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UCONN - PNB 3260 - Class Notes - Week 2

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UCONN - PNB 3260 - Class Notes - Week 2

School: University of Connecticut
Department: Physiology
Course: Stem Cell Biology
Professor: Conover
Term: Spring 2016
Tags: PNB 3260
Name: PNB 3260 Stem Cell Bio
Description: Week 2 notes from Stem Cell bio
Uploaded: 01/29/2016
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background image PNB 3260 Stem Cell Bio Week 2 Notes  Human Embryonic Stem Cells  Radmila Filipovic, PhD 
To make embryonic stem cells (ESCs) 
  need to derive the cells in the most similar manner that happens in vivo  use antibodies to detect different cells types and parts of neurons 
 
3D culture is making clumps of tissue from hESCs 
Hierarchy of stem cells 
  totipotent --> pluripotent --> blood stem cells (-->Red Blood Cell and White Blood Cell)  and other stem cells (-->Muscle, nerve, bone, other tissues)    ESCs are pluripotent, undifferentiated, able to make all 3 germ layers  Stem cell applications    test different drugs    generate tissues/cells for transplantation     research cell differentiation   Currently (2016) there are 363 eligible stem cell lines  1998 - first hESC isolated from blastocyst 
Gastrulation 
  differentiation into  1)ectoderm - neural, skin  2) endoderm - lung, liver and pancreas  3) mesoderm - heart, blood, vascular and skeletal muscle  Growth factors    FGF shifts cells to neuronal cleavage    can stimulate or inhibit growth  Transplantation of cells into infracted heart improved heart's beating ability   Neurogenesis in vitro from ES    CNS cell types:   1)neurons  2)glia -supporting cells that are harder to make from hESCs than neurons    astrocytes, oligodendrocytes, microglia  Sources of stem cells    adult brain has some stem cells, fetal brain cells, ESCs    adult stem cells are located in rats in   1) around lateral ventricles  2) hippocampus  The nervous system develops from a neural plate 
the notochord, the aggregate of mesodermal cells which extend along the midline of the embryo, 
induces overlaying structures 
Neurulation = formation of the neural tube 
Neural tube forms the entire CNS 
  Different parts of the neural tube differentiate into different parts of the CNS and require 
different growth factors/inhibitors 
  use these specific growth factors/inhibitors to generate specific parts of the CNS  Cortex    6 layers    project to different areas, long distance and local    promote activity of certain genes to promote growth of certain neuronal layers    2 classes of cells in cortex  1)projection neurons, mostly in deep layers and send long distance projections  to other parts of the brain  2) interneurons, mostly local connections  Use fibroblasts (found in skin) 
background image   will proliferate and grow small colonies (neurorosettes)    need to change to fresh media everyday to avoid differentiation, some spontaneous 
differentiation may occur (use neurospheres) 
  then introduce factors that will promote differentiation to a specific cell type    then start to make structure similar to neural tube  Immature neurons and adult neurons have different markers    Pax6 marker is only in forebrain neurons, typically in immature neurons here  After 5-10 passages you need to send for genetic testing to check for mutations using karyotyping  
Formation of the neural tube in vitro from hESC 
  FoxG1 is a forebrain marker    beta III tubulin is a neuronal marker  Growth requirements of pyramidal neurons from hESC    FGF2, BDNF, GDNF, ascorbic acid, cAMP    Sox5 gene is present after 5 wks - shows level of development (deep layer neurons first  then much later upper layer neurons) and differentiation    more difficult to generate actually functional neurons (can fire action potential)    After 10 wks - more function observed    astrocytes help maturation of neuron  2D culture: monolayer of cell growth  3D culture: organoid - mini organs generated, "mini brain" 
Progenitor cells are found in the more upper layers of the cortex 
Derivation of optic cup and retina from hESC 
  retina is a derivative of the CNS  Organoids    mini brain    grow organs in suspension    3D cultures    different parts of the brain can be seen in vivo    neural induction from a single layer to 3D  from embryonic bodies - multilayered self-organization structure with apical 
and basal membrane and specification of the anterior-posterior axis 
KO certain genes and observe   Microcephaly    genetic disease    smaller brain size and more simple brain folding    modeled the disease by  1) KO gene associated with disease  2) restore phenotype by reintroducing proteins by electroporation (can be used 
to promote or silence genes) 
Additional concerns    2D and 3D models for corticogenesis  Accuracy   polarization/cell division   cell connectivity in vitro, ability to generate cortical columnar organization in 
vitro 
issues with producing aging models  Applications for treating neurodegenerative disease    Alzheimer’s, Parkinson’s, stroke    1) proliferative ability in petri dish    2) specificity     3)migration, correct location    4) integration: correct connections    cells transplanted into the cortex may integrate to other brain locations 

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School: University of Connecticut
Department: Physiology
Course: Stem Cell Biology
Professor: Conover
Term: Spring 2016
Tags: PNB 3260
Name: PNB 3260 Stem Cell Bio
Description: Week 2 notes from Stem Cell bio
Uploaded: 01/29/2016
3 Pages 10 Views 8 Unlocks
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