ChenLab_Rotation_Presentation
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Transcript of ChenLab_Rotation_Presentation
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Rotation 1 Presentation: Chen Lab
By Amaris Castanon
Adapted from Chen Lab webpage
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Background: Cerebral Organoids• Human brain development: dramatic size
expansion, unique cell types, and distinct neural stem cell behaviors
• 3D tissues generated from human pluripotent stem cells that allow modelling of human brain development in vitro
• Novel methods for restoring brain function after damaged by combining aspects of stem cell biology, neural tissue engineering, and neural interface technologies Neural Stem Cells: Red; Neurons: Green
Molecular Research Council, Lancaster Lab, Cambridge, UK
Translational research goal: Improve the outcomes of patients suffering from a variety of brain disorders and diseases.
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Problem & SignificanceProblem: The organoids have cortical layer specific neurons and progenitor zones, however they do not recapitulate the structure of developing brain as multiple loci of progenitor zones and layered structures are generated in a single organoid
Significance : Transplanting the organoid which matches the host cortical structure ensuring better functional integration of the organoid
TujPax6
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Multiple progenitor zones
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Aim & Hypothesis• Aim: Find the initial number of cells needed to generate an
organoid with a single progenitor zone
• Hypothesis: Larger cell number--larger organoids & more progenitor zones
smaller cell number--smaller organoids & less progenitor zones-Factors that may lead to more/less progenitor zones
with larger/less number of cells: Cell adhesion, organoid fusion, hypoxia, and apoptosis
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Methods• Embryoid bodies with specific cell
numbers were formed by reaggregation of dissociated cells in U-bottom 96-well plates • Cell Seeding Trial 1 (3K, 6K, 9K, 12K)• Cell Seeding Trial 2 (100, 500, 1K, 2K)• Metrics: Organoid size, hypoxia stain,
progenitor markers, neural induction markers
Lancaster & Knoblich, 2014
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Cell Seeding Trial 1: iPSC WT2.3Da
y 5
Day
93K 6K 9K 12K
Scale Bar: 500 µm
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DAPI Cleaved Caspase-3
iPSC
WT2
.3: 3
KiP
SC W
T2.3
: 12K
Cell Seeding Trial 1: Apoptosis
Scale Bar: 500 µm
Sox2Sox2 + Cleaved
Caspase-3
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Cell Density Trial 1: HypoxiaiP
SC W
T2.3
:12K
DAPI Tuj + Sox2 HIF-1 𝛼iP
SC W
T2.3
:3K
Sox2+ HIF-1 𝛼
Scale Bar: 500 µm
Sox2 HIF-1 𝛼
Sox2 HIF-1 𝛼 Sox2
Tuj
Sox2 Tuj
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Improving health & differentiation
Ming et al., 2016
Cells in Cell Seeding Trial 2 were transferred to a 24-well plate on Day 6 using the spinning bioreactor to promote nutrient exchange, prevent
aggregation, and adhesion
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Cell Seeding Trial 2: iPSC WT2.3100 500 1K 2K
Day
5Da
y 9
Scale Bar: 500 µm
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Cell Seeding Trial 2: ApoptosisDAPI Cleaved Caspase-3
iPSC
WT2
.3: 1
00iP
SC W
T2.3
: 2K
Scale Bar: 500 µm
Sox2Sox2 + Cleaved
Caspase-3
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Cell Seeding Trial 2: HypoxiaDAPI Tuj + Sox2 HIF-1 𝛼
iPSC
WT2
.3:1
00
Sox2 + HIF-1 𝛼
iPSC
WT2
.3:2
K
Scale Bar: 500 µm
Sox2HIF-1 𝛼
Sox2HIF-1 𝛼
Sox2 Tuj
Sox2 Tuj
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Discussion• Organoid size was observed to be larger in higher cell numbers
vs. lower cell numbers
• Organoid fusion was prevalent among all cell seeding trials by Day 9
• Both hypoxic and apoptotic markers seem to have accumulated at center of organoids (except for 100 cells)
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Future Steps• Conduct further cell seeding trials with lower cell numbers and longer
development periods, fix, and stain
• Utilize a V-bottom plate rather than a U-bottom plate for cellular seeding to promote cellular aggregation and prevent adhesion
Scale Bar: 500 µm
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Acknowledgements
Denis Jgamadze Ashley Nemes
James Lim Chao Ji
Isaac Chen Principal Investigator
TOWNE BUILDING, UPENN ENGINEERING