SYSU-CHINA@iGEM PRESENT

iPSCs

SafeGuard

Fang Yiming He Dawei Zhao Yuchen Chen Haoqi Sun Mengyi

3

Possibility of

Regeneration

4

Promising Prospect in Medical Application

5

Stem Cell Technology

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

6

In Yamanaka’s experiment in 2009, the tumor formation rate is 30%among 100 mice transplanted with iPS cells, much higher thannorman 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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iPS Safeguard

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Part I: Killer---Suicide gene

To prevent

Sensor

Part II: Sensor

iPS Safeguard

Switch

Part III: Switch

Unwanted Cells

wanted Cells

Killer

9

Now let me show you our design & results

Killer Sensor Switch

Design & Results 10

Promoter

PCMV

Suicide gene

SensorKiller 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

Design & Results 11

Suicide genes RIP1 RIP3and Apoptinsuccessfully induce cell death

0

25

50

75

100

Mock Rip1 Rip3 Apoptin GFP

HepG2 Cell Survival Rate (%)

0

25

50

75

Mock Rip1 Rip3 Apoptin

iPSC Survival Rate (%)

Data is from Flow Cytometry Method(FCM)

GFP RIP 1 RIP3

HE

K2

93

(BL

)H

ep

G2

(BL

)H

ep

G2

(DA

PI

sta

inin

g)

100μm100μm 100μm

100μm 100μm 100μm

0h 48h

BL

48

8n

m

Apoptin

SensorKiller Switch

Design & Results 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: one type of somatic cell which represent the normally differentiated cells in our project

SensorKiller Switch

Design & Results 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

SensorKiller Switch

Design & Results 14

Strategy B : Post-transcriptional level Signals: tissue-specific miRNA Sensor: miRNA binding targets on mRNA

Suicide gene miRNA targetsmRNA

miRNA

Suicide gene miRNA targetsmRNA

Wanted cells

Unwanted cells

mRNA

degradation

Suicide gene

expression

SensorKiller Switch

Design & Results 15

A model is found

Human miRNA-122 (endogenous)

Human liver cells

(hepatocyte)

SensorKiller Switch

miRNA-122 miRNA-122

Liver cells Non-liver cells

Data from www.microrna.org

Design & Results 16

PROJECT DECISION

PromoterSuicide gene miRNA-122 target

Liver cells Liver tissue

iPS cells Non-liver cells have included all

unwanted cells: undifferentiated

iPS cells and cancer cellsNon-liver cells suicide

Model : Human Liver cell (hepatocyte)

Molecular marker: miRNA-122

Sensor: miRNA-122 targets

SensorKiller Switch

17DESIGN & RESULTS

Promoter

GFP miRNA-122 target

Completely complementary

binding sequence of

miRNA-122

Natural miRNA-122 binding

sequence

(Partially complementary)

CONSTRUCTION OF SENSOR

SensorKiller Switch

18DESIGN & RESULTS

How to test the sensor?Promoter

GFP miRNA-122 target

Low transfection

efficiency

High transfection

efficiency

Liver cells HEK 293T cells

Endogenous

miRNA-122Endogenous

miRNA-122

SensorKiller Switch

19DESIGN & RESULTS

miRNA-122 gradients by exogenous expression

pMiR-122

HEK 293T

cells

miRNA-122 gradient

miRNA-122

expressing

plasmid

SensorKiller Switch

20DESIGN & RESULTS

miRNA-122 Target Responds Accordingly with miR-122 Level

0 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

SensorKiller Switch

21DESIGN & RESULTS

22DESIGN & RESULTS

Switch: Tet-off system

ON OFF

iPScells Liver cells Cancer cells

SensorKiller Switch

23DESIGN & RESULTS

Leaky Expression of Different TRE

SensorKiller Switch

Switching Performance of PTIGHT

ONOFF OFF

24DESIGN & RESULTS

0

25

50

75

100

Mock Mock+Dox

iPSC survival rate (%)

+ DOX- DOX

Dox has little or no toxicity on m iPSCs

SensorKiller Switch

DESIGN & RESULTS 25

SensorKiller Switch

All Parts Assembled

DESIGN & RESULTS 26

SensorKiller Switch

tTAPef-1α

Pmincmv Suicide gene miRNA-122 targetTRE

DOXtTA

tTA

ASSEMBLY WORK 27

SensorKiller Switch

Chemical reactions of gene expression

𝑿𝟏 +𝑫𝒌𝟏

𝑿𝟏𝑫

𝑿𝟏𝑫𝒌𝟐

𝑿𝟐

𝑫𝒌𝟑

𝑿𝟐

𝑿𝟐𝒌𝟒

∅

𝑿𝟏 = 𝑿𝟐 =𝑫 = ∅ =

ASSEMBLY WORK 28

SensorKiller Switch

𝒌𝟏 𝑿𝟏 𝑫 = 𝒌−𝟏𝑿𝟏𝑫

𝒅 𝑿𝟐𝒅𝒕

= 𝒌𝟐 𝑿𝟏𝑫 + 𝒌𝟑 𝑫 − 𝒌𝟒 𝑿𝟐

𝑫 + 𝑿𝟏𝑫 = 𝑫𝟎 = 𝒄𝟎

𝑿𝟏 = 𝒄𝟏

𝒚 = −𝜷

𝜶𝒆−𝒂𝒕 +

𝜷

𝜶

𝞪 = 𝒌𝟒

𝞫 = −𝒌𝟐𝒉𝒄𝟎𝒄𝟏𝟏 + 𝒉𝒄

+𝒌𝒄𝟎

𝟏 + 𝒉𝒄𝟏

⟹

1. SolutionofODEs:proteinconcentra-tionversustime.

2. Dynamicsandsteadystatearedeter-minedbyreaction parameters

𝞪

𝞫

Protein of interest

Time

ASSEMBLY WORK 29

SensorKiller Switch

2XComplete

4XComplete

2XCUTL

2XCUTL 2XComplete

PEF-𝞪

PminCMVTRE PminCMV

Knockdown effect of different targets0.7

0.75

0.8

0.85

2*com 2*com+2*CUTL 4*com

Knockdown Efficiency

Target Type

PEF-𝞪(weak)

PminCMVTight

PminCMVTRE2

PminCMVTRE3G

PEF-𝞪(Strong)

4XCUTL

DOX DOX

ASSEMBLY WORK 30

SensorKiller Switch

2XComplete

4XComplete

2XCUTL

2XCUTL 2XComplete

PEF-𝞪

PminCMVTRE PminCMV

PEF-𝞪(weak)

PminCMVTight

PminCMVTRE2

PminCMVTRE3G

PEF-𝞪(Strong)

4XCUTL

𝒚 = −𝜷

𝜶𝒆−𝒂𝒕 +

𝜷

𝜶

𝞪 = 𝒌𝟒

𝞫 = −𝒌𝟐𝒉𝒄𝟎𝒄𝟏𝟏 + 𝒉𝒄

+𝒌𝒄𝟎

𝟏 + 𝒉𝒄𝟏

ASSEMBLY WORK 31

SensorKiller Switch

ASSEMBLY WORK 32

SensorKiller Switch

iPSCs Cultivation

hiPSC on feeder hiPSC on matrigel

miPSC on feeder miPSC on feeder

ASSEMBLY WORK 33

SensorKiller Switch

Day 0 Day 2 Day 3 Day 7

HepG2 stable cell line

miPSC

HepG 2

HEK293

Hela

U2OS

HTC75

Infection of different cell lines

34

A brief sum up

Summary & Future 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

Summary & Future work 36

Future work

Test the circuit in vivo;

Induce our engineered iPSc into liver cells.

Summary & Future work 37

Extensions

Protect other organs;

Extend the device to gene therapy.

Summary & Future 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

Summary & Future work 39

Acknowlegement Instructors

Advisor

Sponsor

Summary & Future work 40

Thank you all!!!

Design & Results 41

Experiments on iPSCs

hiPSC on feeder hiPSC on matrigel

Design & Results 42

Mouse Primary Hepatocytes

11.29

1.00

7.02

8.22

0

2

4

6

8

10

12

14

— — 0.25ug 1ugpMiR-122

HEK293Hepatocytes