Stem cell

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Stem cell
4UFNDFMM
Mouse embryonic stem cells with fluorescent marker
Human embryonic stem cell colony on mouse embryonic fibroblast feeder layer
Latin cellula precursoria
Code TH H2.00.01.0.00001
Stem cells are biological cells found in all multicellular organisms, that can divide through mitosis and differentiate
into diverse specialized cell types and can self renew to produce more stem cells. In mammals, there are two broad
types of stem cells: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem
cells that are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for
the body, replenished in adult tissues. In a developing embryo, stem cells can differentiate into all the specialized
cells, but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.
Stem cells can now be artificially grown and transformed into specialized cell types with characteristics consistent
with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells are
routinely used in medical therapies. Stem cells can be taken from a variety of sources, including umbilical cord
blood and bone marrow. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic
cloning have also been proposed as promising candidates for future therapies.
[1]
Research of stem cells grew out of
findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s.
[2]

[3]
Stem cell
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Properties
The classical definition of a stem cell requires that it possess two properties:
Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated
state.
Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to
be either totipotent or pluripotent - to be able to give rise to any mature cell type, although multipotent or
unipotent progenitor cells are sometimes referred to as stem cells.
Self-renewal
Two mechanisms exist to ensure that the stem cell population is maintained:
1. Obligatory asymmetric replication - a stem cell divides into one father cell that is identical to the original stem
cell, and another daughter cell that is differentiated
2. Stochastic differentiation - when one stem cell develops into two differentiated daughter cells, another stem cell
undergoes mitosis and produces two stem cells identical to the original.
Potency definitions
Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. The
stem cells can become any tissue in the body, excluding a placenta. Only the morula's
cells are totipotent, able to become all tissues and a placenta.
Potency specifies the differentiation
potential (the potential to differentiate
into different cell types) of the stem
cell.
[4]
Totipotent (a.k.a omnipotent) stem
cells can differentiate into
embryonic and extraembryonic cell
types. Such cells can construct a
complete, viable organism.
[4]
These
cells are produced from the fusion
of an egg and sperm cell. Cells
produced by the first few divisions
of the fertilized egg are also
totipotent.
[5]
Pluripotent stem cells are the
descendants of totipotent cells and
can differentiate into nearly all
cells,
[4]
i.e. cells derived from any
of the three germ layers.
[6]
Multipotent stem cells can
differentiate into a number of cells,
but only those of a closely related family of cells.
[4]
Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
[4]
Stem cell
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Human embryonic stem cells
A: Cell colonies that are not yet differentiated.
B: Nerve cell
Unipotent cells can produce only one cell type, their own,
[4]
but
have the property of self-renewal which distinguishes them from
non-stem cells (e.g. muscle stem cells).
Identification
The practical definition of a stem cell is the functional definition - a
cell that has the potential to regenerate tissue over a lifetime. For
example, the gold standard test for a bone marrow or hematopoietic
stem cell (HSC) is the ability to transplant one cell and save an
individual without HSCs. In this case, a stem cell must be able to
produce new blood cells and immune cells over a long term,
demonstrating potency. It should also be possible to isolate stem
cells from the transplanted individual, which can themselves be
transplanted into another individual without HSCs, demonstrating
that the stem cell was able to self-renew.
Properties of stem cells can be illustrated in vitro, using methods
such as clonogenic assays, where single cells are characterized by
their ability to differentiate and self-renew.
[7]

[8]
As well, stem cells
can be isolated based on a distinctive set of cell surface markers.
However, in vitro culture conditions can alter the behavior of cells,
making it unclear whether the cells will behave in a similar manner
in vivo. Considerable debate exists whether some proposed adult
cell populations are truly stem cells.
Embryonic
Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass
(ICM) of a blastocyst or earlier morula stage embryos.
[9]
A blastocyst is an early stage embryo`approximately four
to five days old in humans and consisting of 50a150 cells. ES cells are pluripotent and give rise during development
to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can
develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation
for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta. The endoderm is
composed of the entire gut tube and the lungs, the ectoderm gives rise to the nervous system and skin, and the
mesoderm gives rise to muscle, bone, blood, basically everything else that connects the endoderm to the ectoderm.
Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells
(hES). Both have the essential stem cell characteristics, yet they require very different environments in order to
maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of
Leukemia Inhibitory Factor (LIF).
[10]
Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts
(MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2).
[11]
Without optimal culture
conditions or genetic manipulation,
[12]
embryonic stem cells will rapidly differentiate.
A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface
proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the
suppression of genes that lead to differentiation and the maintenance of pluripotency.
[13]
The cell surface antigens
most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens
Stem cell
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Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a
topic of research.
[14]
There are currently no approved treatments using embryonic stem cells. The first human trial was approved by the
US Food & Drug Administration in January 2009.
[15]
However, as of August 2010, the first human trial had not yet
been initiated. The first human medical trial for embryonic stem cells started in Atlanta on October 13, 2010 for
spinal injury victims. ES cells, being pluripotent cells, require specific signals for correct differentiation - if injected
directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma.
Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that
embryonic stem cell researchers still face.
[16]
Many nations currently have moratoria on either ES cell research or the
production of new ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency,
embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after
injury or disease.
Fetal
The primitive stem cells located in the organs of fetuses are referred to as fetal stem cells.
[17]
Adult
Stem cell division and differentiation. A -
stem cell; B - progenitor cell; C -
differentiated cell; 1 - symmetric stem cell
division; 2 - asymmetric stem cell division;
3 - progenitor division; 4 - terminal
differentiation
Also known as somatic (from Greek 2cuottxc, "of the body") stem cells
and germline (giving rise to gametes) stem cells, they can be found in
children, as well as adults.
[18]
Pluripotent adult stem cells are rare and generally small in number but can
be found in a number of tissues including umbilical cord blood.
[19]
A great
deal of adult stem cell research has focused on clarifying their capacity to
divide or self-renew indefinitely and their differentiation potential.
[20]
In
mice, pluripotent stem cells are directly generated from adult fibroblast
cultures. Unfortunately, many mice don't live long with stem cell
organs.
[21]
Most adult stem cells are lineage-restricted (multipotent) and are generally
referred to by their tissue origin (mesenchymal stem cell, adipose-derived
stem cell, endothelial stem cell, dental pulp stem cell, etc.).
[22]

[23]
Adult stem cell treatments have been successfully used for many years to
treat leukemia and related bone/blood cancers through bone marrow
transplants.
[24]
Adult stem cells are also used in veterinary medicine to treat
tendon and ligament injuries in horses.
[25]
The use of adult stem cells in research and therapy is not as controversial as
embryonic stem cells, because the production of adult stem cells does not
require the destruction of an embryo. Additionally, because in some
instances adult stem cells can be obtained from the intended recipient (an
autograft), the risk of rejection is essentially non-existent in these
situations. Consequently, more US government funding is being provided
for adult stem cell research.
[26]
An extremely rich source for adult mesenchymal stem cells is the developing tooth bud of the mandibular third
molar.
[27]
The stem cells eventually form enamel (ectodrm), dentin, periodontal ligament, blood vessels, dental pulp,
Stem cell
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nervous tissues, including a minimum of 29 different unique end organs. Because of extreme ease in collection at
8a10 years of age before calcification and minimal to no morbidity, these will probably constitute a major source for
personal banking, research and multiple therapies. These stem cells have been shown capable of producing
hepatocytes.
Amniotic
Multipotent stem cells are also found in amniotic fluid. These stem cells are very active, expand extensively without
feeders and are not tumorigenic. Amniotic stem cells are multipotent and can differentiate in cells of adipogenic,
osteogenic, myogenic, endothelial, hepatic and also neuronal lines.
[28]
All over the world, universities and research
institutes are studying amniotic fluid to discover all the qualities of amniotic stem cells, and scientists such as
Anthony Atala
[29]

[30]
and Giuseppe Simoni
[31]

[32]

[33]
have discovered important results.
From an ethical point of view, stem cells from amniotic fluid can solve a lot of problems, because it is possible to
catch amniotic stem cells without destroying embryos. For example, the Vatican newspaper "Osservatore Romano"
called amniotic stem cell "the future of medicine".
[34]
It is possible to collect amniotic stem cells for donors or for autologuous use: the first US amniotic stem cells bank
[35]

[36]
opened in 2009 in Medford, MA, by Biocell Center Corporation
[37]

[38]

[39]
and collaborates with various
hospitals and universities all over the world.
[40]
Induced pluripotent
These are not adult stem cells, but rather reprogrammed cells (e.g. epithelial cells) given pluripotent capabilities.
Using genetic reprogramming with protein transcription factors, pluripotent stem cells equivalent to embryonic stem
cells have been derived from human adult skin tissue.
[41]

[42]

[43]
Shinya Yamanaka and his colleagues at Kyoto
University used the transcription factors Oct3/4, Sox2, c-Myc, and Klf4
[41]
in their experiments on cells from human
faces. Junying Yu, James Thomson, and their colleagues at the University of WisconsinaMadison used a different
set of factors, Oct4, Sox2, Nanog and Lin28,
[41]
and carried out their experiments using cells from human foreskin.
As a result of the success of these experiments, Ian Wilmut, who helped create the first cloned animal Dolly the
Sheep, has announced that he will abandon nuclear transfer as an avenue of research.
[44]
Frozen blood samples can be used as a source of induced pluripotent stem cells, opening a new avenue for obtaining
the valued cells.
[45]
Lineage
To ensure self-renewal, stem cells undergo two types of cell division (see Stem cell division and differentiation
diagram). Symmetric division gives rise to two identical daughter cells both endowed with stem cell properties.
Asymmetric division, on the other hand, produces only one stem cell and a progenitor cell with limited self-renewal
potential. Progenitors can go through several rounds of cell division before terminally differentiating into a mature
cell. It is possible that the molecular distinction between symmetric and asymmetric divisions lies in differential
segregation of cell membrane proteins (such as receptors) between the daughter cells.
[46]
An alternative theory is that stem cells remain undifferentiated due to environmental cues in their particular niche.
Stem cells differentiate when they leave that niche or no longer receive those signals. Studies in Drosophila
germarium have identified the signals dpp and adherens junctions that prevent germarium stem cells from
differentiating.
[47]

[48]
The signals that lead to reprogramming of cells to an embryonic-like state are also being investigated. These signal
pathways include several transcription factors including the oncogene c-Myc. Initial studies indicate that
transformation of mice cells with a combination of these anti-differentiation signals can reverse differentiation and
Stem cell
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may allow adult cells to become pluripotent.
[21]
However, the need to transform these cells with an oncogene may
prevent the use of this approach in therapy.
Challenging the terminal nature of cellular differentiation and the integrity of lineage commitment, it was recently
determined that the somatic expression of combined transcription factors can directly induce other defined somatic
cell fates; researchers identified three neural-lineage-specific transcription factors that could directly convert mouse
fibroblasts (skin cells) into fully functional neurons. This "induced neurons" (iN) cell research inspires the
researchers to induce other cell types implies that all cells are totipotent: with the proper tools, all cells may form all
kinds of tissue.
[49]
Treatments
Diseases and conditions where stem cell treatment is promising or emerging.
[50]
Bone
marrow transplantation is, as of 2009, the only established use of stem cells.
Medical researchers believe that stem
cell therapy has the potential to
dramatically change the treatment of
human disease. A number of adult
stem cell therapies already exist,
particularly bone marrow transplants
that are used to treat leukemia.
[51]
In
the future, medical researchers
anticipate being able to use
technologies derived from stem cell
research to treat a wider variety of
diseases including cancer, Parkinson's
disease, spinal cord injuries,
Amyotrophic lateral sclerosis, multiple
sclerosis, and muscle damage, amongst
a number of other impairments and
conditions.
[52]

[53]
However, there still
exists a great deal of social and
scientific uncertainty surrounding stem
cell research, which could possibly be overcome through public debate and future research, and further education of
the public.
One concern of treatment is the possible risk that transplanted stem cells could form tumors and have the possibility
of becoming cancerous if cell division continues uncontrollably.
[54]
Stem cells, however, are already studied extensively. While some scientists are hesitant to associate the therapeutic
potential of stem cells as the first goal of the research, they find the investigation of stem cells as a goal worthy in
itself.
[55]
Contrarily, supporters of embryonic stem cell research argue that such research should be pursued because the
resultant treatments could have significant medical potential. It is also noted that excess embryos created for in vitro
fertilization could be donated with consent and used for the research.
The recent development of iPS cells has been called a bypass of the legal controversy. Laws limiting the destruction
of human embryos have been credited for being the reason for development of iPS cells, but it is still not completely
clear whether hiPS cells are equivalent to hES cells. Recent work demonstrates hotspots of aberrant epigenomic
reprogramming in hiPS cells (Lister, R., et al., 2011).
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Research patents
The patents covering a lot of work on human embryonic stem cells are owned by the Wisconsin Alumni Research
Foundation (WARF). WARF does not charge academics to study human stem cells but does charge commercial
users. WARF sold Geron Corp. exclusive rights to work on human stem cells but later sued Geron Corp. to recover
some of the previously sold rights. The two sides agreed that Geron Corp. would keep the rights to only three cell
types. In 2001, WARF came under public pressure to widen access to human stem-cell technology.
[56]
A request for reviewing the WARF patents 5,843,780; 6,200,806; 7,029,913 US Patent and Trademark Office were
filed by non-profit patent-watchdogs The Foundation for Taxpayer & Consumer Rights
[57]
, and the Public Patent
Foundation as well as molecular biologist Jeanne Loring of the Burnham Institute. According to them, two of the
patents granted to WARF are invalid because they cover a technique published in 1993 for which a patent had
already been granted to an Australian researcher. Another part of the challenge states that these techniques,
developed by James A. Thomson, are rendered obvious by a 1990 paper and two textbooks. Based on this challenge,
patent 7,029,913 has been rejected in 2010. The two remaining hES WARF patents are due to expire in 2015.
The outcome of this legal challenge is particularly relevant to the Geron Corp. as it can only license patents that are
upheld.
[58]
Key research events
1908 - The term "stem cell" was proposed for scientific use by the Russian histologist Alexander Maksimov
(1874a1928) at congress of hematologic society in Berlin. It postulated existence of haematopoietic stem cells.
1960s - Joseph Altman and Gopal Das present scientific evidence of adult neurogenesis, ongoing stem cell
activity in the brain; like Andr Gernez, their reports contradict Cajal's "no new neurons" dogma and are largely
ignored.
1963 - McCulloch and Till illustrate the presence of self-renewing cells in mouse bone marrow.
1968 - Bone marrow transplant between two siblings successfully treats SCID.
1978 - Haematopoietic stem cells are discovered in human cord blood.
1981 - Mouse embryonic stem cells are derived from the inner cell mass by scientists Martin Evans, Matthew
Kaufman, and Gail R. Martin. Gail Martin is attributed for coining the term "Embryonic Stem Cell".
1992 - Neural stem cells are cultured in vitro as neurospheres.
1997 - Leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem
cells.
1998 - James Thomson and coworkers derive the first human embryonic stem cell line at the University of
WisconsinaMadison.
[59]
1998 - John Gearhart (Johns Hopkins University) extracted germ cells from fetal gonadal tissue (primordial germ
cells) before developing pluripotent stem cell lines from the original extract.
2000s - Several reports of adult stem cell plasticity are published.
2001 - Scientists at Advanced Cell Technology clone first early (four- to six-cell stage) human embryos for the
purpose of generating embryonic stem cells.
[60]
2003 - Dr. Songtao Shi of NIH discovers new source of adult stem cells in children's primary teeth.
[61]
2004a2005 - Korean researcher Hwang Woo-Suk claims to have created several human embryonic stem cell lines
from unfertilised human oocytes. The lines were later shown to be fabricated.
2005 - Researchers at Kingston University in England claim to have discovered a third category of stem cell,
dubbed cord-blood-derived embryonic-like stem cells (CBEs), derived from umbilical cord blood. The group
claims these cells are able to differentiate into more types of tissue than adult stem cells.
2005 - Researchers at UC Irvine's Reeve-Irvine Research Center are able to partially restore the ability of mice
with paralyzed spines to walk through the injection of human neural stem cells.
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August 2006 - Mouse Induced pluripotent stem cells: the journal Cell publishes Kazutoshi Takahashi and Shinya
Yamanaka.
[62]
October 2006 - Scientists at Newcastle University in England create the first ever artificial liver cells using
umbilical cord blood stem cells.
[63]

[64]
January 2007 - Scientists at Wake Forest University led by Dr. Anthony Atala and Harvard University report
discovery of a new type of stem cell in amniotic fluid.
[65]
This may potentially provide an alternative to
embryonic stem cells for use in research and therapy.
[66]
June 2007 - Research reported by three different groups shows that normal skin cells can be reprogrammed to an
embryonic state in mice.
[67]
In the same month, scientist Shoukhrat Mitalipov reports the first successful creation
of a primate stem cell line through somatic cell nuclear transfer
[68]
October 2007 - Mario Capecchi, Martin Evans, and Oliver Smithies win the 2007 Nobel Prize for Physiology or
Medicine for their work on embryonic stem cells from mice using gene targeting strategies producing genetically
engineered mice (known as knockout mice) for gene research.
[69]
November 2007 - Human induced pluripotent stem cells: Two similar papers released by their respective journals
prior to formal publication: in Cell by Kazutoshi Takahashi and Shinya Yamanaka, "Induction of pluripotent stem
cells from adult human fibroblasts by defined factors",
[70]
and in Science by Junying Yu, et al., from the research
group of James Thomson, "Induced pluripotent stem cell lines derived from human somatic cells":
[71]
pluripotent
stem cells generated from mature human fibroblasts. It is possible now to produce a stem cell from almost any
other human cell instead of using embryos as needed previously, albeit the risk of tumorigenesis due to c-myc and
retroviral gene transfer remains to be determined.
January 2008 - Robert Lanza and colleagues at Advanced Cell Technology and UCSF create the first human
embryonic stem cells without destruction of the embryo
[72]
January 2008 - Development of human cloned blastocysts following somatic cell nuclear transfer with adult
fibroblasts
[73]
February 2008 - Generation of pluripotent stem cells from adult mouse liver and stomach: these iPS cells seem to
be more similar to embryonic stem cells than the previously developed iPS cells and not tumorigenic, moreover
genes that are required for iPS cells do not need to be inserted into specific sites, which encourages the
development of non-viral reprogramming techniques.
[74]
March 2008-The first published study of successful cartilage regeneration in the human knee using autologous
adult mesenchymal stem cells is published by clinicians from Regenerative Sciences
[75]
October 2008 - Sabine Conrad and colleagues at Tbingen, Germany generate pluripotent stem cells from
spermatogonial cells of adult human testis by culturing the cells in vitro under leukemia inhibitory factor (LIF)
supplementation.
[76]
30 October 2008 - Embryonic-like stem cells from a single human hair.
[77]
1 March 2009 - Andras Nagy, Keisuke Kaji, et al. discover a way to produce embryonic-like stem cells from
normal adult cells by using a novel "wrapping" procedure to deliver specific genes to adult cells to reprogram
them into stem cells without the risks of using a virus to make the change.
[78]

[79]

[80]
The use of electroporation is
said to allow for the temporary insertion of genes into the cell.
[81]

[82]

[83]

[84]
28 May 2009 Kim et al. announced that they had devised a way to manipulate skin cells to create patient specific
"induced pluripotent stem cells" (iPS), claiming it to be the 'ultimate stem cell solution'.
[85]
11 October 2010 First trial of embryonic stem cells in humans.
[86]
25 October 2010 - Ishikawa et al. write in the Journal of Experimental Medicine that research shows that
transplanted cells which contain their new host's nuclear DNA could still be rejected by the invidual's immune
system due to foreign mitochondrial DNA. Tissues made from a person's stem cells could therefore be rejected,
because mitochondrial genomes tend to accumulate mutations.
[87]
Stem cell
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External links
General
Stem Cell Basics (http:/ / stemcells.nih. gov/ info/ basics/ )
Nature Reports Stem Cells: Introductory material, research advances and debates concerning stem cell research.
(http:/ / www. nature. com/ stemcells)
Understanding Stem Cells: A View of the Science and Issues from the National Academies (http:/ / dels.nas. edu/
bls/ stemcells/ booklet. shtml)
Scientific American Magazine (June 2004 Issue) The Stem Cell Challenge (http:/ / www. scientificamerican.
com/ article. cfm?id=the-stem-cell-challenge)
Scientific American Magazine (July 2006 Issue) Stem Cells: The Real Culprits in Cancer? (http:/ / www.
scientificamerican. com/ article. cfm?id=stem-cells-the-real-culpr-2006-07)
Ethics of Stem Cell Research (http:/ / plato. stanford. edu/ entries/ stem-cells) entry by Andrew Siegel in the
Stanford Encyclopedia of Philosophy
Isolation of amniotic stem cell lines with potential for therapy (http:/ / www. nature. com/ nbt/ journal/ v25/ n1/
abs/ nbt1274. html)
Children's Hospital Stem Cell Research (http:/ / stemcell. childrenshospital.org)
Stem Cell Research and Industry Directory (http:/ / stemcelllist. com)
Stem Cell Research and Cloning (http:/ / uiuc.libguides. com/ stemcellcloning/ ) LibGuide resources from the
University of Illinois at UrbanaaChampaign Library
Corneal endothelial and epithelial stem cell research and application (http:/ / www.cesbank. org)
Peer-reviewed journals
Cytotherapy (http:/ / www.tandf.co. uk/ journals/ titles/ 14653249.asp)
Cloning and Stem Cells (http:/ / www.liebertpub. com/ products/ product.aspx?pid=9)
Journal of Stem Cells and Regenerative Medicine (http:/ / www.pubstemcell.com)
Stem Cells and Development (http:/ / www.liebertpub. com/ products/ product.aspx?pid=125)
Regenerative Medicine (http:/ / www. futuremedicine. com/ loi/ rme)
Stem Cell Research (http:/ / www.elsevier. com/ wps/ find/ journaldescription. cws_home/ 711630/
description#description)
Article Sources and Contributors
14
Article Sources and Contributors
Stem cell Source: http://en.wikipedia.org/w/index.php?oldid=432845251 Contributors: 041744, 04spicerc, 203.170.3.xxx, 217.99.96.xxx, 5 albert square, 9Nak, A.V., AHands, AVM, Aaron
Schulz, Aaron charles, Abb615, Abbaroodle, Abbasbeyli, Accuruss, Acgator09, Adam.douggie, AdamJacobMuller, AdamRetchless, Adashiel, Adenosine, Adraeus, Adrian, Aeon221, Aetheling,
Agathman, Agricolae, Aircorn, Ajournaleditor, Alakey2010, Alan Liefting, Alanliddell, Alansohn, Alasdair.g1998, Alchewizzard, Aldux, Alenshrew, Alex.tan, Alexei Kouprianov,
AlexiusHoratius, Alextill95, Alienus, Allen4names, Allmightyduck, AllyUnion, Alphachimp, Altenmann, Altonbr, AmiDaniel, Andcarne, Andre Engels, Andres, Andrewbadr, Androstachys,
Anetode, Angus Lepper, Anonymous Dissident, Antandrus, Antonrojo, Arcadian, Arcus, Argon233, Arkantosstevius, Arkrishna, ArneLH, Arthena, Artichoker, Arx Fortis, Astoman,
AstroHurricane001, Astronaut, Astronautics, Austinbond, Autonova, Avb, Averross, Avoided, AxelBoldt, Axl, Aymatth2, Bachrach44, Badanagram, Badarkace, Banman1004, Bartimaeus666,
Baughnie, BballJones, Benbread, Bender235, Bento00, Berlickshane, BesigedB, Betacommand, Bethelizebeth, Bfiggis, Bgpaulus, Big Smooth, Big iron, Bigal78, Bigminisachin1231, Bill52270,
Bioinformin, Bisyork, Blake-, Blankfaction.tk, Blazersguy, Blkpowernig2, Blonde chik819, Blunub, Bob Wiyadabebe-Iytsaboi, Bobblewik, Bobo192, Boghog, Bomac, Bookandcoffee,
Boothy443, Boots bklyn, Boredzo, BorgHunter, BorgQueen, Borisblue, Bradeos Graphon, Brainfood, Brainhell, Brendan Moody, Brendanconway, Brian Crawford, Brian Kendig, Brian0918,
Brim, Brossow, Brown boi5, Bryan Derksen, Bubbles3, Bucaro1975, Bucketsofg, Buhuzu, Bushytails, Butros, Byornski, C45207, CBFan, CMacMillan, CRUCIFYKEVIN, Caelarch,
Calliopejen1, Caltas, CambridgeBayWeather, Can't sleep, clown will eat me, Canderson7, Capecodeph, Captain-n00dle, Cassivs, Cburnett, Cellmedsociety, Centrx, Change93, Chimera16,
ChipsAhoya, Choach, Chocolatelana, Chodorkovskiy, Cholerashot, Chowbok, Chris 73, ChrisGlew, Chrisdale92, Chrislk02, Chrismeehan, Christopher Parham, Circeus, ClockworkSoul,
Closedmouth, Cntras, CoJaBo, Cobi, Cohesion, Colin marks, Conversion script, Corporate.legal, Corvus cornix, Cosmic Latte, Courcelles, Cpt.squeaky, Cquan, Crazycomputers, Crazydog115,
Cryptic, CyrilleDunant, Cyrus Andiron, D. Wu, D3monicVeng3ance, DARTH SIDIOUS 2, DMacks, Dabomb87, Dan100, Danandlollie, Daniel5127, DanielCD, Danny, Dantheman531, Darena
mipt, Dark.ranger, Darqcyde, Dave Nelson, Davewho2, Davfoster88, David Peacham, David Shankbone, DavidCary, Daycd, Dbsteve100, Dbtfz, DeadEyeArrow, Debatesensei, Decumanus,
Deepakpurang, Defunkt, Delldot, Demong, DerHexer, Derek Ross, Deving, Dharmabum420, Dimitmant, Discospinster, DividedByNegativeZero, Dlh-stablelights, Dobett, DocWatson42,
DonSiano, Dougofborg, Dr Aaron, Dr d12, Dr.enh, Draeco, Dragostanasie, Drbohlav, Drianmcniece, Drumman91, Drunken Whale, Duckzilla2000, Dwayne, ERcheck, ESkog, Ed Poor, EdBever,
Edgar181, Egandrews, ElKevbo, Elektrik Shoos, Ellmist, Ellwyn, Eloquence, EncMstr, Encephalon, Epbr123, Espetkov, Esprit15d, Etparle, Eubanks718, Eurobas, Europa284, Everard Proudfoot,
Evercat, Everyking, Everyone Dies In the End, Evil Monkey, Evil saltine, Excirial, Extro, FF2010, Fama Clamosa, Fang Aili, Fatalityduff, Favonian, Fenice, Feverinlove, Fic-in, Fingerz,
Flatterworld, Fleminra, Florentino floro, Flux.books, Flyingdream, Forluvoft, Frazzydee, Fredrik, Free Bear, Freeze42084, FreplySpang, Freshyill, FrozenUmbrella, Fstorino, Funky Monkey,
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