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Stem Cell
Jim Huettner
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.
• Buganim Y, Faddah DA, Jaenisch R. Mechanisms and models of somatic cell
reprogramming. Nat Rev Genet. 2013 Jun;14(6):427-39.
• Buganim Y, Markoulaki S, van Wietmarschen N, et al. (2014) The
developmental potential of iPSCs is greatly influenced by reprogramming
factor selection. Cell Stem Cell. 15:295-309..
• De Los Angeles et al. (Daley GQ). (2015) Hallmarks of pluripotency. Nature
• Fox IJ, Daley GQ, Goldman SA, Huard J, Kamp TJ, Trucco M. (2014) Stem cell
therapy. Use of differentiated pluripotent stem cells as replacement therapy
for treating disease. Science 345(6199):1247391.
• Schwartz SD, Regillo CD, Lam BL et al., (2014) Human embryonic stem cellderived retinal pigment epithelium in patients with age-related macular
degeneration and Stargardt’s macular dystrophy: follow-up of two open-label
phase 1/2 studies. Lancet. e-pub October 15.
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,
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)
• 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
Inner cell mass
Epiblast: embryo
Hypoblast: yolk sac
Early Embryology
making a knockout mouse
First Isolation of ES cells
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.
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
• Oct3/4, Sox2, Nanog – transcription factors
involved with maintaining pluripotency
• Normal karyotype, and pre-X-inactivation?
Two types of ES cells?
Blastocyst chimera (+)
High cloning efficiency
Short doubling time
Xa Xa
Distal Oct4 enhancer
High Nanog, Klf2/4, Rex1
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
Positive Regulators
LIF - Stat3
BMP4 - Smad1/5
Wnt (GSK-3 inhibitors)
TGFb/activin – Smad2/3
Wnt (GSK-3 inhibitors)
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
For serum free growth also need:
Insulin, transferrin, progesterone,
putrescine, selenium
Human (2013)
LIF – Stat3
GSK-3 inhibitors (Wnt)
ERK1/2 inhibitors
PKC inhibitor
p38 inhibitor
JNK inhibitor
ROCK inhibitor
but see: Takashima et al., Cell 158:1254-1269 (2014)
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
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?
Pancreatic β cells:
•Pagliuca FW, et al. (2014) Generation of Functional Human Pancreatic β Cells In Vitro.
Cell. Oct, 159:428-39.
•Rezania A, et al. (2014) Reversal of diabetes with insulin-producing cells derived in vitro
from human pluripotent stem cells. Nat Biotechnol. Nov, 32:1121-33.
ES cells
• pluripotent
• functionally immortal
• genetically &
developmentally normal
Serum Free Medium
+ RA
Kim et al., 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
• 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
Reprogramming Firsts: SCNT
Gurdon JB. (1962) Adult frogs derived from the nuclei of single
somatic cells. Dev Biol. 4:256-73.
Campbell KH, McWhir J, Ritchie WA, Wilmut I. (1996) Sheep
cloned by nuclear transfer from a cultured cell line. Nature.
(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
Takahashi K, Yamanaka S. (2006) Induction of pluripotent stem
cells from mouse embryonic and adult fibroblast cultures by
defined factors. Cell. 126:663-76
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, et al., (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.
Skipping the bottle neck?
• Jaenisch lab (Cell Stem Cell 15:295-309, 2014) used
SNEL factors from the deterministic phase (Sall4,
Nanog, Esrrb and Lin28).
• Obtained fewer but “higher quality” mouse iPSC
colonies as judged by production of all-iPSC mice
from 4n blastocyst injections, and lack of trisomy 8.
• Has not worked yet in humans
• Is this de-differentiation or 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
• Brn2 Pou-Homeodomain transcription factor is
recruited by Asc1 to a subset of locations
• SCNT vs iPSCs from isogenic cells:
Ma H, Morey R, O'Neil RC, et al., (2014) Abnormalities in human pluripotent cells
due to reprogramming mechanisms. Nature 511:177-83.
Johannesson B, Sagi I, Gore A, et al., (2014) Comparable frequencies of coding
mutations and loss of imprinting in human pluripotent cells derived by nuclear
transfer and defined factors. Cell Stem Cell 15:634-642.
• Origin-dependence after iPSC differentiation:
Hargus G, Ehrlich M, Araúzo-Bravo MJ, et al., (2014) Origin-dependent neural cell
identities in differentiated human iPSCs in vitro and after transplantation into the
mouse brain. Cell Reports 8:1697-703.
• An optimization strategy?
Morris SA, Cahan P, Li H, et al., (2014) Dissecting engineered cell types and
enhancing cell fate conversion via CellNet. Cell 158:889-902.
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