Bhaskar Thyagarajan

Creation of Engineered Human Embryonic Stem Cell Lines Using phiC31 Integrase

It has previously been shown that the phage‐derived phiC31 integrase can efficiently target native pseudo‐attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC‐derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1α promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo‐attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1α promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo‐attP sites in hESC and show that integrase‐mediated site‐specific integration can efficiently create stably expressing engineered human embryonic stem cell clones.

Generation of Platform Human Embryonic Stem Cell Lines That Allow Efficient Targeting at a Predetermined Genomic Location

Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency, allowing for long-term expression of transgenes. In the present work, we describe a retargeting system where we used phiC31 integrase to target a plasmid to a pseudo-attP site in the cellular genome. The integration site was mapped and the chromosomal location evaluated for potential to be transcriptionally active in differentiated cells. The target plasmid, thus inserted, carried a wild-type R4 attB site that acts as a target for further integration of expression constructs. We engineered 2 hESC lines, BG01V and H9, to contain the target and showed that genetic elements such as promoter-reporter pairs can be inserted at the target efficiently and specifically. The retargeting construct has been adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture, upon random differentiation, and directed induction into neural lineages. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in undifferentiated as well as in differentiated cells.

Generation of Site-Specific Retargeting Platform Cell Lines for Drug Discovery Using phiC31 and R4 Integrases

One of the challenges in developing cell lines for high-throughput screening in drug discovery is the labor- and time-intensive process required to create stable clonal cell lines that express specific reporters or drug targets. The authors report here the generation of a site-specific retargeting platform in 3 different cell lines: adherent HEK293, suspension CHO-S, and a human embryonic cell line (BGO1V). These platform cell lines were generated by using a combination of 2 site-specific integrases to develop a system that allows one to efficiently target a gene of interest to a specific locus and generates rapid production of homogeneous cell pools that stably express the gene of interest. The phiC31 integrase was used to create a platform line by placing a target site for the R4 integrase into a pseudo attP site, and then the R4 integrase was used to place a gene of interest into specific R4 target site. The authors demonstrate the successful and rapid retargeting of a G-protein-coupled receptor (cholecystokinin receptor A, CCKAR), an ion channel (the transient receptor potential cation channel, subfamily M, member 8, TRPM8), and a GFP-c-Jun(1-79) fusion protein into the specific loci in these cell lines and show that these retargeted cell lines exhibit functional and pharmacological responses consistent with those reported in the literature.

Micro RNA Profiling

MicroRNAs (miRNAs) are small regulatory RNAs varying in length between 20 and 24 nucleotides. They are thought to play a key role during development by negative gene regulation at the post-transcriptional level. Recent studies using quantitative polymerase chain reaction (QPCR) and northern blot analysis have reported the presence of several miRNA unique to specific cell types. The NCode multispecies miRNA array provides a means for simultaneously profiling the expression patterns of hundreds of known miRNAs in a given cell type or biological sample. Using this method, miRNA expression patterns in embryonic and adult stem cell lines can be characterized and compared with each other. The accuracy of NCode miRNA array data can be further confirmed by QPCR analysis of putative array hits. This array-based screening platform is a fast and easy to use analytical tool that allows one to asses the state of stem cell lines following multiple passages in culture as well as a discovery tool that eliminates the need to screen large numbers of candidate regulatory miRNAs by northern blot or PCR. In this chapter, we describe in detail the method to carry out miRNA array analysis in human embryonal carcinoma cells and confirm the array results using QPCR.

Emerging technology platforms for stem cells

Foreword Current state of stem cell field: Overview (Mahendra S. Rao). Chapter 1: Derivation methods for human embryonic stem cells: Past, present & future Necati Findikli. Mohan Vemuri. Chapter 2: Isolation of human ESCs from various stages of the human embryo (Yuri Verlinsky, N. Strelchenko, V. Kukharenko, A. Shkumatov, S. Rechitsky, O. Verlinsky, and A. Kuliev). Chapter 3: Derivation of stem cells from epiblasts (Michal Amit). Chapter 4: Derivation of Embryonic Stem Cells from Parthenogenetic Eggs (Jose Cibelli). Chapter 5: Reprogramming Developmental Potential (Costas A. Lyssiotis Cradley D. Charette, and Luke L. Lairson). Chapter 6: Adult stem cells and their role in endogenous tissue repair (N. Sachewsky and Cindi Morshead). Chapter 7: Greater differentiation potential of adult stem cells (Carlos Clavel and Catherine Verfaillie). Chapter 8: Cancer stem cells (Scott Dylla, In-Kyung Park and Austin L. Gurney). Chapter 9: Large scale production of adult stem cells for clinical use (Kristin Goltry, Brian Hampson, Naia Venturi and Ronnda Bartel). Chapter 10: Genetic and epigenetic features of stem cells (Jonathan Auerbach and Richard Josephson). Chapter 11: Directed differentiation of embryonic stem cells (Marjorie Pick). Chapter 12: Identification of signaling pathways involved during differentiation Takumi Miura. Chapter 13 Media and extracellular matrix requirements for large scale ESC growth (Allan J. Robins and Tom Schultz). Chapter 14: Automated method for culturing ES cells (S. Terstegge and Oliver Brustle). Chapter 15: Quantitative 2D Imaging of Human Embryonic Stem Cells (Steven K.W. Oh, Allen K. Chen, Andre B.H. Choo and Ivan Reading). Chapter 16: Nanobiotechonology for stem cell culture and Maintenance (Minseok S. Kim, Wonhye Lee and Je-Kyun Park). Chapter 17: Engineering Microenvironments to Control Stem Cell Functions (Anielle An-Chi Tsou and Song Li). Chapter 18: Improved lentiviral gene delivery tools for stem cells (Sanjay Vasu, Jian-Ping Yang and Wieslaw Kudlicki). Chapter 19: Sleeping Beauty-mediated Transposition in Stem Cells (Andrew Wilbur, Jakub Tolar, Bruce R Blazar, Catherine M Verfaillie, Uma Lakshmipathy, Dan S Kaufman and Scott McIvor). Chapter 20: PhiC31 Integrase for Modification of Stem Cells (W. Edward Jung and Michelle Calos). Chapter 21: Cell Engineering using Integrase and Recombinase systems (Takefumi Sone, Fumiko Nishi, Kazuhide Yahata, Yukari Sasaki, Hiroe Kishine, Taichi Andoh, Ken Inoue, Bhaskar Thyagarajan, Jonathan D. Chesnut and Fumio Imamoto). Chapter 22: hESC derived cardiomyocytes for cell therapy and drug discovery (William Sun and Robert Zweigerdt). Chapter 23: hESC in Drug discovery (Catharina Ellerstrom, Petter Bjorquist, Peter Sartipy, Johan Hyllner and Raimund Strehl). Chapter 24: Characterization and Culturing of Adipose-Derived Precursor Cells (Dietmar Hutmacher, Joanna Olkowska-Truchanowicz, David Leong, Johannes Reichert and Thiam Chye Lim). Chapter 25: Bringing Mesenchymal stem cells to clinic (Robert Deans).

hESC Engineering by Integrase-Mediated Chromosomal Targeting

Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency allowing for long-term expression of transgenes. In this chapter, we describe a retargeting system where phiC31 integrase is used to deliver a chromosomal target for a second integrase, R4. The engineered hESC line can be adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture and upon differentiation. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in pluripotent as well as in differentiated lineages there from.

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells

AIM Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. METHODS The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. RESULTS Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. CONCLUSIONS Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Role of DNA Methylation and Epigenetics in Stem Cells

In recent years, great strides have been made in our understanding of the biology of human embryonic stem cells and their ability to differentiate into multiple lineages. Although it has always been obvious that the differentiation of stem cells does not come about due to changes to the pri- mary sequence of the genome, only now are we beginning to understand the mechanisms involved in this process. Chro- matin modification has been shown to control the expression of key genes involved in the progression of stem cells into their differentiated progeny. In this chapter, some of the key regulatory mechanisms involved in epigenetic modification of the genome are discussed.

Lentiviral Vector Systems for Transgene Delivery

The powerful delivery capabilities of viral vectors have made them a prime tool in gene delivery for both basic research and therapy applications. Viral delivery tools combine both the cloning vector and delivery mechanism and they can target a wide variety of cell types both in vitro and in vivo with high efficiency. The lentivirus, specifically, has become a favored gene therapy tool for its unique ability to transduce dividing as well as terminally differentiated, non-dividing cells and to generate stable gene expression. These features are especially critical when attempting to genetically modify stem cells, hematopoetic cells, neurons and various tissues in vivo . Here we describe procedures to generate high titer lentivirus and transduce hPSCs. We then detail a method for site-specific integration of lentiviral vectors.

Primary and Stem Cells: Gene Transfer Technologies and Applications

This book describes basic cell engineering methods, emphasizing stem cell applications, and use of the genetically modified stem cells in cell therapy and drug discovery. Together, the chapters introduce and offer insights on new techniques for engineering of stem cells and the delivery of transgenes into stem cells via various viral and non-viral systems. The book offers a guide to the types of manipulations currently available to create genetically engineered stem cells that suit any investigators purpose, whether its basic science investigation, creation of disease models and screens, or cells for therapeutic applications.