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160 DISEASE MODELING USING HUMAN IPS CELLS
S70 applications, we have taken a systems biology approach. In order to identify features characteristic of pluripotent cells, we have performed high-resolution genomic, epigenomic, and gene expression analyses on diverse pluripotent, multipotent, and differentiated cell types. Integrative analysis of these complex datasets has allowed us to identify networks of interacting factors that maintain the pluripotent state. In the course of our work, we have also discoveredmechanisms regulating such fundamental cellular activities as transcription, splicing, and silencing of repetitive elements. 159 ASSESSMENT OF STRA8 GENE EXPRESSION FOLLOWING BMP4 TREATMENT IN MOUSE DIFFERENTIATED EMBRYONIC STEM CELLS Z. Makoolati1 , M. Movahedin1 , M. Forouzandeh-Moghadam2 . 1 Department of Anatomical sciences, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran, 2 Department of Biotechnology, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran Introduction: Under appropriate culture conditions, stem cells can differentiate into germ cell lineage. The aim of this study was to evaluate the effect of bone morphogenetic protein 4 (BMP4) on the expression of Stra8, the germ cell-specific gene, in mouse embryonic stem cell (ESC) derived germ cells. Materials and Methods: Following the pluripotency confirmation of the CCE mouse ESC line by OCT-4 immunocytochemistry, XY cytogenetic verification was done through Sry gene expression. ESCs were cultured in order to 1-day-old EB formation (the first step of germ cell induction) and then cultured for 4 days in the presence or absence of 5 ng/ml BMP4 (the second step of germ cell induction). The expression of Stra8 was calculated in ESC, the first and the second steps of germ cell induction by quantitative RT-PCR. Data analysis was done with ANOVA and Tukey posttest. Results: The mean normalized expression of Stra8 was increased significantly in 1-day-old EB relative to ESCs. Also, Quantitative RT-PCR results showed that Stra8 was expressed in a higher significant rate in BMP4-treated cells compared to BMP4-free group, ESC and 1-day-old EB (P 0.05). Conclusion: The results confirmed that the EB system and the presence of BMP4 in a culture system with ESCs improve the differentiation of ESCs to germ cell. 160 DISEASE MODELING USING HUMAN IPS CELLS D. Sareen1 , C.N. Svendsen2 . 1 Cedars-Sinai Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, USA, 2 Department of Neurology, University of Wisconsin-Madison, Madison, USA Spinal muscular atrophy (SMA) Types I, II, and III belong to a group of recessively inherited pediatric neuromuscular diseases caused by a deletion or mutation in the survival motor neuron 1 (SMN1) gene and characterized by degeneration of spinal motor neurons, resulting in progressive muscle wasting, paralysis, and often death. Recently, we reported the generation of induced pluripotent stem cells (iPSCs) from a child with SMA (iPSSMA) [1]. These iPS-SMA cells maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child’s unaffected mother (iPS-WT). SMN is a ubiquitously expressed protein known to be important in small nuclear ribonucleoprotein (SnRNP) biogenesis,
WARM 2010 but it is unclear how low SMN levels specifically affect motor neurons and muscle cells. We have identified an increased apoptosis in the iPS-SMA cells from Type I patient lines compared to the iPS-WT control cells. Motor neuron (MN) patterned iPSSMA cells showed significant chromatin condensation as well as activation of initiator caspases-2 and -8. Consequently, we also found increased levels of Fas ligand (FasL/Apo-1L/CD95L), a potent activator of the extrinsic apoptotic pathway, in conditioned media from the differentiated iPS-SMA cells. Preliminary data also suggests that differentiated iPS-SMA MN cultures may show selective loss of mitochondrial membrane potential. Together our data suggest that cell death in iPS-SMA MN cultures may be triggered by cross-talk between FasL/Apo-1L signaling and mitochondria-mediated events. Activation of extrinsic death-receptor mediated apoptosis may be involved in motor neuron degeneration in SMA, and we are currently investigating the role of intermediary players downstream of Fas receptor activation. We have also generated Down Syndrome (DS) patient and normal control episomal iPSC lines from human fetal neural progenitors (hNPC) with high efficiency using a transgene integration-free method of reprogramming. Premature aging, dementia, or memory loss and impaired judgment similar to that occurring in Alzheimer disease patients, appears in adults with Down syndrome. Therefore, we are investigating DS iPS cells as a model of “aging-in-a-dish”. Reference(s) [1] Ebert, A.D. et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277 280 (2009). 161 HUMAN ES AND IPS CELL DIFFERENTIATION TO NEURAL PHENOTYPES D. Sareen1 , C.N. Svendsen2 . 1 Cedars-Sinai Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, USA, 2 Department of Neurology, University of Wisconsin-Madison, Madison, USA We have developed a simple method to generate multipotent neural stem cells (termed EZ spheres) from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) without utilizing embryoid body formation [1]. After gently lifting hESC or hiPSC colonies and culturing in suspension medium containing high concentrations of EGF and bFGF, cell aggregates, termed EZ spheres, form within 1 week with a propensity towards the neural lineage. EZ spheres allow simple and rapid proliferation of multipotent NSC populations from iPSC colonies in the absence of feeder layers, expensive substrates or complex serum components. EZ spheres show chromosomal stability over at least 30 40 passages, unlike some of the neural progenitor cell lines that we have extensively expanded for clinical grade cell banks [2]. EZ spheres can subsequently be differentiated into neural and glial fates including dopamine, motor and striatal neurons, oligodendrocytes, and astrocytes. Using a transgene integration-free method of reprogramming normal episomal iPSC lines have been generated from human fetal neural progenitors (hNPC) with high efficiency. EZ spheres derived from hNPC-iPSCs have a significantly greater yield of neural cells upon differentiation. Using episomal pig iPSCderived EZ sphere lines, we are establishing and validating the autologous engraftment of iPSC-derived motor neurons. EZ sphere cultures do not require complex combinations of substrates or morphogens, and need only twice weekly feedings. This allows for rapid growth of neural cells for developmental studies, high-content drug screening, or regenerative therapies. In addition, we are establishing episomal pig iPSC-derived EZ sphere lines to validate autologous engraftment of iPSCderived motor neurons in large animals for cellular regenerative
WARM 2010 but it is unclear how low SMN levels specifically affect motor neurons and muscle cells. We have identified an increased apoptosis in the iPS-SMA cells from Type I patient lines compared to the iPS-WT control cells. Motor neuron (MN) patterned iPSSMA cells showed significant chromatin condensation as well as activation of initiator caspases-2 and -8. Consequently, we also found increased levels of Fas ligand (FasL/Apo-1L/CD95L), a potent activator of the extrinsic apoptotic pathway, in conditioned media from the differentiated iPS-SMA cells. Preliminary data also suggests that differentiated iPS-SMA MN cultures may show selective loss of mitochondrial membrane potential. Together our data suggest that cell death in iPS-SMA MN cultures may be triggered by cross-talk between FasL/Apo-1L signaling and mitochondria-mediated events. Activation of extrinsic death-receptor mediated apoptosis may be involved in motor neuron degeneration in SMA, and we are currently investigating the role of intermediary players downstream of Fas receptor activation. We have also generated Down Syndrome (DS) patient and normal control episomal iPSC lines from human fetal neural progenitors (hNPC) with high efficiency using a transgene integration-free method of reprogramming. Premature aging, dementia, or memory loss and impaired judgment similar to that occurring in Alzheimer disease patients, appears in adults with Down syndrome. Therefore, we are investigating DS iPS cells as a model of “aging-in-a-dish”. Reference(s) [1] Ebert, A.D. et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277 280 (2009). 161 HUMAN ES AND IPS CELL DIFFERENTIATION TO NEURAL PHENOTYPES D. Sareen1 , C.N. Svendsen2 . 1 Cedars-Sinai Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, USA, 2 Department of Neurology, University of Wisconsin-Madison, Madison, USA We have developed a simple method to generate multipotent neural stem cells (termed EZ spheres) from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) without utilizing embryoid body formation [1]. After gently lifting hESC or hiPSC colonies and culturing in suspension medium containing high concentrations of EGF and bFGF, cell aggregates, termed EZ spheres, form within 1 week with a propensity towards the neural lineage. EZ spheres allow simple and rapid proliferation of multipotent NSC populations from iPSC colonies in the absence of feeder layers, expensive substrates or complex serum components. EZ spheres show chromosomal stability over at least 30 40 passages, unlike some of the neural progenitor cell lines that we have extensively expanded for clinical grade cell banks [2]. EZ spheres can subsequently be differentiated into neural and glial fates including dopamine, motor and striatal neurons, oligodendrocytes, and astrocytes. Using a transgene integration-free method of reprogramming normal episomal iPSC lines have been generated from human fetal neural progenitors (hNPC) with high efficiency. EZ spheres derived from hNPC-iPSCs have a significantly greater yield of neural cells upon differentiation. Using episomal pig iPSCderived EZ sphere lines, we are establishing and validating the autologous engraftment of iPSC-derived motor neurons. EZ sphere cultures do not require complex combinations of substrates or morphogens, and need only twice weekly feedings. This allows for rapid growth of neural cells for developmental studies, high-content drug screening, or regenerative therapies. In addition, we are establishing episomal pig iPSC-derived EZ sphere lines to validate autologous engraftment of iPSCderived motor neurons in large animals for cellular regenerative