Allogeneic stem cells:

Stem cells that are used clinically to augment, repair, replace or regenerate organs and tissues and are derived from a cell source that is genetically different from the host/recipient.

A hematopoietic stem cell transplant (bone marrow transplant) is an example of a clinical allogeneic transplant in which the patient receives blood-forming hematopoietic stem cells from a genetically similar, but not identical, donor. Allogeneic hematopoietic stem cells are derived from donated bone marrow or umbilical cord blood.

An autologous stem cell transplant uses the patient's own cells to augment, repair, replace or regenerate diseased and/or damaged organs and tissues. There are currently no autologous stem cell therapies in clinical use.

Anaphase:

The stage of mitosis during which the two sets of chromosomes in the cell separate and move away from each other in preparation for cell division.

Antibody:

A protein produced by a specific white blood cell, known as a B-cell, in response to a foreign molecule or invading microorganism. Antibodies bind to proteins with high specificity, thereby inactivating the molecule or microorganism or marking it for destruction by the body’s immune system. Because antibodies bind to specific proteins, they are commonly used in the lab to detect particular proteins of interest.

Cell biologists isolate stem cells from heterogenous cell populations by engineering antibodies that are coupled to a microscopic flourescent probe. The engineered antibody is designed to bind a specific protein marker on the surface of the cell/stem cell of interest. The cells that bind to the antibodies will fluoresce, allowing the cells to be detected and isolated using a fluorescence activated cell sorter (FACS).

Antigen:

A molecule that is able to provoke an immune response; a short stretch of peptide in a protein that an antibody recognizes

Apoptosis:

Programmed cell death, where the cell initiates its own death. Apoptosis leads to fragmentation of the DNA, shrinkage of the cytoplasm, membrane changes and cell death without lysis. Apoptosis is a normal phenomenon that occurs frequently in multicellular organisms. Apoptosis is often initiated by ubiquitin proteins--highly conserved regulatory proteins that aid in apoptosis and the labeling of proteins for degradation.

Asymmetric Cell Division:

The process where a stem cell undergoes mitotic cell division yielding two daughter cells. One daughter cell has much developmental potential as the mother cell to create another stem cell. The second daughter cell is more differentiated than the mother cell, and therefore has less developmental potential than the mother cell.

Asymmetric division (self-renewal) is a necessary cellular function that allows the body to maintain a sufficient number of stem cells in the different tissues and organs of the body for the maintenance/repair of lost or damaged cells/tissue. Without self-renewal, through both asymmetric and symmetric cell division, the body would exhaust its population of stem cells.

Symmetric division, on the other hand, is the process whereby a stem cell undergoes mitotic cell division that yields two identical daughter cells with the same developmental potential as the mother cell (e.g. self-renewal). During embryonic development and/or in response to an injury, certain stem cells will undergo symmetric cell division instead of asymmetric division.

Autologous stem cells:

Stem cells that are used to augment, repair, replace or regenerate organs and tissues and are genetically identical to the recipient (e.g. using your own cells).

A hematopoietic stem cell transplant (bone marrow transplant) that uses the patient's own hematopoietic stem cells is a clinical example of an autologous transplant in which the patient uses his or her own blood-forming hematopoietic stem cells to treat the disease. Autologous hematopoietic stem cells are derived from the patient's own bone marrow or umbilical cord blood.

An allogeneic stem cell transplant uses cells from a donor other than the patient to augment, repair, replace or regenerate diseased and/or damaged organs and tissues

Blastocoel

The fluid-filled cavity of the blastocyst that contains the inner cell mass of the blastocyst. The cells from the inner cell mass are extracted to establish embryonic stem cell lines.

Blastocyst:

A preimplantation embryo of 30-150 cells. The blastocyst consists of a sphere made up of an outer layer of cells (the trophectoderm), a fluid-filled cavity (the blastocoel), and a cluster of cells on the interior (the inner cell mass).

Pluripotent embryonic stem cells are located within the inner cell mass of the 3-5 day old blastocyst. An embryonic stem cell line is established by extracting one or more pluripotent stem cells from the inner cell mass of the blastocyst. The embryonic stem cell(s) can then be expanded indefinitely under proper culture conditions in vitro to produce large quantities of embryonic stem cells for research purposes.

Biotechnology:

The manipulation of organisms or their components to produce useful products.

c-Myc:

A well known proto-oncogene. The c-Myc gene codes for a transcription factor that regulates the expression of many genes involved in the control of cell proliferation, growth, differentiation and apoptosis. Abberant expression of c-Myc on the other hand is associated with tumor formation and cancer.

c-Myc is one of four factors originally used by the Yamanaka group to reprogram somatic cells into pluripotent stem cells. Recent studies have demonstrated that c-Myc is not an essential reprogramming factor, but does greatly improve reprogramming efficiency.

Cell Culture :

Cells growing in a petri dish in laboratory conditions.

Stem cells are grown, expanded and differentiated in defined culture conditionsin vitro. Each type of stem cell requires unique growth media that mimics the in vivo niche. Various reagents are added to the media to provide the proper molecular cues to promote either differentiation or self-renewal.

Cell cycle:

The cell cycle, or cell-division cycle, is the series of events that takes place in a cell that lead to the replication of its DNA and the division of the parent cell into two daughter cells. In cells without a nucleus (prokaryotes), the cell cycle occurs via a process termed binary fission. In cells with a nucleus (eukaryotes), the cell cycle consists of four phases: G1 phase (growth), S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis).One of the hurdles facing the therapeutic use of iPS cells is their tumorigenic potential, which is caused by the up-regulation or over-expression of the known proto-oncogene (increasing cell cycle) and reprogramming factor, c-Myc. Recent scientific evidence has shown that c-Myc is a dispensable reprogramming factor, bringing the field one step closer to therapeutic application. Many research groups have demonstrated that reprogramming efficiency is greatly improved when tumor suppressor proteins, such as p53, are temporarily down-regulated. This finding further supports the importance of an active cell cycle for the reprogramming process.

Cell Fate:

In developmental biology, cell fate describes what a particular cell at a given stage of development will eventually give rise to.

Cellular reprogramming:

Cellular reprogramming describes the process where a fully differentiated, specialized cell type is induced to transform into a different cell type that they would not otherwise become under normal physiological conditions. Cellular reprogramming has been achieved using a variety of methods, including somatic cell nuclear transfer, cell-cell fusion and, most recently, through the introduction of four transcription factors. Most scientists have focused on reprogramming somatic cells into pluripotent stem cells, but recently some researchers have begun to focus on reprogramming somatic cells into multipotent stem cells, which have a more restricted developmental potential and are closer to the cell population the researcher ultimately wants to engineer.

Chromatin:

The complex of DNA and DNA-binding protein that makes up a eukaryotic chromosome. The DNA is wrapped around histone proteins, each of which is composed of four core histones, H2A, H2B, H3 and H4. The wrapping of the DNA around histones effectively packages over 2 meters of human DNA into compacted nucleosomes. The chromatin can exist in two forms: tightly coiled chromatin called heterochromatin that is not actively transcribed or loosely coiled chromatin called euchromatin that is actively transcribed.

In mature, fully differentiated somatic cells, several of the genes that encode for the iPS reprogramming factors are located within the heterchromatin and are therefore considered transcriptionally silent. Because the reprogramming factors are not actively expressed, scientists must induce the expression of these genes artificially using transgenic strategies. International research efforts are currently exploring alternative methods to help unlock necessary reprograming genes that are located within the tightly packed heterochromatin.

Chromosomes:

The structures within the nucleus that consist mostly of DNA and associated proteins.

Chromosomes contain genes, the stretches of DNA that carry the genetic code for specific proteins, interspersed with large stretches of DNA of unknown function. A normal human cell contains 46 chromosomes, half of which are contributed by the father and half by the mother.

Collagen:

A glycoprotein of the extracellular matrix in animal cells that forms strong fibers and is found extensively in connective tissue and bone. Collagen is the most abundant protein in the animal kingdom.

Cytoplasm:

The cytoplasm is found in a compartment of the cell that is enclosed within the plasma membrane , excluding the nucleus. In eukaryotic cells, the cell nucleus is a distinct compartment of the cell that is encased by the nuclear membrane. The cytoplasm is comprised mostly of fluid called the cytosol. Also contained within the cytoplasm are cytoskeletal proteins and membrane-bound organelles such as the mitochondria, endoplasmic reticulum, and Golgi bodies. The cytoplasm is the site where most cellular activities occur, including glycolysis and protein translation.

Differentiation

The process whereby an unspecialized stem cell or progenitor cell acquires the features of a specialized cell during multicellular organismal development or in specific laboratory conditions. Differentiation is controlled by epigenetic factors that regulate chromatin structure and, therefore, gene expression

Diploid Cell:

A cell containing two sets of chromosomes (2n), one set inherited from each parent.

DNA (deoxyribonucleic acid):

Deoxyribonucleic acid; the genetic material of chromosomes that is transcribed into mRNA and then translated into a corresponding protein.

DNA is comprised of nucleotide monomers that each contain a deoxyribose sugar backbone and a nitrogenous base: adenine (A), cytosine (C), guanine (G), or thymine (T).

DNA Polymerase:

An enzyme involved in the synthesis of DNA during cell division and DNA replication. The DNA polymerase enzyme reads an intact strand of DNA which serves as a template to synthesize a new complimentary strand of DNA.

Dolly the sheep (July 5, 1996-Feb. 14, 2003):

The first mammal cloned using somatic cell nuclear transfer (SCNT). Dolly was a female domestic sheep born on July 5, 1996 and was cloned by Ian Wilmut and his team at the Roslin Institute, which is located just outside Edinburgh, Scotland. The succes of SCNT and the cloning of Dolly proved that a somatic cell's genetic material can be reprogrammed to an embryonic state by epigenetic factors.

This hypothesis that cells only differentiate unidirectionally towards terminal differentiation was challenged in 1962 by John Gurdon's pioneering work in nuclear transfer and also with the historic birth of Dolly the Sheep in February of 1997 via somatic cell nuclear transfer (SCNT). Somatic cell nuclear transfer, or SCNT, requires the injection of a nucleus from a somatic cell into an enucleated oocyte to form a pluripotent cell capable of developing into an entire organism. The success of SCNT stimulated developmental biologists to begin exploring the possibility of creating pluripotent stem cells (e.g. induced pluripotent stem cells) through cellular reprogramming of fully differentiated somatic cells.

Ectoderm:

The outermost of the three primary germ layers in animal embryos. Ectoderm gives rise to the outer covering and, in some phyla, the nervous system, inner ear, and lens of the eye. Generally speaking, the ectoderm differentiates to form the nervous system, tooth enamel and the epidermis (skin).

Embryo :

The developing human organism from the time of fertilization until the end of the eighth week of gestation. After eight weeks, the developing organism is called a fetus.

Embryonic germ (EG) cells:

Pluripotent stem cells derived from cells in the fetal gonad that would normally develop into mature gametes.

Embryonic stem (ES) cells:

Pluripotent stem cells derived from the inner cell mass of a blastocyst-stage embryo. Embryonic stem cells can become all cell types found in an implanted embryo, fetus, or developed organism, but not the trophoblast or placenta.

Endocytosis:

Cellular uptake of extracellular biological molecules via formation of new vesicles by the plasma membrane.

Genetically engineered retroviral vectors containing reprogramming machinery--integrase, reverse transcriptase and the reprogramming factors Oct-4, c-Myc, Klf-4 and SOX2-- enter the host fibroblast cell by endocytosis.

Endoderm:

The innermost of the three primary germ layers in animal embryos. The endoderm gives rise to the liver, pancreas, lungs, and the lining of the digestive track in species that have these structures.

Induced pluripotent stem cells and embryonic stem cells are capable of differentiating into cell types of all three germ layers--endoderm, ectoderm and mesoderm.

Epigenetics:

A mode of genetic regulation that is caused by mechanisms other than changes in the primary DNA sequence.

Euchromatin:

The less condensed form of eukaryotic chromatin that is available for transcription.

International research efforts are currently exploring alternative methods to uncoil the tightly wound DNA into less condensed euchromatin to unlock necessary reprograming genes located within the tightly packed heterochromatin.

Eukaryote:

A eukaryotic cell contains complex structures within a membrane. The major membrane-bound structures that differentiates a eukaryotic cell from prokaryotes is the presence of a nucleus and nuclear envelope, which contain the genetic material for the organism that house the eukaryote's genetic material. All mammals, plants and other organisms made of cells with nuclei are eukaryotes.

Extracellular Matrix:

A network of proteins and polysaccharides that are secreted by cells. The polysaccharides and proteins provide a highly adhesive substrate for cells to attach to as they organize into complex tissues. Major components of the extracellular matrix are proteoglycans, hyaluronic acid, collagen, elastin and fibronectin.

Fertilization

The union of haploid gametes to produce a diploid zygote.

Fetus:

A developing organism from the end of the embryonic period up to birth. After 8 weeks, the developing human embryo is called a fetus.

Fibroblast :

A type of cell in loose connective tissue that secretes the protein ingredients of the extracellular matrix.

Many somatic cells have been successfully reprogrammed. However, fibroblast cells are commonly utilized to create induced pluripotent stem cells because they can be easily extracted from a patient using a safe and non-invasive skin biopsy and are easy to culture in a lab.

Gamete

A specialized haploid cell required for sexual reproduction. The functional, mature, male gamete is called a sperm, while the female gamete is called the ovum, or egg.

Gametocyte:

A diploid cell that divides by meiosis to give rise to haploid gametes. Spermatocytes give rise to haploid sperm cells and oocytes give rise to ova.

Genome:

All of the genetic information (hereditary information) of an organism.

Haploid

A cell containing only one set of chromosomes (n).

Hematopoietic stem cell:

Tissue-specific stem cells found in mature blood, bone marrow and umbilical cord blood that give rise to all red and white blood cells and platelets.

Heterochromatin:

Eukaryotic chromatin that remains highly compacted during interphase and is generally not transcribed.

As embryonic stem cells differentiate into mature specialized cell types, many pluripotency-related genes are silenced or down regulated because of their location within the highly compacted heterochromatin. Research efforts are currently exploring alternative methods to uncoil the tightly wound DNA into less condensed euchromatin to unlock necessary reprogramming genes located within the tightly packed heterochromatin.

Histone:

A small protein with a high proportion of positively charged amino acids that binds to the negatively charged DNA and plays a key role in chromatin structure and gene expression.

iPS technology and cellular reprogramming will be greatly impacted by understanding the epigenetic mechanisms that act upon histones. Histone proteins can be enzymatically modified by proteins in the nucleus, resulting in overall changes in chromatin structure. Various enzymes catalyze the acetylation, methylation, phosphorylation and ubiquitylation of histone proteins at specific amino acid residues. Generally, the acetylation of histone proteins is associated with euchromatin formation leading to the activation of transcription, while methylation is associated with heterochromatin formation that causes genes to become transcriptionally silenced.

Disassociating the histone proteins from DNA may uncoil the tightly wound heterochromatin into less condensed euchromatin and increase transcriptional efficiency.

Human embryonic stem cell (hESC):

A type of pluripotent stem cell which is derived from the inner cell mass of a developing human blastocyst. Pluripotent hESCs are capable of producing cell types found within an implanted embryo, fetus, or developed organism, but not tissues that support pregnancy (trophoblast and placenta).

The new NIH federal guidelines for human embryonic stem cell research allow for funding of research using hESCs derived from embryos created using in vitro fertilization (IVF) for reproductive purposes and no longer needed for these purposes, assuming the research has scientific merit and the embryos were donated after proper informed consent was obtained from the donor(s).

In vitro

Literally, "in glass." Used to describe the culture of cells in a laboratory setting outside the living body.

In vitro fertilization (IVF):

Fertilization of an egg by a sperm outside a woman's body followed by implantation of the early embryo in the mother or another woman's uterus.

In vitro fertilization techniques are not 100% efficient; therefore, IVF clinics harvest and attempt to fertilize multiple oocytes (eggs). Often, more embryos are created by IVF clinics than are needed for reproductive purposes. The surplus embryos (blastocyst stage) that are not needed for reproductive purposes must either be discarded as medical waste, stored indefinitely, or donated to scientific research with proper informed consent. The stem cells derived from the left over embryos are extracted from the inner cell mass of the blastocyst and are not capable of developing into a complete organism(s) because they are no longer totipotent stem cells. These pluripotent stem cells lack the ability to produce the trophoblast and placenta, which are required to support embryonic/fetal development and birth. The new NIH federal guidelines for embryonic stem cell research allow for funding of research using hESCs derived from embryos created using in vitro fertilization (IVF) for reproductive purposes and no longer needed for these purposes, assuming the research has scientific merit and the embryos were donated after proper informed consent was obtained from the donor(s). NIH Guidelines for Human Embryonic Stem Cell Research

In vivo:

Literally, "in the body." This term is used to describe something within the living organism. For example, a human clinical trial is performed "in vivo" because the researchers are studying the effect of the treatment on the living human subject.

Induced pluripotent stem cell:

A somatic cell that has been reprogrammed to exhibit pluripotent stem cell properties. Many somatic cells have been successfully reprogrammed; however, fibroblast cells are commonly utilized because they can be easily extracted from a patient using a safe and non-invasive skin biopsy and are easy to culture in a lab.

The successful creation of induced pluripotent stem cells proved to the world that cellular differentiation is not a unidirectional process--with the proper instruction, cellular differentiation is bidirectional. However, the hypothesis that cells only differentiate unidirectionally towards terminal differentiation was first challenged in 1962 by John Gurdon's pioneering work in nuclear transfer and also with the historic birth of Dolly the Sheep in February of 1997 via somatic cell nuclear transfer (SCNT). The success of SCNT stimulated developmental biologists to begin exploring the possibility of creating pluripotent stem cells (e.g. induced pluripotent stem cells) by reprogramming fully differentiated somatic cells to an embryonic-like state.

The groundbreaking discovery came in 2006 when Kazutoshi Takahashi and Shinya Yamanaka demonstrated that mouse fibroblasts can be reprogrammed to pluripotent "embryonic like" stem cells by overexpressing specific genetic factors. The team hypothesized that a select group of 24 pluripotency-related genes, when over-expressed in mouse somatic cells, can induce pluripotency. Of the 24 genes screened, only four were necessary to reprogram mouse fibroblasts into pluripotent stem cells—otherwise known as induced pluripotent stem (iPS) cells. The Yamanaka team determined the essential stemness genes to be Oct-4, SOX2, Klf-4 and c-Myc -- four genes that have important functions in the regulation of pluripotency in embryonic stem cells. One year later, the team reported that the same four factors are capable of inducing pluripotency in human somatic cells. This discovery revolutionized the field of stem cell science and regenerative medicine because it opened the door for development of patient-specific autologous pluripotent stem cells for clinical use.

A number of methods are used to artificially over-express genes in a cell. Stem cell scientists are also exploring the use of small molecules and other technologies to reprogram cells without the use of viral vectors. The Yamanaka group utilized retroviral vectors to deliver the transgenes encoded for the four reprogramming factors: Oct-4, SOX2, Klf-4 and c-Myc. The retroviral vectors deliver the four transgenes into the cells. The transgenes are then integrated into the host’s genome, thereby permitting long term expression of the transgenes. If all four transgenes successfully integrate into the fibroblast’s genome, they will then begin to express the transgenes as functional proteins. The four reprogramming factors, Oct-4, SOX2, Klf-4 and c-Myc function as transcription factors, meaning that they are capable of binding to the DNA to control the transcription of a unique set of genes. Together, Oct-4, SOX2, Klf-4 and c-Myc induce the expression of genes that are not normally expressed in the fibroblast but are expressed in pluripotent stem cells. The four transcription factors continue to drive transcription of their downstream genes, leading to the activation of other transcriptional networks, inducing a cascade of transcriptional activity. Over the period of two to three weeks, the gene expression profile of the fibroblast changes and begins to express a repertoire of genes that are commonly identified in pluripotent embryonic stem cells. At approximately the same time, the reprogrammed fibroblasts undergo morphological changes. Soon after, the reprogrammed cells begin to grow as a tightly packed cluster of cells known as a colony, which mirrors how undifferentiated embryonic stem cells grow in culture. The emergence of these colonies is the first sign that the fibroblasts have been reprogrammed into induced pluripotent stem cells. From here, the iPS cell colonies are isolated and expanded so that they may be used to further our understanding of human development and mechanisms involved in many devastating human diseases.

Inner cell mass:

A small cluster of cells within the blastocyst that subsequently develops into the embryo proper and some of the extraembryonic membranes.

Pluripotent embryonic stem cells are located within the inner cell mass of the 3-5 day old blastocyst. An embryonic stem cell line is established by extracting one or more cells from the inner cell mass of the blastocyst. The extracted cell(s) can then be expanded indefinitely under proper culture conditions in vitro to produce large quantities of embryonic stem cells for research purposes.

iPS Cell Colony:

Refers to the tightly packed homogenous cluster of induced pluripotent cells. Undifferentiated embryonic stem cells and induced pluripotent stem cells grow in colonies in vitro. As the pluripotent cells begin to differentiate, the cells will no longer form colonies and will instead appear flattened. The emergence of colonies is one of the first signs that somatic cells have been successfully reprogrammed into iPS cells.

Kruppel-like factor 4 (Klf-4)

A transcription factor that is highly expressed in undifferentiated embryonic stem cells and is also expressed elsewhere in the adult organism, including the gut, testis and lungs, and regulates proliferation, differentiation and cell survival.

In 2006, the Yamanaka lab identified Klf-4 as one of the four factors that, when co-transfected and expressed in mouse adult fibroblasts, caused the fibroblasts to revert to a pluripotent-like state. One year later, the same four factors where used to successfully reprogram human adult fibroblast cells into induced pluripotent stem cells. These four factors are Oct-4, SOX2, Klf-4 and c-Myc.

Meiosis

Meiosis is process of cellular division that reduces the number of chromosomes in a cell by half. This process always results in the formation of gametes (sperm and eggs) in animals. Meiosis results in four haploid cells because the genome of the diploid germ cell undergoes DNA replication, which is followed by TWO round of cell division.

Mesoderm:

The middle primary germ layer in an animal embryo. The mesoderm develops into the notochord, the linings of the coelom, muscles, skeleton, gonads, kidneys, and most of the circulatory system in species that have these structures.

Messenger RNA (mRNA):

Messenger RNA is transcribed from the DNA by RNA polymerase. mRNA carries the genetic information required for protein synthesis. mRNA attaches to ribosomes, where it becomes the template for protein translation. The genetic information in mRNA is organized into codons, each consisting of three nucleotide sequences (e.g. AUG, GGA). Each codon corresponds to a single amino acid. During translation, the mRNA is read in the 5' to 3' direction and the appropriate amino acid is attached to form the polypeptide chain.

Mitosis:

A process of nuclear division in eukaryotic cells that is divided into five stages: prophase, prometaphase, metaphase, anaphase and telophase. Mitosis conserves chromosome numbers by allocating replicated chromosomes equally to each daughter cell.

Morula:

Latin for morus or mulberry, a morula is a very early embryo produced by embryonic cleavage or the rapid division of the zygote. Cells of the morula are called blastomeres and represent totipotent stem cells. Each blastomere has the potential to develop into all cell types found in an implanted embryo, fetus, or developed organism, including the trophoblast and placenta, which are required to support fetal development and birth. The inner blastomeres will develop into the inner cell mass and the blastomeres on the surface will later flatten to form the trophoblast during the the blastocyst stage of embryo development.

Multipotent:

The ability of a cell type to change into more than one type of cell within the body, but not into cells of all three germ layers. Multipotent stem cells have less differentiation potential than pluripotent stem cells. Tissue-specific stem cells such as hematopoietic stem cells, mesechymal stem cells (bone marrow-derived meschymal stromal cells) and neural stem cells are examples of multipotent cells.

Nucleolus

A specialized structure in the nucleus, consisting of chromatin regions containing ribosomal RNA genes, along with ribosomal proteins imported from the cytoplasmic site of rRNA synthesis and ribosomal subunit assembly.

Nucleosome:

A subunit of chromatin. The nucleosome is made of a short length of DNA that is wrapped around a core of histone proteins.

Nucleus:

The organelle of a eukaryotic cell that contains the genetic material.

Octamer-4 (Oct-4):

Oct-4, encoded by the gene POU5F1, is a transcription factor that is highly expressed in undifferentiated embryonic stem cells compared to other somatic cells. Oct-4 expression in embryonic stem cells is critical to maintain pluripotency. In fact, when Oct-4 experession is experimentally knocked out, ES cells spontaneously differentiate.

In 2006, the Yamanaka lab identified Oct-4 as one of the four factors that, when co-transfected and expressed in mouse adult fibroblasts, caused fibroblasts to revert to an embryonic-like state. One year later, the same four factors where used to successfully reprogram human adult fibroblast cells into induced pluripotent stem cells. These four factors are Oct-4, SOX2, Klf-4 and c-Myc.

Oncogene:

A gene found in viral or cellular genomes that is involved in triggering molecular events that can lead to cancer.

c-Myc, one of the four reprogramming factors originally used by the Yamanaka group to reprogram somatic cells into induced pluripotent stem cells, is a well known proto-oncogene. The c-Myc gene codes for a transcription factor that regulates the expression of many genes involved in the control of cell proliferation, growth, differentiation and apoptosis. Abberant expression of c-Myc on the other hand is associated with tumor formation and cancer.

Oocyte:

The female gamete (n).

Placenta:

A structure in the pregnant uterus that nourishes a viviparous (developing organism that will be live-born) fetus with the mother's blood supply. The placenta is formed from the uterine lining and embryonic membranes.

Pluripotent stem cell:

A stem cell with the capacity to differentiate into cells of all three germ layers (endoderm, ectoderm, and mesoderm) in the implanted embryo, fetus, or developed organism, but not cells of the trophoblast or placenta.

Pluripotent embryonic stem cells are located within the inner cell mass of the 3-5 day old blastocyst. An embryonic stem cell line is established by extracting one or more cells from the inner cell mass of the blastocyst. The extracted cell(s) can then be expanded indefinitely under proper culture conditions in vitro to produce large quantities of embryonic stem cells for research purposes.

In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated that mouse fibroblasts can be reprogrammed to pluripotent "embryonic like" stem cells by overexpressing four genetic factors, Oct-4, SOX2, c-Myc and Klf-4.

Plasma membrane:

The plasma membrane is made up of a lipid bilayer that acts as a selective barrier that regulates the cell's chemical composition and also plays an important role in cell transport and communication.

Potency:

A general term that describes the capability of a cell (stem cell or progenitor) to differentiate into another more committed cell type.

Promoter:

A specific nucleotide sequence in DNA that binds transcriptional machinery in the proper position to initiate transcription or the production of mRNA.

Proto-oncogene:

A normal cellular gene that has the potential to become an oncogene.

c-Myc, one of the four reprogramming factors originally used by the Yamanaka group to reprogram somatic cells into pluripotent stem cells, is a well known proto-oncogene. The c-Myc gene codes for a transcription factor that regulates the expression of many genes involved in the control of cell proliferation, growth, differentiation and apoptosis. Abberant expression of c-Myc, on the other hand, is associated with tumor formation and cancer.

Progenitor cell:

A cell type that can differentiate, but cannot self-renew. As a stem cell begins to differentiate into a progenitor cell, potency and self renewal begin to lessen and cellular senescence increases. When a stem cell can no longer self-renew, but can still differentiate into multiple cell types, the stem cell has now transitioned into a progenitor cell. For example, a hematopoietic stem cell will first differentiate into a hematopoietic progentitor cell that maintains the same differentiation potential, but loses the ability to self-renew. The hematopoietic progenitor cell will then differentiate into one of two more specialized progenitor cell types: 1) lymphoid progenitor cells that will further differenitate into cells of the lymphoid lineage (b-cells, t-cells, NK cells) or 2) myloid progenitor cells that will further differentiate into cells of the myloid lineage (erythrocytes, platelets, macrophages, neutrophils, eosinophil, basophil).

Regenerative Medicine:

A rapidly evolving interdisciplinary field in health care that translates fundamental knowledge in biology, chemistry and physics into materials, devices, systems and therapeutic strategies, including cell-based therapies, which augment, repair, replace or regenerate organs and tissues. (Definition provided by the Alliance for Regenerative Medicine)

Repressor:

A DNA-binding protein that inhibits gene transcription by binding to the operator and blocking the attachment of RNA polymerase to the promoter region of the gene.

Reprogramming Factors:

In 2006, the Yamanaka lab identified four factors that, when co-transfected and expressed in mouse adult fibroblast cells, caused those fibroblasts to revert back to a pluripotent like state. One year later, the same four factors were used to successfully reprogram human adult fibroblast cells into induced pluripotent stem cells. These four factors are Oct-4, SOX2, c-Myc and Klf-4.

Octamer-4 (Oct-4) encoded by the gene POU5F1 is a transcription factor that is highly expressed in undifferentiated embryonic stem cells compared to other somatic cells. Oct-4 expression in embryonic stem cells is critical to maintain them in an undifferentiated, pluripotent state. In fact, if Oct-4 experession is experimentally knocked out, this causes embryonic stem cells to spontaneously differentiate.

SOX2 is a transcription factor critical for the maintenance of pluripotency in embryonic stem cells. SOX2 and Oct-4 work in parallel to co-regulate expression of target genes involved in the maintenance of pluripotency.

c-Myc is a well known proto-oncogene. The c-Myc gene codes for a transcription factor that regulates the expression of many genes involved in the control of cell proliferation, growth, differentiation and apoptosis. Abberant expression of c-Myc on the other hand is associated with tumor formation and cancer. Recent studies have demonstated that c-Myc is a dispensable reprograming factor; however, the transcription factor has been shown to greatly improve reprogramming efficiency.

Kruppel-like factor 4 (Klf-4) is a transcription factor that is highly expressed in undifferentiated ES cells and is also expressed elsewhere in the adult organism including the gut, testis and lungs and functions to regulate proliferation, differentiation and cell survival.

Retrovirus:

An RNA virus that fuses to surface receptors located within the host cell's plasma membrane. After binding, the membrane of the retrovirus (the envelope protein) and the membrane of the host cell fuse, transmitting the genetic material of the virus into the host cell. The retroviral genetic material is then replicated in the host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA. Retroviruses are enveloped protein that belong to the viral family Retrovirida.

The Yamanaka group utilized retroviral vectors to deliver transgenes that encoded for the four reprogramming factors, Oct-4, SOX2, Klf-4 and c-Myc. The retroviral vectors deliver the four transgenes into fibroblasts to reprogram them into induced pluripotent stem cells. The transgenes are then integrated into the host’s genome thereby permitting its long term gene expression. If all four transgenes successfully integrate into the fibroblast’s genome, they will they begin to express the transgenes as functional proteins. The four reprogramming factors, Oct-4, SOX2, Klf-4 and c-Myc function as transcription factors meaning that they are capable of binding to the DNA to control the transcription of a unique set of genes. Together Oct-4, Sox2, Klf-4 and c-Myc induce the expression of genes that are not normally expressed in the fibroblast but are expressed in pluripotent stem cells. The four transcription factors continue to drive transcription of their downstream genes leading to the activation of other transcriptional networks inducing a cascade of transcriptional activity.

Over the period of two to three weeks, the gene expression profile of the fibroblast changes and begins to express a repertoire of genes that are commonly identified in pluripotent embryonic stem cells. At approximately the same time, the reprogrammed fibroblasts undergo morphological changes. Soon after, the reprogrammed cells begin to grow as a tightly packed cluster of cells known as a colony, which mirrors how undifferentiated embryonic stem cells grow in culture. The emergence of these colonies is the first sign that the fibroblasts have been reprogrammed into induced pluripotent stem cells. From here the iPS colonies are isolated and expanded so that they may be used to further our understanding of human development and the onset of many devastating human diseases.

Reverse Transcriptase:

A DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA which is then integrated into the host genome by integrase proteins. This process is the reverse of normal transcription.

The retroviral vectors (retroviruses) are genetically engineered by scientists to encapsulate and deliver reverse transcriptase, intergrase and the four reprogramming factors, Oct-4, SOX2, c-Myc, Klf-4 into the fibroblasts to reprogram them into induced pluripotent stem cells. These transgenes are then reverse transcribed from RNA into DNA by the enzyme reverse transcriptase and finally integrated into the host’s genome by integrase proteins.

RNA (ribonucleic acid):

A type of nucleic acid consisting of nucleotide monomers with a ribose sugar and the nitrogenous base adenine (A), cytosine (C), guanine (G), or uracil (U). RNA is usually single-stranded and functions in protein synthesis, gene regulation, and often makes up the genetic material of viruses.

RNA polymerase:

An enzyme involved in the synthesis of messenger RNA during gene transcription. RNA polymerase moves along a gene in the 5' to 3' direction and uses the genetic information encoded by the DNA to add the complementary ribonucleotides to synthesize messenger RNA.

Self-Renewal:

Self-renewal describes the process where a stem cell undergoes mitotic cell division that yields at least one daughter cell with equivalent developmental potential as the mother cell - i.e. creating another stem cell. The capacity of stem cells to self-renew varies based on the type of stem cell. Induced pluripotent stem cells and embryonic stem cells maintain the ability to self-renew indefinitely under proper culture conditions. Adult stem cells have limited capacity to self-renew and proliferate. Self-renewal is a necessary cellular function that allows the body to maintain a sufficient number of stem cells within its different tissues and organs for the maintenance/repair of lost or damaged cells/tissues.

Shinya Yamanaka:

Shinya Yamanaka and his team demonstrated that mouse fibroblasts can be reprogrammed into pluripotent "embryonic like" stem cells by overexpressing genetic factors. The team hypothesized that a select group of 24 pluripotency related genes, when over-expressed in mouse somatic cells, can induce pluripotency. Of the 24 genes screened, only four were necessary to reprogram mouse fibroblasts into pluripotent stem cells—otherwise known as induced pluripotent stem (iPS) cells. The Yamanaka team determined the essential stemness genes to be Oct-4, SOX2, Klf-4 and c-Myc, four genes that have important functions in the regulation of pluripotency in embryonic stem cells. One year later, the team reported that the same four factors are capable of inducing pluripotency in human somatic cells. This discovery revolutionized the field of stem cell science and regenerative medicine because it opened the door for the development of patient-specific autologous pluripotent stem cells. Today, stem cell scientists are working to reprogram cells using small molecules and other safer technologies to develop iPS technology for use in clinical therapies.

Somatic Cell:

Any cell in a multicellular organism that is not a gamete. Somatic cells include all diploid cells (2n) of an organism.

Somatic cell nuclear transfer (SCNT):

A technique that combines an enucleated egg and the nucleus of a diploid somatic cell to make an embryo. Since the genetic material of the SCNT embryo is derived from a diploid somatic cell, the embryo will be an identical clone of the source somatic cell. Therapeutic cloning is the use of SCNT to produce pluripotent embryonic stem cells with an exact match to the donor.

SCNT can be used by scientists to create disease models in vitro. For example, a patient-specific ALS cell could be enucleated and then transfered into an enucleated egg. Once the oocyte (egg) is reprogrammed, scientists could differentiate the SCNT-derived stem cells into ALS cells that mirror the patient's diseased cells. This technique has the potential to allow scientists to study human disease cells outside the body in controlled laboratory conditions, allowing us to better understand the root cause of disease and hopefully develop better medical technology.

Reproductive cloning is used to create a fetus through somatic cell nuclear transfer from a cell that is genetically identical to the cell donor. An example of reproductive cloning is the creation of Dolly the Sheep.

Sperm:

The male gamete (n).

Stem cell:

There are many different types of stem cells within our body. Each type of stem cell is defined by the group of specialized cells it gives rise to. However, there are two fundamental properties that distinguish all stem cells, including induced pluripotent stem cells, from other somatic cells: self-renewal and potency.

Self-renewal describes the process where a stem cell undergoes mitotic cell division that yields at least one daughter cell with equivalent developmental potential as the mother cell - i.e. another stem cell. The capacity to self-renew varies based on the type and age of the stem cells. Induced pluripotent stem cells and embryonic stem cells maintain the ability to self-renew indefinitely under proper culture conditions. Multipotent stem cells, such as hematopoietic stem cells, have more limited capacity for self-renewal. Self-renewal is a necessary cellular function to maintain a sufficient number of stem cells within different tissues and organs of the body for the maintenance/repair of lost or damaged cells/tissue.

The second distinguishing property of a stem cell is the ability to differentiate into mature specialized cell types. The number of specialized cell types a stem cell can produce determines its potency (e.g. totipotent, pluripotent, multipotent, unipotent). Stem cells that give rise to all cell types of the body, including the embryonic components of the trophoblast and placenta, are totipotent stem cells. These stem cells are derived from the pre-implantation embryo at the morula stage of embryonic development.

SOX2:

SOX2 is a transcription factor critical for the maintenance of pluripotency in embryonic stem cells. SOX2 and Oct-4 work in parallel to co-regulate expression of target genes involved in the maintenance of pluripotency.

In 2006, the Yamanaka lab identified Oct-4 as one of the four factors that, when co-transfected and expressed in mouse adult fibroblasts, caused fibroblasts to revert to an "embryonic-like" state. One year later, the same four factors where used to successfully reprogram human adult fibroblast cells into induced pluripotent stem cells. These four factors are Oct-4, SOX2, c-Myc, Klf-4.

Symmetric cell division:

The process by which a stem cell undergoes mitotic cell division, yielding two identical daughter cells with the same developmental potential as the mother cell (e.g. self-renewal). During embryonic development and/or in response to an injury, certain stem cells will undergo symmetric cell division instead of asymmetric cell division.

On the other hand, asymmetric cell division is the process whereby a stem cell undergoes mitotic cell division, yielding one daughter cell with equivalent developmental potential as the mother cell - i.e. another stem cell - and one daughter cell with less developmental potential than the mother cell - i.e. a progenitor cell.

Symmetric and asymmetric cell division (self-renewal) are necessary cellular functions that allow the body to maintain a sufficient number of stem cells within its different tissues and organs. The stem cells are important for the maintenance/repair of lost or damaged cells/tissue. Without self-renewal, through asymmetric and symmetric cell division, the body would exhaust its population of stem cells.

Telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells. Embryonic stem cells have been shown to express high levels of telomerase activity, allowing embryonic stem cells to divide indefinitely or be "immortal" under proper culture conditions.

Telomere:

The tandemly repetitive DNA at the end of a eukaryotic chromosome's DNA molecule that protects the organism's genes from being eroded during successive rounds of replication.

Tissue-specific stem cells:

(also referred to as somatic stem cells or adult stem cells) Undifferentiated cells found in various tissues within the human body that have the ability to self-renew and give rise to specialized cell types and tissues needed by the body. Most tissue-specific stem cells are multipotent, meaning the cells have the capacity to change into more than one type of cell within the body but not cells of all three germ layers. Multipotent stem cells have less differentiation potential than pluripotent stem cells. Examples of tissue-specific stem cells include hematopoietic stem cells, mesechymal stem cells (bone marrow-derived meschymal stromal cells) and neural stem cells.

Totipotent stem cells:

Stem cells that give rise to all cell types found in the embryo, fetus or developed organism, including the embryonic components of the trophoblast and placenta, are totipotent stem cells. These stem cells are derived from the pre-implantation embryo at the morula stage of embryonic development.

Transcription:

The synthesis of RNA using a DNA template. During transcription, a DNA sequence is read by RNA polymerase and then transcribed into a complementary, antiparallel strand of mRNA. Transcription occurs in the nucleus of eukaryotic cells.

Transcription Factor:

A regulatory protein that binds to DNA and affects the transcription of specific genes.

The four reprogramming factors, Oct-4, SOX2, Klf-4 and c-Myc function as transcription factors- they are capable of binding to the DNA to control the transcription of a unique set of genes. Together, Oct-4, SOX2, Klf-4 and c-Myc induce the expression of genes that are not normally expressed in the fibroblast, but are expressed in pluripotent stem cells. The four transcription factors continue to drive transcription of their downstream genes leading to the activation of other transcriptional networks, inducing a cascade of transcriptional activity.

Transgene:

A gene that has been artificially introduced into a cell through genetic engineering strategies.

In 2006, Shinya Yamanaka and his team determined that expression of only four genes (Oct-4, SOX2, Klf-4 and c-Myc) were necessary to reprogram mouse fibroblasts into pluripotent stem cells—otherwise known as induced pluripotent stem cells. However, these genes are highly down-regulated in fully differentiated somatic cells. To increase the expression of Oct-4, SOX2, c-Myc, and Klf-4, the team engineered Oct-4, SOX2, c-Myc, and Klf-4 transgenes and delivered them into the cell nuclei using retroviral vectors.

Translation:

The process whereby the genetic information encoded in messenger RNA is used to synthesize a polypeptide. The polypeptide will eventually be folded into a functional protein.

Trophectoderm:

The outer epithelium of a mamalian blastocyst that is made up of trophoblast cells. The trophectoderm forms the fetal part of the placenta, supporting embryonic development but not forming part of the embryo proper. Stem cells that give rise to all cell types of the body, including the embryonic components of the trophoblast and placenta, are totipotent stem cells. These stem cells are derived from the pre-implantation embryo at the morula stage of embryonic development.

Tumor-suppressor gene:

A gene whose protein product inhibits cell division, thereby preventing the uncontrolled cell growth that contributes to cancer.

Umbilical cord blood stem cells

Small amounts of hematopoietic stem cells which may be harvested from the umbilical cord at birth. These cells are similar to those residing within the bone marrow, and may be used for the treatment of leukemia, and other blood disorders. Often, multiple cord blood units are needed to treat hematopoietic disorders of children and adults since the number of hematopoietic stem cells found within 1 unit of umbilical cord blood is limited and hematopoietc stem cells are difficult to expand (1 unit = 1 umbilical cord). Research are focusing on expansion techniques for hematopoietic stem cells which will hopefully increase the clinical use and application of these cells.

Unipotent:

Stem cells that maintain the capacity to self-renew, but give rise to only one mature cell type and therefore have the lowest potency.

Vector

An organism that transmits pathogens from one host to another. Cell biologists use vectors as genetic delivery tools that transfer genetic material into a target cell.

In 2006, the Yamanaka team modified viruses called retroviruses to serve as vectors that delivered the four transgenes, Oct-4, SOX2, c-Myc and Klf-4, into the fibroblast cells. The transgenes are then integrated into the host’s genome, thereby permitting its long term gene expression. If all four transgenes successfully integrate into the fibroblast’s genome, they will they begin to express the transgenes as functional proteins and induce pluripotency.

Zygote

The diploid product of the union of haploid gametes during fertilization; a fertilized egg..