Rebecca Marton
<p class="MsoNormal" >MARTON, Rebecca, Oligodendrocytes, Stanford, Feb 21, 2019
this is her <b>thesis defense </b>at Stanford. this is at 2PM in S360 at the Clark Center.
SRO crowd.</p>
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intro by Sergiu Pasca her PI.</p>
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slide one: millions of people affected by neuropsych disorders.</p>
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1 in 6 people affected by neuropsych disorders.</p>
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and 6.8 M people died each year of these.</p>
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goal of lab: develop 3d cell models to study these.</p>
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1) overview: brain has many cell types.</p>
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major celll types in brain:</p>
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neurons; astrocytes (metabolic and trophic support and involved in neuron synapse formation;</p>
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oligodendrocytes: insulation of axons but also buffering extracell fluid. etc.</p>
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human brains have unique characteristics...</p>
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<b>human astrocytes are much bigger and more complex than those in mouse. </b>
hugely different!</p>
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we use <b>pluripotent stem cells...</b></p>
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we use iPSCs...</p>
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<b>we reprogram skin cells ...</b>
<b>reprogram into iPSCs.</b></p>
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brain cells can be made from stem cells in culture.</p>
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advantages of 3d culture:</p>
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with similar mechanosensory factors => morpho similarities...</p>
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<b>hCSs human cortical spheroids or organoids...</b></p>
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at day 26 they are 4 mm in diameter.</p>
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we can watch devel. over time of labeled cells, eg neurons and glia.</p>
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oligos are made later! (and prone to die easily)</p>
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previous organoid models did not have oligos.</p>
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her goal: want 3d models that have oligos in addition to neurons and astros.</p>
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we need combos of small molecules and difft concentrations; length of rx and type of media.</p>
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and difft % of CO2.... she tried 20 different protocols.</p>
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<b>culture each for > 100 days</b>.</p>
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markers: start with <b>OPC oligo progenitor cells.</b></p>
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Sox10 and Olig2 expressed thruout dev of oligos.</p>
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and PDGFR alpha.</p>
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hOLS human oligodendrocyte spheroids...</p>
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she shows her ultimate protocol.</p>
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add in various factors at day zero; day 4, 6, 25, day 37.</p>
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NKX2.2 stains late OPC/immature olgios.</p>
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we looked for O4 and O1 and MBP markers.</p>
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yes, getting oligos at difft stages of devel.</p>
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and the hOLS also contain neurons and astros.</p>
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want all three to be able to study the interaction.</p>
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next... how similar are these oligos to native, natural oligos.</p>
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we did single cell RNAseq to study these oligos from hOLS and compare
these to brain biopsy specimens from patients.</p>
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yes, similar... shows clustering of RNA for difft cell populations (eg neurons vs oligos vs astros)</p>
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how abt their functional properties?</p>
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she looked at migration and ephys properties...</p>
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oligo progenitors migrate during development.</p>
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at day 200... not migrating anymore;</p>
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ephys properties....</p>
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<b>oligos have voltage gated ion channels</b>....</p>
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properties change over time....</p>
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use patch clamping by Omer Revah...</p>
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progentior cells have channels but mature cells do not.</p>
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could we achieve myelination?</p>
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we looked at interaction btwn oligos and neurons....</p>
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we used EM to look for myelination... in hOLS.</p>
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yes, can see myelination....</p>
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topic four: Vanishing White Matter disease results from loss of myelin.</p>
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this has progressive cystic degeneration of white matter;</p>
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most common onset is at age 2 to 6 years of age.</p>
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wheel chair bound....</p>
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this is autosomal recessive with mutation from EIF2B</p>
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EIF2B influences the rate of protein synthesis in all cell types.</p>
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get delayed oligo dev. and foamy soma in these cells.</p>
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we got skin cells from patients and from controls.</p>
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three patients and five controls....</p>
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yes, fewer mature oligos in vanishing WM patients (in their hOLS)</p>
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future:</p>
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single cell RNA seq of neurons, astros, and oligos in control and patient hOLS.</p>
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can we rescue with EIF2B stabilizer and try addition of stressors</p>
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and is it envionmental... try mix of vWM and control stem cells (does that correct the problem</p>
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her comm: Sergiu, Theo Palmer; Michelle Monje,
Aaron Gitler and Tony Wyss-Coray. (all here today)</p>
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