Download April 22, 2009

Stem Cells
Sarah Holton
April 22, 2008
April 22, 2009
Stem Cells:
What are they?
✤
Unique cells with the capacity
for self-renewal
✤
Progenitor Cells:
✤
Capable of forming at least
one, or often many
specialized cell types
✤
Present in many adult tissues
✤
Important in tissue repair and
homeostasis
http://www.brown.edu/Courses/BI0032/adltstem/adult-stem-cell.gif
Stem Cells: Types
✤
✤
✤
✤
Unipotent
✤ Can give rise to one cell type
✤ spermatogonial stem cells in testis differentiate to form
spermatozoon
Multipotent
✤ Can give rise to multiple cell types
✤ hematopoietic stem cells produce erythrocytes and all types of
WBCs
Pluripotent
✤ Can give rise to every cell type (from all 3 germ layers: ectoderm,
mesoderm, endoderm)
✤ Derived from embryonic tissues
Totipotent
✤ Fertilized egg is totipotent because it can form all cells and tissues
Pluripotent
Stem Cells
✤
✤
✤
First discovered in
teratocarcinoma
✤ gonadal tumors containing
tissues derived from 3 primary
germ layers
✤ differentiated tissues derived
from pluripotent embryonic
cells (EC)
Cultured embryonic cell lines
derived from tumors
grown in medium containing
serum in presence of feeder layer
of fibroblasts
http://www.pathconsultddx.com/pathCon/diagnosis?pii=S1559-8675(06)70552-6
Pluripotent Stem Cells
✤
✤
Embryonic Stem Cells (ES)
✤ derived from inner cell mass (ICM) cells of pre-implanted
blastocyst-stage embryo
✤ undifferentiated cells sub-cultured onto feeder layers and
expanded into established ES cell lines (seemingly
immortal)
Embryonic Germ Cells (EG)
✤ derived from cultured PGCs isolated directly from
embryonic gonad
✤ when plated onto feeder layers in presence of serum, forms
colonies of cells morphologically different from EC and ES
Pluripotent Stem Cells
✤
Classical markers of pluripotent stem cells
✤
isozyme of alkaline phosphatase
✤
high telomerase activity
✤
POU-domain transcription factor Oct4
✤
✤
Oct4 critical in establishing/maintaining pluripotentcy
cell surface markers recognized to monoclonal antibodies
Pluripotent
Stem Cells
✤
For application, separate out
differentiated cells from
undifferentiated stem cells
✤
Fluorescence-activated cell
sorting (FACS)
✤
Favorable culture conditions
✤
Use of Selectable Markers
Adult Stem Cells
✤
✤
✤
✤
Ethical problems related to obtaining and using embryonic tissuederived stem cells
Multipotent Stem cells exist in most adult tissues
✤ Can be derived from germ cells or somatic cells
How useful are they?
✤ Neural stem cells can form blood-forming and muscle tissue
✤ Mesenchymal stem cells can form differentiated cell types in the brain
✤ Skin stem cells can make neurons, glia, smooth muscle, and
adipocytes
May be extremely useful for treatment of some types of disease but
unable to treat others
Adult Stem Cells
✤
Problems:
✤
Not all can be grown
indefinitely in culture while
maintaining karyotype
(hematopoietic cannot,
oligodendrocyte precursor can)
✤
conditions not established to
allow multipotent cells to
expand in culture without losing
differentiation potential
Adult stem cell from bone marrow (http://www.rochester.edu/pr/Review/V69N1/feature1.ht
Progress
✤
✤
Animal model studies:
✤ cardiomycytes from mouse ES cells form stable, functioning intracardiac
grafts in mice
✤ mouse ES cell derived glial precursors interact with host neurons to
produce myelin in CNS
✤ Retinoic-acid (RA) treated mouse ES cells injected into a rat spinal cord
9 days after traumatic injury, differentiated into astrocytes,
oligodendrocytes, and neurons and promoted motor recovery
✤ genetically selected, insulin-producing cell line derived from mouse ES
cells injected into spleen of streptozotocin-induced diabetic mice
resulted in normal glycemia
Transplanted cells substitute directly for lost populations of cells or provide
factors that facilitate regeneration of host cells
Progress
✤
Clinical Trial:
✤ Biotech company Geron
✤ Phase I: human embryonic stem cell derived oligodendrocytes
to treat spinal cord injury
Product
Description
Disease Treatment
Stage
GRNOPC1
hESC-derived Oligodendrocytes
Spinal Cord Injury
Clinical (Phase I)
GRNCM1
hESC-derived cardiomyocytes
Heart Disease
Preclinical
GRNIC1
hESC-derived Islets
Osteoblasts
Chondrocytes
hESC-Derived cells for drug screening
Immature dendritic cells
Type I Diabetes
Osteoporosis
Osteoarthritis
Liver disease
Immune Rejection
Research
GRNVAC2
Mature Dendritic Cells
Cancer Immunotherapy
Product Research
Therapeutic Challenges
✤
✤
✤
Will derived cells be histocompatible with each individual?
✤ short term: immune suppression or tolerance induction
✤ solution:
✤ therapeutic cloning: isolate somatic nucleus from patient and grow in
oocyte. embryo is genetically identical to patient
✤ stem cell line modified by homologous recombination
Will the transplanted pluripotent cell form a tumor or otherwise differentiate
improperly?
✤ EC, ES, EG cells form tumors when implanted in animals
✤ solution: use differentiated stem cells, but how can we control this?
Will infectious agents possibly present in embryo-derived pluripotent stem
cells or contracted through feeder-culture dependent on bovine serum affect
the patient?
✤ solution: establish conditions for growing pluripotent human stem cells in
serum-free medium
Controlling Differentiation
✤
Pluripotent stem cell
differentiation has been
directed by manipulating the
environment by trial and error
✤
One of the ways to control
stem cell differentiation is by
changing the elasticity of the
growth matrix
Introduction
✤
✤
During normal regenerative
processes, adult stem cells leave
their “niche” and engraft and
differentiate in a range of tissue
microenvironments
Mesenchymal stem cells (MSCs)
✤ marrow-derived
✤ differentiate into anchoragedependent cell types
✤ neurons, myoblasts,
osteoblasts, etc.
http://www.umdnj.edu/gsbsnweb/stemcell/scofthemonth/2007/msc/1.jpg
Effect of microenvironment
✤
✤
Effect is well defined for differentiated cells
✤ for fibroblasts, response to growth factors is coupled with
anchorage to surrounding matrix
✤ matrix stiffness influences focal-adhesion structure and
cytoskeleton
✤ cells committed to a specific lineage respond to physical state of
matrix
✤ fibroblasts respond differently to floating collagen gels and
wrinkling-silicone sheets
What about the effect on naïve stem cells?
Effect of matrix elasticity
✤
[...] Tissue-level matrix
stiffness is distinct and
shown here in sparse
cultures to exert very strong
effects on the lineage
specification and
commitment of naïve MSCs,
as evident in cell
morphology, transcript
profiles, marker proteins, and
the stability of responses
✤
How do the MSCs sense
matrix elasticity?
✤ Ability to pull against
matrix
✤ Requirement of cellular
mechano-transducer to
Mechanotransduction of endothelial shear stress
(http://content.onlinejacc.org/cgi/content-nw/full/j.jacc.2007.02.059v1/FIG4)
How?
✤
✤
✤
✤
✤
✤
One or all of the nonmuscle myosin II isoforms (NMM IIA, B, and/or C)
implicated in tensioning cortical actin structures
Actin structures linked to focal adhesions
✤ provide pathway of force transmission from inside cell to
extracellular elastic matrix
Focal adhesions associated with a number of signaling molecules
which can act as mechano-transducers
In this article, show one or all of the NMM IIA-C likely involved in
matrix-elasticity sensing that drives lineage specification
Blebbistatin blocks branching, elongation, spreading of MSCs on any
substrate and inhibits actin activation of NMM II ATPase activity
Matrix Elasticity
✤
Cell feels resistance as it deforms
extracellular matrix
✤
resistance related to elastic
constant, E
✤
Consider: brain, muscle, and
osteoid precursors of bone
✤
Matrix mimicked in vitro with inert
polyacrylamide gels
✤
degree of elasticity altered by
changing amount of bisacrylamide crosslinking
✤
adhesion controlled by using
collagen I coating
Results
✤
Matrix can specify lineage of MSCs toward neurons, myoblasts, and
osteoblasts
✤
When NMM IIs inhibited with blebbistatin, differentiation is blocked
✤
Soluble induction factors less selective than matrix stiffness
✤
Soluble induction factors cannot reprogram MSCs that have been
grown for weeks on a given matrix
✤
Controlling gel thickness, h, establish how far stem cells can feel and
physically define their microenvironment
Matrix can
specify lineage
✤
MSCs differentiate into cell type
with morphology consistent with
neurons, myoblasts, and
osteoblasts
✤
✤
✤
✤
E(brain) = 0.1-1 kPa
E(muscle) = 8-17 kPa
E(bone) = 25-40 kPa
Microarray:
✤ neurogenic markers highest on
0.1-1 kPa gels, myogenic
markers highest on 11 kPa gels,
osteogenic markers highest on
34 kPa gels
✤ Blebbistatin blocks specification
Soluble factors
✤
✤
✤
In culture, MSC differentiation
is usually induced by soluble
factors (i.e. Dexamethasone)
to directly activate lineage
programs
myoblast system:
✤ soluble factors (MIM)MyoD, Myogenin, skeletal
muscle myosin heavy chain
✤ MIM stimulates myogenesis
regardless of cell shape or
active NMM II
✤ Matrix-driven expression
changes depend on active
NMM II
ECM elasticity + active NMM
II + soluble induction factors
Soluble Factors
✤
✤
✤
✤
MSCs plated in standard growth
media for 1 or 3 weeks on soft
‘neurogenic’ gels and then
switched to induction media
Without induction media, cells
maintain neurogenic marker β3
Tubulin
When induction media is added
after 1 week, result is ‘mixedphenotype’ MSCs (B3 tubulin
levels decline and MyoD levels
increase)
When induction media is added
after 3 weeks, levels of β3
Tubulin stay high: MSCs lose
their plasticity
Stiff substrates
promote
Focal
Adhesions
focal adhesion growth and
✤
✤
✤
elongation (paxillin
immunofluorescence)
✤ increased expression of
components: nonmuscle
α-actinin, filamin, talin,
and focal adhesion
kinase (FAK)
MSCs ‘feel’ their
environment on the length
scale of their adhesions
✤ cell spreading on thin (h:
0.5-1 μm) gel similar to
stiffer gels
Focal adhesions provide
force transmission
pathways through actinmyosin pathways
Conclusions
✤
Stem Cells (both embryonic and adult) have therapeutic potential
✤
Controlled differentiation of multipotent stem cells can be achieved
using engineered matrices of defined elasticities
✤
‘Precommitting’ stem cells to a specific lineage using in vitro matrix
conditions may help overcome an inappropriate or comprimised in
vivo microenvironment
References/Citations
✤
✤
✤
✤
“The end of the beginning for pluripotent stem cells”, Peter J. Donovan and John Gearhart, Nature Vol 414,
November 1, 2001
“Matrix elasticity directs stem cell lineage specification”, Adam J. Engler, Shamik Sen, H. Lee Sweeney, and Dennis
E. Discher, Cell Vol 126, August 25, 2006
Cover picture: “Stem Cell Research: Medical Miracle or Moral Morass?”, Gotham Gazette, March 20, 2006
(http://www.gothamgazette.com/article/issueoftheweek/20060320/200/1794)
“Human Embryonic Stem Cells” (http://www.geron.com/technology/stemcell/stemcellprogram.aspx)