ES-derived Cells Research Articles

Increased gp130 Signaling in Combination with Inhibition of the MEK/ERK Pathway Facilitates Embryonic Stem Cell Isolation from Normally Refractory Murine CBA Blastocysts

The standard method for isolation of ES cells from strain 129 mice does not give rise to ES lines of CBA origin. We investigated the effect of inhibition of MEK/ERK signaling in combination with increased stimulation of gp130 signaling on derivation of ES cells from CBA blastocysts. Inhibition of MEKI and MEKII using the drug U0126 and stimulation of gp130 signaling by elevating the level of LIF present gave rise to ES-like lines in 22.6% of explants. No lines arose when MEK was inhibited in the absence of additional LIF stimulation, nor when additional LIF stimulation occurred in the absence of MEK inhibition. Typically for ES cell lines, CBA-derived cells contributed to chimeric mice and differentiated broadly in vitro. Increased levels of gp130 signaling led to similar levels of STAT3 activation in strain 129 and CBA ES cells. We conclude that CBA ES cells have a requirement for additional STAT3 activation.

Oct-3/4 and Sox2 Regulate Oct-3/4 Gene in Embryonic Stem Cells

Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in the inner cell mass and ES cells. However, little is known about the cis-elements and trans-factors required for DE activity. In this study, we identified a novel cis-element, called Site 2B here, located approximately 30 bp downstream from Site 2A, which was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In an electrophoretic mobility shift assay we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for Site 2B-mediated DE activity using the inducible knock-out system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF-4, UTF1, Sox2, and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This autoregulatory circuit of the Sox2.Oct-3/4 complex may contribute to maintaining robustly the precise expression level of Oct-3/4 in primitive cells.

A germ cell origin of embryonic stem cells?

Because embryonic stem (ES) cells are generally derived by the culture of inner cell mass (ICM) cells, they are often assumed to be the equivalent of ICM cells. However, various evidence indicates that ICM cells transition to a different cell type during ES-cell derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm cells derived directly from the ICM. However, differences between ES cells and primitive ectoderm cells have caused developmental biologists to question whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue culture environment. Here, we review recent evidence that the closest in vivo equivalent of an ES cell is an early germ cell.

173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

Scalable production of embryonic stem cell-derived cells.

Embryonic stem (ES) cells have the ability to self-renew as well as differentiate into any cell type in the body. These traits make ES cells an attractive "raw material" for a variety of cell-based technologies. However, uncontrolled cell aggregation in ES cell differentiation culture inhibits cell proliferation and differentiation and thwarts the use of stirred suspension bioreactors. Encapsulation of ES cells in agarose microdrops prevents physical interaction between developing embryoid bodies (EBs) that, in turn, prevents EB agglomeration. This enables use of stirred suspension bioreactors that can generate large numbers of ES-derived cells under controlled conditions.

Identification of a Novel Stromal Cell Membrane Protein that Binds to Megakaryocytes.

In bone marrow, hematopoiesis-supporting cells or stromal cells govern the growth, survival and differentiation of hematopoietic stem cells. We previously reported the method to produce the functional platelets from murine embryonic stem (ES) cells in vitro, using the coculture system with OP9 stromal cells. Also in this system, the cell contact between ES-derived cells and stromal cells is essential for megakaryocyte differentiation, and even after growth factors including TPO were supplied, ES-derived megakaryocytes cannot be maintained without stromal cells. In this study, we searched membrane proteins that were expressed in the stromal cells and were involved in the cell contact with megakaryocytes. We employed the retrovirus-based signal sequence trap (SST) method to identify such membrane proteins from murine stromal cells. 117 signal sequence-containing clones were identified, and the clones were further screened by the binding capacity to megakaryocytic cell lines. We finally identified a novel protein that contains two Ig domains and belongs to the transmembrane-type immunoglobulin superfamily. RT-PCR analysis showed that this protein is expressed in bone marrow stromal cell lines including OP9, and vascular endothelial cell lines, whereas no expression was detected in any hematopoietic cell lines. We next created a cDNA construct in which the extracellular domain of the protein was linked to IgG Fc domain, and the construct was transfected into 293 cells. The fusion protein was purified from the culture supernatant. By flow cytometer, the purified recombinant protein bound to megakaryocytic cell lines and CD41-positive bone marrow megakaryocytes. The binding to murine Sca-1-positive and human CD34-positive immature cells was also detected. The binding was dependent on calcium, but was not inhibited by RGD peptide. The effect on in vitro differentiation system of ES cells was further tested. The addition of the fusion protein showed no influences on the maintenance of undifferentiated state of ES cells. However, after the differentiation induction, the protein facilitated morphological change of the ES cells. Further analysis will contribute to understanding of the mechanisms by which the stromal cell contact regulates megakaryocyte differentiation and platelet production.

Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector.

Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.

Erythroblasts derived in vitro from embryonic stem cells in the presence of erythropoietin do not express the TER-119 antigen

Objective In this study, we analyzed murine primitive erythropoiesis by coculturing Flk-1 + ES-derived cells with OP9 to find efficient culture conditions for erythroid cell induction. We utilized a nonserum culture system and EPO (erythropoietin) and found that this cytokine had unique properties. Materials and methods ES cells (E14.1) were first differentiated to Flk-1 + cells and then cocultured with OP9 stromal cells. BIT9500 was used as a serum replacement. The erythroid morphology, hemoglobin types, and TER-119 expression levels were analyzed. Results Primitive erythroid cells with embryonic hemoglobin were generated very efficiently when the serum-containing culture was converted to the nonserum system. In this serum-free culture, TER-119 + erythroblasts appeared first on day 2 and maturation proceeded until day 7. When EPO was added to this coculture, the number of induced floating cells increased twofold to threefold. Unexpectedly, the erythroid-specific antigen TER-119 expression of these cells was drastically reduced. Since reduced TER-119 expression is usually interpreted as maturation arrest, we examined the phenotypic features of the EPO-treated cells. We found, however, no evidence of maturation arrest in the aspects of morphology and hemoglobin content. EPO did not suppress TER-119 expression of erythroblasts derived from fetal liver or adult bone marrow. Conclusions Our results showed that EPO had the unusual property of inducing TER-119 − erythroblasts in ES-derived primitive erythropoiesis. It is likely that this effect is unique to primitive erythropoiesis.

Progress with nonhuman primate embryonic stem cells.

Embryonic stem cells hold potential in the fields of regenerative medicine, developmental biology, tissue regeneration, disease pathogenicity, and drug discovery. Embryonic stem (ES) cell lines are now available in primates, including man, rhesus, and cynomologous monkeys. Monkey ES cells serve as invaluable clinically relevant models for studies that can't be conducted in humans because of practical or ethical limitations, or in rodents because of differences in physiology and anatomy. Here, we review the current status of nonhuman primate research with ES cells, beginning with a description of their isolation, characterization, and availability. Substantial limitations still plague the use of primate ES cells, such as their required growth on feeder layers, poor cloning efficiency, and restricted availability. The ability to produce homogenous populations of both undifferentiated as well as differentiated phenotypes is an important challenge, and genetic approaches to achieving these objectives are discussed. Finally, safety, efficiency, and feasibility issues relating to the transplantation of ES-derived cells are considered.

Quantitative gene expression profiling reveals a fetal hepatic phenotype of murine ES-derived hepatocytes.

To use embryonic stem (ES) cells in future therapeutical applications, differentiated hepatic phenotypes with specific liver functions would be necessary. We analyzed albumin (ALB), alpha-fetoprotein (AFP) and hepatic transcription factor (TF) gene expression in tissues derived from embryonic, fetal and adult liver, and compared the gene expression profiles with those from mouse ES cells after hepatic differentiation and from cultured adult hepatocytes. The mRNA expression of hepatocyte nuclear factor (HNF)-1alpha,beta, -3alpha,beta, -4alpha, -6, CCAAT/enhancer binding protein (C/EBP) alpha,beta, ALB and AFP relative to glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) were studied by "real time" RT-PCR. ALBand AFP-expression was also determined by in situ hybridization (tissue) and immunofluorescence (ES-derived cells after hepatic differentiation, ES-HPC). Peak levels for HNF-1alpha, -3alpha, -4alpha and -6 were detected in early liver development at d9.5 and d11.5. C/EBPalpha and beta were most abundantly expressed in adult liver. ALB mRNA increased steadily from d10.5 on and was maximally present in adult liver. AFP was present at d9.5, peaked at d15.5 and dramatically declined in mature liver tissue. Based on immunofluorescence, ALB and AFP were expressed in approximately 20% of ES-HPC. While expression of HNF-3, 4 and 6 reached levels similar to adult hepatocytes, ALB and AFP expression was several orders of magnitude lowerthan in adult tissue or cells. Stages of liver organogenesis are characterized by specific expression patterns of developmentally regulated genes. With sophisticated differentiation protocols, hepatic gene expression can be induced in a proportion of ES cells with gene expression patterns similar to early fetal liver.

Induction of hepatic differentiation in embryonic stem cells by co-culture with embryonic cardiac mesoderm

Background. Modifications in vitro have been used to direct embryonic stem (ES) cells toward endodermal phenotypes including hepatocytes; however, developmental correlates and evidence of biologic activity is lacking, and critical cell-cell interactions have not been investigated. In this study, we hypothesized that cardiac mesoderm (CM) signals ES cells in co-culture to undergo differentiation toward early hepatocyte lineage as determined by morphology and induction of genes essential for endodermal competence and hepatocyte development. Methods. Green fluorescent protein ES derived from A129 mice were cultured with or without embryonic chick cardiac mesoderm. Cultures from day 1, 2, and 4 were analyzed for colony formation and ES morphology and 106 ES-derived cells were isolated for mRNA analysis. Results. ES in co-culture with CM displayed colony formation, polymorphic appearance, and definitive interface with CM. In addition, ES + CM co-culture activated crucial transcription factors (sox 17α, HNF3β, and GATA 4) required for hepatocyte development by day 1. mRNA for albumin and especially a-fetoprotein were also increased by culture days 2 and 4. Conclusions. ES cells co-cultured with CM display morphology and gene expression pattern required for hepatocyte differentiation and appear to recapitulate the molecular events of hepatogenesis. (Surgery 2003;134:189-96.)

Embryonic stem cells and cell replacement therapies in the nervous system

Embryonic stem (ES) cells are pluripotential cells derived from the pre-implantation embryo. They can proliferate indefinitely in vitro while retaining pluripotency. ES cells can also be made to differentiate into a large variety of cell types in vitro. This has paved the way to research aimed at using ES-derived cells for cell replacement therapies. Hence, mouse ES cells can efficiently differentiate into neural precursors which can further generate functional neurons, astrocytes, and oligodendrocytes. Methods have also been developed to coax mouse ES-derived neural stem cells to differentiate into either dopaminergic neurons or motoneurons. Mouse ES-derived neural stem cells, or their fully differentiated progeny, have been shown to survive, integrate, and to some extent, function following transplantation within appropriate rodent host tissue. Research on human ES cells is still in its infancy. Considerable work has to be done: (1) to master growth and genetic manipulation of human ES cells; (2) to master their differentiation into specific cell types; and (3) to demonstrate that they can provide long term therapeutical benefits upon grafting into damaged tissues in humans. From the ethical point of view, the establishment of appropriate primate model will be an obligatory prerequisite to clinical trials based on ES cells derivatives grafting.

Differentiation of embryonic stem cells into hepatocytes: Biological functions and therapeutic application

Embryonic stem (ES) cells provide a unique source for tissue regeneration. We examined whether mouse ES cells can efficiently differentiate into transplantable hepatocytes. ES cells were implanted into mouse livers 24 hours after carbon tetrachloride intoxication; ES-derived cells with several hepatocyte-cell-markers were generated. They were able to grow in vitro and showed morphology consistent with typical mature hepatocytes and expressed hepatocyte-specific genes. After transplantation into the carbon tetrachloride-injured mouse liver, ES-derived green fluorescent protein-positive cells were incorporated into liver tissue and rescued mice from hepatic injury. No teratoma formation was observed in the transplant recipients. In conclusion, ES cells can provide a valuable tool for studying the molecular basis for differentiation of hepatocytes and form the basis for cell therapies. (H epatology 2003;37:983-993.)

A dual role of the GTPase Rac in cardiac differentiation of stem cells.

The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.

Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells.

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro. We show that constitutive expression of Pax4 (Pax4(+)), and to a lesser extent Pdx1 (Pdx1(+)), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin+ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult beta cells. In response to glucose, Pax4(+) and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.

Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes

Embryonic stem (ES) cells have a potential to differentiate into various progenitor cells. Here we investigated the differentiation capacity of mouse ES cells into hepatocytes both in vitro and in vivo. During the culture of embryoid bodies (EBs) derived from ES cells, albumin (ALB) messenger RNA (mRNA) was expressed within 12 days after removal of leukemia inhibitory factor, and α-fetoprotein (AFP) mRNA was observed within 9 days without additional exogenous growth factors. In ES cells and early EBs, by contrast, neither ALB mRNA nor AFP mRNA was observed. ALB protein was first detected at day 15 and the level increased with the culture period. The differentiation of EBs facilitated the synthesis of urea with the culture period, whereas early EBs and ES cells produced no urea. These results suggest that cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from EBs were transplanted through portal vein to the liver after 30% partial hepatectomy of female mice pretreated with 2-acetylaminofluorene. Four weeks after transplantation with isolated cells from day-9 EBs, ES-derived cells containing Y-chromosome in the liver were positive for ALB (0.2% of total liver cells), whereas teratoma was found in mice transplanted with ES cells or EBs up to day 6. The incidence of teratoma was decreased with the culture duration and no teratoma was observed in the liver transplanted with isolated cells from day-9 EBs. In conclusion, our in vitro and in vivo experiments revealed that cultured EBs contain functional hepatocytes or hepatocyte-like cells. (H EPATOLOGY 2002;36:22-29.)

Establishment of ES Cell Lines and Regenerative Medicine

Stem cells are divided into three types. ES cells show most potent activities. It can be differentiate into many types of organs, such as liver, lung etc. Then umblical stem cells are known. It will support the growth of tissue and organs. The bone marrow stem cells can be differentiated to form many types of blood cells, and also be used for leukemia therapy. Even though stem cells and somatic cells are presenting different function, their genome structure should be the same. But gene action are most probably different at each steps. Then stem cell research provides further genetic and molecular biology research and also will open a evolutionary frontier clinical medicine. Establishment of human embryonic stem (ES) cell lines has opened great potential and expectation for regenerative medicine and tissue engineering, because many types of human cells could be produced by their unlimited growth and differentiation in culture. ES cell lines have been established from mouse blastocysts and used for gene targeting studies to produce mouse strains with specific genetic alterations. Such pluripotent stem cells show extensive ability to differentiate into many types of functional cells and thy also enable the production of chimeric mice, in which they can contribute to all tissues and organs including germ cells. Primate and human ES cell lines have been established from blastocysts of monkey and surplus human blastocysts from fertility clinics. They showed several differences compared to mouse ES cells, including a tendency to produce the trophectoderm lineage and a different expression pattern of surface antigens. This may reflect species-specific differences, or these primate ES cells could represent earlier stages of pluripotent cell development than mouse ES cells. Also, they show no response to the LIF and gp130 signals, which are widely used to repress spontaneous differentiation of mouse ES cell colonies. We have established several ES cell lines from blastocysts of the cynomolgus monkey. They can be maintained in culture as stem cell colonies, and also, they produce several differentiated cell types in culture. When such ES cells were transplanted into SCID mice, they produced teratomas containing many differentiated tissues (Suemori H, Tada T, Torii R, Hosoi Y, Kobayashi K, Imahie H, Kondo Y, Iritani A, and Nakatsuji N. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev. Dynamics, 222, 273-279, 2001). The unlimited proliferation capacity of ES cells in culture and subsequent differentiation into various types of functional cells could be used for cell transplantation therapy. Promising results have been obtained so far, mostly by using mouse ES cells for cell types such as neuron, glia, cardiac muscle, hematopoietic cells, endothelial cells and insulin-producing pancreatic cells. Combination of these ES-derived cells and various aspects of the tissue engineering should greatly expand the therapeutic scope in the future.

Human Pluripotential Stem Cells

After completing this article, readers should be able to: 1. List the three cells from which mouse pluripotential stem cells can be derived. 2. List the types of specific cells that have been differentiated from murine embryonic stem cells in laboratory investigations. 3. Delineate the potential methods of using human stem cells to minimize immunologic rejection after transplantation. 4. Describe some of the issues requiring resolution before human stem cells can be used in therapies. The dream of one day being able to provide an unlimited supply of human tissues for transplantation came one step closer 2 years ago when two teams of scientists from Johns Hopkins University and the University of Wisconsin announced the successful derivation of human pluripotential stem cells (PSCs). This research immediately caught the public’s eye because of its enormous impact on transplantation therapies and the sources of tissues. Human stem cells are renewable in culture and are capable of differentiating into a wide variety of tissue types. The unlimited ability to divide and the capability to form into almost every cell type provide the source of replacement cells for transplantation and raise the hopes of numerous patients who have debilitating conditions, such as Parkinson disease, Alzheimer disease, stroke, and type I diabetes. Human stem cells will be important for in vitro studies of human gene discovery, for pharmaceutical research such as drug toxicology studies for screening and testing, and as a renewable source of cells for tissue transplantation and gene therapies. In addition to clinical applications, human stem cells provide a powerful tool for biomedical research into human embryogenesis, specific gene functions, and lineage development. PSCs, primarily embryonic stem (ES) cells, have been used extensively in studies of embryogenesis, gene function, and development in the mouse. Present in the early stages of embryo development, PSCs can generate all of …

Functions of c-Jun in liver and heart development.

Mice lacking the AP-1 transcription factor c-Jun die around embryonic day E13.0 but little is known about the cell types affected as well as the cause of embryonic lethality. Here we show that a fraction of mutant E13.0 fetal livers exhibits extensive apoptosis of both hematopoietic cells and hepatoblasts, whereas the expression of 15 mRNAs, including those of albumin, keratin 18, hepatocyte nuclear factor 1, beta-globin, and erythropoietin, some of which are putative AP-1 target genes, is not affected. Apoptosis of hematopoietic cells in mutant livers is most likely not due to a cell-autonomous defect, since c-jun-/- fetal liver cells are able to reconstitute all hematopoietic compartments of lethally irradiated recipient mice. A developmental analysis of chimeras showed contribution of c-jun-/- ES cell derivatives to fetal, but not to adult livers, suggesting a role of c-Jun in hepatocyte turnover. This is in agreement with the reduced mitotic and increased apoptotic rates found in primary liver cell cultures derived from c-jun-/- fetuses. Furthermore, a novel function for c-Jun was found in heart development. The heart outflow tract of c-jun-/- fetuses show malformations that resemble the human disease of a truncus arteriosus persistens. Therefore, the lethality of c-jun mutant fetuses is most likely due to pleiotropic defects reflecting the diversity of functions of c-Jun in development, such as a role in neural crest cell function, in the maintenance of hepatic hematopoiesis and in the regulation of apoptosis.

Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts.

This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the alpha-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures (> 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed. These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells. Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.