Stem Cells

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Stem Cells
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The Fertilized Egg is The Ultimate Stem Cell
Comparison of Normal Development, Reproductive
Cloning Therapeutic Cloning
Hochedlinger, K. Jaenisch, R. N Engl J Med
2003 349275-286
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Dolly!
Dolly at her press conference explaining her
importance
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Many different Cell Types Arise From 3 Germ Layers
Epithelia
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The Generation of Embryonic Stem Cells after
Somatic-Cell Nuclear Transfer
Early Development Generates a Pluripotent Stem
Cell Population
Red White blood cells
Snyder, E. Y. et al. N Engl J Med 2006354321-324
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Cardiomyocytes From Human ESCs
The scheme shows the directed differentiation of
human ESCs to cardiomyocytes and their
application for cardiac repair in a rat model of
cardiac infarct (Laflamme et al., 2007).
Undifferentiated human ESC colonies are replated
as high-density monolayers, expanded, and then
induced to differentiate by sequential treatment
with activin A (day 0) and BMP4 (day 1).
Differentiation along the cardiac lineage can be
further enhanced by activating the Wnt/ß-catenin
pathway. Cultures typically exhibit vigorous
beating activity 1014 days postinduction. These
populations are then subjected to heat shock and
treated with IGF-1 24 hr prior to transplantation
to enhance viability, and then enriched for
cardiomyocytes using Percoll density-gradient
centrifugation. They are then suspended in a
prosurvival cocktail to block cell-death
pathways, and are delivered to the infarcted
heart by direct injection. Experimental endpoints
are assessed by microscopy and magnetic resonance
imaging. By combining these advances,
researchers have generated significant amounts of
human myocardium in the infarcted rat heart,
reaching up to 11 of the infarct's volume
(Figure 4) (Laflamme et al., 2007). The human
myocardium prevented the progression to heart
failure seen in untreated rats and in control
animals receiving noncardiac derivatives of human
ESCs.
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(A) Shown is a confocal fluorescent micrograph of
a human myocardial graft in an infarcted rat
heart. The peri-infarct zone is stained with
human-specific ß-myosin heavy chain (red) and
pan-cardiac marker cardiac troponin I (green)
revealing immature human cardiomyocytes (yellow)
in close apposition to host cardiomyocytes
(green). (Nature Biotechnology 25, 1015,
2007). (B) Human cardiomyocyte engraftment and
cardiac contractile function. Magnetic resonance
imaging demonstrates a 2.5-fold enhancement of
systolic wall thickening in the infarct region of
the rat heart receiving a human cardiomyocyte
graft. Control groups received noncardiac human
ESC derivatives in prosurvival cocktail (PSC),
PSC only, or serum-free media (SFM only). NS, no
significant difference. (Adapted from Laflamme
et al., 2007 M.A. Laflamme et al., Biotechnol. 25
(2007)
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Strategies to Induce Reprogramming of Somatic
Cells(1) Nuclear transfer involves the injection
of a somatic nucleus into an enucleated oocyte,
which, upon transfer into a surrogate mother, can
give rise to a clone (reproductive cloning),
or, upon explanation in culture, can give rise to
genetically matched embryonic stem (ES) cells
(somatic cell nuclear transfer, SCNT). (2) Cell
fusion of somatic cells with ES cells results in
the generation of hybrids that show all features
of pluripotent ES cells. (3) Explantation of
somatic cells in culture selects for immortal
cell lines that may be pluripotent or
multipotent. At present, spermatogonial stem
cells are the only source of pluripotent cells
that can be derived from postnatal animals.
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Tests For Pluripotency
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Yamanakas Bombshell
Takahashi Yamanaka Cell 2006
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Characterization of iPS Cells Derived from Adult
Mouse Tail-Tip Fibroblasts(A) Morphology of
iPS-TTFgfp4-3 on STO feeder cells.(B) RT-PCR
analysis of ES marker gene expression in
iPS-TTFgfp4 cells (clones 15 and 7). We used
primer sets that amplified endogenous but not
transgenic transcripts.(C) Contribution of
iPS-TTFgfp4-7 and iPS-TTFgfp4-3 cells to mouse
embryonic development. iPS cells were
microinjected into C57/BL6 blastocysts. Embryos
were analyzed with a fluorescence microscope at
E7.5 (upper panels, iPS-TTFgfp4-7) or E13.5
(lower panels, iPS-TTFgfp4-3). Scale bars 200
µm (upper panels) and 2 mm (lower panels).(D) The
E13.5 chimeric embryo was sectioned and stained
with anti-GFP antibody (brown). Cells were
counterstained with eosin (blue).
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But Expression of Some Genes May be Better
Indicators than Others
Mice from iPS cells
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Derivation of autologous iPS cells from hßS/hßS
mice and correction of the sickle allele by gene
targeting. (A) Scheme for in vitro reprogramming
of skin fibroblasts with defined transcription
factors combined with gene and cell therapy to
correct sickle cell anemia in mice. (B)
Representative images of various steps of
deriving hßS/hßS iPS line 3. (C) Southern blot
for c-Myc viral integrations in (i) ES cells,
(ii) hßS/hßS iPS line 3 and (iii) its derived
subclone hßS/hßS iPS 3.3 obtained after
infection with adeno-Cre virus and deletion of
the viral c-Myc copies. indicates endogenous
c-Myc band. Arrows point to transgenic copies of
c-Myc. (D) hßS/hßS iPS3.3 displayed normal
karyotype 40XY (upper left), was able to generate
viable chimeras (upper right), and formed
teratomas (bottom). (E) Replacement of the hßS
gene with a hßA globin gene in sickle iPS cell
line 3.3. Homologous recombinants were
identified by PCR to identify correct 5' and 3'
end replacement. PCR with primers 3 and 4
followed by Bsu36I digestion was used to
distinguish hßS and hßA alleles. Correctly
targeted clone 11 displayed identical pattern to
that previously obtained for correctly targeted
ES cell clone
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Yet Another Example!
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a, Schematic diagram of the experimental
strategy. Adenoviruses encoding bicistronic
transcription factor (TF) and nGFP linked by an
IRES element (I) were injected into the pancreas
of an adult mouse (Rag-/-). CMV, cytomegaloviral
promoter. b, Wild type (WT) pancreas is
predominantly exocrine tissue with insulin
-cells in the islet (outlined). Nuclei were
stained blue with DAPI. c, One month after
infection with a combination of Ngn3, Pdx1 and
Mafa viruses (pAd-M3), numerous insulin cells
appear outside of islets. d, e, Quantification of
induction one month after infection. M9, M6
mixture of 9 and 6 different viruses,
respectively. Data are presented as mean  s.d.
n 3 animals. 1,000 nGFP cells were counted
per animal. Asterisk, P lt 0.05 two asterisks,
P lt 0.01 three asterisks, P lt 0.001.