Turning straw into gold directing cell fate for regenerative medicine

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 NatureReviews|Genetics
 “Turning Straw Into Gold: Directing Cell Fate
          For Regenerative Medicine”
     Volume 12|April 2011
     Pages 243-252
     Authors: Dena E. Cohen & Douglas Melton

 Google   for Images
 Introduction
 Directed Differentiation
 Spontaneous Differentiation
 Co-culture Systems
 Reprogramming
 Transdetermination
 Functional Analysis of Differentiated cells
 Key Challenges
 Conclusion
   Many diseases in humans are caused by deficits in the
    quantity or functionality of particular cells.
     Ex: Neurodegenerative disorders, certain forms of
    blindness and deafness, diabetes and some other
    types of liver and heart disease.

   Creating and delivery of replacement cells to
    patients facilitates disease cure.

   Novel biological studies can be performed on these

   Such cells have promise as tools for drug discovery
    and toxicology testing.
      for disease research and therapy might be
 Cells
 created from readily available sources.

 Three main approaches to convert cells available
 to desired cell types.

 They    are:
           a) In vitro Directed Differentiation
           b) Reprogramming ( Transdifferentiation)
           c) Transdetermination
 Cultured pluripotent stem cells coaxed
 through a series of steps usually designed
 to mimic those that produce the desired
 cell type in vivo.

            cells are derived from the
 Pluripotent
 patient hence there is no risk of immune
 Productionof a particular cell type is
 studied using genetic mutants.

 These mutants help in the
 identification of key transcription
 factors, signaling molecules and
 proteins required for the development
 of the cell type.
 Recombinant  growth factors (involved in
 differentiation of desired cell type in
 vivo)are added to the differentiation

     strategy has been extremely
 This
 successful in mimicking earliest steps in
 embryonic development.
 The first step in the differentiation of the
 pluripotent cells of the inner cell mass occurs
 during gastrulation; which results in the
 formation of three germ layers: Ectoderm,
 Mesoderm and Endoderm.

 The first step is the conversion of ESCs or
 iPSCs into cells of the germ layer that gives
 rise to the desired cell type.

 Developmental  studies demonstrated that
 only a handful of signaling-molecule families
 are required to specify these germ layers.
Family name (number of   Family members used in directed differentiation   Sample applications           Sample refs
members in mammals)

TGFβ superfamily (33)    Activators                    Activin A           Induction of endoderm         87

                                                                           Induction of mesoderm         16,17

                                                       BMP4                Induction of endoderm         29

                                                       TGFβ3               Dopaminergic neuron           53
                                                       TGFβ1               Retinal pigment epithelium    84
                         Inhibitors                    LeftyA              Ectoderm specification        19,28

                                                       Cerberus            Ectoderm specification        28

                                                       Follistatin         Mesoderm specification        16

                                                       Noggin              Anterior neural induction     18

FGF family (23)          Activators                    bFGF                Retinal determination; otic   18,19
                                                       FGF2                Hepatocyte differentiation    29,56

                                                       FGF8                Dopaminergic neuron           27
                                                       FGF10               Hepatocyte differentiation;   19,29
                                                                           otic induction
WNT family (19)          Activators                    WNT3A               Induction of endoderm         20,31

                                                                           Induction of mesoderm         16

                         Inhibitors                    DKK1, Frizzled 8    Induction of ectoderm         18,19

Hedgehog family (3)      Activators                    SHH                 Induction of motor neurons    24

                                                                           Induction of dopamine         53

bFGF-basic fibroblast growth factor;

BMP-bone morphogenetic protein;

DKK1-Dickkopf-related protein 1;

FGF-fibroblast growth factor;

SHH-sonic hedgehog;

TGF-transforming growth factor.
 Optimizationof growth-factors treatments is
 extremely laborious.

 Recombinant  factors are produced in
 engineered bacterial or mammalian cells,
 traces of which may contaminate the final

 Extremely   high cost of recombinant growth
 Offer   an alternative to protein factors.

 Lessexpensive, have less lot-to-lot
 variability, non-immunogenic and more

 Asa result, there is intense interest in the
 idea of replacing biological factors with
 chemical ones in different protocols.

 Threemain approaches can be considered to
 approach this goal.
 Small molecule agonists and antagonists of
  Hedgehog Pathway proved to be effective for
  both neuron differentiation and cancer

 SB-431542 can substitute for protein
 antagonists of TGF-β signaling in
 differentiation of neurons and hepatocytes
 from human ESCs.
 Small molecules are used to inhibit
 signaling through a pathway for
 which an endogenous inhibitor is not

 Thisis used either to direct
 differentiation or as a tool to verify
 the importance of a particular
 signaling pathway in direction a
 given cell-fate decision.
 No protein antagonist of the Hedgehog
  Pathway is known; instead, KAAD-
  cyclopamine has been used to diminish
  Hedgehog signilling to allow the
  formation of gut tube endoderm from
  human ESCs.

 Similarlyno protein antagonist of FGF
 signaling are known; therefore SU5402
 was used as a chemical tool to verify
 the importance of FGF signilling in
 mouse otic lineage induction.
 Usedin cases when the target biological
 pathway is not known or molecules targeting
 the desired pathway have not been

 Howevera limitation of this approach is the
 extreme difficulty in determining the
 mechanism of action of the identified
 Chemical screening has been
  extensively applied to the search for
  molecules that can induce endoderm
  from ESCs in the hope for replacing
  protein activinA.
 Ex:
  Retinoic acid, a form of Vitamin A is an
  endogenous morphogen that is important in
  the patterning of the Central Nervous

 Ithas been successfully used to generate
  neural or retinal cells from ESCs.
Molecule name               Function                                  Effect/use                            Refs
Ascorbic acid               Not known                                 Dopamine and motor neuron             27

                                                                      Cardiac differentiation               34

Nicotinamide                Not known                                 Retinal pigment epithelium            84

Retinoic acid               Endogenous small molecule                 Neuronal protocols                    24

                                                                      Retinal protocols                     35
Taurine                     Endogenous small molecule                 Retinal differentiation               35

PD173074                    FGF inhibitor                             Blocks endogenous caudalizing         86
                                                                      signals in motor neuron
SU5402                      FGF inhibitor                             Blocks otic induction                 19

Hh.Agf.3                    Hedgehog agonist                          Induces motor neurons                 24

C61414                      Hedgehog antagonist                       Blocks motor neuron induction         86

KAAD–cyclopamine            Hedgehog antagonist                       Induces pancreatic cells from         20,31

LY294002                    Phosphoinositide 3-kinase inhibitor       Enhances activin A signalling to      29,33
                                                                      generate endoderm

Indolactam V                Protein kinase C inhibitor                Induces pancreatic progenitors from   31

ALK inhibitor (SB-431542)   TGFβ signalling inhibitor (inhibitor of   Neuron and hepatocyte                 27,28,29
                            activin/Nodal signalling)                 differentiation

SIS3                        TGFβ signalling inhibitor (inhibits       Otic induction                        19

ALK-activin receptor-like kinase;

FGF-fibroblast growth factor;


SIS3-specific inhibitor of SMAD3;

TGF-transforming growth factor.
 ESCs and iPSCs of human or mouse
 origin give rise to desired cell types
 through spontaneous differentiation
 of floating clumps of cells, called
 embryoid bodies.

       for the production of
 Useful
 neuronal cells and cardiomyocytes.
 Differentiationvaries depending on
 the starting size of the embryoid

 Desiredcell type will be in mixed
 population of many other cell types.
                cells are plated on a
 Differentiating
 layer of supporting cells.

 These cells provide appropriate
 environment (cell-cell contacts,
 secretion of a complex mixture of
 factors, or both) to guide
        from the physical location in the
 Derived
 embryo from which the desired cell type

  To guide neuroectodermal cells to become
 hair cells, a co-culture system was used in
 which differentiating mouse iPSCs were
 grown atop cells isolated from embryonic
 chicken utricle(a region of the inner ear).
 Differentiatedcells cannot be
 transplanted into humans owing to the
 risk of contamination with animal
 pathogens or potentially tumorigenic co-
 culture cells.

 Hencethey are useful only for research
 Onefully differentiated cell type is directly
 converted to another.

 No   multipotent or pluripotent intermediate.

 Achieved   by overexpression of key transcription

            are generally used as the starting
 Fibroblasts
 material for production of desired cell types like
 neurons and cardiomyocytes.
 The first step is the identification of
  transcription factors.

 Viralexpression constructs designed to over
  express each factor are produced.

 These are used to transduce a starting
  population of cells.

 Thereprogrammed cells are evaluated by
  immunodetection of a marker gene.
 An adult multipotent stem cell is
 induced to switch from its normal
 lineage to a closely related lineage.

 Throughdifferentiation of its
 progeny it gives rise to desired cell
 Adult multipotent stem cells are
 relatively scarce and difficult to
 Expression   of the marker gene.

 The Transcription Profile, DNA Methylation and
  Histone Modifications of differentiated cells
  and their in vivo counterparts are compared.

 Physiological   behaviour of both cells are

 Abilityto functionally replace the same cell
  type in vivo.
 Relativelylow efficiency of desired cell
  generation with these processes.

           in adapting methods developed using
 Difficulty
  mouse cells for use with human cells.

 Safetyand cost concerns posed by the
  reagents used to direct cell fate.

 Separating desired cells from other cells in the
  preparation is difficult.
 Greatpotential to generate cells for
 transplantation therapy and study.

 Two Phase I clinical trials of human ESC
 derivatives are currently under way.

Description: Many diseases in humans are caused by deficits in the quantity or functionality of particular cells. Ex: Neurodegenerative disorders, certain forms of blindness and deafness, diabetes and some other types of liver and heart disease.Creating and delivery of replacement cells to patients facilitates disease cure.Novel biological studies can be performed on these cells.Such cells have promise as tools for drug discovery and toxicology testing.