SYSU-CHINA@iGEM PRESENT iPSCs SafeGuard F ang Yiming H e Dawei Z - - PowerPoint PPT Presentation

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SYSU-CHINA@iGEM PRESENT iPSCs SafeGuard F ang Yiming H e Dawei Z - - PowerPoint PPT Presentation

Jan 30, 2024 109 likes •538 views

SYSU-CHINA@iGEM PRESENT iPSCs SafeGuard F ang Yiming H e Dawei Z hao Yuchen C hen Haoqi S un Mengyi Possibility of Regeneration 3 Promising Prospect in Medical Application 4 (http://www2.estrellamountain.edu/) Stem

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SYSU-CHINA@iGEM PRESENT

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iPSCs SafeGuard

Fang Yiming He Dawei Zhao Yuchen Chen Haoqi Sun Mengyi

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3

Possibility of

Regeneration

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4

 

Promising Prospect in Medical Application

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5

Stem Cell Technology

(http://www2.estrellamountain.edu/)

 

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6 In Yamanaka’s experiment in 2009, the tumor formation rate is 30% among 100 mice transplanted with iPS cells, much higher than norman ES cells. That’s due to:  Reactivation of transcription factor c-Myc, also an oncogene  Wrong insertion of viral vectors

 

(Keisuke Okita, et al. Nature, 2007)

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7

iPS Safeguard

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8

 Part I: Killer---Suicide gene

To prevent Sensor

 Part II: Sensor

iPS Safeguard

Switch

 Part III: Switch

Unwanted Cells wanted Cells Killer

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9

Now let me show you our design & results

Killer Sensor Switch

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Design ign & Results ts 10

Promoter PCMV Suicide gene Sensor Killer Switch

 hBax  hBax -184  RIP1  RIP3  Apoptin  Caspase family

Candidates are chosen from genes playing important roles in cell apoptosis pathways

CANDIDATES for SUICIDE GENE

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Design ign & Results ts 11

Suicide genes RIP1 RIP3and Apoptinsuccessfully induce cell death

25 50 75 100

Mock Rip1 Rip3 Apoptin GFP

HepG2 Cell Survival Rate (%)

25 50 75

Mock Rip1 Rip3 Apoptin

iPSC Survival Rate (%)

Data is from Flow Cytometry Method(FCM)

GFP RIP 1 RIP3 HEK293(BL) HepG2(BL) HepG2 (DAPI staining)

100μm 100μm 100μm 100μm 100μm 100μm

0h 48h BL 488nm

Apoptin

Sensor Killer Switch

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Design ign & Results ts 12

Normally differentiated cells

SELECTIVE KILLING

Undifferentiated ips cells & cancer cells

WHAT IS NEEDED?

 Signal: endogenous and distinguishing molecular markers  Pre-transcriptional level  Post-transcriptional level  Sensor:

to sense the signal and determine the expression of suicide gene

 A model:

  • ne type of somatic cell which

represent the normally differentiated cells in our project Sensor Killer Switch

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Design ign & Results ts 13

Strategy A : Pre-transcriptional level

 Signals: transcription factors epigenetic modifications, etc  Sensor: tissue-specific promoter

However…

 Escape of cancer cells: Tissue-specific promoters cannot be universally activated in all types of cancer cells, which may all be differentiated from iPS cells.

Wanted cells Unwanted cells iPS cells Suicide gene Tissue-specific Promoter

Sensor Killer Switch

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Design ign & Results ts 14

Strategy B : Post-transcriptional level

 Signals: tissue-specific miRNA  Sensor: miRNA binding targets on mRNA

Suicide gene miRNA targets mRNA miRNA Suicide gene miRNA targets mRNA

Wanted cells Unwanted cells mRNA degradation Suicide gene expression

Sensor Killer Switch

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Design ign & Results ts 15

A model is found

Human miRNA-122 (endogenous)

Human liver cells (hepatocyte)

Sensor Killer Switch

miRNA-122 miRNA-122

Liver cells Non-liver cells

Data from www.microrna.org

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Design ign & Results ts 16

PROJECT DECISION

Promoter Suicide gene miRNA-122 target

Liver cells Liver tissue iPS cells

Non-liver cells have included all unwanted cells: undifferentiated iPS cells and cancer cells

Non-liver cells suicide Model : Human Liver cell (hepatocyte) Molecular marker: miRNA-122 Sensor: miRNA-122 targets

Sensor Killer Switch

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17 DESIGN IGN & RESUL ULTS TS

Promoter GFP miRNA-122 target

Completely complementary binding sequence of miRNA-122 Natural miRNA-122 binding sequence (Partially complementary)

CONSTRUCTION OF SENSOR

Sensor Killer Switch

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18 DESIGN IGN & RESUL ULTS TS

How to test the sensor?

Promoter GFP miRNA-122 target

Low transfection efficiency High transfection efficiency

Liver cells HEK 293T cells

Endogenous miRNA-122 Endogenous miRNA-122

Sensor Killer Switch

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19 DESIGN IGN & RESUL ULTS TS

miRNA-122 gradients by exogenous expression

pMiR-122

HEK 293T cells

miRNA-122 gradient

miRNA-122 expressing plasmid

Sensor Killer Switch

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20 DESIGN IGN & RESUL ULTS TS

miRNA-122 Target Responds Accordingly with miR-122 Level

0.025 0.05 0.1 0.25

GFP GAPDH miRNA-122 pmiR-122/ug

p miR-122/ug

GFP-target /ug 0.75

0.75 0.75 0.75 0.75

Sensor Killer Switch

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21 DESIGN IGN & RESUL ULTS TS

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22 DESIGN IGN & RESUL ULTS TS

Switch: Tet-off system

ON OFF

iPScells Liver cells Cancer cells Sensor Killer Switch

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23 DESIGN IGN & RESUL ULTS TS Leaky Expression of Different TRE

Sensor Killer Switch

Switching Performance of P

TIGHT

ON OFF OFF

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24 DESIGN IGN & RESUL ULTS TS

25 50 75 100 Mock Mock+Dox

iPSC survival rate (%)

+ DOX + DOX

  • DOX

DOX

 Doxhas little or no toxicity on m iPSCs

Sensor Killer Switch

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DESIGN IGN & RESUL ULTS TS 25

Sensor Killer Switch

All Parts Assembled

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DESIGN IGN & RESUL ULTS TS 26

Sensor Killer Switch

tTA Pef-1α Pmincmv Suicide gene miRNA-122 target TRE DOX tTA tTA

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ASSEM EMBLY BLY WORK 27

Sensor Killer Switch

Chemical reactions of gene expression

𝒀𝟐 + 𝑬

𝒍𝟐

𝒀𝟐𝑬 𝒀𝟐𝑬

𝒍𝟑

𝒀𝟑 𝑬

𝒍𝟒

𝒀𝟑 𝒀𝟑

𝒍𝟓

∅

𝒀𝟐 = 𝒀𝟑 = 𝑬 = ∅ =

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ASSEM EMBLY BLY WORK 28

Sensor Killer Switch

𝒍𝟐 𝒀𝟐 𝑬 = 𝒍−𝟐𝒀𝟐𝑬 𝒆 𝒀𝟑 𝒆𝒖 = 𝒍𝟑 𝒀𝟐𝑬 + 𝒍𝟒 𝑬 − 𝒍𝟓 𝒀𝟑 𝑬 + 𝒀𝟐𝑬 = 𝑬𝟏 = 𝒅𝟏 𝒀𝟐 = 𝒅𝟐

𝒛 = − 𝜸 𝜷 𝒇−𝒃𝒖 + 𝜸 𝜷 𝞫 = 𝒍𝟓 𝞬 = − 𝒍𝟑𝒊𝒅𝟏𝒅𝟐 𝟐 + 𝒊𝒅 + 𝒍𝒅𝟏 𝟐 + 𝒊𝒅𝟐 ⟹ 1. SolutionofODEs:proteinconcentra- tionversustime. 2. Dynamicsandsteadystatearedeter- minedbyreactionparameters

𝞫 𝞬 Protein of interest Time

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ASSEM EMBLY BLY WORK 29

Sensor Killer Switch

2XComplete 4XComplete 2XCUTL 2XCUTL 2XComplete

PEF-𝞫 PminCMV TRE PminCMV

Knockdown effect of different targets

0.7 0.75 0.8 0.85 2*com 2*com+2 +2*C *CUTL 4*com

Knockdown Efficiency

Target Type

PEF-𝞫(weak) PminCMV Tight PminCMV TRE2 PminCMV TRE3G PEF-𝞫(Strong)

4XCUTL

DOX DOX

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ASSEM EMBLY BLY WORK 30

Sensor Killer Switch

2XComplete 4XComplete 2XCUTL 2XCUTL 2XComplete

PEF-𝞫 PminCMV TRE PminCMV PEF-𝞫(weak) PminCMV Tight PminCMV TRE2 PminCMV TRE3G PEF-𝞫(Strong)

4XCUTL

𝒛 = − 𝜸 𝜷 𝒇−𝒃𝒖 + 𝜸 𝜷 𝞫 = 𝒍𝟓 𝞬 = − 𝒍𝟑𝒊𝒅𝟏𝒅𝟐 𝟐 + 𝒊𝒅 + 𝒍𝒅𝟏 𝟐 + 𝒊𝒅𝟐

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ASSEM EMBLY BLY WORK 31

Sensor Killer Switch

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ASSEM EMBLY BLY WORK 32

Sensor Killer Switch

iPSCs Cultivation

hiPSC on feeder hiPSC on matrigel miPSC on feeder miPSC on feeder

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ASSEM EMBLY BLY WORK 33

Sensor Killer Switch

Day 0 Day 2 Day 3 Day 7 HepG2 stable cell line

 miPSC  HepG2  HEK293  Hela  U2OS  HTC75

Infection of different cell lines

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A brief sum up

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Summa mmary y & Futu ture e work 35

Achivements

 Test every part independently  Set up the foundation for future iGEM teams

to work about iPS cells.

 BBa_K1061001  BBa_K1061002  BBa_K1061003  BBa_K1061006

 Submit several biobricks and use multiple

methods to thoroughly characterized them

 BBa_K1061011  BBa_K1061012  BBa_K1061012  BBa_K1061014  BBa_K1061021

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Summa mmary y & Futu ture e work 36

Future work

 Test the circuit in vivo;  Induce our engineered iPSc into liver cells.

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Summa mmary y & Futu ture e work 37

Extensions

 Protect other organs;  Extend the device to gene therapy.

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Summa mmary y & Futu ture e work 38

Human Practice

Have heard about it 25% have never heard about it 75%

People's knowledge about iPS safeguard

Have heard about it have never heard about it

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Summa mmary y & Futu ture e work 39

Acknowlegement

Instructors Advisor Sponsor

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Summa mmary y & Futu ture e work 40

Thank you all!!!

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Design ign & Results ts 41

Experiments on iPSCs

hiPSC on feeder hiPSC on matrigel

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Design ign & Results ts 42

Mouse Primary Hepatocytes

11.29 1.00 7.02 8.22 2 4 6 8 10 12 14 — — 0.25ug 1ug pMiR-122

HEK293 Hepatocytes