Advanced Aminal Cell Culture

2013 2nd Semester

Department of Animal Science

Chungbuk National University

6th Lecture

Syllabus

Date TopicsSeptember 5, 2013 Introduction : What is Cell Culture?

September 12, 2013 Cell Culture As Model System For Research

September 26, 2013 Cell Culture For Antibody / Protein Production

October 17, 2013 Protein Production/Purification

October 31, 2013 Stem Cell INovember 14, 2013 Stem Cell IINovember 28, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS

December 12, 2013 Final Exam

DateSeptember 26, 2013 Cell Culture For

Antibody / Protein Production

조유미 , Madhusumida

October 17, 2013 Protein Production/Purification 이미진 , 정용호

October 31, 2013 이영 , 윤준호November 14, 2013 Stem Cell I Jia Jia Lin, 염동현

November 28, 2013 Stem Cell II Zhao MingHui,권정우December 5, 2013 Transgenic Animals Lin Zili, 이상배December 12, 2013 Genome

Engineering/NGS 조유진 , 김상욱

Stem Cell II

Today’s topic : Reprogramming

Differentiated Cell vs Stem Cell

Cell Differentation

The process by which cells or parts of an organism become different from one and other and also from their previous state.

The process by which cells or tissues of an organism acquire the ability to perform their special functions.

How is this accomplished?

Selective gene transcription

In any given cell, only the genes necessary for basic metabolism

and that cell’s special functions are active.

* muscle cells - actin, myosin

* pancreatic acinar cells - digestive enzymes

* neurons – neurotransmitters

Mechnism of Cell Differentitation

How selective gene transcription could be achieved?

Messiner, Nature Biotechnol. 2010

Changes in epigenetic marks during Development

Bock et al., Mol.Cell 2012

Reprograming

How to return back to pluripotent state?

Things to do :

- Reset all of epigenetic marks which repress pluripotent factors maintenance genes(Activation)

- Reset all of epigenetic marks which activate lineage specific gene expression(Repression)

Natural Reprogramming : Sperm & Oocyte

Sperm is highly differentated Cell.After fertillization, DNA derived from sperm is demethylated rapidly

Smith et al., Nature 2012

Conclusion : Oocyte have ability to reprogramming terminally differentiated cell

Experiments by John Gurdon (1962)

Somatic Cell Nuclear Transfer (SCNT)

Animal Cloning

Stem Cell from Somatic Cell Nuclear Transfer (SCNT)

Drawbacks of Stem Cell Generation using SCNT

- Ethical Problems

• ES cell should be generated from Cloned Blastocyst• Human Oocyte is needed• Human Cloning?

- Technical Problems : Low efficiency

iPSC (induced Pluripotent Stem Cell)

‘Yamanaka Factor’

- Oct4, Sox2, Klf-4, c-Myc (OSKM)- Transcription Factors which express abundantly in Embryonic Stem Cell- Screened from 24 transcription factors expressed in ESC- Retroviral expression of these 4 genes in embryonic/Adult fibroblast transformCells into ‘Stem Cell Like’ cells

Molecular event of induced pluripotency

Cell Lineage Specific Gene

Pluripotency Maintenance genes

Limitation of iPSC

- Although iPSC is much similar with ESC, its properties is not exactly same with those of ESC

• Source of Original Cell• Condition used for the generation of iPSCDifferent epigenetic states Different tumorgenicity…

- Potential Tumorgencity of iPSC

- Retroviral vector : Danger in the tumor inducing

- Known oncogene (c-Myc) was one of component

- Low efficiency for reprogramming

- Less factors (Sometime Only Oct4 can induce pluripotency)

-Protein-mediated generation of iPSC

- Chemical mediated?

Efforts to overcome these Limitation

Methodologic Limitation

‘OSKM’ is not enough? Presence of ‘Break’

Rais et al., Nature 2013

Current Application of iPSC

- Research Tools : Patient specific cell derived from iPSC

ES Cell

ES Cell

High-throughput screening of drug candidate using iPSC derived cells