Rotation 1 Presentation: Chen Lab

By Amaris Castanon

Adapted from Chen Lab webpage

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.

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

Multiple progenitor zones

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

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

Cell Seeding Trial 1: iPSC WT2.3Da

y 5

Day

93K 6K 9K 12K

Scale Bar: 500 µm

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

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

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

Cell Seeding Trial 2: iPSC WT2.3100 500 1K 2K

Day

5Da

y 9

Scale Bar: 500 µm

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

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

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)

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

Acknowledgements

Denis Jgamadze Ashley Nemes

James Lim Chao Ji

Isaac Chen Principal Investigator

TOWNE BUILDING, UPENN ENGINEERING