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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

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:

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”


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 cells types and terminology
Stem cells-types and terminology

Can form all tissues

including placenta


Can form any embryonic tissue but not placenta


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

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







Chen et al, 2007

Differentiation of human es cells
Differentiation of human ES cells




Neuronal cells







Sk muscle



Neural tube

Chen et al, 2007

Embryonic or adult stem cells for cell replacementtherapy advantages and disadvantages

Embryonic SC


Stable. Can undergo many cell divisions.

Easy to obtain but blastocyst is destroyed (Ethics)

Possibility of immune rejection

High potential for tumours

Adult SC


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

Re programming the nucleus
Re-programming the nucleus Candidate?

Stem cell

Differentiation is not an irreversible commitment

Stem cell

Differentiated cell

Nuclear reprogramming - functional or molecular changes

in cells undergoing fate changes

Transcription factors for reprogramming
Transcription factors for reprogramming fusion

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 fusion

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: fusion

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

Were these ips cells identical to the es cells
Were these iPS cells identical to the ES cells? fusion


- 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


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

Nanog selected ips cells
Nanog-selected iPS cells fusion

-Expressed all markers and characteristics of ES cells

-Chimera formation when injected into blastocysts


20% of the mice developed tumours

Reprogramming 2 stage process
Reprogramming: 2-stage process fusion

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 fusion

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

Proposed mechanism of ips cell reprogramming
Proposed mechanism of iPS cell reprogramming fusion

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 fusion

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 fusion

Embryonic fibroblasts

Adult tail fibroblasts


Gastric epithelial cell

Pancreatic cell

Neural stem cell

B lymphocyte



Skin fibroblast


Bone marrow stem cell

Peripheral blood cell

iPS Cells- Starting cells

Efficiency of re programming is poor
Efficiency of re-programming is poor fusion

Hochdelinger and Plath, 2009

Derivation of human ips cells
Derivation of human iPS cells fusion

In human cells efficiency of reprogramming ranges

between 0.02% to 0.002%

Potentials of ips cells
Potentials of iPS cells fusion

- 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 fusion

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: generation of iPS cells

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: generation of iPS cells

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 generation of iPS cells

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, generation of iPS cells

non-tumorigenic and

efficiently differentiated


“Lead will be turned into Gold”

Work plans overview
Work Plans-overview generation of iPS cells

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 generation of iPS cells