I-PS CELLS

Capt Rishi Pokhrel

JOURNAL CLUB

iPS cell

‘Induced pluripotent stem

cell is a type of pluripotent

stem cell artificially derived

from a non-pluripotent cell -

typically an adult somatic

cell - by inducing a "forced"

expression of specific genes’.Baker, Monya (2007-12-06).

"Adult cells reprogrammed to pluripotency, without tumors".

Nature Reports Stem Cells. doi:10.1038/stemcells.2007.124

Nobel Prize for

Medicine 2012

Shinya Yamanaka

President of the International Society for Stem Cell Research (ISSCR).

MBBS

MD (1987)

Ph. D (1993)

Residency in Orthopedic surgery

Post doctoral fellowship in Cardiovascular disease

Professor of anatomy University of California,

San Francisco, USA

Nobel prize awarded for

• Generation of induced pluripotent stem cells from adult mouse

fibroblasts (2006)

• Closedly resembled embryonic stem cells (in vivo equivalent of

blastocyst)

• iPSC were pluripotent – could generate whole iPSC mice

• iPSC cells from human adult fibroblasts for the first time (2007)

• Initially used 24 transcription factors for inducing pluri-potency

• Successful in narrowing down the number of factors to just 4

Sox2, Oct4, Klf 4 and c-Myc

Yamanaka SCell stem cellJune 14, 2012Volume 10, No 6Page no 678-684

Literature review• Stem cells• Transcription factors

• Stem cells• Application• Problem

Transcription factor

A regulatory protein that binds to DNA and affects the transcription of specific genes.

Introduction

iPSC: Past, Present and future

How was iPSC possible?• Reprogramming by nuclear transfer• Tadpoles from unfertilized eggs that

received nucleus from intestinal cells of adult frogs (Gurdon J 1962)

• Cloning of Dolly (Wilmut W 1997)

• Adult somatic cells contain all genetic information

• Oocyte contain factors that can reprogram somatic cell nuclei, so do ESC (Tada T 2001)

1Past

How was iPSC possible?

• Discovery of transcription factors– Genes of drosophila coding for antenna could

form legs when ‘antennapeda’ was introduced (Schneuwly 1987)

– Mammalian fibroblasts converted to myocyte using MyoD (Davis 1987)

2

How was iPSC possible?

• Generation of ESC, mouse (Evans 1981), human (Thomson 1988) and culture media

• Long term maintenance of pluripotency using LIF (Smith 1988)

• Optimal cultural conditions with bFGF

3

• iPSC : simplicity and reproducibility• Poor efficacy: success rate 1% (?)• Integrated vectors used for introducing

transcription factors -> retroviruses, can cause mutagenesis & other adverse effects

• Use of non-integrated vectors: plasmid, Sendai virus, adenovirus, synthetic RNA and proteins

• Technology development -> applications

Present

Current works focused in

Regenerative medicine–Parkinson's disease–Platelet deficiency– Spinal cord injury–Macular degeneration

Future

Disease models

• Patient derived iPSC used for testing of drugs & toxins

• Found useful for creating models of late onset diseases like Parkinson’s, Alzheimer’s, Schizophrenia

• Analysis of disease mechanisms

Use in animals

• Genetic engineering• Production of deficient proteins e.g.

enzymes• Preservation or recreation of endangered

or extinct animals

Direct reprogramming• In vivo conversion of exocrine pancreatic cells to

endocrine using 3 transcription factors (Zhou

2008)

• In vitro conversion of adult mature fibroblasts to

neural cells, hepatocyte, cardiomyocyte or

hematopoietic progenitor cells

• Problem: source of cells?

A step ahead

iPSC Vs ESC

• Similar and different Source of tissue– Culture medium– Source of clone e.g. labs– Vectors used– Both are basically artificial cells

Dark side

• Variation in– gene expression– DNA methylation– Pluripotent potential– Somatic mutations– Copy number variations– Immunogenicity

So is it just another hoax?

DARK SIDEUNDER ATTACK

FLAWED TROUBLESOME

GROWING PAINS

Not really• Genetic defects preexisted in source cells

• Cloning magnified the defects

• Immunogenicity is very weak - its effects nil in animal experiments

• ESC not gold standard for comparisons of iPSC

Conclusions

• iPSC technology ready for applications• Necessity of establishment of in-advance

stocks of clones• Source of tissue: healthy donors, cord blood,

HLA homozygous donors

CRITICAL APPRAISAL

DISCUSSION