Engineered Embryonic Stem Cells Research Articles

Transcription Factor Competition Allows Embryonic Stem Cells to Distinguish Authentic Signals from Noise

Stem cells occupy variable environments where they must distinguish stochastic fluctuations from developmental cues. Here, we use optogenetics to investigate how the pluripotency network in embryonic stem (ES) cells achieves a robust response to differentiation cues but not to gene expression fluctuations. We engineered ES cells in which we could quantitatively ontrol the endogenous mechanism of neural differentiation through a light-inducible Brn2 transgene and monitor differentiation status through a genome-integrated Nanog-GFP reporter. By exposing cells to pulses of Brn2, we find that the pluripotency network rejects Brn2 inputs that are below specific magnitude or duration thresholds, but allows rapid differentiation when both thresholds are satisfied. The filtering properties of the network arise through its positive feedback architecture and the intrinsic half-life of Nanog, which determines the duration threshold in the network. Together our results suggest that the dynamic properties of positive-feedback networks might determine how inputs are classified as signal or noise by stem cells.

Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells.

Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10¹³-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.

Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.

Identification of a lectin causing the degeneration of neuronal processes using engineered embryonic stem cells

Unlike the mechanisms involved in the death of neuronal cell bodies, those causing the elimination of processes are not well understood owing to the lack of suitable experimental systems. As the neurotrophin receptor p75(NTR) is known to restrict the growth of neuronal processes, we engineered mouse embryonic stem (ES) cells to express an Ngfr (p75(NTR)) cDNA under the control of the Mapt locus (the gene encoding tau), which begins to be active when ES cell-derived progenitors start elongating processes. This caused a progressive, synchronous degeneration of all processes, and a prospective proteomic analysis showed increased levels of the sugar-binding protein galectin-1 in the p75(NTR)-engineered cells. Function-blocking galectin-1 antibodies prevented the degeneration of processes, and recombinant galectin-1 caused the processes of wild-type neurons to degenerate first, followed by the cell bodies. In vivo, the application of a glutamate receptor agonist, a maneuver known to upregulate p75(NTR), led to an increase in the amount of galectin-1 and to the degeneration of neurons and their processes in a galectin-1-dependent fashion. Section of the sciatic nerve also rapidly upregulated levels of p75(NTR) and galectin-1 in terminal Schwann cells, and the elimination of nerve endings was delayed at the neuromuscular junction of mice lacking Lgals1 (the gene encoding galectin-1). These results indicate that galectin-1 actively participates in the elimination of neuronal processes after lesion, and that engineered ES cells are a useful tool for studying relevant aspects of neuronal degeneration that have been hitherto difficult to analyze.

ES cells mend broken hearts

Heart cells made from embryonic stem (ES) cells improve the function of injured mouse hearts, report Kolossov et al. (page 2315). Figure 1 ES-derived cardiomyocytes (green) fix a damaged mouse heart. Cell replacement is an attractive therapy for heart failure, which is currently treatable only by organ transplant. But which source of cells makes the best starting material has been a matter of debate. Bone marrow cells were reported to provide some improvement in heart failure patients, but their benefit remains controversial. Kolossov et al. found no improvement of heart function when these cells were injected into mice with ischemic heart injury. Cardiomyocytes derived from ES cells, on the other hand, improved the contractility and blood pumping ability of the injured hearts. The use of ES cells as starting material for this kind of therapy runs the risk of inducing tumors in the recipient due to small numbers of undifferentiated, pluripotent cells in the population. To limit this risk, Kolossov et al. used genetically engineered ES cells that allowed them to select for a virtually pure population of cardiomyocytes. This selection trick, however, would have to be modified to be used in humans, as it introduces foreign DNA into the cells. In the meantime, the team plans to see whether similar success is achievable with ES-derived heart cells in larger animals, in which the contractile forces of heart beats are considerably stronger.

Cardiomyocyte production in mass suspension culture: Embryonic stem cells as a source for great amounts of functional cardiomyocytes

Transplantation of cardiomyocytes derived from embryonic stem (ES) cells into damaged or non-functional areas of the myocardium might be a valuable approach to treat heart failure, provided that they can be generated in sufficient quantity and with sufficient purity. Cultivation of ES cells in a standard stirred tank bioreactor makes it possible to generate large numbers of therapeutically useful cells such as cardiomyocytes. We have used this technique to expand genetically engineered ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MyHc) promotor driving the aminoglycoside phosphotransferase gene (neomycin resistance). Addition of G418 to the growth medium allowed an efficient enrichment of cardiomyocytes. We developed a draw-and-fill process in a controlled stirred tank reactor at the 2L scale to facilitate long-term cultivation under stable, reproducible conditions. Throughout the culture period cell viability and EB numbers were measured monitored by flow cytometry, immunohistochemistry and fluorescence microscopy of cardiac specific antigens. About 9e8 cardiomyocytes in 2e5 cardiac bodies were generated. The purity of the population was about 99% measured by MF20 immunohistochemistry and fluorescence microscopy. To analyze the ability of ES-cell derived cardiomyocytes to reconstitute damaged heart tissue cardiac bodies were transplanted into female ICR-mice 7 days after myocardial infarction induced by LAD ligation. Both echocardiographic follow-up and histological analysis 28d after transplantation showed an improved left ventricular function of hearts that received a transplant in comparison to sham-treated mice. We here demonstrate that it is feasible to generate functional cardiomyocytes derived from embryonic stem cells in a bioreactor at a large-scale that retain their functionally in vivo and improve cardiac functions of infarcted hearts. With only one bioreactor process it is possible to provide sufficient cardiomyocytes for a cell based therapy.

Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control

It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 x 10(6) cells/mL after 9 days of differentiation. Approximately 30%-40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (alphaMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 x 10(9) cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications.

Scalable production of embryonic stem cell-derived cardiomyocytes.

Cardiomyocyte transplantation could offer a new approach to replace scarred, nonfunctional myocardium in a diseased heart. Clinical application of this approach would require the ability to generate large numbers of donor cells. The purpose of this study was to develop a scalable, robust, and reproducible process to derive purified cardiomyocytes from genetically engineered embryonic stem (ES) cells. ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MHC) promoter driving the aminoglycoside phosphotransferase (neomycin resistance) gene were used for cardiomyocyte enrichment. The transfected cells were aggregated into embyroid bodies (EBs), inoculated into stirred suspension cultures, and differentiated for 9 days before selection of cardiomyocytes by the addition of G418 with or without retinoic acid (RA). Throughout the culture period, EB and viable cell numbers were measured. In addition, flow cytometric analysis was performed to monitor sarcomeric myosin (a marker for cardiomyocytes) and Oct-4 (a marker for undifferentiated ES cells) expression. Enrichment of cardiomyocytes was achieved in cultures treated with either G418 and retinoic acid (RA) or with G418 alone. Eighteen days after differentiation, G418-selected flasks treated with RA contained approximately twice as many cells as the nontreated flasks, as well as undetectable levels of Oct-4 expression, suggesting that RA may promote cardiac differentiation and/or survival. Immunohistological and electron microscopic analysis showed that the harvested cardiomyocytes displayed many features characteristic of native cardiomyocytes. Our results demonstrate the feasibility of large-scale production of viable, ES cell-derived cardiomyocytes for tissue engineering and/or implantation, an approach that should be transferable to other ES cell derived lineages, as well as to adult stem cells with in vitro cardiomyogenic activity.

Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.

Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system (CNS). They develop from proliferating oligodendrocyte precursor cells (OPCs), which arise in germinal zones, migrate throughout the developing white matter and divide a limited number of times before they terminally differentiate. Thus far, it has been possible to purify OPCs only from the rat optic nerve, but the purified cells cannot be obtained in large enough numbers for conventional biochemical analyses. Moreover, the CNS stem cells that give rise to OPCs have not been purified, limiting one's ability to study the earliest stages of commitment to the oligodendrocyte lineage. Pluripotent, mouse embryonic stem (ES) cells can be propagated indefinitely in culture and induced to differentiate into various cell types. We have genetically engineered ES cells both to positively select neuroepithelial stem cells and to eliminate undifferentiated ES cells. We have then used combinations of known signal molecules to promote the development of OPCs from selected, ES-cell-derived, neuroepithelial cells. We show that the earliest stages of oligodendrocyte development follow an ordered sequence that is remarkably similar to that observed in vivo, suggesting that the ES-cell-derived neuroepithelial cells follow a normal developmental pathway to produce oligodendrocytes. These engineered ES cells thus provide a powerful system to study both the mechanisms that direct CNS stem cells down the oligodendrocyte pathway and those that influence subsequent oligodendrocyte differentiation. This strategy may also be useful for producing human cells for therapy and drug screening.

Targeted disruption of adrenergic receptor genes.

AbstractThe use of targeted gene disruptions, or knockouts, has become commonplace in many basic research laboratories. In its most common application, this technique enables the researcher to disrupt expression of a specific gene product selectively. This approach has been particularly useful for studies on adrenergic receptor (AR) function, having been used to effect disruption of at least six of the nine known AR genes (1–6). As presented here, the knockout strategy is essentially a synthesis of two separate techniques. The first utilizes the phenomenon of homologous recombination, a process whereby foreign DNA (homologous at least in part to portions of the host genome) introduced into cells undergoes strand exchange and integration into the host genome at a specific locus. The second utilizes the pluripotent nature of embryonic stem (ES) cells, which can be cultured and manipulated ex vivo and reintroduced into host embryos. Engineered ES cells having undergone homologous recombination can be incorporated into such “chimeric” embryos, potentially giving rise to all cell types of the adult mouse, including germ cells. Through simple mating experiments, one can then transmit the ES cell-derived disrupted allele to progeny, and eventually intercross heterozygotes to generate mice homozygous for the disrupted allele.KeywordsEmbryonic Stem CellEmbryonic Stem Cell LineMouse Embryonic Fibroblast CellEmbryonic Stem Cell CultureTarget Gene DisruptionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Inactivation of the FGF-4 Gene in Embryonic Stem Cells Alters the Growth and/or the Survival of Their Early Differentiated Progeny

Previous studies have shown that early mouse embryos with both FGF-4 alleles inactivated are developmentally arrested shortly after implantation. To understand the roles of FGF-4 during early development, we prepared genetically engineered embryonic stem (ES) cells, which are unable to produce FGF-4. Specifically, we describe the isolation and characterization of ES cells with both FGF-4 alleles inactivated. The FGF-4−/−ES cells do not require FGF-4 to proliferatein vitro,and addition of FGF-4 to the medium has little or no effect on their growth. Thus, FGF-4 does not appear to act as an autocrine growth factor for cultured ES cells. We also demonstrate that FGF-4−/−ES cells, like their unmodified counterparts, are capable of forming highly complex tumors in syngeneic mice composed of a wide range of differentiated cells types, including neural tissue, glandular epithelium, and muscle. In addition, we demonstrate that the FGF-4−/−ES cells can differentiatein vitroafter exposure to retinoic acid; however, the growth and/or survival of the differentiated cells is severely compromised. Importantly, addition of FGF-4 to the culture medium dramatically increases the number of differentiated cells derived from the FGF-4−/−ES cells, in particular cells with many of the properties of parietal extraembryonic endoderm. Finally, we demonstrate that there are differences in the RNA profiles expressed by the differentiated progeny formedin vitrofrom FGF-4−/−ES cells and FGF-4+/+ES cells when they are cultured with FGF-4. Taken together, the studies described in this report indicate that certain lineages formedin vitroare affected by the inactivation of the FGF-4 gene, in particular specific cells that form during the initial stage of ES cell differentiation. Thus, ES cells with both FGF-4 alleles inactivated should shed light on the important roles of FGF-4 during the early stages of mammalian development and help determine why FGF-4−/−embryos die shortly after implantation.