Advances in Embryonic Stem Cell Research
The studies cited below are a sampling of the
published and peer-reviewed material available on the subject. The
list will be updated on a regular basis.
Production of pancreatic hormone-expressing
endocrine cells from human embryonic stem cells.
D'Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick
AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE.
Novocell Inc., 3550 General Atomics Ct., San Diego, California 92121,
USA., Nat Biotechnol. 2006 Nov;24(11):1392-401. Epub 2006 Oct 19.
Abstract: Of paramount importance
for the development of cell therapies to treat diabetes is the production
of sufficient numbers of pancreatic endocrine cells that function
similarly to primary islets. We have developed a differentiation
process that converts human embryonic stem (hES) cells to endocrine
cells capable of synthesizing the pancreatic hormones insulin, glucagon,
somatostatin, pancreatic polypeptide and ghrelin. This process mimics
in vivo pancreatic organogenesis by directing cells through stages
resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm
and endocrine precursor--en route to cells that express endocrine
hormones. The hES cell-derived insulin-expressing cells have an
insulin content approaching that of adult islets. Similar to fetal
beta-cells, they release C-peptide in response to multiple secretory
stimuli, but only minimally to glucose. Production of these hES
cell-derived endocrine cells may represent a critical step in the
development of a renewable source of cells for diabetes cell therapy.
Distinct differentiation characteristics of
individual human embryonic stem cell lines.
Mikkola M, Olsson C, Palgi J, Ustinov J, Palomaki T, Horelli-Kuitunen
N, Knuutila S, Lundin K, Otonkoski T, Tuuri T. , Program of Developmental
and Reproductive Biology, Biomedicum Helsinki, PO Box 63, 00014
University of Helsinki, Helsinki, Finland. email@example.com,
BMC Dev Biol. 2006 Aug 8;6:40
ABSTRACT: Individual differences
between human embryonic stem cell (hESC) lines are poorly understood.
Here, we describe the derivation of five hESC lines (called FES
21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare
their individual differentiation characteristic. RESULTS: The cell
lines were cultured either on human or mouse feeder cells. The cells
grew significantly faster and could be passaged enzymatically only
on mouse feeders. However, this was found to lead to chromosomal
instability after prolonged culture. All hESC lines expressed the
established markers of pluripotent cells as well as several primordial
germ cell (PGC) marker genes in a uniform manner. However, the cell
lines showed distinct features in their spontaneous differentiation
patterns. The embryoid body (EB) formation frequency of FES 30 cell
line was significantly lower than that of other lines and cells
within the EBs differentiated less readily. Likewise, teratomas
derived from FES 30 cells were constantly cystic and showed only
minor solid tissue formation with a monotonous differentiation pattern
as compared with the other lines. CONCLUSION: hESC lines may differ
substantially in their differentiation properties although they
appear similar in the undifferentiated state.
Functional engraftment of human ES cell-derived
dopaminergic neurons enriched by coculture with telomerase-immortalized
Roy NS, et al., Nat Med. 2006 Nov;12(11):1259-68. Epub 2006
Abstract: To direct human embryonic
stem (HES) cells to a dopaminergic neuronal fate, we cocultured
HES cells that were exposed to both sonic hedgehog and fibroblast
growth factor 8 with telomerase-immortalized human fetal midbrain
astrocytes. These astrocytes substantially potentiated dopaminergic
neurogenesis by both WA09 and WA01 HES cells, biasing them to the
A9 nigrostriatal phenotype. When transplanted into the neostriata
of 6-hydroxydopamine-lesioned parkinsonian rats, the dopaminergic
implants yielded a significant, substantial and long-lasting restitution
of motor function. However, although rich in donor-derived tyrosine
hydroxylase-expressing neurons, the grafts exhibited expanding cores
of undifferentiated mitotic neuroepithelial cells, which can be
tumorigenic. These results show the utility of recreating the cellular
environment of the developing human midbrain while driving dopaminergic
neurogenesis from HES cells, and they demonstrate the potential
of the resultant cells to mediate substantial functional recovery
in a model of Parkinson disease. Yet these data also mandate caution
in the clinical application of HES cell-derived grafts, given their
potential for phenotypic instability and undifferentiated expansion.
Recovery from paralysis in adult rats using
embryonic stem cells.
Deshpande DM, et al., Ann Neurol. 2006 Jul;60(1):32-44.
OBJECTIVE: We explored the potential
of embryonic stem cell-derived motor neurons to functionally replace
those cells destroyed in paralyzed adult rats. METHODS:
We administered a phosphodiesterase type 4 inhibitor and dibutyryl
cyclic adenosine monophosphate to overcome myelin-mediated repulsion
and provided glial cell-derived neurotrophic factor within the sciatic
nerve to attract transplanted embryonic stem cell-derived axons
toward skeletal muscle targets. RESULTS: We found
that these strategies significantly increased the success of transplanted
axons extending out of the spinal cord into ventral roots. Furthermore,
transplant-derived axons reached muscle, formed neuromuscular junctions,
were physiologically active, and mediated partial recovery from
paralysis. INTERPRETATION: We conclude that restoration
of functional motor units by embryonic stem cells is possible and
represents a potential therapeutic strategy for patients with paralysis.
To our knowledge, this is the first report of the anatomical and
functional replacement of a motor neuron circuit within the adult
Transplantation of human embryonic stem cell-derived
cells to a rat model of Parkinson's disease: effect of in vitro
differentiation on graft survival and teratoma formation.
Brederlau A, et al., Stem Cells. 2006 Jun;24(6):1433-40. Epub 2006
Abstract: Human embryonic stem cells
(hESCs) have been proposed as a source of dopamine (DA) neurons
for transplantation in Parkinson's disease (PD). We have investigated
the effect of in vitro predifferentiation on in vivo survival and
differentiation of hESCs implanted into the 6-OHDA (6-hydroxydopamine)-lesion
rat model of PD. The hESCs were cocultured with PA6 cells for 16,
20, or 23 days, leading to the in vitro differentiation into DA
neurons. Grafted hESC-derived cells survived well and expressed
neuronal markers. However, very few exhibited a DA neuron phenotype.
Reversal of lesion-induced motor deficits was not observed. Rats
grafted with hESCs predifferentiated in vitro for 16 days developed
severe teratomas, whereas most rats grafted with hESCs predifferentiated
for 20 and 23 days remained healthy until the end of the experiment.
This indicates that prolonged in vitro differentiation of hESCs
is essential for preventing formation of teratomas.
Dopaminergic neurons generated from monkey
embryonic stem cells function in a Parkinson primate model.
Takagi Y, et al., J Clin Invest. 2005 Jan;115(1):102-9.
Abstract: Parkinson disease (PD)
is a neurodegenerative disorder characterized by loss of midbrain
dopaminergic (DA) neurons. ES cells are currently the most promising
donor cell source for cell-replacement therapy in PD. We previously
described a strong neuralizing activity present on the surface of
stromal cells, named stromal cell-derived inducing activity (SDIA).
In this study, we generated neurospheres composed of neural progenitors
from monkey ES cells, which are capable of producing large numbers
of DA neurons. We demonstrated that FGF20, preferentially expressed
in the substantia nigra, acts synergistically with FGF2 to increase
the number of DA neurons in ES cell-derived neurospheres. We also
analyzed the effect of transplantation of DA neurons generated from
monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated
(MPTP-treated) monkeys, a primate model for PD. Behavioral studies
and functional imaging revealed that the transplanted cells functioned
as DA neurons and attenuated MPTP-induced neurological symptoms.
Human embryonic stem cell-derived oligodendrocyte
progenitor cell transplants remyelinate and restore locomotion after
spinal cord injury.
Keirstead HS, et al, J Neurosci. 2005 May 11;25(19):4694-705.
Abstract: Demyelination contributes
to loss of function after spinal cord injury, and thus a potential
therapeutic strategy involves replacing myelin-forming cells. Here,
we show that transplantation of human embryonic stem cell (hESC)-derived
oligodendrocyte progenitor cells (OPCs) into adult rat spinal cord
injuries enhances remyelination and promotes improvement of motor
function. OPCs were injected 7 d or 10 months after injury. In both
cases, transplanted cells survived, redistributed over short distances,
and differentiated into oligodendrocytes. Animals that received
OPCs 7 d after injury exhibited enhanced remyelination and substantially
improved locomotor ability. In contrast, when OPCs were transplanted
10 months after injury, there was no enhanced remyelination or locomotor
recovery. These studies document the feasibility of predifferentiating
hESCs into functional OPCs and demonstrate their therapeutic potential
at early time points after spinal cord injury.
Transplantation of apoptosis-resistant embryonic
stem cells into the injured rat spinal cord.
Howard MJ, et al., Somatosens Mot Res. 2005 Mar-Jun;22(1-2):37-44.
Abstract: Murine embryonic stem cells
were induced to differentiate into neural lineage cells by exposure
to retinoic acid. Approximately one million cells were transplanted
into the lesion site in the spinal cords of adult rats which had
received moderate contusion injuries 9 days previously. One group
received transplants of cells genetically modified to over-express
bcl-2, which codes for an anti-apoptotic protein. A second group
received transplants of the wild-type ES cells from which the bcl-2
line was developed. In the untransplanted control group, only medium
was injected. Locomotor abilities were assessed using the Basso,
Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was
no incremental locomotor improvement in either transplant group
when compared to control over the survival period. Morbidity and
mortality were significantly more prevalent in the transplant groups
than in controls. At the conclusion of the 6-week survival period,
the spinal cords were examined. Two of six cords from the bcl-2
group and one of 12 cords from the wild-type group showed gross
evidence of abnormal growths at the site of transplantation. No
similar growth was seen in the control. Pathological examination
of the abnormal cords showed very large numbers of undifferentiated
cells proliferating at the injection site and extending up to 1.5
cm rostrally and caudally. These results suggest that transplanting
KD3 ES cells, or apoptosis-resistant cells derived from the KD3
line, into the injured spinal cord does not improve locomotor recovery
and can lead to tumor-like growth of cells, accompanied by increased
debilitation, morbidity and mortality.
Human embryonic stem cells differentiate into
oligodendrocytes in high purity and myelinate after spinal cord
Nistor GI, et al., Glia. 2005 Feb;49(3):385-96.
Abstract: Human embryonic stem cells
(hESCs) demonstrate remarkable proliferative and developmental capacity.
Clinical interest arises from their ability to provide an apparently
unlimited cell supply for transplantation, and from the hope that
they can be directed to desirable phenotypes in high purity. Here
we present for the first time a method for obtaining oligodendrocytes
and their progenitors in high yield from hESCs. We expanded hESCs,
promoted their differentiation into oligodendroglial progenitors,
amplified those progenitors, and then promoted oligodendroglial
differentiation using positive selection and mechanical enrichment.
Transplantation into the shiverer model of dysmyelination resulted
in integration, differentiation into oligodendrocytes, and compact
myelin formation, demonstrating that these cells display a functional
phenotype. This differentiation protocol provides a means of generating
human oligodendroglial lineage cells in high purity, for use in
studies of lineage development, screening assays of oligodendroglial-specific
compounds, and treating neurodegenerative diseases and traumatic
injuries to the adult CNS. Copyright 2004 Wiley-Liss, Inc.
Origin of insulin secreted from islet-like
cell clusters derived from murine embryonic stem cells.
Paek HJ, et al., Cloning Stem Cells. 2005;7(4):226-31.
Abstract: Islet-like cell clusters
(ILCCs) were derived from murine embryonic stem cells using a slightly
modified version of the protocol originally described by Lumelsky
et al. in 2001. Analysis with enzyme-linked immunosorbent assays
(ELISAs) that distinguish human from murine insulin demonstrated
that insulin released from these ILCCs, upon initial in vitro glucose
challenge, was of non-murine origin and in fact corresponded to
the species of insulin, human or bovine, that had been added to
the culture media used to derive ILCCs. This finding convincingly
supports the hypothesis that ILCCs are not synthesizing insulin
de novo, but rather simply regurgitating insulin taken up during
tissue culture. In further experiments, ILCCs were derived in media
in which insulin had been replaced by IGF-I with which it shares
a common signaling pathway. These ILCCs failed to release any detectable
insulin. In contrast, ILCCs produced by various protocols stained
positive (dithizone and immunoselective antibodies) for intracellular
insulin and, in some cases, C-peptide. Despite the presence of at
least some level of de novo, synthesized insulin in ILCCs, the majority
of insulin released by ILCCs was sequestered from the exogenous
Teratoma formation leads to failure of treatment
for type I diabetes using embryonic stem cell-derived insulin-producing
Fujikawa T, et al., Am J Pathol. 2005 Jun;166(6):1781-91.
Abstract: Embryonic stem (ES) cells
have been proposed to be a powerful tool in the study of pancreatic
disease, as well as a potential source for cell replacement therapy
in the treatment of diabetes. However, data demonstrating the feasibility
of using pancreatic islet-like cells differentiated from ES cells
remain controversial. In this study we characterized ES cell-derived
insulin-expressing cells and assessed their suitability for the
treatment of type I diabetes. ES cell-derived insulin-stained cell
clusters expressed insulin mRNA and transcription factors associated
with pancreatic development. The majority of insulin-positive cells
in the clusters also showed immunoreactivity for C-peptide. Insulin
was stored in the cytoplasm and released into the culture medium
in a glucose-dependent manner. When the cultured cells were transplanted
into diabetic mice, they reversed the hyperglycemic state for approximately
3 weeks, but the rescue failed due to immature teratoma formation.
Our studies demonstrate that reversal of hyperglycemia by transplantation
of ES cell-derived insulin-producing cells is possible. However,
the risk of teratoma formation would need to be eliminated before
ES cell-based therapies for the treatment of diabetes are considered.
Artifactual insulin release from differentiated
embryonic stem cells.
Hansson M, et al., Diabetes. 2004 Oct;53(10):2603-9.
Abstract: Several recent reports
claim the generation of insulin-producing cells from embryonic stem
cells via the differentiation of progenitors that express nestin.
Here, we investigate further the properties of these insulin-containing
cells. We find that although differentiated cells contain immunoreactive
insulin, they do not contain proinsulin-derived C-peptide. Furthermore,
we find variable insulin release from these cells upon glucose addition,
but C-peptide release is never detected. In addition, many of the
insulin-immunoreactive cells are undergoing apoptosis or necrosis.
We further show that cells cultured in the presence of a phosphoinositide
3-kinase inhibitor, which previously was reported to facilitate
the differentiation of insulin(+) cells, are not C-peptide immunoreactive
but take up fluorescein isothiocyanate-labeled insulin from the
culture medium. Together, these data suggest that nestin(+) progenitor
cells give rise to a population of cells that contain insulin, not
as a result of biosynthesis but from the uptake of exogenous insulin.
We conclude that C-peptide biosynthesis and secretion should be
demonstrated to claim insulin production from embryonic stem cell
Transplantation of embryonic stem cells into
the infarcted mouse heart: formation of multiple cell types.
Singla DK, et al., J Mol Cell Cardiol. 2006 Jan;40(1):195-200.
Epub 2005 Nov 8.
Abstract: Initial studies have suggested
that transplantation of embryonic stem (ES) cells following myocardial
infarction (MI) in animal models is beneficial; however, the mechanism
of benefit is largely unknown. The present study investigated the
fate of mouse ES cells transplanted post-MI to determine if the
ES cells give rise to the range of major cell types present in the
native myocardium. MI was produced by coronary artery ligation in
C57BL/6 mice. Two different mouse ES cell lines, expressing eGFP
and beta-galactosidase, respectively, were tested. Post-MI intramyocardial
injection of 3 x 10(4) ES cells was compared to injection of media
alone. Histochemistry and immunofluorescence were used to track
the transplanted ES cells and identify the resulting cell types.
Echocardiography assessed the cardiac size and function in a blinded
fashion. Two weeks post-MI, engraftment of the transplanted ES cells
was demonstrated by eGFP or beta-galactosidase-positive cells in
the infarct region without evidence for tumor formation. Co-immunolabeling
demonstrated that the transplanted ES cells had become cardiomyocytes,
vascular smooth muscle, and endothelial cells. Echocardiographic
analysis showed that ES cell transplantation resulted in reduced
post-MI remodeling of the heart and improved cardiac function. In
conclusion, transplanted mouse ES cells can regenerate infarcted
myocardium in part by becoming cardiomyocytes, vascular smooth muscle,
and endothelial cells that result in an improvement in cardiac structure
and function. Therefore, ES cells hold promise for myocardial cellular
They are not stealthy in the heart: embryonic
stem cells trigger cell infiltration, humoral and T-lymphocyte-based
host immune response.
Kofidis T, et al., Eur J Cardiothorac Surg. 2005 Sep;28(3):461-6.
OBJECTIVE: The in vivo immunogenicity
of Embryonic Stem Cells is controversial. At present, there is only
in vitro evidence of MHC I expression by this cell population but
vivid speculation about their immune-privileged state. The immunology
aspect of ESC transplantation deserves thorough investigation. METHODS:
We injected mouse ESC (expressing Green Fluorescent Protein, GFP)
into injured myocardium of syngeneic, allogeneic and SCID recipients.
Furthermore, we monitored host response for up to 4 weeks post cell
transfer. We determined local response (CD 3, CD 11c expression
by host cells), MHC I expression by donor cells, MHC-II expression
within and around the graft, humoral response of allogeneic hosts
using Flow Cytometry and evaluated the hosts' cytokine response
using stimulated spleenocytes by means of ELISPOT. Cell survival
was estimated by morphometry, by calculating the area of the GFP+
graft over the area of infarction at multiple sections of the harvested
heart. RESULTS: There was significant cellular infiltration into
and around the graft consisting of T-lymphocytes (CD3+) and dendritic
cells (CD 11c). Infiltration was detectable at 1 week and progressed
through 4 weeks following cell transplantation. The humoral Ab response
was moderate at 2 weeks but frank at 4 weeks. ELISPOT demonstrated
a Th1 pathway of donor specific T-lymphocyte response with strong
IFN-gamma and Il-2 production (figure A). MHC I expression was significant
within the graft and maximal in the allogeneic groups. CONCLUSIONS:
An immune response against transplanted ESC was demonstrated and
the future use of ESC will likely require the use of systemic immunosuppression.
Stable benefit of embryonic stem cell therapy
in myocardial infarction.
Hodgson DM, et al., Am J Physiol Heart Circ Physiol. 2004 Aug;287(2):H471-9.
Abstract: Conventional therapies
for myocardial infarction attenuate disease progression without
contributing significantly to repair. Because of the capacity for
de novo cardiogenesis, embryonic stem cells are considered a potential
source for myocardial regeneration, yet limited information is available
on their ultimate therapeutic value. We treated infarcted rat hearts
with CGR8 embryonic stem cells preexamined for cardiogenicity, serially
probed left ventricular function, and determined final pathological
outcome. Stem cell delivery generated new cardiomyocytes of embryonic
stem cell origin that integrated with host myocardium within infarct
regions. This resulted in a functional benefit within 3 wk that
remained sustained over 12 wk of continuous follow-up and included
a vigorous inotropic response to beta-adrenergic challenge. Integration
of stem cell-derived cardiomyocytes was associated with normalized
ventricular architecture, little scar, and a decrease in signs of
myocardial necrosis. In contrast, sham-treated infarcted hearts
exhibited ventricular cavity dilation and aneurysm formation, poor
ventricular function, and a lack of response to beta-adrenergic
stimulation. No evidence of graft rejection, ectopy, sudden cardiac
death, or tumor formation was observed after therapy. These findings
indicate that embryonic stem cells, through differentiation within
the host myocardium, can contribute to a stable beneficial outcome
on contractile function and ventricular remodeling in the infarcted
The proteasome restricts permissive transcription
at tissue-specific gene Loci in embryonic stem cells.
Szutorisz H, et al, Cell. 2006 Dec 29;127(7):1375-88.
Abstract: The ability of stem cells
to activate different gene expression programs requires the choreographed
assembly of trans-acting factors at enhancers and promoters during
cell differentiation. In this study, we show that the proteasome
acts on specific regulatory regions in embryonic stem (ES) cells
to prevent incorrect transcriptional initiation. Chemical or siRNA-mediated
inhibition of proteasome activity results in increased transcription
factor and RNA polymerase II binding and leads to activation of
cryptic promoters. Analysis of the binding profiles of different
proteasome subunits in normal ES cells and following RNAi knockdown
of individual subunits provides evidence for a targeted assembly
of the 26S proteasome at specific regulatory elements. Our results
suggest that the proteasome promotes a dynamic turnover of transcription
factor and Pol II binding at tissue-specific gene domains in ES
cells, thereby restricting permissive transcriptional activity and
keeping the genes in a potentiated state, ready for activation at
F0 generation mice fully derived from gene-targeted
embryonic stem cells allowing immediate phenotypic analyses.
Poueymirou WT, et al, Nat Biotechnol. 2006 Dec 24; [Epub ahead
Abstract: A useful approach for exploring
gene function involves generating mutant mice from genetically modified
embryonic stem (ES) cells. Recent advances in genetic engineering
of ES cells have shifted the bottleneck in this process to the generation
of mice. Conventional injections of ES cells into blastocyst hosts
produce F0 generation chimeras that are only partially derived from
ES cells, requiring additional breeding to obtain mutant mice that
can be phenotyped. The tetraploid complementation approach directly
yields mice that are almost entirely derived from ES cells, but
it is inefficient, works only with certain hybrid ES cell lines
and suffers from nonspecific lethality and abnormalities, complicating
phenotypic analyses. Here we show that laser-assisted injection
of either inbred or hybrid ES cells into eight cell-stage embryos
efficiently yields F0 generation mice that are fully ES cell-derived
and healthy, exhibit 100% germline transmission and allow immediate
phenotypic analysis, greatly accelerating gene function assignment.
Persistent dopamine functions of neurons derived
from embryonic stem cells in a rodent model of Parkinson's disease.
Rodriguez-Gomez JA, et al., Stem Cells. 2006 Dec 14; [Epub ahead
Abstract: The derivation of dopamine
neurons is one of the best examples of the clinical potential of
embryonic stem (ES) cells but the long-term function of the grafted
neurons has not been established. Here we show that after transplantation
into an animal model, neurons derived from mouse ES cells survived
for over 32 weeks, maintained midbrain markers and had sustained
behavioral effects. Microdialysis in grafted animals showed DA release
was induced by depolarization and pharmacological stimulants. Positron
emission tomography (PET) measured the expression of pre-synaptic
dopamine transporters (DAT) in the graft and also showed that the
number of postsynaptic DA D2 receptors was normalized in the host
striatum. These data suggest that ES-cell derived neurons show DA
release, reuptake and stimulate appropriate post-synaptic responses
for long periods after implantation. This work supports continued
interest in ES cells as a source of functional DA neurons.
Derivation of human embryonic stem cells in
Ludwig TE, et al., Nat Biotechnol. 2006 Feb;24(2):185-7. Epub
2006 Jan 1.
Abstract: We have previously reported
that high concentrations of basic fibroblast growth factor (bFGF)
support feeder-independent growth of human embryonic stem (ES) cells,
but those conditions included poorly defined serum and matrix components.
Here we report feeder-independent human ES cell culture that includes
protein components solely derived from recombinant sources or purified
from human material. We describe the derivation of two new human
ES cell lines in these defined culture conditions.
Gut-like structures from mouse embryonic stem
cells as an in vitro model for gut organogenesis preserving developmental
potential after transplantation.
Torihashi S, et al., Stem Cells. 2006 Dec;24(12):2618-26. Epub
2006 Aug 3.
Abstract: Recently, we reported the
formation of gut-like structures from mouse ESCs in vitro. To determine
whether ESCs provide an in vitro model of gastrointestinal (GI)
tracts and their organogenesis, we investigated the morphological
features, formation process, cellular development, and regional
location within the GI tract by immunohistochemistry, electron microscopy,
and reverse transcription-polymerase chain reaction. We also examined
the developmental potential by transplantation into kidney capsules.
The results demonstrated that Id2-expressing epithelium developed
first, alpha-smooth muscle actin appeared around the periphery,
and finally, the gut-like structures were formed into a three-layer
organ with well-differentiated epithelium. A connective tissue layer
and musculature with interstitial cells of Cajal developed, similar
to organogenesis of the embryonic gut. Enteric neurons appeared
underdeveloped, and blood vessels were absent. Many structures expressed
intestinal markers Cdx2 and 5-hydroxytryptamine but not the stomach
marker H(+)/K(+) ATPase. Transplants obtained blood vessels and
extrinsic nerve growth from the host to prolong life, and even grafts
of premature structures did not form teratoma. In conclusion, gut-like
structures were provided with prototypical tissue components of
the GI tract and are inherent in the intestine rather than the stomach.
The formation process was basically same as in gut organogenesis.
They maintain their developmental potential after transplantation.
Therefore, gut-like structures provide a unique and useful in vitro
system for development and stem cell studies of the GI tract, including
BMP-4 is required for hepatic specification
of mouse embryonic stem cell-derived definitive endoderm.
Gouon-Evans V, et al., Nat Biotechnol. 2006 Nov;24(11):1402-11.
Epub 2006 Nov 5.
Abstract: When differentiated in
the presence of activin A in serum-free conditions, mouse embryonic
stem cells efficiently generate an endoderm progenitor population
defined by the coexpression of either Brachyury, Foxa2 and c-Kit,
or c-Kit and Cxcr4. Specification of these progenitors with bone
morphogenetic protein-4 in combination with basic fibroblast growth
factor and activin A results in the development of hepatic populations
highly enriched (45-70%) for cells that express the alpha-fetoprotein
and albumin proteins. These cells also express transcripts of Afp,
Alb1, Tat, Cps1, Cyp7a1 and Cyp3a11; they secrete albumin, store
glycogen, show ultrastructural characteristics of mature hepatocytes,
and are able to integrate into and proliferate in injured livers
in vivo and mature into hepatocytes expressing dipeptidyl peptidase
IV or fumarylacetoacetate hydrolase. Together, these findings establish
a developmental pathway in embryonic stem cell differentiation cultures
that leads to efficient generation of cells with an immature hepatocytic
Reversal of mouse hepatic failure using an
implanted liver-assist device containing ES cell-derived hepatocytes.
Soto-Gutierrez A, et al., Nat Biotechnol. 2006 Nov;24(11):1412-9.
Epub 2006 Nov 5.
Abstract: Severe acute liver failure,
even when transient, must be treated by transplantation and lifelong
immune suppression. Treatment could be improved by bioartificial
liver (BAL) support, but this approach is hindered by a shortage
of human hepatocytes. To generate an alternative source of cells
for BAL support, we differentiated mouse embryonic stem (ES) cells
into hepatocytes by coculture with a combination of human liver
nonparenchymal cell lines and fibroblast growth factor-2, human
activin-A and hepatocyte growth factor. Functional hepatocytes were
isolated using albumin promoter-based cell sorting. ES cell-derived
hepatocytes expressed liver-specific genes, secreted albumin and
metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized
mice with a subcutaneously implanted BAL seeded with ES cell-derived
hepatocytes or primary hepatocytes improved liver function and prolonged
survival, whereas treatment with a BAL seeded with control cells
did not. After functioning in the BAL, ES cell-derived hepatocytes
developed characteristics nearly identical to those of primary hepatocytes.
Differentiation of human embryonic stem cells
to hepatocytes using deleted variant of HGF and poly-amino-urethane-coated
nonwoven polytetrafluoroethylene fabric.
Soto-Gutierrez A, et al., Cell Transplant. 2006;15(4):335-41.
Abstract: Human embryonic stem (hES)
cells have recently been studied as an attractive source for the
development of a bioartificial liver (BAL). Here we evaluate the
differentiation capacity of hES cells into hepatocytes. hES cells
were subjected to suspension culture for 5 days, and then cultured
onto poly-amino-urethane (PAU)-coated, nonwoven polytetrafluoroethylene
(PTFE) fabric in the presence of fibroblast growth factor-2 (bFGF)
(100 ng/ml) for 3 days, then with deleted variant of hepatocyte
growth factor (dHGF) (100 ng/ml) and 1% dimethyl sulfoxide (DMSO)
for 8 days, and finally with dexamethasone (10(-7) M) for 3 days.
The hES cells showed gene expression of albumin in a time-dependent
manner of the hepatic differentiation process. The resultant hES-derived
hepatocytes metabolized the loaded ammonia and lidocaine at 7.8%
and 23.6%, respectively. A million of such hepatocytes produced
albumin and urea at 351.2 ng and urea at 7.0 microg. Scanning electron
microscopy showed good attachment of the cells on the surface of
the PTFE fabric and well-developed glycogen rosettes and Gap junction.
In the present work we have demonstrated the efficient differentiation
of hES cells to functional hepatocytes. The findings are useful
to develop a BAL.
Human embryonic stem cells are immunogenic
in allogeneic and xenogeneic settings.
Grinnemo KH, et al., Reprod Biomed Online. 2006 Nov;13(5):712-24.
Abstract: Recent studies have suggested
that human embryonic stem cells (HESC) are immune-privileged and
may thereby circumvent rejection. The expression of immunologically
active molecules was studied by DNA microarray analysis and by flow
cytometry. HESC were transplanted into immunologically competent
mice and traced by fluorescence in-situ hybridization (FISH) and
immunohistochemistry. The ability of HESC to directly and indirectly
induce immune responses in CD4+ T-cells from naive and transplanted
mice was studied. Their ability to induce immune responses of human
CD4+ T-cells, when cultured in the presence of dendritic cells (DC)
syngeneic to responder T-cells, was also analysed. HESC demonstrated
expression of HLA class I and HLA class II genes, but the cell surface
expression of HLA class II molecules was low even after incubation
with IFNgamma. In wild-type mice, HESC could be demonstrated by
FISH until 3 days after transplantation and were surrounded by heavy
infiltrates of T-cells and macrophages. HESC induced a similar immune
response as human fibroblast cells (HFib) on naive and immunized
T-cells, both directly and in the presence of syngeneic DC. A similar
response was observed in the allogeneic setting. It is concluded
that HESC are immunologically inert and do not inhibit immune responses
during direct or indirect antigen presentation, and they were acutely
rejected in a xenogeneic setting.
Human embryonic stem cell-derived cells rescue
visual function in dystrophic RCS rats.
Lund RD, et al., Cloning Stem Cells. 2006 Fall;8(3):189-99.
Abstract: Embryonic stem cells promise
to provide a well-characterized and reproducible source of replacement
tissue for human clinical studies. An early potential application
of this technology is the use of retinal pigment epithelium (RPE)
for the treatment of retinal degenerative diseases such as macular
degeneration. Here we show the reproducible generation of RPE (67
passageable cultures established from 18 different hES cell lines);
batches of RPE derived from NIH-approved hES cells (H9) were tested
and shown capable of extensive photoreceptor rescue in an animal
model of retinal disease, the Royal College of Surgeons (RCS) rat,
in which photoreceptor loss is caused by a defect in the adjacent
retinal pigment epithelium. Improvement in visual performance was
100% over untreated controls (spatial acuity was approximately 70%
that of normal nondystrophic rats) without evidence of untoward
pathology. The use of somatic cell nuclear transfer (SCNT) and/or
the creation of banks of reduced complexity human leucocyte antigen
(HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive
drugs and/or immunomodulatory protocols.