INDUCED PLURIPOTENT STEM CELLS

Presented by:

VIDUR BHATIA

M. Sc. (F), KUK.

ContentsContents IntroductionIntroduction HistoryHistory Reprogramming of Reprogramming of

somatic cellssomatic cells What are iPSCsWhat are iPSCs Genes responsibleGenes responsible ProductionProduction

first generationfirst generation

Second Second generationgeneration

Human iPSCsHuman iPSCs

ComplicationsComplications Identity to Identity to

natural natural pluripotent stem pluripotent stem cellscells

Applications Applications ConclusionsConclusions ReferencesReferences

Introduction Most of the cells of a multicellular organism become more and more restricted to specific cell lineages.

For the treatment of many genetic diseases Human

embryonic stem cells can be used , But due to some ethics we can’t use embryo for this purpose.

To avoid this problem artificially induced pluripotent stem cells came in picture, which can be created from normal somatic cells by the ectopic expression of some genes which are responsible for the pluripotency.

HistorHistory y

First generated by Shinya Yamanaka et al. First generated by Shinya Yamanaka et al.

At Kyoto in Japan in 2006.; At Kyoto in Japan in 2006.;

Second generated in mice in 2006 by same group.Second generated in mice in 2006 by same group.

Alexender Meissner showed that induction of pluripotency is a Alexender Meissner showed that induction of pluripotency is a slow and gradual process, 2008.slow and gradual process, 2008.

Yang Chao showed that p53 siRNA and UTF1 enhances the Yang Chao showed that p53 siRNA and UTF1 enhances the efficiency of pluripotency, in Nov.2008.efficiency of pluripotency, in Nov.2008.

Conti…..Conti…..

Cesor A. Sommer used a single lentivirus for all the Cesor A. Sommer used a single lentivirus for all the genes required pluripotency in 2008genes required pluripotency in 2008

Yomiuri Shimbun has created the mouse kidney by Yomiuri Shimbun has created the mouse kidney by the use of iPS cells 10 march,2009.the use of iPS cells 10 march,2009.

James A Thomson made the use of plasmid for James A Thomson made the use of plasmid for pluripotency induction 26 march 2009.pluripotency induction 26 march 2009.

Embryonic Stem cells

Totipotent

Pluripotent

Multipotent

Unipotent

Reprogramming of somatic Reprogramming of somatic cells to ES cellscells to ES cells

Somatic cell nuclear transfer

Cell fusion

Treatment with the extract of the pluripotent stem cells

Stable expression of defined factors

(Cowan et al.,2005)

(Wilmut et al.,1997)

(Takahashi and Yamanaka, 2006.)

Cell Fusion TechnologyCell Fusion Technology

Treatment with the extract of the Treatment with the extract of the pluripotent cellspluripotent cells

Permeabilised cells are exposed to cell-free extract of Permeabilised cells are exposed to cell-free extract of pluripotent cells.pluripotent cells.

LimitationsLimitations :- :- a. Limited experience with primary cells. a. Limited experience with primary cells. b. Reprogrammed cells regain only some ofb. Reprogrammed cells regain only some of the properties of pluripotent cellthe properties of pluripotent cell

No. No. SymbolSymbol

11

22

33

44

55

66

77

88

99

1010

1111

1212

1313

1414

1515

1616

1717

1818

1919

2020

Ecat1Ecat1

Dppa5(Esg1)Dppa5(Esg1)

Fbox15Fbox15

NanogNanog

ErasEras

Dnmt31Dnmt31

Ecat8Ecat8

Gdf3Gdf3

Sox15Sox15

Dppa4Dppa4

Dppa2Dppa2

Fthl17Fthl17

Sall4Sall4

Oct3/4Oct3/4

Sox2Sox2

Rex1Rex1

Utf1Utf1

Tcl1Tcl1

Dppa3Dppa3

Klf4Klf4

Twenty four candidate genes play pivotal roles in the maintenance of ES cell identity base on their hypothesis.

(Takahashi and Yamanaka,2006.)

Myb, Kit, Gdf3, Esrrb

21

22

23

24

Stable expression of Stable expression of defined factorsdefined factors

Exogenous expression of Oct3/4, Sox2, Exogenous expression of Oct3/4, Sox2,

Klf4 and c-Myc and factors are essentiallyKlf4 and c-Myc and factors are essentially

required.required.

But large number of factors are also required But large number of factors are also required for pluripotency …..for pluripotency …..

(Takahashi and Yamanaka, August 25, 2006.)

iPSCsiPSCs, are a type of , are a type of pluripotentpluripotent stem cellstem cell artificially derived from a artificially derived from a non-non-pluripotentpluripotent cell, typically an cell, typically an adult adult somatic cellsomatic cell, by inducing a , by inducing a "forced" expression of certain "forced" expression of certain genesgenes..

first produced in 2006 from mouse first produced in 2006 from mouse cells and in 2007 from human cellscells and in 2007 from human cells

WHAT ARE iPSCs ?

Genes responsible for Genes responsible for pluripotencypluripotency

Group 1Group 1

ES cell-Specific transcription factorsES cell-Specific transcription factors

Essential for pluripotency in ES cell & early embryosEssential for pluripotency in ES cell & early embryos

Oct¾, Sox2, Nanog…Oct¾, Sox2, Nanog…

Group 2Group 2 (Proto-oncogene's)(Proto-oncogene's)

Important for proliferation of ES cells, but not in early embryosImportant for proliferation of ES cells, but not in early embryos

TCL1, Stat3, c-Myc, ERas, Klf4…TCL1, Stat3, c-Myc, ERas, Klf4…

Group 3Group 3

Less famousLess famous Specifically expressed in ES cellSpecifically expressed in ES cell

But less defined functionBut less defined function

ECAT1, Esg1,Fbx15, …ECAT1, Esg1,Fbx15, …

(Takahashi and Yamanaka, 2006.)

Oct3/4:Oct3/4: Involve in the maintenance of self renewal of pluripotent Involve in the maintenance of self renewal of pluripotent

cells.cells. Repression in ES cells leads to the formation of Repression in ES cells leads to the formation of

trophoectoderm.trophoectoderm. Overexpression leads to the formation of various lineages Overexpression leads to the formation of various lineages

includingincluding primitive endodermprimitive endoderm..

Sox2:Sox2: Essential for embryonic developmentEssential for embryonic development Downregulation by siRNA silencing leads to the Downregulation by siRNA silencing leads to the

differentiation of cell in murine ES cellsdifferentiation of cell in murine ES cells..

Klf4Klf4

Klf4 repress p53 directlyp53 protein suppress Nanog during ES cell differentiation Klf4 contributes to activation of Nanog and other ES cell-specific genes (Rowland et al., 2005; Lin et al., 2004)

Klf4 acts as an inhibitor of c-Myc-induced apoptosis through the repression of p53 (Zindy et al., 1998)

Klf4 activates p21CIP1, thereby suppressing cell proliferation .This antiproliferation function of Klf4 inhibited by c-Myc, which suppresses the expression of p21CIP1 (Zhang et al., 2000; Seoane et al., 2002)

NanogNanog:: In embryonic stem cells, In embryonic stem cells, Nanog, along with Oct-3/4 and Sox2, is Nanog, along with Oct-3/4 and Sox2, is necessary in promoting pluripotency. necessary in promoting pluripotency.

LIN28LIN28:: LIN28 is an LIN28 is an mRNA binding proteinmRNA binding protein expressed in expressed in embryonic stem cellsembryonic stem cells and and embryonic carcinoma cellsembryonic carcinoma cells associated associated with differentiation and proliferation. with differentiation and proliferation. (Thomson et al.) (Thomson et al.)

Production of iPSCsProduction of iPSCs Typically derived by transfection of Typically derived by transfection of

certain stem cell-associated genes into certain stem cell-associated genes into non-pluripotent cells, such as adult non-pluripotent cells, such as adult fibroblasts. fibroblasts.

Transfection is typically achieved Transfection is typically achieved through viral vectors, such as through viral vectors, such as retroviruses.retroviruses.

After 3–4 weeks, small numbers of After 3–4 weeks, small numbers of transfected cells begin to become transfected cells begin to become morphologically and biochemically morphologically and biochemically similar to pluripotent stem cells, and are similar to pluripotent stem cells, and are typically isolated through morphological typically isolated through morphological selection, doubling time, or through a selection, doubling time, or through a reporter gene and antibiotic selection.reporter gene and antibiotic selection.

(1)Isolate and culture donor cells. (2)Transfect stem cell-associated genes into the cells by viral vectors. (3)Harvest and culture the cells according to ES cell culture, (4)A small subset of the transfected cells become iPS cells and generate ES-like colonies.

First generationFirst generation

First generated by First generated by Shinya YamanakaShinya Yamanaka's 's team at team at Kyoto UniversityKyoto University, Japan in 2006., Japan in 2006.

four key pluripotency genes essential four key pluripotency genes essential for the production of pluripotent stem for the production of pluripotent stem cells were used; Oct-3/4, Sox2, c-cells were used; Oct-3/4, Sox2, c-MycMyc, , and and Klf4Klf4. .

RetrovirusesRetroviruses was used to transfect was used to transfect mouse fibroblasts. mouse fibroblasts.

Cells were isolated by antibiotic Cells were isolated by antibiotic selection of selection of Fbx15Fbx15+ cells. + cells.

LimitationLimitations:-s:-

This iPS line showed DNA This iPS line showed DNA methylation errors compared to methylation errors compared to original patterns in ESC lines and original patterns in ESC lines and failed to produce viable failed to produce viable chimeraschimeras if if injected into developing embryosinjected into developing embryos

Second generation in Second generation in micemice

In June 2007, by the same group.In June 2007, by the same group. These cell lines were also derived from These cell lines were also derived from

mouse fibroblast by retroviral mediated mouse fibroblast by retroviral mediated reactivation of the same four endogenous reactivation of the same four endogenous pluripotent factors, but Instead of Fbx15, pluripotent factors, but Instead of Fbx15, they used they used NanogNanog which is an important which is an important gene in ESCs. gene in ESCs.

DNA methylation patterns and producing DNA methylation patterns and producing viable chimeras (and thereby contributing viable chimeras (and thereby contributing to subsequent germ-line production) to subsequent germ-line production) indicated that Nanog is a major indicated that Nanog is a major determinant of cellular pluripotency.determinant of cellular pluripotency.

LimitatioLimitations:-ns:-

One of the four genes used (namely, One of the four genes used (namely, c-Myc) is c-Myc) is oncogeniconcogenic, and 20% of the , and 20% of the chimeric mice developed cancer. chimeric mice developed cancer.

(In a later study, Yamanaka reported (In a later study, Yamanaka reported that one can create iPSCs even that one can create iPSCs even without c-Myc, although process without c-Myc, although process takes longer and is not as efficient, takes longer and is not as efficient, but the resulting chimeras didn't but the resulting chimeras didn't develop cancer).develop cancer).

Human induced pluripotent Human induced pluripotent stem cellsstem cells

Produced in November 2007. Produced in November 2007.

With the same principle used earlier With the same principle used earlier in mouse models, Yamanaka had in mouse models, Yamanaka had successfully transformed human successfully transformed human fibroblasts into pluripotent stem cells fibroblasts into pluripotent stem cells using the same four pivotal genes: using the same four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc with a Oct3/4, Sox2, Klf4, and c-Myc with a retroviralretroviral system. system.

Pluripotency induction is a slow and Pluripotency induction is a slow and gradual processgradual process

After transfecting the cells with all of the four factors After transfecting the cells with all of the four factors (Sox2,OCct3/4,Klf4 and c-Myc) initially only(Sox2,OCct3/4,Klf4 and c-Myc) initially only

eight colonies were picked at 11th day and ten eight colonies were picked at 11th day and ten colonies on 16th day.colonies on 16th day.

Only one out of eight colonies from 11 day and four Only one out of eight colonies from 11 day and four colonies from ten colonies from 16 day old colonies colonies from ten colonies from 16 day old colonies gave rise the ES like cells.gave rise the ES like cells.

(Alexender Meissner et al.;2007)

ComplicationsComplications Because of viral transfection systems,Because of viral transfection systems, the the

created cells might be created cells might be prone to form prone to form tumors.tumors.

However However Konrad HochedlingerKonrad Hochedlinger and his and his Harvard University research team Harvard University research team successfully used an successfully used an adenovirusadenovirus to to transport the requisite four genes into the transport the requisite four genes into the DNA of skin and liver cells of mice.DNA of skin and liver cells of mice.

Since the adenovirus does not combine any Since the adenovirus does not combine any of its own genes with the targeted host, the of its own genes with the targeted host, the danger of creating tumors is eliminated.danger of creating tumors is eliminated.

IdentityIdentity

The generated iPSCs were remarkably The generated iPSCs were remarkably similar to naturally-isolated pluripotent similar to naturally-isolated pluripotent stem cellsstem cells

Cellular biological properties: Cellular biological properties: MorphologyMorphology, , Growth propertiesGrowth properties, , Stem Cell Stem Cell

MarkersMarkers, , Stem Cell GenesStem Cell Genes, , Telomerase Telomerase Activity:Activity:

Pluripotency of iPSCs :Pluripotency of iPSCs : Neural Differentiation, Cardiac DifferentiationNeural Differentiation, Cardiac Differentiation, ,

Teratoma FormationTeratoma Formation, , Embryoid Body, Embryoid Body,

Conti…Conti…....

Epigenetic reprogramming :Epigenetic reprogramming :

Promoter Promoter DemethylationDemethylation,,Histone Histone

demethylationdemethylation etc. etc.

APPLICATIONS APPLICATIONS

iPS Cells - the Wave of iPS Cells - the Wave of FutureFuture

iPSC regarded as holy grail stem cell researchiPSC regarded as holy grail stem cell research Studying disease models Studying disease models in vitroin vitro Drug screeningDrug screening Toxicological testing of new drugsToxicological testing of new drugs Generating patient specific & disease specific pleuripotent stem Generating patient specific & disease specific pleuripotent stem

cellscells Allow unprecedented access to all stages of human biologyAllow unprecedented access to all stages of human biology Studying development & function of human tissueStudying development & function of human tissue Regenerative medicineRegenerative medicine

iPS CELLS TO CURE SC ANEAMIAiPS CELLS TO CURE SC ANEAMIA

Cell replacement therapy seems particularly suitable for Cell replacement therapy seems particularly suitable for Parkinson’s disease,Parkinson’s disease,

A common neurodegenerative disease caused by loss of A common neurodegenerative disease caused by loss of midbrain dopamine neurons . Transplantation of fetal midbrain midbrain dopamine neurons . Transplantation of fetal midbrain cells has been shown to restore dopamine function in animal cells has been shown to restore dopamine function in animal models and in human patients . models and in human patients .

((Parish CL, Parish CL, et alet al. (2008)). (2008))

iPS can be used for treatment of

Parkinson’s disease

Mouse kidneys created using iPS cells

A team of scientists has successfully used induced pluripotent stem (iPS) cells to create kidneys inside a mouse whose parents were genetically engineered so their offspring would not be born with the organ.

(Hiromitsu Nakuchi; Mar. 10, 2009))

Stem cells scientists at UCLA showed for the first Stem cells scientists at UCLA showed for the first time that human induced pluripotent stem (iPS) cells time that human induced pluripotent stem (iPS) cells can be differentiated into electrically active motor can be differentiated into electrically active motor neurons, a discovery that may aid in studying and neurons, a discovery that may aid in studying and treating neurological disorders.treating neurological disorders.

The motor neurons derived from the iPS cells The motor neurons derived from the iPS cells appeared to be similar in function and efficiency to appeared to be similar in function and efficiency to those derived from human embryonic stem cells, those derived from human embryonic stem cells, although further testing needs to be done to confirm although further testing needs to be done to confirm that. that.

iPSc can be used to create

Electrically Active Neuron

(Michael Scott on February - 25 – 2009)

Somatic cells can be used Somatic cells can be used to generate the to generate the β-cells-cells

Pancreatic Exocrine cells can be converted into β-cells closely related to the islet β-cells.

Can be used to cure diabetes.

(Qiao Zhou et al.,2008)

ConclusionConclusion

SCNT and cell fusion may use to SCNT and cell fusion may use to produce the pluripotent cells but can be produce the pluripotent cells but can be used only for animals, these processes used only for animals, these processes can’t be shifted to human beings.can’t be shifted to human beings.

iPS may be the answer of the all iPS may be the answer of the all question of ethics and may avoid the question of ethics and may avoid the problem of transplant rejection. In problem of transplant rejection. In future these technique may help us in future these technique may help us in the field genetic study, research and to the field genetic study, research and to fight against disease. fight against disease.

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

Yamanaka S. & et al. ; Generation of germline- competent induced pluripotent stem cells, Nature 2007;448:313-317.

Maherali N & et. al.; Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution, Cell Stem Cell 2007;1:55–70 .

References

Stadtfeld M., Nagaya M., Utikal J., Weir G., Hochedlinger K.Induced Pluripotent Stem Cells Generated without Viral Integration. Science 2008 Sep. 25. pp.212-220.

Okita K., Nakagawa M., Hyenjong H., Ichisaka T,Yamanaka S. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors, Science, 2008 Oct 9. pp.167-178.

Thank Thank youyou