Induced Pluripotent Stem Cell FAQs and Glossary of Terms

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• Glossary

How are the induced pluripotent stem (iPS) cells produced?
Currently, iPS cells are produced by inserting copies of four stem cell-associated genes; Oct 3/4, Sox 2, Klf4, and c-Myc (or Oct 3/4, Sox 2, Nanog, and LIN28) into specialized cells using viral vectors. Shinya Yamanaka produced the first iPS cells from mouse cells in 2006. This was followed in 2007 by the generation of iPS cells from human somatic cells by both the Yamanaka lab and the lab of James Thomson.

What are the advantages of iPS cells over embryonic stem cells?
The advantage of iPS cells is that they are not derived from human embryos, which is the ethical concern in this field. By removing the bioethical issues, the scientists are more likely to obtain more federal funding and support. Another significant benefit of iPS cell technology would permit for creation of cell lines that are genetically tailored to a patient. This could eliminate the concern of immune rejection, where the body’s immune system identifies implanted cells or tissues as unknown and attacks them.

What are the risks associated with iPS cell use in humans?
The retroviruses used in the generation of iPSc are associated with cancer because they insert DNA anywhere in a cell's genome, which could potentially trigger the expression of cancer-causing genes. Another risk associated with iPS cell technology applied to humans is the fact that c-Myc, which is one of the genes used in reprogramming, is a known oncogene whose overexpression could also cause cancer.

How are iPS cells similar to ES cells?
iPS cells are similar to ES cells in morphology, teratoma formation, proliferation, as well as their ability to differentiate along a given lineage. They also express cell surface markers and genes that characterize ES cells.

Does iPS cell technology eliminate the need for embryonic stem cell research?
Recent advances do not eliminate the need for ES cell research since it is not yet quite clear whether iPS cells differ extensively from the embryonic stem cells. To bring stem cell research to clinical realization, it is necessary to investigate all the aspects in this field.

What are disease specific iPS cells?
Disease specific iPS cells are iPS cells generated from subjects with a genetic disease.

Is there an iPS cell bank and where?
The University of Wisconsin and James Thomson at the WiCell Research Institute started the first iPS cell bank. There, the scientist grow, test, store and distribute iPS cell lines.

Which media should be used for culturing iPS cells?
The recipe for media for culturing iPS cells was acquired from WiCell MEF-based iPS Cell Culture Protocols and Sigma's catalog numbers have been included for your convenience;

IPS Cell Culture Medium (250 ml)

• 200 ml — DMEM-F12 (D8900, D2906)
• 50 ml — Knockout Serum Replacement
• 2.5 ml — 100 mM L-Glutamine (G8540) + BME Solution (73 mg of L-Glutamine + 5 ml of PBS (P5493) + 3.5 ml 2-Mercatoethanol (M7522))
• 2.5 ml — Non-Essential Amino Acids (M7145)
• 0.5 ml — 50 mg/ml Basic FGF solution (100 mg of bFGF (F0291, F9786) + 2 ml of 0.1% BSA (A2153) in PBS (P5368)).

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iPS Glossary of Terms

Adenovirus
A nonenveloped (naked) icosahedral virus composed of a nucleocapsid and a double-stranded linear DNA genome.

Chimera
A single animal organism with genetically distinct cells from two different zygotes.

Degenerative disease
A disease in which the function or structure of the affected tissues or organs will progressively deteriorate over time, whether due to normal bodily wear or lifestyle choices such as exercise or eating habits.

DNA methylation
A type of chemical modification of DNA that can be inherited and subsequently removed without changing the original DNA sequence.

Ectopic expression
The expression of a gene in an abnormal place in an organism. This can be caused by a disease, or it can be artificially produced as a way to help determine what the function of that gene is.

Expression
The process by which the information in a gene is used to create proteins.

Fibroblast
A type of cell that synthesizes and maintains the extracellular matrix of many animal tissues. Fibroblasts provide a structural framework (stroma) for many tissues, and play a critical role in wound healing.

Gag
A lentiviral packaging element that encodes for a structural precursor protein.

Gene silencing
Targeting or interfering with a specific gene and preventing its expression.

Gene target
A gene or its product (protein) which plays a critical role in disease.

Genomics
The systematic study of genes and their function.

Histones
The chief protein components of chromatin. They act as spools around which DNA winds, and they play a role in gene regulation.

Induced pluripotent stem cells
Adult cells reprogrammed to an embryonic stem cell–like state by being forced to express factors important for maintaining the "stemness" of embryonic stem cells.

Klf
The Krüppel-like family of transcription factors, named for their homology to the Drosophila Krüppel protein. All KLF family members are characterized by their three Cys2 His2 zinc fingers located at the C-terminus, separated by a highly conserved H/C link.

Knock-down
Reduction of gene expression via RNA interference, mediated by short interfering RNA (siRNA) or short hairpin (shRNA).

Lentivirus
A genus of slow viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector.

Lin-28 homolog
Also known as LIN28, is a human gene. It is marker of undifferentiated human embryonic stem cells and has been used to enhance the efficiency of the formation of induced pluripotent stem (iPS) cells from human fibroblasts. It encodes a cytoplasmic mRNA-binding protein that binds to and enhances the translation of the Igf2 mRNA. Lin28 has also been shown to bind to the let-7 pre-miRNA and block production of the mature let-7 microRNA in mouse embryonic stem cells.

Multiplicity of infection (MOI)
Represents the average number of viral particles per single cell. The MOI is calculated by dividing the total number of transducing unites by the number of cells plated.

Myc (c-Myc)
An oncogene that codes for a protein that binds to the DNA of other genes. When c-Myc is mutated, or overexpressed, the protein doesn't bind correctly, and often causes cancer.

Nanog
A transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells.

Oct-4
An abbreviation of Octamer-4. It is a homeodomain transcription factor of the POU family. This protein is critically involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is frequently used as a marker for undifferentiated cells. Oct-4 expression must be closely regulated; too much or too little will actually cause differentiation of the cells.

Packaging plasmid
Lentiviral vectors that contain all necessary elements to efficiently generate active viral particles. For improved safety, third generation packaging plasmids have necessary element seperated between 2 or 3 plasmids, eliminating the posibility of homologous recombination and generation of wild-type virus.

Phenotype
The observable physical characteristics of an organism or cell.

Plasmid
An extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.

Pol
A lentiviral packaging element that encodes for a structural precursor protein.

Regulatory viral protein (Rev)
A lentiviral packaging element that binds to Rev Response Element (RRE) sequences, allowing the cytoplasmic export of viral RNAs.

Replication incompetent
Lentiviral particles that are incapable of producing additional viral particles, due to the elimination of wild-type enhancers in the long terminal repeat region.

Reprogramming
Induced experimentally in mammalian cells by nuclear transfer, cell fusion, genetic manipulation or in vitro culture.

Retrovirus
Any virus belonging to the viral family Retroviridae. They are enveloped viruses possessing an RNA genome, and replicate via a DNA intermediate. Retroviruses rely on the enzyme reverse transcriptase to perform the reverse transcription of its genome from RNA into DNA, which can then be integrated into the host's genome with an integrase enzyme. The virus then replicates as part of the cell's DNA.

Self-inactivating (SIN) long terminal repeat
The basal/enhancer control elements from wild-type lentiviral promoters were replaced in the long terminal repeat by transcriptional control elements from heterologous viral or cellular promoters. This replacement renders the virus replication incompetent.

Somatic cell nuclear transfer (SCNT)
A laboratory technique for creating an ovum with a donor nucleus. In SCNT the nucleus, which contains the organism's DNA, of a somatic cell is removed and the rest of the cell discarded. At the same time, the nucleus of an egg cell is removed. The nucleus of the somatic cell is then inserted into the enucleated egg cell. After being inserted into the egg, the somatic cell nucleus is reprogrammed by the host cell. The egg, now containing the nucleus of a somatic cell, is stimulated with a shock and will begin to divide. After many mitotic divisions in culture, this single cell forms a blastocyst with almost identical DNA to the original organism.

SOX 2
SRY (sex determining region Y)-box 2, is a transcription factor that is essential to maintain self-renewal of undifferentiated embryonic stem cells. This intronless gene encodes a member of the SRY-related HMG-box (SOX) family of transcription factors involved in the regulation of embryonic development and in the determination of cell fate.

TAT
A lentiviral packaging element required for the efficient elongation of nascent viral transcripts.

Teratoma
A tumor with tissue or organ components resembling normal derivatives of all three germ layers.

Titer
The number of transducing units per mL.

Transfection
Describes the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer.

Transduction
The integration of exogenous genetic sequences into a host genome through viral particles.

Vector
Any vehicle used to transfer foreign genetic material into another cell. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transfer genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell.

Viral infectivity factor (Vif)
A lentiviral packaging element that blocks a cellular inhibitor of viral replication.

Viral protein r (Vpr)
A lentiviral packaging element that participates in the viral RNA into the nucleus for chromosomal integration.

Viral protein u (Vpu)
A lentiviral packaging element that is localized at the cell membrane, facilitating the viral release.

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