Somatic Cell Lines Research Articles

Variation in the maternal corticotrophin releasing hormone-binding protein (CRH-BP) gene and birth weight in Blacks, Hispanics and Whites.

BackgroundGiven the unique role of the corticotrophin-releasing hormone (CRH) system in human fetal development, the aim of our study was to estimate the association of birth weight with DNA sequence variation in three maternal genes involved in regulating CRH production, bioavailability and action: CRH, CRH-Binding Protein (CRH-BP), and CRH type 1 receptor (CRH-R1), respectively, in three racial groups (African-Americans, Hispanics, and non-Hispanic Whites).MethodsOur study was carried out on a population-based sample of 575 mother–child dyads. We resequenced the three genes in mouse–human hybrid somatic cell lines and selected SNPs for genotyping.ResultsA significant association was observed in each race between birth weight and maternal CRH-BP SNP genotypes. Estimates of linkage disequilibrium and haplotypes established three common haplotypes marked by the rs1053989 SNP in all three races. This SNP predicted significant birth weight variation after adjustment for gestational age, maternal BMI, parity, and smoking. African American and Hispanic mothers carrying the A allele had infants whose birth weight was on average 254 and 302 grams, respectively, less than infants having C/C mothers. Non-Hispanic White mothers homozygous for the A allele had infants who were on average 148 grams less than those infants having A/C and C/C mothers.ConclusionsThe magnitudes of the estimates of the birth weight effects are comparable to the combined effects of multiple SNPs reported in a recent meta-analysis of 6 GWAS studies and is quantitatively larger than that associated with maternal cigarette smoking. This effect was persistent across subpopulations that vary with respect to ancestry and environment.

LIN28 Binds Messenger RNAs at GGAGA Motifs and Regulates Splicing Factor Abundance

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

A System for the Measurement of Gene Targeting Efficiency in Human Cell Lines Using an Antibiotic Resistance—GFP Fusion Gene

Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.

CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification

High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types.

Assessment of the long-term transcriptional activity of a 550-bp-long human β-actin promoter region

β-actin (ACTB) is one of the genes expressed most abundantly and ubiquitously in human non-muscular tissues. Here, we investigated the long-term activity of a 550-bp-long human ACTB promoter region in human cells in comparison with other commonly used constitutive promoters. We first constructed plasmid vectors expressing enhanced green fluorescent protein (GFP) driven by one of the 5 promoters, human ACTB, human elongation factor-1α (EF1α), cytomegalovirus early enhancer/chicken β-actin (CAG), cytomegalovirus (CMV), and herpes simplex virus thymidine kinase, and transfected them into multiple human somatic cell lines. Stable transfectants were maintained for 45days, and GFP signals from the cells were quantified by fluorescence flow cytometry. GFP signals driven by the human ACTB and the CMV promoters were also compared over time for up to 60days following transfection. We observed robust, prolonged transcriptional activity with the human ACTB promoter that is comparable to the human EF1α and the CAG promoters and significantly more stable than the CMV promoter.

Genetic consequences of programmed genome rearrangement.

The lamprey (Petromyzon marinus) undergoes developmentally programmed genome rearrangements that mediate deletion of∼20% of germline DNA from somatic cells during early embryogenesis. This genomic differentiation of germline and soma is intriguing, because the germline plays a unique biological role wherein it must possess the ability to undergo meiotic recombination and the capacity to differentiate into every cell type. These evolutionarily indispensable functions set the germline at odds with somatic tissues, because factors that promote recombination and pluripotency can potentially disrupt genome integrity or specification of cell fate when misexpressed in somatic cell lineages (e.g., in oncogenesis). Here, we describe the development of new genomic and transcriptomic resources for lamprey and use these to identify hundreds of genes that are targeted for programmed deletion from somatic cell lineages. Transcriptome sequencing and targeted validation studies further confirm that somatically deleted genes function both in adult (meiotic) germline and in the development of primordial germ cells during embryogenesis. Inferred functional information from deleted regions indicates that developmentally programmed rearrangement serves as a (perhaps ancient) biological strategy to ensure segregation of pluripotency functions to the germline, effectively eliminating the potential for somatic misexpression.

Induced Pluripotent Stem–Induced Cells Show Better Constitutive Heterochromatin Remodeling and Developmental Potential After Nuclear Transfer Than Their Parental Cells

Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.

Skin micro-organs from several frog species secrete a repertoire of powerful antimicrobials in culture

This work is an attempt to take advantage of the rich biodiversity that exists in Colombia in order to start a systematic analysis of antimicrobial substances that have emerged through amphibian evolution. For this purpose we have developed a technique to grow intact frog skin derived micro-organs (SMOs) in vitro in the absence of serum. We show that in SMOs, the skin glands remain intact and continue to secrete into the medium substances with potent antibacterial activity, for several days in culture. Our strategy has been to create a bank of substances secreted by amphibian skin from different species. This bank contains at present around 50 species and is of particular importance as some of the species are in danger of disappearing. We show that some of the species tested displayed very strong antibacterial activity without being toxic to somatic cell lines, even at 10-fold higher concentration.

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

Self-renewal of embryonic stem cells through culture on nanopattern polydimethylsiloxane substrate

Embryonic stem (ES) cells can undergo continual proliferation and differentiation into cells of all somatic cell lineages in vitro; they are an unlimited cell source for regenerative medicine. However, techniques for maintaining undifferentiated ES cells are often inefficient and result in heterogeneous cell populations. Here, we determined effects of nanopattern polydimethylsiloxane (PDMS) as a culture substrate in promoting the self-renewal of mouse ES (mES) cells, compared to commercial plastic culture dishes. After many passages, mES cells efficiently maintained their undifferentiated state on nanopattern PDMS, but randomly differentiated on commercial plastic culture dishes, as indicated by partially altered morphologies and decreases in alkaline phosphatase activity and stage-specific expression of embryonic antigen-1. Under nanopattern PDMS conditions, we found increased activities of STAT3 and Akt, important proteins involved in maintaining the self-renewal of mES cells. The substrate-cell interactions also enhanced leukemia inhibitory factor (LIF)-downstream signaling and inhibited spontaneous differentiation, concomitant with reduced focal adhesion kinase (FAK) signaling. This reduction in FAK signaling was shown to be important for promoting mES cell self-renewal. Thus, our data demonstrates that nanopattern PDMS contributes to maintaining the self-renewal of mES cells and may be applicable in the large-scale production of homogeneously undifferentiated mES cells.

Abstract 1997: Resveratrol modifies the activity and expression of antioxidant enzymes in somatic cancer cells

Abstract Background & Objectives: Resveratrol has been reported as a potent anti-cancer compound, but it's mode of action is not completely understood. In this study, we investigated the effects of resveratrol treatment on the activity and expression of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) in three somatic cancer cell lines: PC-3 (prostate cancer), MCF-7 (breast cancer) and HepG2 (hepatocellular cancer). Methods: The cells were treated with different concentration (0-200 μM) of resveratrol for 24, 48 and 72 hours.Cell growth was studied by cell counting; cell homogenate was taken for the measurement of enzyme activities; proteins were isolated for western blotting to measure the genetic expression. Results: Resveratrol showed dose-dependent growth inhibitory activity in all cells. It was significantly effective against PC-3 and HepG2 even in 10 μM, and 25 μM concentration, but not in MCF-7 cells. However 50 μM showed significant growth inhibitory effect in MCF-7, and also in HEK293T (human embryonic kidney cell line as control) cells. 25 μM was identified as most effective dosage against PC-3 and HepG2 cells, without affecting HEK293T cells. Treatment of 25 μM for 48 hours increased the activity of SOD enzymes by 3-4 folds in PC-3; 2-3 folds in HepG2; 1.5-1.8 folds in MCF-7; and 1.2-1.5 folds in HEK293T cells. Resveratrol treatment also significantly increased the activity of CAT in HepG2, moderately in MCF-7, but non-significantly in PC-3 and HEK293T cells. However, the treatment of resveratrol showed no significant change in GPX activity in any cell line. Western blotting also revealed the increased expression of SOD2 in PC-3, and HepG2 cells at 25 μM resveratrol treatment. CAT expression was moderate, but no change in GPX1 expression was found in the tested cancer cells. Conclusions: The results of this study could confirm the dose dependent growth inhibitory potential of resveratrol; however, the dose of resveratrol in treatment of the cancer cells should be limited, as higher dose (Δ50 μM) has also been found dangerous for normal cells (HEK293T). Resveratrol at low dose (25 μM) potentially increases the activity and expression of SOD enzyme in cancer cells while inhibiting cell growth. Moderate increase of CAT activity and expression is also observed, but no significant change in GPX activity and expression in cell lines used in this study. These findings may contribute to understanding the mechanisms of resveratrol action in treatment of cancer. The work was supported by the National Basic Research Program of China (No. 2011CB910700-704) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1997. doi:1538-7445.AM2012-1997

Establishment of epigenetic patterns in development

The distinct cell types of the body are established from the fertilized egg in development and assembled into functional tissues. Functional characteristics and gene expression patterns are then faithfully maintained in somatic cell lineages over a lifetime. On the molecular level, transcription factors initiate lineage-specific gene expression programmmes and epigenetic regulation contributes to stabilization of expression patterns. Epigenetic mechanisms are essential for maintaining stable cell identities and their disruption can lead to disease or cellular transformation. Here, we discuss the role of epigenetic regulation in the early mouse embryo, which presents a relatively well-understood system. A number of studies have contributed to the understanding of the function of Polycomb group complexes and the DNA methylation system. The role of many other chromatin regulators in development remains largely unexplored. Albeit the current picture remains incomplete, the view emerges that multiple epigenetic mechanisms cooperate for repressing critical developmental regulators. Some chromatin modifications appear to act in parallel and others might repress the same gene at a different stage of cell differentiation. Studies in pluripotent mouse embryonic stem cells show that epigenetic mechanisms function to repress lineage specific gene expression and prevent extraembryonic differentiation. Insights into this epigenetic “memory” of the first lineage decisions help to provide a better understanding of the function of epigenetic regulation in adult stem cell differentiation.

A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.

Germ cell DNA-repair systems—possible tools in cancer research?

A major dogma in cancer research is that cancer begins at the cellular level. Because of this single-cell origin, evolutionary principles have often been used to explain how somatic cancer cells are selected at a sub-individual level. The traditional application of Darwinian theory, however, in which the colony of cells constituting an individual is regarded as a whole, has not been applied extensively to the understanding of cancer until recently. Two proponents for this view, Breivik and Gaudernack, have suggested that in certain situations the cost of DNA repair might exceed the cost of errors. This model predicts that genetic stability is configured for an optimal cost-benefit relationship. Natural selection is not expected to have produced the best genetic stability available in the human body, merely the best compromise of DNA repair and costs. Repair and maintenance of the vast human genome is thermodynamically expensive, and an optimal balance between DNA repair and dietary needs is likely to have originated. Furthermore, fast growth conveys significant advantages such as early maturation or cognitive development, but usually at the expense of replication accuracy. Thus, a compromise between growth speed and cancer risk is likely to have taken place. These and other ecological mechanisms have probably prevented genomic stability to reach its full potential in the human body. In contrast, germ lines express near perfect DNA maintenance. Although germ cells are specialized DNA-conserving cells with few other functions, it's not given that their proteins will all be incompatible with the somatic cell. One approach to study this would be to systematically explore which DNA-stability and -repair systems are unique in germ cells, and induce their expression in invertebrate and mammalian model organisms. This could unveil which DNA-repair systems are switched off in the somatic cell lines, as they are incompatible, and which are absent due to evolution. The present review discuss different DNA-repair systems and cell cycle check point control mechanisms shown to be different or unique in the germ cell, and how they may be utilized in cancer therapy.

Age-Related Somatic Structural Changes in the Nuclear Genome of Human Blood Cells

Structural variations are among the most frequent interindividual genetic differences in the human genome. The frequency and distribution of de novo somatic structural variants in normal cells is, however, poorly explored. Using age-stratified cohorts of 318 monozygotic (MZ) twins and 296 single-born subjects, we describe age-related accumulation of copy-number variation in the nuclear genomes in vivo and frequency changes for both megabase- and kilobase-range variants. Megabase-range aberrations were found in 3.4% (9 of 264) of subjects ≥60 years old; these subjects included 78 MZ twin pairs and 108 single-born individuals. No such findings were observed in 81 MZ pairs or 180 single-born subjects who were ≤55 years old. Recurrent region- and gene-specific mutations, mostly deletions, were observed. Longitudinal analyses of 43 subjects whose data were collected 7-19 years apart suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberrations suggests that there is a natural self-removal of aberrant cell clones from peripheral blood. In three healthy subjects, we detected somatic aberrations characteristic of patients with myelodysplastic syndrome. The recurrent rearrangements uncovered here are candidates for common age-related defects in human blood cells. We anticipate that extension of these results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our work might also help to explain the cause of an age-related reduction in the number of cell clones in the blood; such a reduction is one of the hallmarks of immunosenescence.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.

235 PRODUCTION OF PORCINE KNOCK-IN FIBROBLASTS EXPRESSING HUMAN DECAY-ACCELERATING FACTOR on α-1,3-GALACTOSYLTRANSFERASE (GGTA1) GENE LOCUS

Galactose-α1,3-galactose (α1,3-gal) epitope is synthesised at a high concentration on the surface of pig cells by α1,3-galactosyltransferase gene (GGTA1). The α1,3-gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesise Galα(1,3)Gal. Knock-in system refers to the integration a foreign gene at a specific target, disrupting the target gene by inserting the transgene. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as complement inhibitor to protect self cells from complement attack. The generation of GGTA1 knockout pigs expressing DAF is necessary to use as organ donors for humans. In this study, we established cell lines of GGTA1 knockout expressing DAF from pig ear fibroblasts to apply somatic cell nuclear transfer. A GGTA1-DAF knock-in vector for positive-negative selection (PNS) consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the diphtheria toxin A fragment (DT-A) gene as a negative selectable marker gene. The DAF gene was subcloned in the GGTA1 gene exon 4 and the endogenous start codon was replaced by that of DAF. The ear fibroblast cell lines were isolated from Chicago miniature pig and used for transfection. Linearized targeting vector DNA was transfected into these cells by electroporation. After selection of G-418 during 11 days, we analysed 242 colonies using PCR and long PCR. We isolated 2 positive colonies for GGTA1 gene disruption. To confirm expression of the hDAF and GGTA1 genes, we isolated total RNA from normal pig cells and the knock-in somatic cell line. A cDNA synthesised using reverse transcriptase progressed to RT-PCR by a pair of DAF-specific primers and α1,3-GT specific primer, respectively. Expression of DAF was detected in DAF knock-in somatic cells, butnot in normal pig cell. Expression of GGTA1 gene was decreased in the knock-in somatic cell line compared with normal pig cell. These cell lines may be used production of α1,3-GT knockout and DAF expression pig for xenotransplantation.

Global Mapping of H3K4me1 and H3K4me3 Reveals the Chromatin State-Based Cell Type-Specific Gene Regulation in Human Treg Cells

Regulatory T cells (Treg) contribute to the crucial immunological processes of self-tolerance and immune homeostasis. Genomic mechanisms that regulate cell fate decisions leading to Treg or conventional T cells (Tconv) lineages and those underlying Treg function remain to be fully elucidated, especially at the histone modification level. We generated high-resolution genome-wide distribution maps of monomethylated histone H3 lysine 4 (H3K4me1) and trimethylated H3K4 (H3K4me3) in human CD4+CD25+FOXP3+ Tregs and CD4+CD25+FOXP3− activated (a)Tconv cells by DNA sequencing-by-synthesis. 2115 H3K4me3 regions corresponded to proximal promoters; in Tregs, the genes associated with these regions included the master regulator FOXP3 and the chemokine (C-C motif) receptor 7 (CCR7). 41024 Treg-specific H3K4me1 regions were identified. The majority of the H3K4me1 regions differing between Treg and aTconv cells were located at promoter-distal sites, and in vitro reporter gene assays were used to evaluate and identify novel enhancer activity. We provide for the first time a comprehensive genome-wide dataset of lineage-specific H3K4me1 and H3K4me3 patterns in Treg and aTconv cells, which may control cell type-specific gene regulation. This basic principle is likely not restricted to the two closely-related T cell populations, but may apply generally to somatic cell lineages in adult organisms.

Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle.

Introgression of the heterologous nuclear DNAs and efficacious compositions from Swertia tetraptera Maxim. into Bupleurum scorzonerifolium Willd. via somatic hybridization

Swertia tetraptera Maxim. is an important source of secoiridoid glucosides. To produce these pharmacologically valuable compounds heterologously in somatic hybrid cell lines, S. tetraptera protoplasts were irradiated with various doses of UV light and fused with protoplasts from a long-term cell line of Bupleurum scorzonerifolium Willd. This recipient was chosen as the cell line is cytogenetically stable and fast growing; furthermore, protoplasts isolated from the cell line are readily regenerable. From a set of 86 putative hybrid calli, only two were able to regenerate viable green plants. The hybridity of the 19 of the 86 selections was revealed by a combined isozyme and RAPD analysis, supported by a karyotypic study based on genomic in situ hybridization (GISH). Clone I-3 contained 0.014% swertiamarin while the regenerants had 0.069% swertiamarin and 0.409% gentiopicroside while the III-4 plants contained only 0.015% gentiopicroside.