The Alchemy of Induced Pluripotent Stem Cells

1
The Alchemy of Induced Pluripotent Stem Cells

• Uma Ladiwala
• UM-DAE Centre for Excellence in Basic Sciences
• Kalina Campus, Mumbai

2
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

3
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

4
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

5
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

6
At the Molecular Level
Differentiated Cell
Stem Cell
Pluripotency genes on Differentiation genes off
Pluripotency genes off Differentiation genes on
7
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

8
Stem cell types and origins
9
Stem cells-types and terminology
Can form all tissues including placenta
Embryonic
Can form any embryonic tissue but not placenta
Adult
10
(No Transcript)
11
(No Transcript)
12
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

13
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 cells are 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 Yamanakas work.
14
Whats 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

15
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

16
Cultivation of ES cells
17
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)

18
Derivation and Characterization of human ES cells
AP
Oct4
SSEA4
SSEA3
TRA-1-60
TRA-1-81
Chen et al, 2007
19
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
20
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

21
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

22
The Induced Pluripotent Stem (iPS) Cell A
Likely Candidate?
23
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
24
Reprogramming by somatic cell nuclear transfer
and cell fusion
Dolly the Sheep
25
(No Transcript)
26
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

27
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
28
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
29
Takahashi and Yamanaka, Cell, Aug 25, 2006
30
Takahashi and Yamanaka, Cell, Aug 25, 2006
31
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

32
Is there a way to improve this and get ES like
iPS cells?
Okita K et al, Nature, 2007
33
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

34
(No Transcript)
35
Proposed explanation for the difference
36
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
37
Mechanism of ES cell pluripotency
Oct4, Sox2m and Nanog form an interconnected
auto-regulatory network
38
(No Transcript)
39
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
40
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
41
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

42
Efficiency of re-programming is poor
Hochdelinger and Plath, 2009
43
Derivation of human iPS cells
In human cells efficiency of reprogramming ranges
between 0.02 to 0.002
44
(No Transcript)
45
(No Transcript)
46
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

47
iPS cell reprogramming Problems

• Use of viral vectors for induction
• Low efficiency of reprogramming
• Risk of tumour formation
• Efficient differentiation protocols required

48
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)

49

• Specific pathways
• TGF-ß 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)

50

• 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),

51
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)

52

• If iPS cells are shown to be safe,
• non-tumorigenic and
• efficiently differentiated
• then
• Lead will be turned into Gold

53
Work Plans-overview

• Generation of adult human neural stem cells and
  differentiated progeny from adult somatic cells
  by non-retroviral reprogramming
• (Collaborator Dr. Jacinta DSouza)
• ?
  ?
• 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
  

54
Thank You
55
(No Transcript)
56
(No Transcript)
57
(No Transcript)
58
(No Transcript)
59
(No Transcript)
60
(No Transcript)
61
(No Transcript)
62
(No Transcript)
63
(No Transcript)