.676 m 581.250 431.676 l 581.250 432.426 l 26.250 432.426 l f q 225.000 0 0 121.500 35.250 543.012 cm /I7 Do Q q 35.250 442.926 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 527.488 Td /F4 9.8 Tf [(Fig. 5: All cells derived from HD-iPS cells contain the same CAG expansion mutation as the parental HD-fibroblasts.)] TJ ET BT 35.250 508.118 Td /F1 9.8 Tf [(Total DNA was extracted from normal iPS cells, HD-iPS cells, NSC and striatal neurons derived from HD-iPS cells, as well )] TJ ET BT 35.250 494.382 Td /F1 9.8 Tf [(as HD-fibroblasts from which the HD-iPS cells were generated. A PCR reaction amplifying the CAG trinucleotide tract )] TJ ET BT 35.250 480.646 Td /F1 9.8 Tf [(containing huntingtin sequence was performed with total DNA samples. cDNAs containing known number of CAG repeats )] TJ ET BT 35.250 466.909 Td /F1 9.8 Tf [(were also included. Unlike Normal iPS cells, HD-iPS cells and cells derived from HD-iPS cells have the exact number of )] TJ ET BT 35.250 453.173 Td /F1 9.8 Tf [(CAG repeat as the HD-Fibroblasts.)] TJ ET Q BT 26.250 414.652 Td /F4 9.8 Tf [(5. Enhanced caspase activity in growth factor deprived HD-NSCs when compared to WT-NSCs)] TJ ET BT 26.250 395.247 Td /F1 9.8 Tf [(One of the most important applications of a HD cell model is to screen for therapeutic compounds with cellular assays. One )] TJ ET BT 26.250 383.343 Td /F1 9.8 Tf [(such assay utilized in the field is the measurement of caspase activity. Caspase activity is elevated in HD cell culture models, )] TJ ET BT 26.250 371.438 Td /F1 9.8 Tf [(mouse models and postmortem tissue when compared to controls )] TJ ET 0.267 0.267 0.267 rg BT 313.446 371.438 Td /F1 9.8 Tf [([11])] TJ ET 0.271 0.267 0.267 rg BT 329.709 371.438 Td /F1 9.8 Tf [( . Because caspase cleavage of huntingtin is an )] TJ ET BT 26.250 359.533 Td /F1 9.8 Tf [(important step to generate toxic fragments, the activity of caspase reflects Htt-mediated cellular toxicity. To test if our human HD )] TJ ET BT 26.250 347.628 Td /F1 9.8 Tf [(iPSC derived model is suitable for assay based drug screenings, we measured caspase activity in HD-NSCs and WT-NSCs )] TJ ET BT 26.250 335.724 Td /F1 9.8 Tf [(\(NSCs derived from normal H9 ES cells )] TJ ET 0.267 0.267 0.267 rg BT 200.161 335.724 Td /F1 9.8 Tf [([11])] TJ ET 0.271 0.267 0.267 rg BT 216.424 335.724 Td /F1 9.8 Tf [( using the same EB method or from iPSC with normal HD repeat length\) after )] TJ ET BT 26.250 323.819 Td /F1 9.8 Tf [(growth factor withdrawal for 24h. In WT-NSCs, 24h of growth factor withdrawal did not affect caspase-3/7 activity significantly. )] TJ ET BT 26.250 311.914 Td /F1 9.8 Tf [(However, in HD-NSCs caspase-3/7 activity was stimulated under the same condition [Figure 6]. This result demonstrates the )] TJ ET BT 26.250 300.009 Td /F1 9.8 Tf [(potential use of this human HD cell model in drug screenings.)] TJ ET 0.965 0.965 0.965 rg 26.250 49.378 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 290.128 m 581.250 290.128 l 581.250 289.378 l 26.250 289.378 l f q 178.500 0 0 225.000 35.250 55.378 cm /I8 Do Q q 35.250 49.378 537.000 0.000 re W n Q Q q 225.000 0 0 121.500 35.250 543.012 cm /I7 Do Q q 178.500 0 0 225.000 35.250 55.378 cm /I8 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 139 0 obj << /Type /Annot /Subtype /Link /A 140 0 R /Border [0 0 0] /H /I /Rect [ 472.7805 766.5743 497.1750 776.4950 ] >> endobj 140 0 obj << /Type /Action >> endobj 141 0 obj << /Type /Annot /Subtype /Link /A 142 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 543.0120 260.2500 664.5120 ] >> endobj 142 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2010/10/figure-5.tiff) >> endobj 143 0 obj << /Type /XObject /Subtype /Image /Width 300 /Height 162 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 10538>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   ," }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Q?J?җ?J?җ?J?җ?J?җ?J?җ?J?җ?J?җ?J?җ?J?җon~g5IDH7;`gw>/Q~c~NΗwicHX+By ގր(?J_֏ր(?J_֏ր(?J_֏ր(?J_֏ր<]c6Wv:}MW)n Lh[ W 5obBvW2u+ Aԯ,ԴYRy'WR`}J(tXR$H}(Q&-~qh4;ewÕi s1fHYU@Mzל{&Ծ^l-^)zyo+*iH&*X<7C迕yǭF+owgOԵlj M2gĈ\#:yxI) h y/.u9RiX\lNB `10 }3={x.AhI47DZW0rH#jr@V$^P/GIQPxW.l- Y.o|*  u 24~-'^K!HfV'#x`(=(=+,hoh}]!``, VWDEbt@:xL{0캹d$7 {ђ= oҏғbx~Եc乔D)bw4R*Ew 5f<ûJP.2¾\M75xO]]e]=*x}>xWc.YZ%Յߗqo}{;/3-.+~vҏғ\C;gjNJ_CCSr\~$ ޻*s6Jnm}RXu+wO>9mtvn/(y-#yM#\-ouo;{?z5R,xG4{zNTsx"xsĺ]<^mlT7R"[UPN>p zlgӼ%As&0qY%Y%vfa9,k{KV?X\Eqo:4Ҽ7+ FIMawUwUXGj1@]&\FckYnB7iw'`1\'6u2_Lh RO*>OLj>+Zl,ʑ 9M$![fXT޸?ߊx]R kxȭ =ݶo2KB(ؿJ?JLQ_?|CxTt)?om06eS#Ep7{&}8k$<Bg|P5^XGoI γ ;,9mbP~ ͧZpb_12ppG=Z=+~4_]OmtɒILkiV7+#7`N q^OG!~u7Z^z֗zDХУ*@N@h׌V`@}>L\'k=+~h aqI{4XGV>\pTݹ88=(=)1F(J?JLQ<7:m֥\B%IdqS o~6!$/rO95jzqj7[ygQ}MroR X*ec̸I|랴j;ULּKiZ[5yK#1l'։vULg8Ya`|˞"?{A4%:46BJ #Z~ O??_±?GYO(;[J3Ұ5_٫Ƕgg<`Z? 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HD-NSCs )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(showed elevated toxicity while normal NSCs did not. \(** P<0.01 and *** P<0.001 in paired t-test\) The upper panel the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(normal NSCs were derived from human H9 embryonic stem cells. The lower panel the NSCs were derived from iPS with )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(normal CAG repeat length \(HD-iPS line 1\) .)] TJ ET Q BT 26.250 651.179 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 631.224 Td /F1 9.8 Tf [(We have demonstrated that HD patient-specific pluripotent stem cells can be cultured over multiple passages without losing )] TJ ET BT 26.250 619.320 Td /F1 9.8 Tf [(CAG repeat length or pluripotent markers such as SSEA-4, NANOG, OCT4 and SOX2. These markers are absent in the )] TJ ET BT 26.250 607.415 Td /F1 9.8 Tf [(fibroblasts in which the iPSCs were derived \(data not shown\). As might be expected, we do detect altered signalling when )] TJ ET BT 26.250 595.510 Td /F1 9.8 Tf [(comparing the normal iPSCs to the HD iPSCs. In this case, we detect altered ERK phosphorylation in resting cells and in )] TJ ET BT 26.250 583.605 Td /F1 9.8 Tf [(response to bFGF.)] TJ ET BT 26.250 564.201 Td /F1 9.8 Tf [(A characteristic property of pluripotent cells is their ability, when plated in suspension culture, to form embryoid bodies \(EB\). We )] TJ ET BT 26.250 552.296 Td /F1 9.8 Tf [(found both the normal and HD iPSCs formed EBs. The hope is that HD iPSCs can be differentiated into the disease relevant )] TJ ET BT 26.250 540.391 Td /F1 9.8 Tf [(cell types. We found that from EBs, HD iPSCs can be differentiated into neuronal precursor cells in high yield. These HD-NSCs )] TJ ET BT 26.250 528.486 Td /F1 9.8 Tf [(expressed markers nestin, SOX1 and PAX6. The HD NSCs could be further differentiated into cells expressing GABAergic )] TJ ET BT 26.250 516.582 Td /F1 9.8 Tf [(neuron marker GABA and a subset of these stained with neuron marker calbindin. Further differentiation yielded DARPP-32 )] TJ ET BT 26.250 504.677 Td /F1 9.8 Tf [(positive neurons. Currently we are optimizing the yield of the DARPP-32 positive cells. However, even this mixed culture may )] TJ ET BT 26.250 492.772 Td /F1 9.8 Tf [(yield clues to cell selective neuronal vulnerability in HD.)] TJ ET BT 26.250 473.367 Td /F1 9.8 Tf [(Finally, we found that normal NSCs when compared to HD NSCs have altered levels of caspase activity during serum )] TJ ET BT 26.250 461.463 Td /F1 9.8 Tf [(withdrawal. This phenotype could be utilized for screening of therapeutic compounds or to optimize differentiation procedures. )] TJ ET BT 26.250 449.558 Td /F1 9.8 Tf [(Although the possible differences in ERK activation and caspase induction are promising they will need to be confirmed in )] TJ ET BT 26.250 437.653 Td /F1 9.8 Tf [(multiple HD iPS from distinct patient fibroblasts.)] TJ ET BT 26.250 418.248 Td /F1 9.8 Tf [(Many insights into molecular pathways in HD come from analysis of post-morterm HD tissue or HD mouse models. With the )] TJ ET BT 26.250 406.344 Td /F1 9.8 Tf [(opportunity to utilized patient specific iPS cells, new tools will be generated to understand mechanisms of HD.)] TJ ET BT 26.250 369.741 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 349.787 Td /F4 9.8 Tf [(Cell Culture)] TJ ET BT 26.250 330.382 Td /F1 9.8 Tf [(All cell culture reagents were from Invitrogen unless otherwise mentioned. HD-iPSCs were cultured like ES cells on either )] TJ ET BT 26.250 318.477 Td /F1 9.8 Tf [(Matrigel \(BD\) or irradiation inactivated mouse embryonic fibroblasts \(MEFs\). When HD-iPSCs were grown on MEFs, the ES )] TJ ET BT 26.250 306.573 Td /F1 9.8 Tf [(culture medium was knockout DMEM/F12 supplemented with 20% knockout serum replacement, 2.48mM L-glutamine, 1X )] TJ ET BT 26.250 294.668 Td /F1 9.8 Tf [(nonessential amino acid, 15.4mM HEPES, 50?M ?-mercaptoethanol, 100U/ml penicillin, 100?g/ml streptomycin \(Cellgro\) and )] TJ ET BT 26.250 282.763 Td /F1 9.8 Tf [(4ng/ml bFGF \(Peprotech\). When HD-iPSCs were grown on Matrigel, the ES medium conditioned by MEFs was used. The HD-)] TJ ET BT 26.250 270.858 Td /F1 9.8 Tf [(iPSCs were regularly passaged with collagenase. HD-NSCs and WT-NSCs were cultured on plates coated with 20?g/ml poly-)] TJ ET BT 26.250 258.954 Td /F1 9.8 Tf [(ornithine \(Sigma\) for 1h at 37 )] TJ ET BT 155.223 262.842 Td /F1 8.7 Tf [(o)] TJ ET BT 160.042 258.954 Td /F1 9.8 Tf [( C followed by 5?g/ml mouse laminin \(Sigma\) for 1h at 37 )] TJ ET BT 410.383 262.842 Td /F1 8.7 Tf [(o)] TJ ET BT 415.201 258.954 Td /F1 9.8 Tf [( C. The neural proliferation medium )] TJ ET BT 26.250 247.049 Td /F1 9.8 Tf [(for NSCs was ENStem-A Neural Expansion Medium \(Millipore, NeuroBasal medium with 1X B27, 10ng/ml LIF\) supplemented )] TJ ET BT 26.250 235.144 Td /F1 9.8 Tf [(with 2mM L-glutamine, 100U/ml penicillin, 100?g/ml streptomycin, and 25ng/ml bFGF. NSCs were regularly passaged with )] TJ ET BT 26.250 223.239 Td /F1 9.8 Tf [(Accutase \(Sigma\).)] TJ ET BT 26.250 203.835 Td /F4 9.8 Tf [(Western blotting)] TJ ET BT 26.250 184.430 Td /F1 9.8 Tf [(HD-iPSCs, normal iPSCs or H9 cells cultured on Matrigel were fed with bFGF containing MEF conditioned medium daily. On the )] TJ ET BT 26.250 172.525 Td /F1 9.8 Tf [(day of experiment, without feeding cells with fresh medium different doses of bFGF \(4ng/ml or 20ng/ml\) were added to these )] TJ ET BT 26.250 160.620 Td /F1 9.8 Tf [(cells which had not been fed for 24h. 30 min after bFGF treatment cells were scraped off, pelleted and washed once with DPBS )] TJ ET BT 26.250 148.716 Td /F1 9.8 Tf [(\(Cellgro\). Cell pellets were lyzed by sonication in mammalian protein extraction reagent \(M-PER, from Thermo Scientific\) )] TJ ET BT 26.250 136.811 Td /F1 9.8 Tf [(containing protease inhibitors \(one Complete mini tablet per 10ml, Roche\) and 1% phosphatase inhibitor cocktail set II )] TJ ET BT 26.250 124.906 Td /F1 9.8 Tf [(\(Calbiochem\). Protein concentration was measured with Pierce BCA protein assay kit \(Thermo Scientific\) to ensure equal )] TJ ET BT 26.250 113.001 Td /F1 9.8 Tf [(sample loading. Protein samples were run on 4-12% bis-tris gel \(Invitrogen\), transferred to nitrocellulose membrane \(Whatman\), )] TJ ET BT 26.250 101.097 Td /F1 9.8 Tf [(probed with anti-phospho-p44/42 \(ERK1/2\) \(Thr202/Tyr204\) antibody \(Cell Signaling\) and reprobed with anti-p44/42 \(ERK1/2\) )] TJ ET BT 26.250 89.192 Td /F1 9.8 Tf [(antibody \(Cell Signaling\).)] TJ ET BT 26.250 69.787 Td /F4 9.8 Tf [(NSC and striatal differentiation)] TJ ET BT 26.250 50.382 Td /F1 9.8 Tf [(HD-NSCs were derived from HD-iPSCs with EB method. Briefly HD-iPSCs were passaged with collagenase and cell clumps )] TJ ET BT 26.250 38.478 Td /F1 9.8 Tf [(were cultured in a low attachment petri-dish \(Kord-Valmark\) in ES medium without bFGF. Medium was replaced every 2 days )] TJ ET Q q 15.000 24.192 577.500 752.808 re W n 0.965 0.965 0.965 rg 26.250 685.400 555.000 91.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 685.400 m 581.250 685.400 l 581.250 686.150 l 26.250 686.150 l f q 35.250 696.650 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 6: Higher cellular toxicity of HD-NSCs compared to normal NSCs.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Both HD and normal NSCs were subject to a caspase-3/7 activity assay 24h after growth factor withdrawal. HD-NSCs )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(showed elevated toxicity while normal NSCs did not. \(** P<0.01 and *** P<0.001 in paired t-test\) The upper panel the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(normal NSCs were derived from human H9 embryonic stem cells. The lower panel the NSCs were derived from iPS with )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(normal CAG repeat length \(HD-iPS line 1\) .)] TJ ET Q BT 26.250 651.179 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 631.224 Td /F1 9.8 Tf [(We have demonstrated that HD patient-specific pluripotent stem cells can be cultured over multiple passages without losing )] TJ ET BT 26.250 619.320 Td /F1 9.8 Tf [(CAG repeat length or pluripotent markers such as SSEA-4, NANOG, OCT4 and SOX2. These markers are absent in the )] TJ ET BT 26.250 607.415 Td /F1 9.8 Tf [(fibroblasts in which the iPSCs were derived \(data not shown\). As might be expected, we do detect altered signalling when )] TJ ET BT 26.250 595.510 Td /F1 9.8 Tf [(comparing the normal iPSCs to the HD iPSCs. In this case, we detect altered ERK phosphorylation in resting cells and in )] TJ ET BT 26.250 583.605 Td /F1 9.8 Tf [(response to bFGF.)] TJ ET BT 26.250 564.201 Td /F1 9.8 Tf [(A characteristic property of pluripotent cells is their ability, when plated in suspension culture, to form embryoid bodies \(EB\). We )] TJ ET BT 26.250 552.296 Td /F1 9.8 Tf [(found both the normal and HD iPSCs formed EBs. The hope is that HD iPSCs can be differentiated into the disease relevant )] TJ ET BT 26.250 540.391 Td /F1 9.8 Tf [(cell types. We found that from EBs, HD iPSCs can be differentiated into neuronal precursor cells in high yield. These HD-NSCs )] TJ ET BT 26.250 528.486 Td /F1 9.8 Tf [(expressed markers nestin, SOX1 and PAX6. The HD NSCs could be further differentiated into cells expressing GABAergic )] TJ ET BT 26.250 516.582 Td /F1 9.8 Tf [(neuron marker GABA and a subset of these stained with neuron marker calbindin. Further differentiation yielded DARPP-32 )] TJ ET BT 26.250 504.677 Td /F1 9.8 Tf [(positive neurons. Currently we are optimizing the yield of the DARPP-32 positive cells. However, even this mixed culture may )] TJ ET BT 26.250 492.772 Td /F1 9.8 Tf [(yield clues to cell selective neuronal vulnerability in HD.)] TJ ET BT 26.250 473.367 Td /F1 9.8 Tf [(Finally, we found that normal NSCs when compared to HD NSCs have altered levels of caspase activity during serum )] TJ ET BT 26.250 461.463 Td /F1 9.8 Tf [(withdrawal. This phenotype could be utilized for screening of therapeutic compounds or to optimize differentiation procedures. )] TJ ET BT 26.250 449.558 Td /F1 9.8 Tf [(Although the possible differences in ERK activation and caspase induction are promising they will need to be confirmed in )] TJ ET BT 26.250 437.653 Td /F1 9.8 Tf [(multiple HD iPS from distinct patient fibroblasts.)] TJ ET BT 26.250 418.248 Td /F1 9.8 Tf [(Many insights into molecular pathways in HD come from analysis of post-morterm HD tissue or HD mouse models. With the )] TJ ET BT 26.250 406.344 Td /F1 9.8 Tf [(opportunity to utilized patient specific iPS cells, new tools will be generated to understand mechanisms of HD.)] TJ ET BT 26.250 369.741 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 349.787 Td /F4 9.8 Tf [(Cell Culture)] TJ ET BT 26.250 330.382 Td /F1 9.8 Tf [(All cell culture reagents were from Invitrogen unless otherwise mentioned. HD-iPSCs were cultured like ES cells on either )] TJ ET BT 26.250 318.477 Td /F1 9.8 Tf [(Matrigel \(BD\) or irradiation inactivated mouse embryonic fibroblasts \(MEFs\). When HD-iPSCs were grown on MEFs, the ES )] TJ ET BT 26.250 306.573 Td /F1 9.8 Tf [(culture medium was knockout DMEM/F12 supplemented with 20% knockout serum replacement, 2.48mM L-glutamine, 1X )] TJ ET BT 26.250 294.668 Td /F1 9.8 Tf [(nonessential amino acid, 15.4mM HEPES, 50?M ?-mercaptoethanol, 100U/ml penicillin, 100?g/ml streptomycin \(Cellgro\) and )] TJ ET BT 26.250 282.763 Td /F1 9.8 Tf [(4ng/ml bFGF \(Peprotech\). When HD-iPSCs were grown on Matrigel, the ES medium conditioned by MEFs was used. The HD-)] TJ ET BT 26.250 270.858 Td /F1 9.8 Tf [(iPSCs were regularly passaged with collagenase. HD-NSCs and WT-NSCs were cultured on plates coated with 20?g/ml poly-)] TJ ET BT 26.250 258.954 Td /F1 9.8 Tf [(ornithine \(Sigma\) for 1h at 37 )] TJ ET BT 155.223 262.842 Td /F1 8.7 Tf [(o)] TJ ET BT 160.042 258.954 Td /F1 9.8 Tf [( C followed by 5?g/ml mouse laminin \(Sigma\) for 1h at 37 )] TJ ET BT 410.383 262.842 Td /F1 8.7 Tf [(o)] TJ ET BT 415.201 258.954 Td /F1 9.8 Tf [( C. The neural proliferation medium )] TJ ET BT 26.250 247.049 Td /F1 9.8 Tf [(for NSCs was ENStem-A Neural Expansion Medium \(Millipore, NeuroBasal medium with 1X B27, 10ng/ml LIF\) supplemented )] TJ ET BT 26.250 235.144 Td /F1 9.8 Tf [(with 2mM L-glutamine, 100U/ml penicillin, 100?g/ml streptomycin, and 25ng/ml bFGF. NSCs were regularly passaged with )] TJ ET BT 26.250 223.239 Td /F1 9.8 Tf [(Accutase \(Sigma\).)] TJ ET BT 26.250 203.835 Td /F4 9.8 Tf [(Western blotting)] TJ ET BT 26.250 184.430 Td /F1 9.8 Tf [(HD-iPSCs, normal iPSCs or H9 cells cultured on Matrigel were fed with bFGF containing MEF conditioned medium daily. On the )] TJ ET BT 26.250 172.525 Td /F1 9.8 Tf [(day of experiment, without feeding cells with fresh medium different doses of bFGF \(4ng/ml or 20ng/ml\) were added to these )] TJ ET BT 26.250 160.620 Td /F1 9.8 Tf [(cells which had not been fed for 24h. 30 min after bFGF treatment cells were scraped off, pelleted and washed once with DPBS )] TJ ET BT 26.250 148.716 Td /F1 9.8 Tf [(\(Cellgro\). Cell pellets were lyzed by sonication in mammalian protein extraction reagent \(M-PER, from Thermo Scientific\) )] TJ ET BT 26.250 136.811 Td /F1 9.8 Tf [(containing protease inhibitors \(one Complete mini tablet per 10ml, Roche\) and 1% phosphatase inhibitor cocktail set II )] TJ ET BT 26.250 124.906 Td /F1 9.8 Tf [(\(Calbiochem\). Protein concentration was measured with Pierce BCA protein assay kit \(Thermo Scientific\) to ensure equal )] TJ ET BT 26.250 113.001 Td /F1 9.8 Tf [(sample loading. Protein samples were run on 4-12% bis-tris gel \(Invitrogen\), transferred to nitrocellulose membrane \(Whatman\), )] TJ ET BT 26.250 101.097 Td /F1 9.8 Tf [(probed with anti-phospho-p44/42 \(ERK1/2\) \(Thr202/Tyr204\) antibody \(Cell Signaling\) and reprobed with anti-p44/42 \(ERK1/2\) )] TJ ET BT 26.250 89.192 Td /F1 9.8 Tf [(antibody \(Cell Signaling\).)] TJ ET BT 26.250 69.787 Td /F4 9.8 Tf [(NSC and striatal differentiation)] TJ ET BT 26.250 50.382 Td /F1 9.8 Tf [(HD-NSCs were derived from HD-iPSCs with EB method. Briefly HD-iPSCs were passaged with collagenase and cell clumps )] TJ ET BT 26.250 38.478 Td /F1 9.8 Tf [(were cultured in a low attachment petri-dish \(Kord-Valmark\) in ES medium without bFGF. Medium was replaced every 2 days )] TJ ET Q q 15.000 24.192 577.500 752.808 re W n 0.965 0.965 0.965 rg 26.250 685.400 555.000 91.600 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 685.400 m 581.250 685.400 l 581.250 686.150 l 26.250 686.150 l f q 35.250 696.650 537.000 80.350 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 6: Higher cellular toxicity of HD-NSCs compared to normal NSCs.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Both HD and normal NSCs were subject to a caspase-3/7 activity assay 24h after growth factor withdrawal. HD-NSCs )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(showed elevated toxicity while normal NSCs did not. \(** P<0.01 and *** P<0.001 in paired t-test\) The upper panel the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(normal NSCs were derived from human H9 embryonic stem cells. The lower panel the NSCs were derived from iPS with )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(normal CAG repeat length \(HD-iPS line 1\) .)] TJ ET Q BT 26.250 651.179 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 631.224 Td /F1 9.8 Tf [(We have demonstrated that HD patient-specific pluripotent stem cells can be cultured over multiple passages without losing )] TJ ET BT 26.250 619.320 Td /F1 9.8 Tf [(CAG repeat length or pluripotent markers such as SSEA-4, NANOG, OCT4 and SOX2. These markers are absent in the )] TJ ET BT 26.250 607.415 Td /F1 9.8 Tf [(fibroblasts in which the iPSCs were derived \(data not shown\). As might be expected, we do detect altered signalling when )] TJ ET BT 26.250 595.510 Td /F1 9.8 Tf [(comparing the normal iPSCs to the HD iPSCs. In this case, we detect altered ERK phosphorylation in resting cells and in )] TJ ET BT 26.250 583.605 Td /F1 9.8 Tf [(response to bFGF.)] TJ ET BT 26.250 564.201 Td /F1 9.8 Tf [(A characteristic property of pluripotent cells is their ability, when plated in suspension culture, to form embryoid bodies \(EB\). We )] TJ ET BT 26.250 552.296 Td /F1 9.8 Tf [(found both the normal and HD iPSCs formed EBs. The hope is that HD iPSCs can be differentiated into the disease relevant )] TJ ET BT 26.250 540.391 Td /F1 9.8 Tf [(cell types. We found that from EBs, HD iPSCs can be differentiated into neuronal precursor cells in high yield. These HD-NSCs )] TJ ET BT 26.250 528.486 Td /F1 9.8 Tf [(expressed markers nestin, SOX1 and PAX6. The HD NSCs could be further differentiated into cells expressing GABAergic )] TJ ET BT 26.250 516.582 Td /F1 9.8 Tf [(neuron marker GABA and a subset of these stained with neuron marker calbindin. Further differentiation yielded DARPP-32 )] TJ ET BT 26.250 504.677 Td /F1 9.8 Tf [(positive neurons. Currently we are optimizing the yield of the DARPP-32 positive cells. However, even this mixed culture may )] TJ ET BT 26.250 492.772 Td /F1 9.8 Tf [(yield clues to cell selective neuronal vulnerability in HD.)] TJ ET BT 26.250 473.367 Td /F1 9.8 Tf [(Finally, we found that normal NSCs when compared to HD NSCs have altered levels of caspase activity during serum )] TJ ET BT 26.250 461.463 Td /F1 9.8 Tf [(withdrawal. This phenotype could be utilized for screening of therapeutic compounds or to optimize differentiation procedures. )] TJ ET BT 26.250 449.558 Td /F1 9.8 Tf [(Although the possible differences in ERK activation and caspase induction are promising they will need to be confirmed in )] TJ ET BT 26.250 437.653 Td /F1 9.8 Tf [(multiple HD iPS from distinct patient fibroblasts.)] TJ ET BT 26.250 418.248 Td /F1 9.8 Tf [(Many insights into molecular pathways in HD come from analysis of post-morterm HD tissue or HD mouse models. With the )] TJ ET BT 26.250 406.344 Td /F1 9.8 Tf [(opportunity to utilized patient specific iPS cells, new tools will be generated to understand mechanisms of HD.)] TJ ET BT 26.250 369.741 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 349.787 Td /F4 9.8 Tf [(Cell Culture)] TJ ET BT 26.250 330.382 Td /F1 9.8 Tf [(All cell culture reagents were from Invitrogen unless otherwise mentioned. HD-iPSCs were cultured like ES cells on either )] TJ ET BT 26.250 318.477 Td /F1 9.8 Tf [(Matrigel \(BD\) or irradiation inactivated mouse embryonic fibroblasts \(MEFs\). When HD-iPSCs were grown on MEFs, the ES )] TJ ET BT 26.250 306.573 Td /F1 9.8 Tf [(culture medium was knockout DMEM/F12 supplemented with 20% knockout serum replacement, 2.48mM L-glutamine, 1X )] TJ ET BT 26.250 294.668 Td /F1 9.8 Tf [(nonessential amino acid, 15.4mM HEPES, 50?M ?-mercaptoethanol, 100U/ml penicillin, 100?g/ml streptomycin \(Cellgro\) and )] TJ ET BT 26.250 282.763 Td /F1 9.8 Tf [(4ng/ml bFGF \(Peprotech\). When HD-iPSCs were grown on Matrigel, the ES medium conditioned by MEFs was used. The HD-)] TJ ET BT 26.250 270.858 Td /F1 9.8 Tf [(iPSCs were regularly passaged with collagenase. HD-NSCs and WT-NSCs were cultured on plates coated with 20?g/ml poly-)] TJ ET BT 26.250 258.954 Td /F1 9.8 Tf [(ornithine \(Sigma\) for 1h at 37 )] TJ ET BT 155.223 262.842 Td /F1 8.7 Tf [(o)] TJ ET BT 160.042 258.954 Td /F1 9.8 Tf [( C followed by 5?g/ml mouse laminin \(Sigma\) for 1h at 37 )] TJ ET BT 410.383 262.842 Td /F1 8.7 Tf [(o)] TJ ET BT 415.201 258.954 Td /F1 9.8 Tf [( C. The neural proliferation medium )] TJ ET BT 26.250 247.049 Td /F1 9.8 Tf [(for NSCs was ENStem-A Neural Expansion Medium \(Millipore, NeuroBasal medium with 1X B27, 10ng/ml LIF\) supplemented )] TJ ET BT 26.250 235.144 Td /F1 9.8 Tf [(with 2mM L-glutamine, 100U/ml penicillin, 100?g/ml streptomycin, and 25ng/ml bFGF. NSCs were regularly passaged with )] TJ ET BT 26.250 223.239 Td /F1 9.8 Tf [(Accutase \(Sigma\).)] TJ ET BT 26.250 203.835 Td /F4 9.8 Tf [(Western blotting)] TJ ET BT 26.250 184.430 Td /F1 9.8 Tf [(HD-iPSCs, normal iPSCs or H9 cells cultured on Matrigel were fed with bFGF containing MEF conditioned medium daily. On the )] TJ ET BT 26.250 172.525 Td /F1 9.8 Tf [(day of experiment, without feeding cells with fresh medium different doses of bFGF \(4ng/ml or 20ng/ml\) were added to these )] TJ ET BT 26.250 160.620 Td /F1 9.8 Tf [(cells which had not been fed for 24h. 30 min after bFGF treatment cells were scraped off, pelleted and washed once with DPBS )] TJ ET BT 26.250 148.716 Td /F1 9.8 Tf [(\(Cellgro\). Cell pellets were lyzed by sonication in mammalian protein extraction reagent \(M-PER, from Thermo Scientific\) )] TJ ET BT 26.250 136.811 Td /F1 9.8 Tf [(containing protease inhibitors \(one Complete mini tablet per 10ml, Roche\) and 1% phosphatase inhibitor cocktail set II )] TJ ET BT 26.250 124.906 Td /F1 9.8 Tf [(\(Calbiochem\). Protein concentration was measured with Pierce BCA protein assay kit \(Thermo Scientific\) to ensure equal )] TJ ET BT 26.250 113.001 Td /F1 9.8 Tf [(sample loading. Protein samples were run on 4-12% bis-tris gel \(Invitrogen\), transferred to nitrocellulose membrane \(Whatman\), )] TJ ET BT 26.250 101.097 Td /F1 9.8 Tf [(probed with anti-phospho-p44/42 \(ERK1/2\) \(Thr202/Tyr204\) antibody \(Cell Signaling\) and reprobed with anti-p44/42 \(ERK1/2\) )] TJ ET BT 26.250 89.192 Td /F1 9.8 Tf [(antibody \(Cell Signaling\).)] TJ ET BT 26.250 69.787 Td /F4 9.8 Tf [(NSC and striatal differentiation)] TJ ET BT 26.250 50.382 Td /F1 9.8 Tf [(HD-NSCs were derived from HD-iPSCs with EB method. Briefly HD-iPSCs were passaged with collagenase and cell clumps )] TJ ET BT 26.250 38.478 Td /F1 9.8 Tf [(were cultured in a low attachment petri-dish \(Kord-Valmark\) in ES medium without bFGF. Medium was replaced every 2 days )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(5)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 173 0 obj << /Type /Page /Parent 3 0 R /Contents 174 0 R >> endobj 174 0 obj << /Length 20827 >> stream 0.271 0.267 0.267 rg q 15.000 51.735 577.500 725.265 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(and at each time of medium change 25% more ES medium was replaced by EB differentiation medium \(DMEM supplemented )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(with 20% fetal bovine serum, 1X nonessential amino acid, 50?M ?-mercaptoethanol, 100U/ml penicillin and 100?g/ml )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(streptomycin\). After 8 days the medium was 100% EB differentiation medium. After 10 days the EBs in suspension were )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(attached onto pO/L coated plates in neural differentiation medium \(DMEM/F12 supplemented with 1X N2, 100U/ml penicillin and )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(100?g/ml streptomycin\) with 25ng/ml bFGF. Medium was replaced every 2 days. After 10-12 days rosettes were manually )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(picked, triturated by P1000 tip and plated on pO/L coated plates in neural proliferation medium. The first passage was )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(performed with 0.05% Trypsin and the following passages were done with Accutase \(Sigma\). WT-NSCs were derived from H9 )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(human ESCs by the same procedure. The striatal differentiation of HD-NSCs was induced by changing neural proliferation )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(medium to neural differentiation medium supplemented with 250ng/ml SHH \(R&D Systems\), 100ng/ml DKK1 \(R&D Systems\), )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(20ng/ml BDNF \(Peprotech\) and 10?M Y27632 \(Calbiochem\). After 8-10 days in the condition above \(Stage 1\), cells were )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(exposed to 0.5mM dibutryl-cyclic AMP \(Sigma\), 0.5?M valpromide \(Alfa Aesar\), 20ng/ml BDNF and 10?M Y27632 for an )] TJ ET BT 26.250 636.524 Td /F1 9.8 Tf [(additional 1-3 days \(Stage 2\).)] TJ ET BT 26.250 617.119 Td /F4 9.8 Tf [(CAG repeats PCR)] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(Total DNA was extracted from different cell samples with DNeasy kit \(Qiagen\) according to manufacturers instructions. The )] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(primers used to amplify the CAG trinucleotide containing fragment are: Forward 5-CCT TCG AGT CCC TCA AGT CCT TC-3, )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Reverse 5-GGC GGG GGC GGC TGC GGC TGA G-3.)] TJ ET BT 26.250 554.500 Td /F4 9.8 Tf [(Caspase-3/7 activity assay)] TJ ET BT 26.250 535.095 Td /F1 9.8 Tf [(The caspase activity assay was performed with Apo3 HTS kit \(Cell Technology\). Either HD-NSCs or WT-NSCs were grown in )] TJ ET BT 26.250 523.191 Td /F1 9.8 Tf [(24 well plates. For growth factor deprived samples cells were first washed once with neural proliferation medium without LIF and )] TJ ET BT 26.250 511.286 Td /F1 9.8 Tf [(bFGF, then cultured in this growth factor free medium for 24h. After 24h of growth factor withdrawal, medium was removed and )] TJ ET BT 26.250 499.381 Td /F1 9.8 Tf [(150?l 1X lysis buffer was added into each well. The plate were placed on an orbital shaker at 700 rpm for 10 min. Then, 30?l of )] TJ ET BT 26.250 487.476 Td /F1 9.8 Tf [(cell lysate was dispensed into one well of a 96 well plate in triplicate for each sample. 70?l of substrate mix \(1X lysis buffer with )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(1X Apo3 HTS Caspase3/7 detection reagent and 20mM DTT\) was added into each well and the plate was shaken at 700rpm for )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(30s. Subsequently the plate was loaded on Fusion-Alpha Universal Microplate Analyzer \(Perkin Elmer\) for the fluorescence )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(based reading \(Ex: 485nm, Em: 530nm\). For each sample 10?l of lysate was dispensed into another 96 well plate in triplicate )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(for protein concentration measurement with Pierce BCA protein assay kit. The caspase activity was normalized against protein )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(concentration for each sample.)] TJ ET BT 26.250 391.350 Td /F4 12.0 Tf [(Competing Interests)] TJ ET BT 26.250 371.396 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 334.793 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 314.839 Td /F1 9.8 Tf [(We thank Dr. Daley for providing us the normal and HD-iPSCs. Correspondence should be sent to lellerby@buckinstitute.org, )] TJ ET BT 26.250 302.934 Td /F1 9.8 Tf [(Buck Institute for Age Research, Novato, CA 94945)] TJ ET BT 26.250 273.832 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 246.378 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 246.378 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 234.473 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83. PubMed PMID:8458085.)] TJ ET BT 26.250 215.068 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 215.068 Td /F1 9.8 Tf [(Duyao M, Ambrose C, Myers R, Novelletto A, Persichetti F, Frontali M, Folstein S, Ross C, Franz M, Abbott M. Trinucleotide )] TJ ET BT 26.250 203.163 Td /F1 9.8 Tf [(repeat length instability and age of onset in Huntington's disease. Nat Genet. 1993 Aug;4\(4\):387-92. PubMed PMID:8401587.)] TJ ET BT 26.250 183.759 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 183.759 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Hackam AS, Margolis RL, Trifiro MA, Singaraja R, McCutcheon K, Salvesen GS, Propp SS, )] TJ ET BT 26.250 171.854 Td /F1 9.8 Tf [(Bromm M, Rowland KJ, Zhang T, Rasper D, Roy S, Thornberry N, Pinsky L, Kakizuka A, Ross CA, Nicholson DW, Bredesen )] TJ ET BT 26.250 159.949 Td /F1 9.8 Tf [(DE, Hayden MR. Caspase cleavage of gene products associated with triplet expansion disorders generates truncated )] TJ ET BT 26.250 148.044 Td /F1 9.8 Tf [(fragments containing the polyglutamine tract. J Biol Chem. 1998 Apr 10;273\(15\):9158-67. PubMed PMID:9535906.)] TJ ET BT 26.250 128.640 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 128.640 Td /F1 9.8 Tf [(Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, Kalchman M, Hayden MR. The influence of )] TJ ET BT 26.250 116.735 Td /F1 9.8 Tf [(huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol. 1998 Jun 1;141\(5\):1097-105. PubMed )] TJ ET BT 26.250 104.830 Td /F1 9.8 Tf [(PMID:9606203.)] TJ ET BT 26.250 85.425 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 85.425 Td /F1 9.8 Tf [(Imarisio S, Carmichael J, Korolchuk V, Chen CW, Saiki S, Rose C, Krishna G, Davies JE, Ttofi E, Underwood BR, )] TJ ET BT 26.250 73.521 Td /F1 9.8 Tf [(Rubinsztein DC. Huntington's disease: from pathology and genetics to potential therapies. Biochem J. 2008 Jun 1;412\(2\):191-)] TJ ET BT 26.250 61.616 Td /F1 9.8 Tf [(209. PubMed PMID:18466116.)] TJ ET Q q 15.000 51.735 577.500 725.265 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(and at each time of medium change 25% more ES medium was replaced by EB differentiation medium \(DMEM supplemented )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(with 20% fetal bovine serum, 1X nonessential amino acid, 50?M ?-mercaptoethanol, 100U/ml penicillin and 100?g/ml )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(streptomycin\). After 8 days the medium was 100% EB differentiation medium. After 10 days the EBs in suspension were )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(attached onto pO/L coated plates in neural differentiation medium \(DMEM/F12 supplemented with 1X N2, 100U/ml penicillin and )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(100?g/ml streptomycin\) with 25ng/ml bFGF. Medium was replaced every 2 days. After 10-12 days rosettes were manually )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(picked, triturated by P1000 tip and plated on pO/L coated plates in neural proliferation medium. The first passage was )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(performed with 0.05% Trypsin and the following passages were done with Accutase \(Sigma\). WT-NSCs were derived from H9 )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(human ESCs by the same procedure. The striatal differentiation of HD-NSCs was induced by changing neural proliferation )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(medium to neural differentiation medium supplemented with 250ng/ml SHH \(R&D Systems\), 100ng/ml DKK1 \(R&D Systems\), )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(20ng/ml BDNF \(Peprotech\) and 10?M Y27632 \(Calbiochem\). After 8-10 days in the condition above \(Stage 1\), cells were )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(exposed to 0.5mM dibutryl-cyclic AMP \(Sigma\), 0.5?M valpromide \(Alfa Aesar\), 20ng/ml BDNF and 10?M Y27632 for an )] TJ ET BT 26.250 636.524 Td /F1 9.8 Tf [(additional 1-3 days \(Stage 2\).)] TJ ET BT 26.250 617.119 Td /F4 9.8 Tf [(CAG repeats PCR)] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(Total DNA was extracted from different cell samples with DNeasy kit \(Qiagen\) according to manufacturers instructions. The )] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(primers used to amplify the CAG trinucleotide containing fragment are: Forward 5-CCT TCG AGT CCC TCA AGT CCT TC-3, )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Reverse 5-GGC GGG GGC GGC TGC GGC TGA G-3.)] TJ ET BT 26.250 554.500 Td /F4 9.8 Tf [(Caspase-3/7 activity assay)] TJ ET BT 26.250 535.095 Td /F1 9.8 Tf [(The caspase activity assay was performed with Apo3 HTS kit \(Cell Technology\). Either HD-NSCs or WT-NSCs were grown in )] TJ ET BT 26.250 523.191 Td /F1 9.8 Tf [(24 well plates. For growth factor deprived samples cells were first washed once with neural proliferation medium without LIF and )] TJ ET BT 26.250 511.286 Td /F1 9.8 Tf [(bFGF, then cultured in this growth factor free medium for 24h. After 24h of growth factor withdrawal, medium was removed and )] TJ ET BT 26.250 499.381 Td /F1 9.8 Tf [(150?l 1X lysis buffer was added into each well. The plate were placed on an orbital shaker at 700 rpm for 10 min. Then, 30?l of )] TJ ET BT 26.250 487.476 Td /F1 9.8 Tf [(cell lysate was dispensed into one well of a 96 well plate in triplicate for each sample. 70?l of substrate mix \(1X lysis buffer with )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(1X Apo3 HTS Caspase3/7 detection reagent and 20mM DTT\) was added into each well and the plate was shaken at 700rpm for )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(30s. Subsequently the plate was loaded on Fusion-Alpha Universal Microplate Analyzer \(Perkin Elmer\) for the fluorescence )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(based reading \(Ex: 485nm, Em: 530nm\). For each sample 10?l of lysate was dispensed into another 96 well plate in triplicate )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(for protein concentration measurement with Pierce BCA protein assay kit. The caspase activity was normalized against protein )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(concentration for each sample.)] TJ ET BT 26.250 391.350 Td /F4 12.0 Tf [(Competing Interests)] TJ ET BT 26.250 371.396 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 334.793 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 314.839 Td /F1 9.8 Tf [(We thank Dr. Daley for providing us the normal and HD-iPSCs. Correspondence should be sent to lellerby@buckinstitute.org, )] TJ ET BT 26.250 302.934 Td /F1 9.8 Tf [(Buck Institute for Age Research, Novato, CA 94945)] TJ ET BT 26.250 273.832 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 246.378 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 246.378 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 234.473 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83. PubMed PMID:8458085.)] TJ ET BT 26.250 215.068 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 215.068 Td /F1 9.8 Tf [(Duyao M, Ambrose C, Myers R, Novelletto A, Persichetti F, Frontali M, Folstein S, Ross C, Franz M, Abbott M. Trinucleotide )] TJ ET BT 26.250 203.163 Td /F1 9.8 Tf [(repeat length instability and age of onset in Huntington's disease. Nat Genet. 1993 Aug;4\(4\):387-92. PubMed PMID:8401587.)] TJ ET BT 26.250 183.759 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 183.759 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Hackam AS, Margolis RL, Trifiro MA, Singaraja R, McCutcheon K, Salvesen GS, Propp SS, )] TJ ET BT 26.250 171.854 Td /F1 9.8 Tf [(Bromm M, Rowland KJ, Zhang T, Rasper D, Roy S, Thornberry N, Pinsky L, Kakizuka A, Ross CA, Nicholson DW, Bredesen )] TJ ET BT 26.250 159.949 Td /F1 9.8 Tf [(DE, Hayden MR. Caspase cleavage of gene products associated with triplet expansion disorders generates truncated )] TJ ET BT 26.250 148.044 Td /F1 9.8 Tf [(fragments containing the polyglutamine tract. J Biol Chem. 1998 Apr 10;273\(15\):9158-67. PubMed PMID:9535906.)] TJ ET BT 26.250 128.640 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 128.640 Td /F1 9.8 Tf [(Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, Kalchman M, Hayden MR. The influence of )] TJ ET BT 26.250 116.735 Td /F1 9.8 Tf [(huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol. 1998 Jun 1;141\(5\):1097-105. PubMed )] TJ ET BT 26.250 104.830 Td /F1 9.8 Tf [(PMID:9606203.)] TJ ET BT 26.250 85.425 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 85.425 Td /F1 9.8 Tf [(Imarisio S, Carmichael J, Korolchuk V, Chen CW, Saiki S, Rose C, Krishna G, Davies JE, Ttofi E, Underwood BR, )] TJ ET BT 26.250 73.521 Td /F1 9.8 Tf [(Rubinsztein DC. Huntington's disease: from pathology and genetics to potential therapies. Biochem J. 2008 Jun 1;412\(2\):191-)] TJ ET BT 26.250 61.616 Td /F1 9.8 Tf [(209. PubMed PMID:18466116.)] TJ ET Q q 15.000 51.735 577.500 725.265 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(and at each time of medium change 25% more ES medium was replaced by EB differentiation medium \(DMEM supplemented )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(with 20% fetal bovine serum, 1X nonessential amino acid, 50?M ?-mercaptoethanol, 100U/ml penicillin and 100?g/ml )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(streptomycin\). After 8 days the medium was 100% EB differentiation medium. After 10 days the EBs in suspension were )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(attached onto pO/L coated plates in neural differentiation medium \(DMEM/F12 supplemented with 1X N2, 100U/ml penicillin and )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(100?g/ml streptomycin\) with 25ng/ml bFGF. Medium was replaced every 2 days. After 10-12 days rosettes were manually )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(picked, triturated by P1000 tip and plated on pO/L coated plates in neural proliferation medium. The first passage was )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(performed with 0.05% Trypsin and the following passages were done with Accutase \(Sigma\). WT-NSCs were derived from H9 )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(human ESCs by the same procedure. The striatal differentiation of HD-NSCs was induced by changing neural proliferation )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(medium to neural differentiation medium supplemented with 250ng/ml SHH \(R&D Systems\), 100ng/ml DKK1 \(R&D Systems\), )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(20ng/ml BDNF \(Peprotech\) and 10?M Y27632 \(Calbiochem\). After 8-10 days in the condition above \(Stage 1\), cells were )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(exposed to 0.5mM dibutryl-cyclic AMP \(Sigma\), 0.5?M valpromide \(Alfa Aesar\), 20ng/ml BDNF and 10?M Y27632 for an )] TJ ET BT 26.250 636.524 Td /F1 9.8 Tf [(additional 1-3 days \(Stage 2\).)] TJ ET BT 26.250 617.119 Td /F4 9.8 Tf [(CAG repeats PCR)] TJ ET BT 26.250 597.714 Td /F1 9.8 Tf [(Total DNA was extracted from different cell samples with DNeasy kit \(Qiagen\) according to manufacturers instructions. The )] TJ ET BT 26.250 585.810 Td /F1 9.8 Tf [(primers used to amplify the CAG trinucleotide containing fragment are: Forward 5-CCT TCG AGT CCC TCA AGT CCT TC-3, )] TJ ET BT 26.250 573.905 Td /F1 9.8 Tf [(Reverse 5-GGC GGG GGC GGC TGC GGC TGA G-3.)] TJ ET BT 26.250 554.500 Td /F4 9.8 Tf [(Caspase-3/7 activity assay)] TJ ET BT 26.250 535.095 Td /F1 9.8 Tf [(The caspase activity assay was performed with Apo3 HTS kit \(Cell Technology\). Either HD-NSCs or WT-NSCs were grown in )] TJ ET BT 26.250 523.191 Td /F1 9.8 Tf [(24 well plates. For growth factor deprived samples cells were first washed once with neural proliferation medium without LIF and )] TJ ET BT 26.250 511.286 Td /F1 9.8 Tf [(bFGF, then cultured in this growth factor free medium for 24h. After 24h of growth factor withdrawal, medium was removed and )] TJ ET BT 26.250 499.381 Td /F1 9.8 Tf [(150?l 1X lysis buffer was added into each well. The plate were placed on an orbital shaker at 700 rpm for 10 min. Then, 30?l of )] TJ ET BT 26.250 487.476 Td /F1 9.8 Tf [(cell lysate was dispensed into one well of a 96 well plate in triplicate for each sample. 70?l of substrate mix \(1X lysis buffer with )] TJ ET BT 26.250 475.572 Td /F1 9.8 Tf [(1X Apo3 HTS Caspase3/7 detection reagent and 20mM DTT\) was added into each well and the plate was shaken at 700rpm for )] TJ ET BT 26.250 463.667 Td /F1 9.8 Tf [(30s. Subsequently the plate was loaded on Fusion-Alpha Universal Microplate Analyzer \(Perkin Elmer\) for the fluorescence )] TJ ET BT 26.250 451.762 Td /F1 9.8 Tf [(based reading \(Ex: 485nm, Em: 530nm\). For each sample 10?l of lysate was dispensed into another 96 well plate in triplicate )] TJ ET BT 26.250 439.857 Td /F1 9.8 Tf [(for protein concentration measurement with Pierce BCA protein assay kit. The caspase activity was normalized against protein )] TJ ET BT 26.250 427.953 Td /F1 9.8 Tf [(concentration for each sample.)] TJ ET BT 26.250 391.350 Td /F4 12.0 Tf [(Competing Interests)] TJ ET BT 26.250 371.396 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET BT 26.250 334.793 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 314.839 Td /F1 9.8 Tf [(We thank Dr. Daley for providing us the normal and HD-iPSCs. Correspondence should be sent to lellerby@buckinstitute.org, )] TJ ET BT 26.250 302.934 Td /F1 9.8 Tf [(Buck Institute for Age Research, Novato, CA 94945)] TJ ET BT 26.250 273.832 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 246.378 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 246.378 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 234.473 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83. PubMed PMID:8458085.)] TJ ET BT 26.250 215.068 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 215.068 Td /F1 9.8 Tf [(Duyao M, Ambrose C, Myers R, Novelletto A, Persichetti F, Frontali M, Folstein S, Ross C, Franz M, Abbott M. Trinucleotide )] TJ ET BT 26.250 203.163 Td /F1 9.8 Tf [(repeat length instability and age of onset in Huntington's disease. Nat Genet. 1993 Aug;4\(4\):387-92. PubMed PMID:8401587.)] TJ ET BT 26.250 183.759 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 183.759 Td /F1 9.8 Tf [(Wellington CL, Ellerby LM, Hackam AS, Margolis RL, Trifiro MA, Singaraja R, McCutcheon K, Salvesen GS, Propp SS, )] TJ ET BT 26.250 171.854 Td /F1 9.8 Tf [(Bromm M, Rowland KJ, Zhang T, Rasper D, Roy S, Thornberry N, Pinsky L, Kakizuka A, Ross CA, Nicholson DW, Bredesen )] TJ ET BT 26.250 159.949 Td /F1 9.8 Tf [(DE, Hayden MR. Caspase cleavage of gene products associated with triplet expansion disorders generates truncated )] TJ ET BT 26.250 148.044 Td /F1 9.8 Tf [(fragments containing the polyglutamine tract. J Biol Chem. 1998 Apr 10;273\(15\):9158-67. PubMed PMID:9535906.)] TJ ET BT 26.250 128.640 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 128.640 Td /F1 9.8 Tf [(Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, Kalchman M, Hayden MR. The influence of )] TJ ET BT 26.250 116.735 Td /F1 9.8 Tf [(huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol. 1998 Jun 1;141\(5\):1097-105. PubMed )] TJ ET BT 26.250 104.830 Td /F1 9.8 Tf [(PMID:9606203.)] TJ ET BT 26.250 85.425 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 85.425 Td /F1 9.8 Tf [(Imarisio S, Carmichael J, Korolchuk V, Chen CW, Saiki S, Rose C, Krishna G, Davies JE, Ttofi E, Underwood BR, )] TJ ET BT 26.250 73.521 Td /F1 9.8 Tf [(Rubinsztein DC. Huntington's disease: from pathology and genetics to potential therapies. Biochem J. 2008 Jun 1;412\(2\):191-)] TJ ET BT 26.250 61.616 Td /F1 9.8 Tf [(209. PubMed PMID:18466116.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(6)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 175 0 obj << /Type /Page /Parent 3 0 R /Contents 176 0 R >> endobj 176 0 obj << /Length 7996 >> stream 0.271 0.267 0.267 rg q 15.000 526.524 577.500 250.476 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(factors. Cell. 2006 Aug 25;126\(4\):663-76. PubMed PMID:16904174.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Fecke W, Gianfriddo M, Gaviraghi G, Terstappen GC, Heitz F. Small molecule drug discovery for Huntington's Disease. Drug )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(Discov Today. 2009 May;14\(9-10\):453-64. PubMed PMID:19429504.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 697.357 Td /F1 9.8 Tf [(Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(specific induced pluripotent stem cells. Cell. 2008 Sep 5;134\(5\):877-86. PubMed PMID:18691744.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 666.048 Td /F1 9.8 Tf [(Aubry L, Bugi A, Lefort N, Rousseau F, Peschanski M, Perrier AL. Striatal progenitors derived from human ES cells mature )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci U S A. 2008 Oct 28;105\(43\):16707-12. )] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(PubMed PMID:18922775.)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Apostol BL, Illes K, Pallos J, Bodai L, Wu J, Strand A, Schweitzer ES, Olson JM, Kazantsev A, Marsh JL, Thompson LM. )] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-)] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(associated toxicity. Hum Mol Genet. 2006 Jan 15;15\(2\):273-85. PubMed PMID:16330479.)] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 579.619 Td /F1 9.8 Tf [(Hermel E, Gafni J, Propp SS, Leavitt BR, Wellington CL, Young JE, Hackam AS, Logvinova AV, Peel AL, Chen SF, Hook V, )] TJ ET BT 26.250 567.714 Td /F1 9.8 Tf [(Singaraja R, Krajewski S, Goldsmith PC, Ellerby HM, Hayden MR, Bredesen DE, Ellerby LM. Specific caspase interactions and )] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(amplification are involved in selective neuronal vulnerability in Huntington's disease. Cell Death Differ. 2004 Apr;11\(4\):424-38. )] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(PubMed PMID:14713958.)] TJ ET Q q 15.000 526.524 577.500 250.476 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(factors. Cell. 2006 Aug 25;126\(4\):663-76. PubMed PMID:16904174.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Fecke W, Gianfriddo M, Gaviraghi G, Terstappen GC, Heitz F. Small molecule drug discovery for Huntington's Disease. Drug )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(Discov Today. 2009 May;14\(9-10\):453-64. PubMed PMID:19429504.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 697.357 Td /F1 9.8 Tf [(Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(specific induced pluripotent stem cells. Cell. 2008 Sep 5;134\(5\):877-86. PubMed PMID:18691744.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 666.048 Td /F1 9.8 Tf [(Aubry L, Bugi A, Lefort N, Rousseau F, Peschanski M, Perrier AL. Striatal progenitors derived from human ES cells mature )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci U S A. 2008 Oct 28;105\(43\):16707-12. )] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(PubMed PMID:18922775.)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Apostol BL, Illes K, Pallos J, Bodai L, Wu J, Strand A, Schweitzer ES, Olson JM, Kazantsev A, Marsh JL, Thompson LM. )] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-)] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(associated toxicity. Hum Mol Genet. 2006 Jan 15;15\(2\):273-85. PubMed PMID:16330479.)] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 579.619 Td /F1 9.8 Tf [(Hermel E, Gafni J, Propp SS, Leavitt BR, Wellington CL, Young JE, Hackam AS, Logvinova AV, Peel AL, Chen SF, Hook V, )] TJ ET BT 26.250 567.714 Td /F1 9.8 Tf [(Singaraja R, Krajewski S, Goldsmith PC, Ellerby HM, Hayden MR, Bredesen DE, Ellerby LM. Specific caspase interactions and )] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(amplification are involved in selective neuronal vulnerability in Huntington's disease. Cell Death Differ. 2004 Apr;11\(4\):424-38. )] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(PubMed PMID:14713958.)] TJ ET Q q 15.000 526.524 577.500 250.476 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(factors. Cell. 2006 Aug 25;126\(4\):663-76. PubMed PMID:16904174.)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Fecke W, Gianfriddo M, Gaviraghi G, Terstappen GC, Heitz F. Small molecule drug discovery for Huntington's Disease. Drug )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(Discov Today. 2009 May;14\(9-10\):453-64. PubMed PMID:19429504.)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 697.357 Td /F1 9.8 Tf [(Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(specific induced pluripotent stem cells. Cell. 2008 Sep 5;134\(5\):877-86. PubMed PMID:18691744.)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 666.048 Td /F1 9.8 Tf [(Aubry L, Bugi A, Lefort N, Rousseau F, Peschanski M, Perrier AL. Striatal progenitors derived from human ES cells mature )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci U S A. 2008 Oct 28;105\(43\):16707-12. )] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(PubMed PMID:18922775.)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Apostol BL, Illes K, Pallos J, Bodai L, Wu J, Strand A, Schweitzer ES, Olson JM, Kazantsev A, Marsh JL, Thompson LM. )] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-)] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(associated toxicity. Hum Mol Genet. 2006 Jan 15;15\(2\):273-85. PubMed PMID:16330479.)] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 579.619 Td /F1 9.8 Tf [(Hermel E, Gafni J, Propp SS, Leavitt BR, Wellington CL, Young JE, Hackam AS, Logvinova AV, Peel AL, Chen SF, Hook V, )] TJ ET BT 26.250 567.714 Td /F1 9.8 Tf [(Singaraja R, Krajewski S, Goldsmith PC, Ellerby HM, Hayden MR, Bredesen DE, Ellerby LM. Specific caspase interactions and )] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(amplification are involved in selective neuronal vulnerability in Huntington's disease. Cell Death Differ. 2004 Apr;11\(4\):424-38. )] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(PubMed PMID:14713958.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(7)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj xref 0 177 0000000000 65535 f 0000000008 00000 n 0000000073 00000 n 0000000119 00000 n 0000000472 00000 n 0000000509 00000 n 0000000761 00000 n 0000001109 00000 n 0000027666 00000 n 0000027773 00000 n 0000027881 00000 n 0000027992 00000 n 0000028105 00000 n 0000028221 00000 n 0000028609 00000 n 0000033090 00000 n 0000033217 00000 n 0000033400 00000 n 0000033527 00000 n 0000033710 00000 n 0000033836 00000 n 0000033936 00000 n 0000034063 00000 n 0000034159 00000 n 0000034287 00000 n 0000034388 00000 n 0000034516 00000 n 0000034615 00000 n 0000034743 00000 n 0000034779 00000 n 0000034907 00000 n 0000034943 00000 n 0000035071 00000 n 0000035107 00000 n 0000035235 00000 n 0000035271 00000 n 0000035397 00000 n 0000035433 00000 n 0000035560 00000 n 0000035596 00000 n 0000035722 00000 n 0000035758 00000 n 0000035885 00000 n 0000036068 00000 n 0000036195 00000 n 0000036378 00000 n 0000036504 00000 n 0000036604 00000 n 0000036731 00000 n 0000036827 00000 n 0000036955 00000 n 0000037056 00000 n 0000037184 00000 n 0000037283 00000 n 0000037411 00000 n 0000037447 00000 n 0000037575 00000 n 0000037611 00000 n 0000037739 00000 n 0000037775 00000 n 0000037903 00000 n 0000037939 00000 n 0000038065 00000 n 0000038101 00000 n 0000038228 00000 n 0000038264 00000 n 0000038390 00000 n 0000038426 00000 n 0000038553 00000 n 0000038736 00000 n 0000038863 00000 n 0000039046 00000 n 0000039172 00000 n 0000039272 00000 n 0000039399 00000 n 0000039495 00000 n 0000039623 00000 n 0000039724 00000 n 0000039852 00000 n 0000039951 00000 n 0000040079 00000 n 0000040115 00000 n 0000040243 00000 n 0000040279 00000 n 0000040407 00000 n 0000040443 00000 n 0000040571 00000 n 0000040607 00000 n 0000040733 00000 n 0000040769 00000 n 0000040896 00000 n 0000040932 00000 n 0000041058 00000 n 0000041094 00000 n 0000041241 00000 n 0000055247 00000 n 0000055375 00000 n 0000055411 00000 n 0000055538 00000 n 0000055645 00000 n 0000071945 00000 n 0000072074 00000 n 0000072181 00000 n 0000081382 00000 n 0000081512 00000 n 0000081549 00000 n 0000081678 00000 n 0000081786 00000 n 0000081915 00000 n 0000082022 00000 n 0000082152 00000 n 0000082189 00000 n 0000082318 00000 n 0000082426 00000 n 0000082555 00000 n 0000082662 00000 n 0000082813 00000 n 0000096993 00000 n 0000097122 00000 n 0000097230 00000 n 0000106505 00000 n 0000106635 00000 n 0000106672 00000 n 0000106801 00000 n 0000106909 00000 n 0000125159 00000 n 0000125288 00000 n 0000125396 00000 n 0000125526 00000 n 0000125563 00000 n 0000125692 00000 n 0000125800 00000 n 0000125929 00000 n 0000126037 00000 n 0000126167 00000 n 0000126204 00000 n 0000126333 00000 n 0000126441 00000 n 0000126640 00000 n 0000141279 00000 n 0000141409 00000 n 0000141446 00000 n 0000141575 00000 n 0000141683 00000 n 0000152391 00000 n 0000152521 00000 n 0000152558 00000 n 0000152688 00000 n 0000152725 00000 n 0000152853 00000 n 0000152960 00000 n 0000163843 00000 n 0000163973 00000 n 0000164010 00000 n 0000164139 00000 n 0000164247 00000 n 0000164377 00000 n 0000164414 00000 n 0000164544 00000 n 0000164581 00000 n 0000164709 00000 n 0000164816 00000 n 0000164946 00000 n 0000164983 00000 n 0000165112 00000 n 0000165220 00000 n 0000165350 00000 n 0000165387 00000 n 0000165517 00000 n 0000165554 00000 n 0000165682 00000 n 0000165789 00000 n 0000165856 00000 n 0000188418 00000 n 0000188485 00000 n 0000209367 00000 n 0000209434 00000 n trailer << /Size 177 /Root 1 0 R /Info 5 0 R >> startxref 217484 %%EOF