JOURNEY TO IPS CELL :OUR FUTURE

Extra-ordinaryGround-Breaking

Noble prize winning discovery

AboutReprogramming somatic cell

Created BySavani Ahmed

Brief Introduction of stem cell What are stem cells Types of stem cell Various terminology Limitation of using ES cell & Adult stem cell

Journey toward iPS cell Background / History Battle between Egg & nucleus for supremacy How Reprograming can be achieved Potential of iPSCs Application of this Technology Future of iPSCs Limitation/ problem

Prospectus What I Realized

Outline

BRIEF INTRODUCTION OF STEM CELL

unspecialized Can proliferate or self renew can be differentiated into specialized

cell

What are Stem Cell

BroadlyTwo Kinds of Stem Cells

• EMBRYONIC (also called “pluripotent”) stem cells are capable of developing into all the cell types of the body. -Discovered actually isolated in 1998

• ADULT STEM cells posses less Plasicity and more difficult to identify, isolate, and purify. - Discovered in 1960

SCAN – Stem Cell Action Network

Totipotent

PluripotentES cell Multipotent

Various TerminologyTOTIPOTENCY :- ability / potency to form whole organism eg. Zygot(fertilized egg) , early cleavage 2-3 cell blastomeres

PLURIPOTENCY :- ability / potency to form all cell types present in your body not the whole organism eg. Embryonic stem cells (ES cells)

MULTIPOTENCY :- ability / potency to form many types but not all type of cell

eg. Neural stem cell , haemopoetic stem cell & so on

UNIPOTENCY :- ability / potency to form one type of cell eg. Germinal cell spermatogonia & oogonia …

1.Embryonic stem cells - are harvested from the inner cell mass of the blastocyst seven to ten days after fertilization.

2.Fetal stem cells - are taken from the germline tissues that will make up the gonads of aborted fetuses.

3.Umbilical cord stem cells - Umbilical cord blood contains stem cells similar to those found in bone marrow.

4.Placenta derived stem cells - up to ten times as many stem cells can be harvested from a placenta as from cord blood.

5.Adult stem cells - Many adult tissues contain stem cells that can be isolated.

Various type on the basis of their Potencey

Phenotypically Characterised Adult Stem Cells

Limitation of using

Embryonic Stem cell Adult stem cell Less plasticity (potency)

Limited potential

Less abundant

Isolating is difficult

As to use Embryonic stem cell for the purpose either for Research or clinical technique one has to destroy the whole developing embryo to isolate those ICM

And it is considered something immoral so it is been ethical issue therefore banned in many countries

However, current research is changing some of these ideas.

Mutations can lead to leukemia

Problems with Adult Stem Cells

- cell replacement therapy

- drug discovery

- Diseases model

- early human development

Why Are Stem Cells Important?

Dopaminergic Neuron Parkinson Disease

Neural stem cells Spinal cord injury

Cardiac cells Cardiac failure

Pancreatic cells Diabetics Hepatic cells Hepatic failure

Bone cells Osteoporosis

Muscle cells Dystrophy

Bone marrow cells Leukemia Skin cells Burn

Regenerative medicine & ES

Journey to iPS cell

Beginning to start

John gurdon Shinya yamanka

Journey of human start from single cell zygote(fertilized egg)

Robert briggs(1911-1983)

Thomas J. king1921-2000

• In 1952, They worked on a frog, Rana pipiens, became the first to successfully transplant living nuclei in multicellular organisms. They transplanted later embryo (blastula ) cell nuclei into enucleated eggs, which then developed into normal embryos.

• They were initiator of using SCNT for first time• However, the successful transplants that Briggs and King performed were of

undifferentiated nuclei• Until it was possible to accomplish the same feat with a differentiated nucleus, it

would remain an open question as to whether the genome itself somehow changed during development

Worked on

Robert briggs(1911-1983)

Thomas J. king1921-2000

Conclusion they drew out• If & only nucleus transplanted is from the same species as the egg

cytoplasm then only egg will cleaves and can develop in to a normal embryo….further to tadpole

• There something happens when the cell get differentiated that make the nucleus unable to reprogramed or participating in normal development whether it was loss of Genes or some permanent inactivation

• But that was also not clear

Do all cells in the Body have same sets of Genes?

Sir John Gurdon

• In 1958, Gurdon, at the University of Oxford successfully transplanted intestinal epithelium-cell nuclei from Xenopus tadpoles into enucleated frog eggs and managed to produce 10 normal tadpoles: Molly and her fellow clones

• This work was an important extension of work of Briggs and King

• It was cleared that all cell have contain Blueprint of life

The logical consequence of Gurdon's success — that the nuclei of differentiated cells retain their totipotency — provided a key conceptual advance in developmental biology.

• Genes were not lost or changed during cell differentiation — they were just differentially expressed.

• It was cleared that all cell have contain same Blueprint of life

• It proved once-and-for-all that the genome remained intact during differentiation and that the epigenetic changes to the somatic-cell nucleus were reversible.

Provide the KeySomatic cells can be reprogrammed into the embryonic state

Sir John GurdonConclusion derived

You might be Interested to Know how many genes undergoes reprogrammed

Diagram really represent that

Analyses difference between Resistance(repression) & activation state of genes

The battle for supremacy

The egg The nucleus

Designed to transform sperm to an embryo active nucleus

Designed to maintain the same pattern of gene expression

Tries to do the same for somatic nuclei

Tries to resist any change

A sperm nucleus is specially designed to yield normal development

Sperm cell Embryo cell Specialized cell

% of normal development after nuclear transfer (to a feeding tadpole)

99% 35% 1%

Images from Dr Kei Miyamoto

Cloning of sheep : Dolly by SCNT 1998

• In 1998, Thomson’s Lab was the first to report the successful isolation of human embryonic stem cells.

• On November 6, 1998, Science published this research in an article titled "Embryonic Stem Cell Lines Derived from Human Blastocysts", results which Science later featured in its “Scientific Breakthrough of the Year” article, 1999

Human ES cells

Dr. James Thomson, 1998

hES cell

Dr. Shinya Yamanaka, PhD

Dr. Kazutoshi Takahashi, PhD Human iPSCs

2006

2007

Reprogramming factor

A combination of several genes can re-program skin fibroblasts into pluripotent cells

induced Pluripotent stem cells (iPSC)

ES like celliPS cellUsing Retrovirus for induction

Shinya Yamanaka:

OCT3/4

SOX2

C-myc

Klf4

James A Thomson

OCT3/4,

SOX2

NANOG

LIN28

iPSCs were 1st produced in 2007 from human cells by Shinya Yamanka team at Kyoto University Japan, and by James Thomson's team at the University of Wisconsin-Madison. independently

Potential of Induced Pluripotent Stem Cells

Retroviruses

• Randomly inserts DNA into genome of cells• The host cell then treats the viral DNA as part of its

own genome, translating and transcribing the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus.

• Can make special retroviruses with whatever gene you want

• Can’t really control how many copies of genes

Takahashi and Yamanaka, Cell, Aug 25, 2006

Four Magic Genes• Sox2- Self Renewal• Oct4- Differentiation switch • Klf4- p53 pathway, Oncogene• c-Myc- Global Histone Acetylation, Oncogene

Reprogramming Factors – Magic Four

10 candidates 4 candidates24 candidates expressed in embryonic stem cells

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Takahashi and Yamanaka. Cell. 126, 663-676, 2006.

Reprogramming Menu

Different cells have different efficiency for reprogramming

iPS cell sources

• Cell type effects reprogramming efficency• Human iPS cells

– Fibroblasts and keratinocytes - most common sources– Neural cells - neural stem cells need only Oct4– WBCs from blood being developed - convient– Amniotic Fluid cells -increased efficency– Melanocytes - increased efficency.

Application of iPSCs

Long-Term Applications

Applications of Human pluripotent stem Cells

• Basic Knowledge of Human Development• Models of Human Disease• Human model for drug screnning• Transplantation-Cell Replacement • Drug Development • Regenerative Medicine / Therapeutic cloning/

Cell replacement therapy• Organogenesis

Human Diseases Model

1987 Weintraub the transcription factor MyoD turns fibroblasts into muscle

In 1962Cloning in FrogGurdon

1997Cloning in SheepWilmut

2001 ESC fusionTada

2006 iPSCsShinya yamanka

Scientific pathway that lead to iPS cells

1981Mouse ESCsEvans Martin 1998

Human ESCsThomson

FUTURE OF IPSCS

2006 iPSCsShinya yamanka

2007 Mouse iPS therapy

?Human iPSC therapy ?

?Drug discovery ?

2009Patient iPSCsDalleyEggan

2008In vivo directReprogrammingMelton

2012 Mouse Therapy byDirect reprogrammingSrivastava

New Pathway originated from iPS cells

iPSCs has been generated from

Mouse (Yamanaka et al., 2006)

Humans (Yamanaka et al., 2007)

Rhesus monkey (Liu et al., 2008)

Rats (Liao et al., 2009; Li et al., 2009)

Canine (Shimada, H. et al, 2010)

Porcine ( Esteban, M. A. et al., 2009)

Marmoset (Wu, Y. et al., 2010)

Rabbit (Honda, A. et al., 2010)

Equine (Kristina Nagy et al., 2011 )

Avian (Lu et al., 2011)

our contributions to iPSC research

• iPSC without viral integration. • Selection of bonafide iPS clone

based on Imprinting pattern.

• Disease specific iPS.• Differentiation bias due to epigenetic memory. • Ease in gene targeting in hiPS with murine ES

cell state.

• Secondary reprogramming system.• Differentiation state of starting cells.• Endogenous level of reprogramming factors.

Efficiency and Kinetics

SafetyDisease Modeling

iPS cell reprogramming: Problems

• Low efficiency of reprogramming• As using of retroviruses for induction of

factors can lead to mutations and cancers• Epigenetic memory• So many changes in the DNA can be harmful• Risk of tumour formation• Efficient differentiation protocols required

What I Myself Realize

“ Always Biological science especially Cell biology is

intricately Designed to the point where the more We Discover the more We realize how much there

is to discover it is like Question which Yield a thousand

Questions”

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