Stem Cells
Comparison of Normal Development, Reproductive Cloning &
The Fertilized Egg is The
Therapeutic Cloning
Ultimate Stem Cell
Hochedlinger, K. & Jaenisch, R. N Engl J Med
2003; 349:275-286
Dolly!
Dolly at her press conference
explaining her importance
Many different Cell Types Arise From 3 Germ Layers
Epithelia
Early
Development Generates a Pluripotent Stem Cell Population
The Generation of Embryonic Stem Cells after Somatic-Cell Nuclear Transfer
Red & White
blood cells
Snyder, E. Y. et al. N Engl J Med 2006;354:321-324
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/β-catenin pathway. Cultures typically exhibit vigorous beating activity 10–14 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.
(A) Shown is a confocal fluorescent micrograph of a human myocardial graft in an infarcted rat heart. The peri-infarct zone is stained with humanspecific β-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)
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.
Tests For Pluripotency
Yamanaka’s Bombshell
Takahashi & Yamanaka
Cell 2006
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 1–5 and 7).
We used primer sets that
amplified endogenous but
not transgenic
transcripts.(C) Contribution
of iPS-TTFgfp4-7 and iPSTTFgfp4-3 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, iPSTTFgfp4-7) 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).
But Expression of Some Genes May be Better
Indicators than Others
Mice from iPS cells
Derivation of autologous iPS cells from hβS/hβS 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 hβS/hβS iPS line #3. (C) Southern
blot for c-Myc viral integrations in (i) ES cells, (ii) hβS/hβS iPS
line #3 and (iii) its derived subclone hβS/hβS iPS #3.3 obtained
after infection with adeno-Cre virus and deletion of the viral cMyc copies. * indicates endogenous c-Myc band. Arrows point
to transgenic copies of c-Myc. (D) hβS/hβS iPS#3.3 displayed
normal karyotype 40XY (upper left), was able to generate
viable chimeras (upper right), and formed teratomas (bottom).
(E) Replacement of the hβS gene with a hβA 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 hβS and hβA alleles. Correctly targeted
clone #11 displayed identical pattern to that previously obtained
for correctly targeted ES cell clone
Yet Another
Example!
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 < 0.05; two asterisks,
P < 0.01; three asterisks, P < 0.001.