Stem Cell
Biology
Jim Huettner
11/26/2013
suggested readings
• Solter D. (2006) From teratocarcinomas to embryonic stem
cells and beyond: a history of embryonic stem cell research.
Nat Rev Genet. 7:319-27.
• Hanna JH, Saha K, Jaenisch R. Pluripotency and cellular
reprogramming: facts, hypotheses, unresolved issues. Cell.
2010 Nov 12;143(4):508-25.
• Buganim Y, Faddah DA, Jaenisch R. Mechanisms and models of
somatic cell reprogramming. Nat Rev Genet. 2013
Jun;14(6):427-39.
• Gafni O. et al., (2013) Derivation of novel human ground state
naive pluripotent stem cells. Nature. Oct 30
• Rais Y. (2013) Deterministic direct reprogramming of somatic
cells to pluripotency. Nature. 502:65-70.
Stem Cells: definition
• Self Renewal - undifferentiated cells that can
divide repeatedly while maintaining their
undifferentiated state.
• Pluripotency – ability to differentiate into a
variety of different cell types
In vitro differentiation:
• Cell/tissue replacement therapies
• Human model systems of
disease and development
Donovan and Gearhart, 2001
Types of Stem Cells
Embryonic – from the inner cell mass of preimplantation embryos, prior to formation of
the 3 germ layers (ectoderm, mesoderm,
endoderm)
Somatic – undifferentiated cells found in specific
locations in “mature” tissues
iPS cells – induced pluripotent stem cells
generated by reprogramming differentiated
cells (or cell nuclei, i.e. therapeutic cloning)
Potency
• Totipotent – able to generate every cell type
including extraembryonic tissues
• Pluripotent – able to generate cells from all
three embryonic germ layers
• Multipotent – able to generate a variety of
cells from a particular somatic structure
• Unipotent – only generate one cell type
Time Line
totipotent
fertilization
zygote
morula
pluripotent
multipotent
implantation
somatic
differentiation
blastocyst
gastrulation
Inner cell mass
Epiblast: embryo
Hypoblast: yolk sac
http://stemcells.nih.gov/info/scireport/pages/chapter1.aspx
Early Embryology
Human
Mouse
http://stemcells.nih.gov/info/scireport/pages/appendixa.aspx
making a knockout mouse
http://en.wikipedia.org/wiki/Knockout_mouse
First Isolation of ES cells
Mouse:
Evans MJ, Kaufman MH. (1981) Establishment in culture of pluripotential cells from mouse
embryos. Nature. 292:154-6.
Martin GR. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in
medium conditioned by teratocarcinoma stem cells. P.N.A.S. U S A. 78:7634-8.
Human:
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM.
(1998) Embryonic stem cell lines derived from human blastocysts. Science. 282:1145-7.
Genetic and Developmental Normality (140 cycles):
Suda Y, Suzuki M, Ikawa Y, Aizawa S. (1987) Mouse embryonic stem cells exhibit
indefinite proliferative potential. J Cell Physiol. 133:197-201.
Pluripotency markers
• Stage-specific antigens: Anti-SSEA 3 and 4
recognize globo-series gangliosides
• Tra1-60 and Tra1-81: keratin sulfate surface
antigens
• Oct3/4, Sox2, Nanog – transcription factors
involved with maintaining pluripotency
• Normal karyotype, and pre-X-inactivation?
Two types of ES cells?
“Naïve”
(ICM-like)
•
•
•
•
•
•
Blastocyst chimera (+)
High cloning efficiency
Short doubling time
Xa Xa
Distal Oct4 enhancer
High Nanog, Klf2/4, Rex1
“Primed”
(Epi-SC)
•
•
•
•
•
•
Blastocyst chimera (-)
Low cloning efficiency
Long doubling time
Xa Xi
Proximal Oct4 enhancer
Low Nanog, Klf2/4, Rex1
Both types can self renew and give rise to cells from all 3 germ
layers in teratomas or following in vitro differentiation
maintenance of pluripotency - 1
• Initial work done on mouse embryonic fibroblast (MEF) feeder
cells in medium supplemented with animal serum
• One factor produced by feeder cells that helps maintain
mouse ES cells in their undifferentiated state is leukemia
inhibitory factor (LIF) which activates the Stat3 pathway.
• Good Manufacturing Process (GMP) – guidelines for isolation
and propagation of cells that would be used for replacement
therapy. Ideally they would be xeno-free.
• The push for xeno-free conditions, combined with work to
optimize reprogramming, has driven screening of factors that
can enable serum-free maintenance of pluripotency
maintenance of pluripotency - 2
“naïve”
•
•
•
•
Positive Regulators
LIF - Stat3
BMP4 - Smad1/5
Wnt (GSK-3 inhibitors)
IGF
•
•
•
•
•
“primed”
TGFb/activin – Smad2/3
FGF2
ERK1/2
Wnt (GSK-3 inhibitors)
IGF
Negative Regulators
• TGFb/activin-Smad2/3
• FGF2
• ERK1/2
• BMP4 – Smad1/5
maintenance of pluripotency - 3
“Current Standard” Conditions
Mouse (2008)
• LIF - Stat3
• GSK-3 inhibitors (Wnt)
• ERK1/2 inhibitors
(2i/LIF)
For serum free growth also need:
Insulin, transferrin, progesterone,
putrescine, selenium
Human (2013)
•
•
•
•
•
•
•
•
•
LIF – Stat3
GSK-3 inhibitors (Wnt)
ERK1/2 inhibitors
p38 inhibitor
JNK inhibitor
FGF2
TGF-b1
ROCK inhibitor
PKC inhibitor
In vitro differentiation
• Different culture conditions alter the fate of ES cells
in vitro
• Protocols exist for all three germ layers
• Many, but not all, protocols involve aggregation of ES
cells in “embryoid bodies”
• Most protocols do not yield a single type of cell
• Selection steps can help to remove undesired cell
types
• Need to ask: How far? & How faithful?
ES cells

• pluripotent
• functionally immortal
• genetically &
developmentally normal
neurons
•
•
•
•
postmitotic
polarized
excitable
heterogeneous
Serum Free Medium
4d
4d
4d
SFD
6d
ESC
+ RA
Kim et al., Developmental Biology 328:456-471, 2009
SF+RA
Vimentin
b-tubulin
Developmental Biology 328:456-471, 2009
b-tubulin nestin Hoechst
GAP43 MAP2 Hoechst
GABA b-tubulin Hoechst
voltage-gated Na+ and K+ currents
Developmental Biology 168:342-357, 1995
Journal of Neuroscience 16:1056-65, 1996
Hierarchical clustering by frequency of Gene Ontology terms
Developmental Biology 328:456-471, 2009
Attempts to define “Stemness”
• Early microarray profiles showed surprising
lack of agreement (limitations in microarray
technology or platform/lab/primary cell or cell
line differences) (Science 302:393, 2003)
• Relatively weak overlap between mouse and
human ES cells (~25%) compared to >90%
typical for differentiated tissues. (Stem Cell
Reviews 1:111-118, 2005) but this may reflect
confusion between naïve and primed ES cells
Reprogramming
• SCNT – somatic cell nuclear transfer (reproductive and
therapeutic cloning) – deterministic and fairly rapid
• iPS – induced pluripotent stem cells – slow and
stochastic (until recently)
• Transdifferentiation – conversion of one terminally
differentiated cell type into another without dedifferentiation to an immature phenotype. Must rule
out cell fusion or other explanations.
Reprogramming: somatic cell nuclear transfer
http://www.biotechnologyonline.gov.au/images/contentpages/scnt.gif
Reprogramming Firsts: SCNT
Frog:
Gurdon JB. (1962) Adult frogs derived from the nuclei of single
somatic cells. Dev Biol. 4:256-73.
Sheep:
Campbell KH, McWhir J, Ritchie WA, Wilmut I. (1996) Sheep
cloned by nuclear transfer from a cultured cell line. Nature.
380:64-6.
Human:
(2004) – Claim of human SCNT that proved to be unfounded!
Tachibana M, et al. (2013) Human embryonic stem cells derived
by somatic cell nuclear transfer. Cell. 153:1228-38.
Reprogramming Firsts: iPS cells
Mouse:
Takahashi K, Yamanaka S. (2006) Induction of pluripotent stem
cells from mouse embryonic and adult fibroblast cultures by
defined factors. Cell. 126:663-76
Human:
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda
K, Yamanaka S. (2007) Induction of pluripotent stem cells from
adult human fibroblasts by defined factors. Cell. 131:861-72.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane
JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II,
Thomson JA. (2007) Induced pluripotent stem cell lines derived
from human somatic cells. Science. 318:1917-20.
Generating iPS cells
• Express transcription factors:
Oct3/4, Sox2, Klf4 and c-Myc OR
Oct3/4, Sox2, Nanog and Lin28
• Initial de-differentiation and proliferation
(day 1-3, enhanced by Myc); histone modification
and chromatin reorganization
• 2nd wave of gene expression - stem cell and
development related genes (day 9-12); DNA
demethylation and X reactivation
Graf T. Cell Stem Cell 9:504-516, 2011
Nature Reviews Genetics 14:427-439, 2013
Removing the bottle neck?
• Rais et al., Nature 502:65-70, 2013 implicate
Mbd3, a component in the NuRD complex that
mediates gene repression via histone deacetylation
and chromatin remodeling.
• Argue that the reprogramming factors recruit both
repressive (Mbd3/NuRD) and de-repressive (Wdr5
and Utx) complexes, and reprogramming only occurs
when the Mbd3/NuRd repression loses.
• Achieve nearly 100% reprogramming within 7 days in
cells with Mbd3 reduced or eliminated.
Transdifferentiation
• Conversion from one differentiated cell type
to another without evident de-differentiation
and re-differentiation
• Must not be confused by cell fusion or
selection for rare pluripotent cells in the
source material.
• Induced by expression of transcription factors
and microRNAs
Graf T. Cell Stem Cell 9:504-516, 2011
Fibroblasts to neurons
• Wernig and colleagues screened 19
transcription factors via lentiviral expression
• Found 5 were most critical Asc1, Brn2, Olig2,
Zic1 and Myt1l, and 3 were sufficient
• 20% conversion within 2 weeks
• For human fibroblast conversion also require
NeuroD1 and it is less efficient (2-4%) and
slower (5-6 weeks for functional synapses)
Yang et al., Cell Stem Cell 9:517-525, 2011
Conversion process
• Asc1 bHLH transcription factor binds to many of the
same genomic loci when expressed in fibroblasts,
myoblasts or neural progenitors.
• These sites are marked by specific histone
modifications (H3K4me1, H3K27acetyl, H3K9me3)
• these sites are not accessible in keratinocytes or
osteoblasts, which resist transdifferentiation into
neurons.
• Brn2 Pou-Homeodomain transcription factor is
recruited by Asc1 to a subset of locations
Goals of Reprogramming:
• Models of human disease
• Isogenic cells for
replacement therapy
Aldhous, 2001
Proof of Concept
Hanna et al., Science 318:1920-1923, 2007