Andrew Fontes

Generation of Human-Induced Pluripotent Stem Cells by a Nonintegrating RNA Sendai Virus Vector in Feeder-Free or Xeno-Free Conditions

The generation of induced pluripotent stem cells (iPSCs) from somatic cells has enabled the possibility of providing unprecedented access to patient-specific iPSC cells for drug screening, disease modeling, and cell therapy applications. However, a major obstacle to the use of iPSC for therapeutic applications is the potential of genomic modifications caused by insertion of viral transgenes in the cellular genome. A second concern is that reprogramming often requires the use of animal feeder layers and reagents that contain animal origin products, which hinder the generation of clinical-grade iPSCs. Here, we report the generation of iPSCs by an RNA Sendai virus vector that does not integrate into the cells genome, providing transgene-free iPSC line. In addition, reprogramming can be performed in feeder-free condition with StemPro hESC SFM medium and in xeno-free (XF) conditions. Generation of an integrant-free iPSCs generated in xeno-free media should facilitate the safe downstream applications of iPSC-based cell therapies.

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 Induced Pluripotent Stem Cells with CytoTune, a Non-Integrating Sendai Virus

One of the major obstacles in generating induced pluripotent stem cells for research or downstream applications is the potential modifications of cellular genome as a result of using integrating viruses during reprogramming. Another major disadvantage of reprogramming cells with integrating vectors is that silencing and activation of transgenes are unpredictable, which may affect terminal differentiation potential and increase the risk of using iPSC-derived cells. Here we describe a protocol for the generation of induced pluripotent stem cells using a non-integrating RNA virus, Sendai virus, to efficiently generate transgene-free iPSCs starting with different cell types as well as in feeder-free conditions.

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.

Advances in genetic modification of pluripotent stem cells

Genetically engineered stem cells aid in dissecting basic cell function and are valuable tools for drug discovery, in vivo cell tracking, and gene therapy. Gene transfer into pluripotent stem cells has been a challenge due to their intrinsic feature of growing in clusters and hence not amenable to common gene delivery methods. Several advances have been made in the rapid assembly of DNA elements, optimization of culture conditions, and DNA delivery methods. This has lead to the development of viral and non-viral methods for transient or stable modification of cells, albeit with varying efficiencies. Most methods require selection and clonal expansion that demand prolonged culture and are not suited for cells with limited proliferative potential. Choosing the right platform based on preferred length, strength, and context of transgene expression is a critical step. Random integration of the transgene into the genome can be complicated due to silencing or altered regulation of expression due to genomic effects. An alternative to this are site-specific methods that target transgenes followed by screening to identify the genomic loci that support long-term expression with stem cell proliferation and differentiation. A highly precise and accurate editing of the genome driven by homology can be achieved using traditional methods as well as the newer technologies such as zinc finger nuclease, TAL effector nucleases and CRISPR. In this review, we summarize the different genetic engineering methods that have been successfully used to create modified embryonic and induced pluripotent stem cells.

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.

Generation of human-induced pluripotent stem cells (hiPSCs) using episomal vectors on defined Essential 8™ Medium conditions.

Human-induced pluripotent stem cells (iPSCs) are an important potential source of cells for regenerative medicine due to their inherent ability to differentiate into all cell types of the three germ layers. Generation of iPSCs with a non-integrating reprogramming method and in culture conditions that are completely absent of animal proteins will be ideal for such regenerative and cell therapy applications. Here we describe a method to generate non-integrating iPSCs using the Episomal iPSC Reprogramming Vectors.

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.

Applications of Quantitative Polymerase Chain Reaction Protein Assays During Reprogramming

The capability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) has opened a new area of biology and provides unprecedented access to patient-specific iPSCs for drug screening, disease models, and transplantation therapies. Although the process of obtaining iPSC lines is technically simple, reprogramming is a slow and inefficient process consisting of a largely uncharacterized chain of molecular events. To date, researchers have reported a wide range of reprogramming efficiencies, from <0.01% to >1%, depending on the specific reprogramming factors used, the mode of delivery of the reprogramming factors, properties of the starting cells, and culture conditions. We have applied a quantitative polymerase chain reaction methodology, TaqMan Protein Assays to directly quantify the kinetics, and cellular levels of crucial transcription factors during the reprogramming process. Further, we have used the assays to ascertain the threshold levels of reprogramming protein factors required to generate iPSC colonies, to characterize the protein expression signatures of different iPSC lines, and to rapidly identify iPS versus non-iPSC colonies based on expression of pluripotency markers. These data demonstrate that TaqMan Protein Assays can be used as tools to dissect and gain greater understanding of the mechanisms guiding reprogramming and to further characterize individual established iPSC lines.

An Alternate Method for Efficient Delivery of Catalyzing Enzymes

Improved cellular engineering tools have enabled scientists to modify genomic sequences at a new pace for a variety of applications. However, in order to create more accurate tools for site specific targeting and editing, improved delivery systems for modifying enzymes are required. These enzymes allow end users to disrupt, insert or edit genes utilizing sequence specific homology. Current applications require co-transfection of an engineering plasmid in tandem with the vector carrying the modifying enzyme. However, co-transfection can negatively affect transfection efficiencies as well as increase probability of randomly integrating DNA. Here we report the transient delivery of catalyzing enzymes via BacMam non-integrating virus at high efficiency. Baculovirus is a non-infectious delivery system that can transduce a variety of mammalian cells. We have shown improved delivery of NLS-Cre recombinase for the excision of regions in disrupted genes to enable expression. In addition, we have tested PhiC31 integrase delivery for the guided insertion of plasmid DNA to a specific locus. The transient expression provided by BacMam, coupled with ease of transfection, creates an ideal system for delivery of catalyzing enzymes in mammalian cells.