The alchemy of induced pluripotent stem cells
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The Alchemy of Induced Pluripotent Stem Cells. Uma Ladiwala UM-DAE Centre for Excellence in Basic Sciences Kalina Campus, Mumbai. Alchemists 300 years ago tried, unsuccessfully, to turn base LEAD into valuable GOLD Cellular Alchemy:

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The Alchemy of Induced Pluripotent Stem Cells

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The alchemy of induced pluripotent stem cells

The Alchemy of Induced Pluripotent Stem Cells

Uma Ladiwala

UM-DAE Centre for Excellence in Basic Sciences

Kalina Campus, Mumbai


Alchemy

Alchemists 300 years ago tried,

unsuccessfully, to turn base

LEAD into valuable GOLD

Cellular Alchemy:

Normally, stem cells give rise to somatic cells of the adult organism

Recent developments have resulted in reversing this process with the production of stem cells from adult somatic cells, eg. skin cells

These stem cells have been termed “Induced Pluripotent Stem (iPS) Cells”

ALCHEMY


What is a stem cell properties

What is a Stem Cell?- Properties

An unspecialized cell with a unique capacity for

- indefinite or prolonged self-renewal and

- ability to give rise to differentiated cells


What is self renewal differentiation

Self-renewal - ability to undergo numerous cycles of cell division while maintaining the undifferentiated or unspecialized state

Clonality – ability of a single cell to form many similar cells

Differentiation - process by which a less specialized cell becomes a more specialized one.

Potency- the potential for differentiation to specialized cell types

What is Self Renewal? Differentiation?


Potency of stem cells

Potency of stem cells

Pluripotent – give rise to cells of all 3 germ layers (ectoderm, endoderm, mesoderm and germ cells)

Multipotent – ability to differentiate into many, related cell types

Progenitors –

oligopotent – few cell types

unipotent – one cell type but can self renew


At the molecular level

At the Molecular Level

Differentiated Cell

Stem Cell

Pluripotency genes on

Differentiation genes off

Pluripotency genes off

Differentiation genes on


Where are stem cells found

Where are stem cells found?

Stem cells have been isolated from the embryo, fetus and adult

Embryonic stem (ES) cells: derived from the inner cell mass of the blastocyst (4-5 day embryo)

Adult stem cells : from adult tissues


Stem cell types and origins

Stem cell types and origins


Stem cells types and terminology

Stem cells-types and terminology

Can form all tissues

including placenta

Embryonic

Can form any embryonic tissue but not placenta

Adult


Division of stem cells

A : Stem cell

B : Progenitor cell

C : Differentiated cell

1 : Symmetric division

2 : Asymmetric division

3 : Progenitor cell division

4 : Terminal differentiation

Division of Stem Cells


Timeline of stem cell research

Timeline of Stem Cell Research

1960s - Joseph Altman and Gopal Das present scientific evidence of adult neurogenesis, ongoing stem cell activity in the brain; their reports are largely ignored.

1978 - Haematopoietic stem cells in human cord blood.

1981 - Mouse embryonic stem cellsare derived from the inner cell mass by scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Gail Martin is attributed for coining the term "Embryonic Stem Cell".

1996 - Cloning of Dolly the sheep by somatic cell nuclear transfer

1997 - Leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem cells.

1998 - James Thomson and coworkers derive the first human embryonic stem cell line

2000s - Several reports of adult stem cell plasticity

2004-2005 - Korean researcher Hwang Woo-Suk human embryonic stem cell line from unfertilised human oocytes by SCNT. The lines were later shown to be fabricated.

August 2006 – Mouse Induced pluripotent stem cells: the journal Cell publishes Takahashi and Yamanaka’s work.


What s so special about stem cells

What’s so special about Stem Cells?

They have the potential to replace cell tissue that has been damaged or destroyed.

They can replicate themselves over and over for a very long time- nearly inexhaustible source

Understanding how stem cells develop into healthy and diseased cells will assist the search for cures.

Drugs and chemicals can be screened and tested on patients’ stem cells and their differentiated tissues


Embryonic stem es cells

Embryonic Stem (ES) cells

Derived from inner cell mass of blastocyst

Capacity for almost unlimited symmetrical divisions without differentiation

Give rise to endoderm, ectoderm, mesoderm

Clonogenic- derived from a single ES cell

Capable of colonizing germline and forming egg and sperm cells


Cultivation of es cells

Cultivation of ES cells


Characterization of human and mouse es cells

Characterization of human and mouse ES cells

Expression of cell surface markers –SSEA-3,SSEA-4 (hESC) SSEA-1 (mESC),TRA-1-60, TRA-1-81, alkaline phosphatase, GTCM-2

- Pluripotency transcription factors – Oct-4, Sox-2, Nanog, Rex1

- High Telomerase activity

- Karyotype- normal (46 XX or XY)

- In vitro pluripotency- embryoid body formation

- Teratoma formation in immune-incompetent mice- tumour contains tissues from all 3 germ layers

- Pluripotency in vivo – Chimera formation (mESC)


Derivation and characterization of human es cells

Derivation and Characterization of human ES cells

AP

Oct4

SSEA4

SSEA3

TRA-1-60

TRA-1-81

Chen et al, 2007


Differentiation of human es cells

Differentiation of human ES cells

In-Vitro

In-Vivo

EB

Neuronal cells

Intestine

Cardiac

Mesoderm

AFP

Germ

Respire

Sk muscle

Ova

Cartilage

Neural tube

Chen et al, 2007

http://www.isscr.org/video/beatingMyocytes.mpg


Embryonic or adult stem cells for cell replacementtherapy advantages and disadvantages

Embryonic SC

“Pluripotent”

Stable. Can undergo many cell divisions.

Easy to obtain but blastocyst is destroyed (Ethics)

Possibility of immune rejection

High potential for tumours

Adult SC

“Multipotent”

Less Stable. Capacity for self-renewal is limited.

No ethical concerns

Difficult to isolate in adult tissue.

Host rejection minimized or absent

Less tumorigenic potential

Embryonic or Adult Stem Cells for Cell ReplacementTherapy: Advantages and Disadvantages


The ideal stem cell the holy grail of cell replacement therapy

The Ideal Stem cell – the “Holy Grail” of Cell Replacement Therapy

- Ability to differentiate into many cell types

- Easily accessible

- Individual-specific i.e. personalized or non-immunogenic

- Vastly renewable

- Demonstrably safe

- Non-tumorigenic


The induced pluripotent stem ips cell a likely candidate

The Induced Pluripotent Stem (iPS) Cell : A Likely Candidate?


Re programming the nucleus

Re-programming the nucleus

Stem cell

Differentiation is not an irreversible commitment

Stem cell

Differentiated cell

Nuclear reprogramming - functional or molecular changes

in cells undergoing fate changes


Reprogramming by somatic cell nuclear transfer and cell fusion

Reprogramming by somatic cell nuclear transfer and cell fusion

Dolly the Sheep


Transcription factors for reprogramming

Transcription factors for reprogramming

Transcription factors are proteins that bind to DNA and regulate gene expression

Oct3/4 and Sox2: transcription factors that function in maintaining pluripotency in both early embryos and ES cells.

c-Myc and Klf4: transcription factors that modify chromatin structure so that Oct3/4 and Sox2 can bind to their target; proto-oncogenes


The making of ips cells

The making of iPS cells

Cell trapping strategy: selection of Fbx15-neomycin-resistant cells

What is fbx15 ? - a transcription factor in ES cells and early embryo but not essential for maintainence of pluripotency

Takahashi and Yamanaka, Cell, Aug 25, 2006


The alchemy of induced pluripotent stem cells

24 candidate genes for pluripotency factors:

Ecat1, Dpp5(Esg1), Fbx015, Nanog, ERas,

Dnmt3l, Ecat8, Gdf3, Sox15, Dppa4, Dppa2,

Fthl17, Sall4, Oct4, Sox2, Rex1, Utf1, Tcl1,

Dppa3, Klf4, b-cat, cMyc, Stat3, Grb2

Takahashi and Yamanaka, Cell, Aug 25, 2006


The alchemy of induced pluripotent stem cells

Takahashi and Yamanaka, Cell, Aug 25, 2006


The alchemy of induced pluripotent stem cells

Takahashi and Yamanaka, Cell, Aug 25, 2006


Were these ips cells identical to the es cells

Were these iPS cells identical to the ES cells?

NO

- The transcriptional profile was somewhere between fibroblasts and ES cells

- No live chimeras produced

So these iPS cells were somewhat similar but not identical to ES cells

WHY?

Because fbx15 was selected for. Fbx15 is a factor that is expressed in ES cells but is not essential for the maintainence of pluripotency


Is there a way to improve this and get es like ips cells

Is there a way to improve this and get ES –like iPS cells?

Okita K et al, Nature, 2007


Nanog selected ips cells

Nanog-selected iPS cells

-Expressed all markers and characteristics of ES cells

-Chimera formation when injected into blastocysts

but

20% of the mice developed tumours


Proposed explanation for the difference

Proposed explanation for the difference


Reprogramming 2 stage process

Reprogramming: 2-stage process

Vit C

ESC morphology

All pluripotent markers

No somatic markers

LIF responsive

Chimera forming

Germline competent

ESC morphology

Some pluripotent markers

Loss of somatic markers

LIF unresponsive

No chimeras

Germline incompetent


Mechanism of es cell pluripotency

Mechanism of ES cell pluripotency

Oct4, Sox2m and Nanog form an interconnected auto-regulatory network


Proposed mechanism of ips cell reprogramming

Proposed mechanism of iPS cell reprogramming

Exogenous Oct4 and Sox2 reactivate endogenous Oct4, Sox2 and Nanog and the auto-regulatory loop then becomes self-sustaining.

Exogenous factors are silenced by

DNA methylation

Scheper, Copray, 2009


Induced pluripotency the two stage switch

Induced pluripotency : the two-stage switch

Stage 2

Stage 1

Activation of auto-regulatory loop

Full reactivation of ES cell transcriptional network

Completion of transgene silencing

Downregulation of lineage genes

Activation of specific ES genes

Chromatin remodelling


Ips cells starting cells

Mouse

Embryonic fibroblasts

Adult tail fibroblasts

Hepatocytes

Gastric epithelial cell

Pancreatic cell

Neural stem cell

B lymphocyte

Keratinocyte

Human

Skin fibroblast

Keratinocyte

Bone marrow stem cell

Peripheral blood cell

iPS Cells- Starting cells


Efficiency of re programming is poor

Efficiency of re-programming is poor

Hochdelinger and Plath, 2009


Derivation of human ips cells

Derivation of human iPS cells

In human cells efficiency of reprogramming ranges

between 0.02% to 0.002%


Potentials of ips cells

Potentials of iPS cells

- Ability to differentiate into many cell types

- Easily accessible

- Individual-specific i.e. personalized or non-immunogenic

- Vastly renewable

- Useful for studying mechanisms of disease

- Useful for drug, toxicity testing


Ips cell reprogramming problems

iPS cell reprogramming: Problems

Use of viral vectors for induction

Low efficiency of reprogramming

Risk of tumour formation

Efficient differentiation protocols required


Further work towards safer and more efficient generation of ips cells

Further work towards “safer” and more efficient generation of iPS cells

Reduced number of transcription factor used:

No myc: Nakagawa and Yamanaka, Nat Biotechnol 2008, Wernig and Jaenisch, Cell Stem Cell 2009

No Sox2: by adding GSK-3 inhibitor, Zhou and Ding, Stem cell 2009, in neural stem cell, Kim and Scholer Nature 2008

No Klf4/myc, by addition of Valproic acid : Huangfu and Melton, Nat Biotech 2008

No Myc and Sox2, by addition of BIX01294 and PD0325901 (Zhou and Ding, Cell Stem Cell 2008).

Klf4 only by adding Kenpaullone (Lyssiotis and Jaenisch, PNAS 2009)


The alchemy of induced pluripotent stem cells

Specific pathways:

TGF-β inhibitor replaces Sox2 and cMyc and induce Nanog (Maherali and Hochedlinger, Curr Biol 2009, Ichida and Eggan 2009 )

p53 inhibition augments iPS efficiency (Hong and Yamanaka, Nature 2009,Utikal and Hochedlinger Nature 2009, Marion and Blastco Nature 2009, Li and Serrano Nature 2009, Kawamura and Belmonte 2009)

Hypoxia stimulates iPS generation – Yoshida and Yamanaka Cell Stem Cell 2009

Wnt signaling stimulates reprogramming efficiency (Marsonm, Jaenisch Cell Stem Cell 2008)


The alchemy of induced pluripotent stem cells

Better vectors:

Drug Inducible vectors (Wernig and Jaenisch, Nat Biotechnol 2008, Hockemeyer and Jaenisch, Cell Stem Cell 2008)

Non-integrating vectors adenovirus in hepatocyte (Stadtfeld and Hochedlinger, Science 2008)

Multi-cistronic vectors: single lentiviral cassette ( Carey and Jaenisch, PNAS 2009, Sommer and Mostoslavsky, Stem Cell 2009)

Vector free (episomes, Yu and Thomson, Science 2009; direct transfection, Okita and Yamanaka Science 2008)

Direct protein induction: poly arginine modification of recombinant protein (Zhou and Ding, Cell Stem Cell 2009),


Parallels between regeneration and reprogramming

Parallels between regeneration and reprogramming

Natural dedifferentiation occurs during regeneration in teleost fish, amphibians

C-Myc, Sox2, Klf-4 expressed during limb regeneration in newts (Maki et al, 2009)

Oct4, Sox2 required for normal fin regeneration in zebrafish, but levels not as high as in pluripotent cells (Christen et al, 2010)


The alchemy of induced pluripotent stem cells

If iPS cells are shown to be safe,

non-tumorigenic and

efficiently differentiated

then

“Lead will be turned into Gold”


Work plans overview

Work Plans-overview

Generation of adult human neural stem cells and differentiated progeny from adult somatic cells by non-retroviral reprogramming

(Collaborator: Dr. Jacinta D’Souza)

 

MEFs Adult human fibroblast/keratinocyte

iPS cell or pre-iPS cell better ?

Can pre-iPS cells give rise to multipotent stem cells?

Most efficient method for induction?

Efficient differentiation ?

Three-dimensional cultures on synthetic scaffolds


Thank you

Thank You


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