Embryonic Stem Cell Technology Research Articles

CRISPR-Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat.

Rationale: Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat. Objective: We sought to generate the mouse lung in a rat. Methods: Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat one-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESCs into Nkx2-1-deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing. Measurements and Main Results: Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9-mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1-/- blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse-rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat. Conclusions: A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors."

The use of fluorescence correlation spectroscopy to monitor cell surface β2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying β2 -adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged β2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface β2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface β2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting β2AR agonists.

Embryoid Body Formation from Mouse and Human Pluripotent Stem Cells for Transplantation to Study Brain Microenvironment and Cellular Differentiation.

Human embryonic stem cell (hESC) and human-induced pluripotent stem cell (hiPSC) technologies have a critical role in regenerative strategies for personalized medicine. Both share the ability to differentiate into almost any cell type of the human body. The study of their properties and clinical applications requires the development of robust and reproducible cell culture paradigms that direct cell differentiation toward a specific phenotype in vitro and in vivo. Our group evaluated the potential of mouse ESCs (mESCs), hESCs, and hiPSCs (collectively named pluripotent stem cells, PSCs) to analyze brain microenvironments through the use of embryoid body (EB)-derived cells from these cell sources. EB are cell aggregates in 3D culture conditions that recapitulate embryonic development. Our approach focuses on studying the midbrain dopaminergic phenotype and transplanting EB into the substantia nigra pars compacta (SNpc) in a Parkinson's disease rodent model. Here, we describe cell culture protocols for EB generation from PSCs that show significant in vivo differentiation toward dopaminergic neurons.

Insights into mammalian morphogen dynamics from embryonic stem cell systems.

Morphogens play an essential role in cell fate specification and patterning including in laying out the mammalian body plan during gastrulation. In vivo studies have shed light on the signaling pathways involved in this process and the phenotypes associated with their disruption, however, several important open questions remain regarding how morphogens function in space and time. Self-organized patterning systems based on embryonic stem cells have emerged as a powerful platform for beginning to address these questions that is complementary to in vivo approaches. Here we review recent progress in understanding morphogen signaling dynamics and patterning in early mammalian development by taking advantage of cutting-edge embryonic stem cell technology.

Consistent Production of Mice with Conditional Knockout Alleles by CRISPR/Cas9-Mediated Genome Editing Using Two Guides/Two Oligos Approach

Gene targeting is extensively used to generate designer mouse mutants and to study gene function in vivo. Knockout mice that harbor a null allele in their germline provide appropriate genetic models of inherited diseases and often exhibit embryonic or early postnatal lethality. To study gene function in adult mice and in selected cell types, a refined strategy for conditional gene inactivation has been developed that relies on the DNA recombinase Cre and its recognition (loxP) sites. This process has traditionally relied on the complex process involving genome editing in embryonic stem (ES) cells despite its limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse ES cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN), which has been questioned by another report combining data from multiple transgenic cores. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of using this method in creating conditional targeted alleles in three genes, phosphatase and actin regulator 1 (Phactr1), apolipoprotein A-I (ApoA1), and actin-related protein T2 (Actrt2). Even though overall success rate was low—about 2.5%—we show that it’s possible to reliably generate conditional knockout alleles using CRISPR/Cas9 on a consistent basis.

Germ cell transplantation in pigs - advances and applications

Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice results in donor-derived spermatogenesis and transmission of the donor haplotype to offspring of recipient animals. In the pig, germ cells can be transplanted to a recipient testis by ultrasoundguided cannulation of the rete testis with delivery of cells by gravity flow. It is important to note that germ cell transplantation was successful between unrelated, immuno-competent pigs, whereas transplantation in rodents requires syngeneic or immuno-compromised recipients. Efficiency of colonization of the recipient testis by donor-derived germ cells can be improved by pretreatment of the recipient animal to deplete endogenous germ cells. Genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in production of transgenic sperm. Transgenesis through the male germ line has tremendous potential in species like pigs where embryonic stem cell technology is not available and current options to generate transgenic animals are inefficient. Introduction of a genetic change prior to fertilization will circumvent problems associated with manipulation of early embryos and developmental abnormalities associated with somatic cell nuclear transfer and reprogramming. Viral transduction of germ cells prior to transplantation has been used to generate transgenic rodents and has also shown early promising results in pigs. Current research is directed toward improving protocols for isolation and culture of porcine male germ cells to increase efficiency of transgene transmission and to allow for gene targeting prior to germ cell transplantation. It is expected that germ cell transplantation will then provide a viable alternate approach to generate germ line transgenic pigs.

Application of induced pluripotent stem cell and embryonic stem cell technology to the study of male infertility.

Stem cells (SCs) are classes of undifferentiated biological cells existing only at the embryonic, fetal, and adult stages that can divide to produce specialized cell types during fetal development and remain in our bodies throughout life. The progression of regenerative and reproductive medicine owes the advancement of respective in vitro and in vivo biological science on the stem cell nature under appropriate conditions. The SCs are promising therapeutic tools to treat currently of infertility because of wide sources and high potency to differentiate. Nevertheless, no effective remedies are available to deal with severe infertility due to congenital or gonadotoxic stem cell deficiency in prepubertal childhood. Some recent solutions have been developed to address the severe fertility problems, including in vitro formation of germ cells from stem cells, induction of pluripotency from somatic cells, and production of patient-specific pluripotent stem cells. There is a possibility of fertility restoration using the in vitro formation of germ cells from somatic cells. Accordingly, the present review aimed at studying the literature published on the medical application of stem cells in reproductive concerns.

Embryonic Stem Cells in Development and Regenerative Medicine.

After progressive improvement in embryonic stem (ES) cell field, several studies have been conducted to explore the usage of ES cells in regenerative medicine. Unlimited self renewal and pluripoteny properties, combined with encouraging preclinical trials, remark that ES cell technology might be promising for clinical practice. ES cells, which can form three germ layers in vitro, are potential candidates to study development at the cellular and molecular level. Understanding the cell fate decision and differentiation processes during development might enable generating functional progenitor cells for tissue restoration. Progression in gene modifications and tissue engineering technology has facilitated the derivation of desired cells for therapy. Success in differentiation protocols and identification the regulatory pathways simplify the research for clinical applications. Although there are established protocols for cell differentiation in vitro and promising preclinical studies in vivo, many challenges need to be adressed before clinical translation. In this review, ES cells are discussed as a model of development in vitro and as a potential candidate for regenerative medicine. This review also dissusses current challenges for ES cell based therapy.

A Novel Mouse Model of iNKT Cell-deficiency Generated by CRISPR/Cas9 Reveals a Pathogenic Role of iNKT Cells in Metabolic Disease

iNKT cells play important roles in immune regulation by bridging the innate and acquired immune systems. The functions of iNKT cells have been investigated in mice lacking the Traj18 gene segment that were generated by traditional embryonic stem cell technology, but these animals contain a biased T cell receptor (TCR) repertoire that might affect immune responses. To circumvent this confounding factor, we have generated a new strain of iNKT cell-deficient mice by deleting the Traj18 locus using CRISPR/Cas9 technology, and these animals contain an unbiased TCR repertoire. We employed these mice to investigate the contribution of iNKT cells to metabolic disease and found a pathogenic role of these cells in obesity-associated insulin-resistance. The new Traj18-deficient mouse strain will assist in studies of iNKT cell biology.

Tracing the knowledge-building dynamics in new stem cell technologies through techno-scientific networks

This study assesses the knowledge-building dynamics of emerging technologies, their participating country-level actors, and their interrelations. We examine research on induced pluripotent stem (iPS) cells, a recently discovered stem cell species. Compared to other studies, our approach conflates the totality of publications and patents of a field, and their references, into single “techno-scientific networks” across intellectual bases (IB) and research fronts (RF). Diverse mapping approaches—co-citation, direct citation, and bibliographic coupling networks—are used, driven by the problems tackled by iPS cell researchers. Besides the study of the field of iPS cells as a whole, we assessed the roles of relevant countries in terms of “knowledge exploration,” “knowledge nurturing,” “knowledge exploitation,” and cognitive content. The results show that a fifth of nodes in IB and edges in RF interconnect science (S) and technology (T). S and T domains tell different, yet complementing stories: S overstresses upstream activities, and T captures the increasing influential role of application domains and general technologies. Both S and T reflect the path-dependent nature of iPS cells in embryonic stem cell technologies. Building on the feedback between IB and RF, we examine the dominating role of the United States. Japan, the pioneer, falls behind in quantity, yet its global influence remains intact. New entrants, such as China, are advancing rapidly, yet, cognitively, the bulk of efforts are still upstream. Our study demonstrates the need for bibliometric assessment studies to account for S&T co-evolution. The multiple data source-based, integrated bibliometric approaches of this study are initial efforts toward this direction.

The patentability of human embryonic stem cell technology in China.

Although China has in the past decade been reluctant to grant patents on human embryonic stem cell technology, it has recently changed its patent policy to loosen some restrictions.

Engineering Embryonic Stem Cell Microenvironments for Tailored Cellular Differentiation

The rapid progress of embryonic stem cell (ESCs) research offers great promise for drug discovery, tissue engineering, and regenerative medicine. However, a major limitation in translation of ESCs technology to pharmaceutical and clinical applications is how to induce their differentiation into tailored lineage commitment with satisfactory efficiency. Many studies indicate that this lineage commitment is precisely controlled by the ESC microenvironment in vivo. Engineering and biomaterial-based approaches to recreate a biomimetic cellular microenvironment provide valuable strategies for directing ESCs differentiation to specific lineages in vitro. In this review, we summarize and examine the recent advances in application of engineering and biomaterial-based approaches to control ESC differentiation. We focus on physical strategies (e.g., geometrical constraint, mechanical stimulation, extracellular matrix (ECM) stiffness, and topography) and biochemical approaches (e.g., genetic engineering, soluble bioactive factors, coculture, and synthetic small molecules), and highlight the three-dimensional (3D) hydrogel-based microenvironment for directed ESC differentiation. Finally, future perspectives in ESCs engineering are provided for the subsequent advancement of this promising research direction.

The Court of Justice of the European Union Clarifies when Human Embryonic Stem Cells Can Be Patented

On 18 December 2014, in International Stem Cell Corporation v. Comptroller General of Patents, Designs and Trade Marks (C-364/13), the Court of Justice of the European Union (CJEU) delivered an important decision regarding the scope of the exclusion from patentability on morality-related grounds under Article 6(2) of the EU Biotech Directive. The Court made an important distinction between embryonic stem cell technologies based on fertilised human ovum and those based on unfertilised human ovum stimulated by parthenogenesis. The CJEU held, in particular, that a human ovum: (i) who is unfertilized and (ii) whose division and further development has been stimulated by parthenogenesis, is not a human embryo under Article 6(2)(c) of the Biotech Directive, if it in itself has not the inherent capacity of developing into a human being, this matter to be ascertained by the national court in the light of current scientific knowledge. By doing so, the Court clarified its previous ruling in Brustle (October 2011).

Is mouse embryonic stem cell technology obsolete?

Injection of recombinant Cas9 protein and synthetic guide RNAs into mouse zygotes has been shown to facilitate gene disruption and knock-ins using the CRISPR system. These technologies may soon displace genetic modification using embryonic stem cells.

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Mouse genome engineering using designer nucleases.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Application of genetically engineered mice model in the research of gastrointestinal tumor

The gene knockout is based on the DNA homologous recombination and embryonic stem cell technology. It is an experimental method for modification of specific gene loci, which is one of the most direct means to clarifying gene functions. The similarity of genomes between mouse and human is as high as up to 95%. The life cycle of mice is relatively short, and easy to breed with stronger reproductive capacity. Therefore, the knockout mice are suitable for observing the whole process of disease pathogenesis. With genetic manipulation technology, researchers can produce the simulations of phenotypic effect by human genetic variation. This article focuses on genetically engineered mice models and their application in gastrointestinal cancer.

Novel Automated Patch-clamp Assays on Stem Cell-derived Cardiomyocytes: Will they Standardize In Vitro Pharmacology and Arrhythmia Research?

Recent progress in embryonic stem cell and human induced pluripotent stem cell technology allowed effective generation of cultured cardiomyocyte preparations with over 99% purity, rendering them suitable for automated patchclamp approaches. Compared to current high-throughput drug screening methods, such as fluorescence assays using calcium-sensitive or transmembrane potential-sensitive dyes, or field potential recordings and activation mapping using multi-electrode arrays, patch-clamp experiments offer the possibility to combine action potential recordings in current-clamp mode with detailed characterization of drug effects on multiple ion current components with carefully designed voltage-clamp protocols, leading to an in-depth understanding of arrhythmogenesis conditions and mechanisms, especially when combined with cellular electrophysiology computerized models. The recently issued Comprehensive in vitro ProArrhythmia Assay (CiPA) guidelines emphasize the importance of pharmacological tests on multiple cardiac ion channels, including at least Nav1.5 (early and late), Cav1.2, hERG1, Kv7.1/minK, and Kir2.1, via voltage-clamp protocols, instead of simple hERG screening, combined with computer modeling, in order to determine the proarrhythmic liability of a drug candidate. In addition, patch-clamp assays on patient-specific induced pluripotent stem cell-derived cardiomyocytes will enhance current molecular diagnosis methods in cardiac channelopathies by identification of the faulty current component and individualized screening of drug sensitivity of mutant channels, a step forward for personalized medicine approaches.

Generation of an allelic series of knock-in mice using recombinase-mediated cassette exchange (RMCE).

Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the function of a gene in mice or to generate a mouse model for a human disease are continuously under development. Next to (conditional) inactivation of genes the application and importance of approaches to generate knock-in mutations are increasing. In this chapter the principle and application of recombinase-mediated cassette exchange (RMCE) are discussed as being a new emerging knock-in strategy, which enables easy generation of a series of different knock-in mutations within one gene. An RMCE protocol, which was used to generate a series of different knock-in mutations in the Lrp1 gene of ES cells, is described in detail as an example of how RMCE can be used to generate highly efficiently an allelic series of differently modified ES cell clones from a parental modified ES cell clone. Subsequently the differently modified ES cell clones can be used to generate an allelic series of mutant knock-in mice.

Culture of Pig Induced Pluripotent Stem Cells without Direct Feeder Contact in Serum Free Media

Background: Reprogramming pig somatic cells into induced pluripotent stem cells (iPSCs) have promising applications in basic biology, disease model development and xenotransplantation. In the mouse, embryonic stem cell (ESC) technology has revolutionized the field enabling gene targeting, complex screening strategies and the creation of animals that show unique characteristics of interest. Recent breakthroughs utilizing induced pluripotent stem cell technology in the pig have made it possible to produce pig pluripotent stem cells that resemble germline chimeric competent mouse ESCs. However, an optimal culture system for piPSC expansion has yet to be developed. Most reports have maintained piPSCs in undefined systems that use xenoproducts and feeder layers, which are potential sources of contamination. Methods: In this study, new lines of pig iPSCs (piPSC) were generated from pig fibroblast cells by overexpressing six reprogramming genes: POU5F1, SOX2, NANOG, LIN28, KLF4 and C-MYC. These new lines were tested for their ability to be maintained on a Matrigel substrate in the established mouse 2i+LIF system, the human mTeSR1 system and variations of a feeder conditioned media system. Analysis and identification of piPSCs were performed using immunocytochemistry, flow cytometry and by examining embryoid body formation and differentiation. Results: The newly generated piPSCs showed morphological features, immunoreactivity and reactivation of endogenous pluripotency networks consistent with iPSCs. Similar to cells cultured on feeders, piPSCs maintained under all 7 feeder-free conditions expressed POU5F1 and NANOG, SSEA-1, SSEA-4 and TRA1-81. However, flow cytometry demonstrated that piPSCs cultured in feeder conditioned media with KnockOut Serum Replacement and basic fibroblast growth factor (FGF2) showed significantly higher levels of SSEA1 and SSEA4 expression than cells cultured in a 2i+LIF or mTeSR1 system. Conclusion: These findings demonstrate that piPSCs can be maintained in defined systems without serum and direct feeder contact, increasing their potential use in both agricultural and biomedical fields.