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The end of the beginning for pluripotent stem cells Peter J. Donovan*

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The end of the beginning for pluripotent stem cells Peter J. Donovan* & John Gearhart *Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania – PowerPoint PPT presentation

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Title: The end of the beginning for pluripotent stem cells Peter J. Donovan*


1
The end of the beginning for pluripotent stem
cellsPeter J. Donovan John GearhartKimmel
Cancer Center, Thomas Jefferson University,
Philadelphia, PennsylvaniaThe Institute of Cell
Engineering, Johns Hopkins University School of
Medicine, Baltimore, Maryland
  • Jason Ip
  • Graduate Student

2
If we do the work that we can do in this
country, the work that we will do when John Kerry
is president, people like Christopher Reeve will
get up out of that wheelchair and walk
again -Senator John Edwards October 12, 2004
3
Background
  • Pluripotent stem cells
  • def. cells in a stem cell line capable of
    differentiating into several different final
    differentiated types
  • First recognized in teratocarinomas
  • Primordial germ layers
  • The ectoderm, endoderm, and mesoderm, are the
    three major cell lineages
  • Formed during gastrulation (cell migration
    resulting in cleavage)

4
Background
  • The three types of embryonic tissue
  • Embryonic stem (ES)
  • Embryonic germ (EG)
  • Embryonal carcinoma (EC)
  • Attributes of EC, ES, and EG cells
  • Transcription factor Oct4
  • Alkaline Phosphatase
  • Telomerase
  • Upregulation sustains pluripotency

5
Agenda
  • The Science of Pluripotency
  • Developmental potential
  • The basic biology of human development
  • Embryonic stem cells vs. adult stem cells
  • Bringing stem cells to the clinic
  • Expansion and differentiation
  • Safety considerations in cell-based therapies
  • The future of stem cells

6
  • The Science of Pluripotency

7
Developmental potential
  • Assessment in three independent assays
  • In vitro differentiation in a Petri dish
  • Differentiation into teratomas or
    teratocarcinomas within histocompatible mice
  • In vivo differentiation within blastocoel cavity
    of a pre-implantation embryo

8
Developmental potential
  • Directing differentiation
  • Manipulation of cellular environment
  • Growth of cells at high density
  • Growth of cells on different types of feeder
    cells
  • Addition of growth factors
  • Growth on crude or defined ECM substrates
  • differentiation varies and lacks robustness
  • Suspended three dimensional aggregation
  • Development of embryoid bodies

9
Developmental potential
  • Embryoid bodies
  • Capable of differentiating into any of the three
    primordial germ layers
  • Germ layer cells are multipotent, as opposed to
    pluripotent

10
The basic biology of human development
  • Pluripotent stem cells can
  • Aid in deciphering developmental gene-expression
  • Survival
  • Proliferation
  • Differentiation
  • Migration
  • Lend insight to tumorigenesis and genetic diseases

11
Embryonic stem cells vs. adult stem cells
  • Ethical consideration
  • What marks the beginning of life?
  • Main differences
  • The number of potential derivatives embryonic gt
    adult
  • Feasibility
  • Lack of publication on adult stem cell research
    (add more?)

12
  • Bringing stem cells to the clinic

13
Successful transplantations of mouse ES cells
  • Cardiomyoctes form stable and functional grafts
  • Glial precursors interact with host neurons to
    replenish lost myelin in the brain and spinal
    cord
  • Embryoid bodies differentiate into neurons in the
    spinal cord, promoting motor recovery
  • Insulin-producing cell line implanted into mice
    resulted in normalized glycemia

14
Successful transplantations of mouse ES cells
  • Rats with motor injury and stroke treated with
    neuronal cells derived from human EC cells
    resulted in partial recovery of motor function

15
Successful transplantations of mouse ES cells
Transplanted cells are replacing lost cells such
as neurons or glia
Transplanted cells providing factors facilitating
the regeneration of host cells
Cell-based therapies may be useful in abating the
effects of injury and disease
16
Stem cell expansion and differentiation
  • Requirements leading to clinical therapy
  • Growth in large quantities
  • Controlled homogeneous differentiation
  • Histocompatibility
  • Limitations
  • Stem cell survival in long-term culture
  • Stem cell genetic mutations

17
Stem cell expansion and differentiation
  • Possible solutions
  • Progenitor cells
  • Advantages
  • Derived from embryo bodies
  • Easier to grow and expand
  • Possess normal karyotype
  • Disadvantages
  • Limited self-renewal capability
  • Can be unipotent or multipotent
  • Example
  • Neural progenitors can be formed from human ES
    cells in high density culture, become neurons

18
Stem cell expansion and differentiation
  • Genomics
  • Microarray technology can reveal expression of
    growth factors, growth-factor receptors, and
    cell-adhesion molecules
  • Expression profiles allow for optimal
    conditioning of stem cell growth environment

19
Safety considerations in cell-based therapies
  • Three key safety issues
  • Histocompatibility
  • Tumorigenesis
  • Infection from serum-containing culture

20
Safety considerations in cell-based therapies
  • Histocompatibility
  • Immune suppression
  • Slows immune response
  • Nonspecific
  • Tolerance induction
  • Antigen-induced
  • Specific
  • Embryo-derived compatible cells
  • Therapeutic Cloning (somatic cell nuclear
    transfer)
  • Genetic recombination of existing stem cell lines
    to match patient

21
Safety considerations in cell-based therapies
Therapeutic cloning
Genetic Recombination
22
Safety considerations in cell-based therapies
  • Tumorigenesis
  • Conflicting arguments
  • Imprinted genetic loci are erased in EG cell
    lines
  • EG cell lines behave normally in chimeras
  • Key questions
  • How can cells be ensured to migrate to designated
    sites?
  • At what stage of differentiation should
    transplantation occur? (hmmm?)

23
Safety considerations in cell-based therapies
  • Infection from serum-containing culture
  • Infection caused by blood-borne bacteria
  • Serum contains necessary growth factors
  • Human ES cells require fetal calf serum or
    conditioned medium via mouse feeder cells for
    growth

24
The future of stem cells
  • Mouse ES cells have contributed much to our
    understanding of embryogenesis
  • Prospects
  • Stem cell therapeutics
  • Deeper understanding of human growth and
    development
  • Treatment on non-human primates likely to be a
    next step before use in the clinic

25
References
  • http//www.biology-online.org/dictionary.asp
  • http//www.csa.com/discoveryguides/stemcell/images
    /pluri.jpg
  • http//en.wikipedia.org/wiki/ImageCloning_diagram
    _english.svg
  • http//alignmap.com/wp-content/Graphics/JohnEdward
    s(098).jpg
  • http//upload.wikimedia.org/wikipedia/commons/d/d4
    /Cell_differentiation.gif
  • http//www.bio.miami.edu/dana/pix/gastrulation.jpg
  • http//abcnews.go.com/Health/wireStory?id4313450
  • http//www.brown.edu/Courses/BI0032/adltstem/stem-
    cell.gif
  • http//www.wormbook.org/chapters/www_germlinegenom
    ics/germlinegenomicsfig1.jpg
  • http//static.howstuffworks.com/gif/stem-cell-ther
    apeutic.gif
  • http//regentsprep.org/Regents/biology/units/repro
    duction/crossingover.gif
  • http//omegascientific.com/catalog/images/fetal-bo
    vine-fam.jpg
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