PNB 3260 Week 4 notes
PNB 3260 Week 4 notes 3260
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This 4 page Class Notes was uploaded by AnnaCiara on Tuesday February 9, 2016. The Class Notes belongs to 3260 at University of Connecticut taught by Dr. Conover in Spring 2016. Since its upload, it has received 14 views. For similar materials see Stem Cell Biology in Physiology at University of Connecticut.
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Date Created: 02/09/16
Week 4 PNB 3260 Lecture Notes Lecture 7 - Student talks on papers Therapeutic Translation of iPSCs for treating neurological disease History neurogenetic disorders modeled first then sporadic disease Patterns in specific diseases important for modeling synaptic defects are different in vivo Bench and Bedside = working with clinicians Targeted Genome Modification goal: find similarities between disease phenotype in the lab (dish) and in vivo Challenge to goal - lower variability worst to best o ZFNs (zinc-finger nucleases) o TALENs (transcription activator-like effector nucleases) o CRISPR/Cas system - clustered regularly interspaced palindromic repeats can use more than one mutation if diseases aren't genetic (they are sporadic) harder to model because it's complex lineage specific cells can be used for sporadic cases Neighboring cells important to consider role of neighboring cells use other fields to learn about entire environment of the cells Microfluidics 3D growth allows multiple lines to be grown at once were able to differentiate half and leave half undifferentiated - ability to choose helps look at bigger picture: structure and function rather than single cell basis Microelectrode Array record activity between dif populations of neurons can be combined with microfluidics useful for long-term culturing limitation: accessibility- can't record intracellular potentials iPSC applications ultimate goal: total regeneration of disease tissue other goals: o drug development only 10% make it past safety concerns to be tested benefits of iPSCs in drug specificity disease specificity human specificity high-throughput screening (HTS) - conduct multiple tests at once o Transplantation therapies human fetal neural stem cells (fNSCs) may not work as well as iPSCs Retinal degeneration differentiated ESC and iPSC to functional rod photoreceptors no tumorgenesis in this study no immunorejection Bench to Bedside Perfecting cell line's safety and efficacy In Vivo reprogramming in humans transdifferentiation of cells fibroblasts and astrocytes from humans o put in 3 growth factors: Ascl!!, Brn2a, Myt1l o became neuronal cells ipscs in clinical and commercial area commercial o mostly based of unsubstantiated data Stem Cell Reports - PAPER #2 iPSCs to treat ALS in mice degeneration of upper and lower motor neurons mostly contralateral but also ipsilateral corticospinal tract mSOD1 mutation associated with familial associated with ~20% sporadic cases 2 main hypotheses 1) Glia play a significant enough role in the ALS phenotype that manipulating these cell types will have an observable effect; 2) hiPSCs can be used to generate glial cells that will effectively replace those damaged in ALS, and restore normal glial functionality. hiPSC-GRNPs glial rich neural progenitors Some mice got hiPSC-GRNPs then littermates got PBS control Male mice had better improvements but died earlier than female mice introduction into in vivo increased amount of neurotrophic factors present mSOD1 induces oxidative stress males may be more vulnerable to this stress VEGF caused enhanced blood vessel production? VEGF may be reason for increased life span Study would have had to continue longer to prove it doesn't result in tumorgenicity Lecture 8: Dr. Radmila Filipovic Reprogramming of somatic cells to iPSCs Need autologous cells to avoid host rejection Often fibroblast cells used, hepatocytes also used use transcription factors reverse steps of programming to reprogram a cell Zygote is totipotent Pluripotent multipotent: gives rise to dif cell types in lineage ICM/ES Unipotent: give rise to only one cell lineage Stages of development are mimicked in reprogramming zygote-->2 cell-->4 cell-->8 cell-->16 cell-->early blastocyst-->late blastocyst Regrogramming strategies somatic cell nuclear transfer into eggs cell fusion -differentiated with undifferentiated introduction of transcription factors into adult cells using retroviruses use of transcription factors in viruses that don't integrate into DNA- e.g. adenovirus small molecules to reprogram RNA reprogramming???? Generation of iPSCs from MEF cultures via 24 factors start with mouse embryonic fibroblasts-MEF (fetal human embryonic cells) introduce transcription factors Test for pluripotency by staining with various biomarkers Factors narrowed down from 24 to 4 Oct4 is rich in the ICM in blastocyst Yamanaka described these 4 factors Validation of iPS 1) teratoma (type of tumor) formation from ipsc ability to form different tissues: ectoderm, endoderm, mesoderm, in vivo 3) germline competent adult mice chimera formation after iPS injection in blastocyst o ipsc are giving rise to embryo Kinetics of iPS reprogramming reprogramming using virally encoded transcription factors takes many days and not all markers of pluripotency are activated together- also low frequency of success originally 0.05% now up to 2% with retroviral infection 10% with 4 factors + valproic acid (VA) Models Elite model o predetermined: small number of cells are competent for reprogramming o induced: only cells with specific viral integration sites are competent for reprogramming Stochastic model: all cells are competent for reprogramming o most supported model Waves of molecular events underlie cellular reprogramming early phase: mesenchymal to epithelial transition, MET, Cell cycle, decrease cell contact, decrease cell adhesion, microRNA expression, decreased differentiated cell markers, chromatin modifications intermediate phase: partially reprogrammed cells, resistant cells late phase: increase in pluripotency markers, DNA methylation, microRNA expression Summary of properties of Oct4, Klf4,Myc, and Sox2 Oct4 critical for making ICM cells pluripotent, active during INTERMEDIATE phase c-Myc enables self-renewal, active during EARLY phase Klf4 helps to upregulate Oct4 in embryoid bodies -i.e. supports self renewal, active in EARLY and LATE phase Sox2 maintains differentiated status, active Will these 4 factors work in humans? done with the 4 Yamanaka factors done with variation of the 4 -Thomson: Oct3/4, Sox2, Nanog, LIN28 o worked to generate first line of human iPSCs Other cells that can be reprogrammed bone marrow, hepatocytes, gastric epithelial cells, pancreatic cells, neural *** Other ways to make iPSCs because retroviruses integrate into the genome there is interest in finding other ways to make iPSCs non integrating viruses carrying transcription factors (adenoviruses) small molecules direct application of proteins plasmids (genes cloned in bacteria) using micro RNAs Direct conversion of fibroblasts to functional neurons by defined factors done without making ipscs then differentiating them 3 factors: BAM: Brn2, Ascl1, Myt1l neuron-like morphology in 3 days form functional synapses efficiency up to 19% o not all are completely mature o some can fire action potentials with other cell types narrow 19 factors down to 3 to be important for the reprogramming Modeling schizophrenia using human induced pluripotent stem cells treatment with antipsychotic drug improves connectivity Advantages of iPSCs over hESCs for preclinical studies disease specific iPSCs provide renewable source of human cells with genetic background sensitive to disease pathology one of the reasons why clinical trials failed to examine the effectiveness of drugs is that the effect of a drug maybe different between patients with different underlying mutations recently iPSC-derived dopaminergic neurons are used to screen group of compounds for neuroprotective Takahashi studies with photoreceptors
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