REVIEWS
THE BIOLOGICAL RESTORATION OF CENTRAL
Azadeh Farin, M.D.
Department of Neurological Surgery,
Keck School of Medicine,
University of Southern California,
Los Angeles, California
Charles Y. Liu, M.D., Ph.D.
Department of Neurological Surgery,
Keck School of Medicine,
University of Southern California,
Los Angeles, California, and
Division of Chemistry and
Chemical Engineering,
California Institute of Technology,
Pasadena, California
James B. Elder, M.D.
Department of Neurological Surgery,
Keck School of Medicine,
University of Southern California,
Los Angeles, California
Iver A. Langmoen, M.D., Ph.D.
Vilhelm Magnus Center,
Institute for Surgical Research,
and Department of Neurosurgery,
Ullevål University Hospital and
Rikshospitalet,
University of Oslo, Norway, and
Department of Clinical Neuroscience,
Karolinska Institute,
Stockholm, Sweden
Michael L.J. Apuzzo, M.D.
Department of Neurological Surgery,
Keck School of Medicine,
University of Southern California,
Los Angeles, California
Reprint requests:
Azadeh Farin, M.D.,
Department of Neurological Surgery,
Los Angeles County-University of
Southern California Medical Center,
1200 North State Street, No. 5046,
Los Angeles, CA 90033.
Email: Azadehfarin@hotmail.com
Received, May 6, 2008.
Accepted, September 16, 2008.
NEUROSURGERY
NERVOUS SYSTEM ARCHITECTURE AND FUNCTION:
PART 1—FOUNDATIONS AND HISTORICAL
LANDMARKS IN CONTEMPORARY STEM CELL BIOLOGY
SINCE THEIR DISCOVERY, stem cells have fascinated scientists with their ultimate poten-
tial: the ability to cure disease, repair altered physiology, and reverse neurological deficit.
Stem cell science unquestionably promises to eliminate many of the tragic limitations con-
temporary medicine must acknowledge, and cloning may provide young cells for an aging
population. Although it is widely believed that stem cells will transform the way medicine
is practiced, therapeutic interventions using stem cell technology are still in their infancy.
The 3 most common stem cell sources studied today are umbilical cord blood, bone mar-
row, and human embryos. Although cord blood is currently used to treat dozens of disor-
ders and bone marrow stem cells have been used clinically since the 1960s, human embry-
onic stem cells have yet to be successfully applied to any disease. Undeniably, stem cell
therapy has the potential to be one of the most powerful therapeutic options available. In
this introductory article of a 5-part series on stem cells, we narrate the evolution of modern
stem cell science, delineating major landmarks that will prove responsible for taking stem
cell technology from the laboratory into revolutionary clinical applications: from the first mile-
stone of identifying the mouse hematopoietic stem cell to the latest feats of producing
pluripotent stem cells without embryos at all. In Part 2, we present the evidence demon-
strating the certainty of adult mammalian neurogenesis; in Parts 3 and 4, we describe neu-
rosurgical applications of stem cell technology; and in Part 5, we discuss the philosophical
and ethical issues surrounding stem cell therapy, as well as future areas of exploration.
KEY WORDS: Cellular neurosurgery, Cloning, Embryo, Molecular neurosurgery, Neurogenesis, Plasticity,
Stem cell
Neurosurgery 64:15–39, 2009 DOI: 10.1227/01.NEU.0000337580.02706.DC www.neurosurgery-online.com
T
he discovery of stem cells is considered a testing, and understanding the earliest stages of
breakthrough that is expected to pro- human development are enormous. These
foundly impact the practice of medicine. notions are just now beginning to emerge as
Although most studies have been historically actualities. In this article, we recount the most
conducted in murine models, advances in critical feats in modern stem cell research that
human embryonic stem (ES) cell research are will lead to definitive clinical applications. A
now a reality. Human ES cells are currently glossary is provided in the Appendix.
derived from 3 sources: blastocysts remaining
after infertility treatments and donated for DEFINITION OF STEM CELLS
research, blastocysts generated from donated
gametes, and nuclear transfer. The potential During the 1800s, the cell was identified as
implications for treating human disease, drug the building block of life. Mammals were then
ABBREVIATIONS: CBE, cord blood-derived embryonic-like stem cell; DNA, deoxyribonucleic acid; ES, embry-
onic stem; HSC, hematopoietic stem cell; iPS, induced pluripotent stem; Oct4, Octamer 4; SCID, severe com-
bined immunodeficiency; SCNT, somatic cell nuclear transfer; SHED, stem cells from human exfoliated decidu-
ous teeth; Sox2, sex-determining region Y-box 2
VOLUME 64 | NUMBER 1 | JANUARY 2009 | 15
FARIN ET AL.
determined to be composed of 3 cell types: germ cells, somatic
cells, and stem cells. Germ cells are the haploid gametes (oocyte
and spermatocyte) responsible for reproductive capacity; oocyte
fertilization by a spermatocyte results in a diploid zygote with
46 chromosomes. The majority of cells in the body are diploid
somatic cells. The word “somatic” is derived from soma the
Greek word for body. In the 1800s, the observation that certain
cells could generate other cells marked the commencement of
stem cell science. This was followed in the early 1900s by the
discovery of the first hematopoietic stem cells (HSC) differen-
tiating into blood cells.
Stem cells, by definition, are primary undifferentiated cells
possessing 2 properties: self-renewal and potency. Self-renewal
is illustrated by the retained ability of stem cells to renew them-
selves through endless mitotic cycles while maintaining the
undifferentiated state. To ensure self- renewal, stem cells
undergo 2 types of cell division. Symmetric division gives rise
to 2 identical daughter cells, both of which are endowed with
stem cell properties. Asymmetric division produces only 1 stem
cell and a progenitor cell with limited self-renewal potential.
Progenitors can undergo several cycles of cell division before
terminally differentiating into a mature cell. The molecular
switch determining symmetric versus asymmetric divisions
may be dependent on differential segregation of cell membrane
proteins, such as receptors, between daughter cells (6).
Alternatively, stem cells may remain undifferentiated owing to
environmental cues in their particular niche and may differen-
tiate when they exit that niche or are deprived of certain signals.
For example, studies in Drosophila germarium have identified
the signals Dpp and adherens junctions that prevent germarium
stem cells from differentiating (110, 141).
Potency is defined as the potential to differentiate into special-
ized cell types (49, 51, 90–92, 94, 101, 106, 112, 114, 135, 137).
Totipotent or pluripotent stem cells, or ES cells, are able to give
rise to any or many mature cell types. Multipotent or unipotent
progenitor cells are sometimes referred to as adult stem cells,
and they are more limited in the variety of progeny they pro-
duce. Considerable debate exists about whether some proposed
adult cell populations are truly stem cells.
Self-renewal and potency are illustrated through multiple
cycles of regeneration in vivo. The “gold standard” test for defin-
ing an HSC is the ability to transplant an HSC into an individual
without HSCs and to rescue that individual; HSCs isolated from
the individual who received the new transplant can subse-
quently be transplanted into another individual without HSCs,
demonstrating self-renewal. HSCs must demonstrate potency
by producing new blood and immune cells. Stem cell properties
can also be illustrated in vitro using clonogenic assays, in which
single cells are characterized by their ability to differentiate and
self-renew (26, 27). However, in vitro culture conditions can alter
cell behavior, rendering it difficult to assess whether the cells will
behave in a similar manner in vivo.
Stem cells’ potential for unlimited versus limited differentia-
tion, or phenotypic maturation along a restricted exclusive lin-
eage, is a fundamental feature that can be used to categorize
them. A morula, or zygote, is the product of fusion between
16 | VOLUME 64 | NUMBER 1 | JANUARY 2009
oocyte and sperm. This totipotent stem cell, or “master cell,”
contains all of the genetic information needed to become any tis-
sue whatsoever, including placenta (Fig. 1). Cells produced by
the first few divisions of the fertilized egg are also totipotent.
The morula becomes the blastocyst, which, in humans, is a 4- to
5-day-old embryo consisting of 50 to 150 cells. The blastocyst
then generates 3 main types of stem cells—ES cells, umbilical
cord blood stem cells, and adult stem cells (Fig. 2)—as it
matures into the human fetus.
Pluripotent ES Cells
ES cells are pluripotent cells derived from either the blasto-
cyst’s undifferentiated inner cell mass epiblast tissue or from the
morula. In contrast to multipotent adult stem cells, ES cells can
differentiate during development into all specialized embry-
onic tissues derived from the 3 primary germ layers (ectoderm,
endoderm, and mesoderm) (Fig. 1). They can further differenti-
ate into each of more than 200 cell types of the adult body when
given sufficient and necessary stimulation for a specific cell lin-
eage (Fig. 3). Unlike the totipotent zygote, however, these cells
do not contribute to the extraembryonic membranes, placenta,
and some uterine tissues.
Most research has involved mouse ES cells and human ES
cells. Differences between these cell types have been extensively
described and are illustrated in Figures 4 and 5 (1, 3, 4, 7, 9, 10,
12, 15, 17, 22–24, 30, 31, 33, 35, 37–40, 45–48, 52, 55–62, 70, 72, 73,
75–78, 81, 87, 89, 97, 98, 100, 103, 104, 108, 109, 113, 115, 117, 118,
120, 123, 139, 142, 144). To maintain an undifferentiated state,
both mouse ES cells and human ES cells require very specific
environments; without optimal culture conditions or genetic
manipulation (11a), ES cells will rapidly differentiate. Mouse
ES cells are grown on a layer of gelatin and require the presence
of leukemia-inhibitory factor (16).
Human ES cells are grown on a feeder layer of mouse embry-
onic fibroblasts and require the presence of basic fibroblast
growth factor (or fibroblast growth factor 2) (79). Human ES
cells maintain pluripotency owing to the action of 3 hallmark
transcription factors, NANOG, Octamer 4 (Oct4), and Sex-
determining region Y-box 2 (Sox2), which suppress genes lead-
ing to differentiation (8). These factors are the subjects of intense
investigation aiming to uncode the mechanisms responsible for
cell pluripotency and to determine how these events can be
manipulated for human benefit. Thus far, all 3 factors are known
to play essential roles in the self-renewal of undifferentiated cells
and maintenance of pluripotency. They also facilitate the tran-
scription of developmentally important homeodomain proteins.
The murine NANOG gene was isolated by Ian Chambers, who
summarized the significance of this gene by calling it the “mas-
ter gene . . . [making ES cells] immortal” (11, 11a). NANOG
overexpression causes mouse ES cells to self-renew in the
absence of leukemia-inhibitory factor and human ES cells to
propagate for multiple passages as pluripotent cells; gene knock-
down of NANOG and Oct4 promotes differentiation. Tumor
suppressor p53 suppresses NANOG expression after deoxyri-
bonucleic acid (DNA) damage in mouse ES cells, inducing differ-
entiation of cells, followed by cell cycle arrest and apoptosis.
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 FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY

specific signals for correct dif-

ferentiation; if injected directly
into the body, ES cells differen-
tiate into many different types
of cells, resulting in a teratoma.
Differentiating ES cells for
practical purposes while avoid-
ing transplant rejection are 2
of the many technical issues
researchers face (140).
Significant ethical contro-
versy has been generated over
human ES cell research. It is
fueled by the fact that starting
an ES cell line has traditionally
required the destruction of a
human embryo or therapeutic
cloning. These techniques are
felt by some to devalue human
life (34, 132). Although ES cells
are considered to have greater
therapeutic potential than adult
stem cells, lack of consensus
with regard to associated com-
plex ethical concerns has prom-
pted some governments to re-
strict state-sponsored ES cell
activities. Significant ES cell re-
search has continued fruitfully
with private funds. However,
given the challenging curative
goals proposed for these cells,
further developments are re-
FIGURE 1. Development and differentiation of cells originating from the zygote (from, National Center for quired before ES cells can be
Biotechnology Information. http://www.ncbi.nlm.nih.gov. Accessed May 1, 2008 [76]). applied safely and effectively to
human disease. Consequently,
Oct4 is active in embryos throughout the preimplantation period after 20 years of research, there have been no approved treat-
and is a marker for both undifferentiated cells and tumors. Its ments or human trials using ES cells. Researchers have begun
exact level determines ES cell fate and regulates pluripotency. exploring approaches that obviate embryo destruction (Fig. 6).
Mouse embryos deficient in Oct4 fail to form the inner cell mass, Recently, it has been shown that ES cell lines can be generated
lose pluripotency, and differentiate into trophectoderm. An using a single-cell biopsy similar to that used in preimplantation
increase in Oct4 causes differentiation into primitive endoderm genetic diagnosis. The differentiated cell is reprogrammed to a
and mesoderm. Because stem cell self-renewal is dependent pluripotent state, and embryos are avoided altogether (83, 122).
upon a critical amount of Oct4 and deviations result in abnormal The ethical issues surrounding ES research will be further dis-
development, Oct4 is considered a primary regulator of pluripo- cussed in Part 5 of this series.
tency. The role of Oct4 in sustaining self-renewal capacity of
adult somatic stem cells requires further study. Sox2 is also Multipotent Adult Stem Cells
involved in regulation of embryonic development and cell fate Adult stem cells are differentiated from pluripotent inner mass
determination. cells. Most adult stem cells are lineage-restricted (multipotent),
The cell surface antigens most commonly used to identify only producing closely related progeny of a given local tissue
human ES cells are the glycolipids SSEA3 and SSEA4 and the ker- source (e.g., HSCs differentiate into red blood cells, white blood
atan sulfate antigens Tra-1-60 and Tra-1-81. The molecular defini- cells, and platelets). These undifferentiated cells reside with dif-
tion of a stem cell includes many more proteins and continues to ferentiated cells of their tissues of origin, with correlative nomen-
be a topic of research (40). Because of their potential for unlimited clature (mesenchymal stem cell, adipose-derived stem cell,
expansion and pluripotency, ES cells remain a theoretically attrac- endothelial stem cell) (5, 32). Adult stem and progenitor cells
tive source for regenerative tissue. However, ES cells require play a reparative role, replenishing cells and maintaining normal

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FARIN ET AL.
FIGURE 2. Classification of human stem cells (from, Bongso A, Lee EH: Stem cells: Their definition, classification
and sources, in Bongso A, Lee EH (eds): Stem Cells: From Bench to Bedside. Singapore, World Scientific,
2005, pp 1–13 [7], at http://www.worldscibooks.com/lifesci/etextbook/5729/5729_chap1.pdf. Accessed May 1, 2008).
turnover of regenerative organs, such as blood, skin, or intestinal
tissues. Given their functions, they are also known as somatic
stem cells or germline stem cells, as they give rise to gametes.
Despite their name, they can be found in children as well (41).
A major research focus has been to clarify their capacity for
indefinite self-renewal and to better characterize their differen-
tiation potential (28). Pluripotent adult stem cells are rare and
generally small in number, but they can be found in a number
of tissues, including umbilical cord blood (69, 99).
Because the production of adult stem cells does not involve
embryo destruction, related research attracts greater govern-
ment funding, and therapeutic applications have been wide-
spread (133). Whereas ES cell potential remains untested, adult
stem cell treatments have been used for many years to success-
fully treat leukemia and related bone and blood cancers
through bone marrow transplants. Promising investigations
involve antidifferentiation transcription factors (oncogene c-
Myc) that signal reprogramming pathways to transform adult
stem cells to an embryonic-like state, reversing differentiation
and recapacitating adult cells with pluripotency (121). How-
ever, oncogenic transformation may limit the clinical appro-
priateness of this technique.
Unipotent Stem Cells
Unipotent cells give rise to only 1 cell type but have the prop-
erty of self-renewal, which distinguishes them from non-stem
cells. Some researchers claim unipotency to be inadequate in
terms of defining a stem cell, arguing that at least multipotency
is required. Examples of unipotent cells are human skin cells.
18 | VOLUME 64 | NUMBER 1 | JANUARY 2009
EVOLUTION
OF MODERN
STEM CELL SCIENCE
1963: Discovery of
the Mouse HSC
The first major discovery in
modern stem cell science is
attributed to Canadian scien-
tists Ernest A. McCulloch and
James E. Till, whose decade-
long experiments culminated
in the 1963 discovery of the
multipotent HSC in mouse
bone marrow (129). The HSC
was actually isolated years
later. Contributing to this dis-
covery was knowledge that
radiation from nuclear bombs
and x-rays can eliminate blood
cell production and that blood
cell production could be res-
cued by bone marrow injec-
tions from healthy mice.
Starting in the 1950s, Mc-
Cullough and Till systemati-
cally measured the quantity of donor bone marrow cells needed
to restore blood cell production in irradiated mice. These meas-
urements enabled them to deduce that hematopoietic rescue
was being carried out by a very small number of donor bone
marrow cells. They further observed that the spleens of the irra-
diated mice receiving replacement bone marrow contained
colonies of red blood cells, white blood cells, and platelets, indi-
cating that something capable of making all blood cell types, a
blood-forming stem cell, resided in the spleen. In a truly elegant
experiment, McCullough and Till imparted distinct genetic sig-
natures to blood-forming stem cells in donor bone marrow and
documented that the blood cells in each spleen colony bore a
particular genetic imprint matching that of the donor stem cell.
This proved that diverse blood cells come from an individual
stem cell. McCullough and Till demonstrated this self-renewal
potential of stem cells by repeatedly taking individual spleen
colonies from a set of mice and injecting them into irradiated
mice to generate new colonies. Their contributions, which initi-
ated the efforts to define how, and in response to what, stem
cells divide and mature, serve as a foundation for all current
research on stem cells (65–68, 80, 93, 126–131).
1968: Bone Marrow Transplant Successfully Treats
Human Severe Combined Immunodeficiency
The next major milestone in stem cell science occurred in 1968
at the University of Minnesota, when severe combined immun-
odeficiency (SCID) was successfully treated by the first success-
ful bone marrow transplant between 2 human siblings (21, 29).
This primary immune deficiency is characterized by a severe
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FIGURE 3. In vitro avenues to differentiation of embryonic stem cells.
Several in vitro embryonic stem cell differentiation protocols show how a
defined microenvironment can enhance various differentiation pathways by
partially imitating early embryonic development. bFGF, basic fibroblast
growth factor (also known as fibroblast growth factor 2); BMP2/4, bone
morphogenetic protein 2/4; dHGF, deleted variant of hepatocyte growth fac-
defect in the T- and B-lymphocyte systems caused by absent or
poorly functioning lymphocytes or defective bone marrow stem
cells, from which mature lymphocytes normally develop. SCID
renders its infant victims prone to life-threatening pneumonia,
meningitis, or sepsis within the first few months of life; even
normal childhood infections and viruses (chicken pox, measles,
and cold sore virus) may be fatal. The term “bubble baby” has
been used to describe such babies because of the sterile environ-
ment used to decrease the risk of infections.
Marrow transplantation is the only treatment that has been
shown to cure SCID and other prematurely lethal primary
immunodeficiencies. A 90% cure rate can be expected for SCID,
NEUROSURGERY
FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY
tor; EB, embryoid body; IGF2, insulin-like growth factor 2; NTF3, neu-
rotrophin 3; SDIA, stromal-derived inducing activity method; SHH, sonic
hedgehog homologue; VEGF, vascular endothelial growth factor (from,
Klimanskaya I, Rosenthal N, Lanza R: Derive and conquer: Sourcing and
differentiating stem cells for therapeutic applications. Nat Rev Drug
Discov 7:131–142, 2008 [47]).
whereas SCID is usually fatal within 1 year after birth without
transplantation. Unfortunately, more than 90% of patients with
primary immunodeficiencies lack suitably histocompatible sib-
ling donors. Alternative sources of stem cells are being investi-
gated to treat these patients. Bone marrow from closely matched
relatives and closely matched unrelated individuals, peripheral
blood stem cells, and cord blood from siblings or unrelated new-
borns have all been used successfully in selected cases.
1978: HSCs Discovered in Human Cord Blood
The treatment of blood disorders was advanced further by
the discovery of stem cells in cord blood (95). Rich in HSCs, cord
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FARIN ET AL.

Gail R. Martin were the first

workers to isolate mouse pluri-
potent ES cells from mouse
embryos and condition them
to differentiate into teratocarci-
nomas when injected in vivo.
Cultures of these ES cells dif-
ferentiated into a wide variety
of cell types, making the full
potential of ES cells a reality.
No longer did it appear that
therapy would be limited by
the multipotency of the HSC
instead of the more desirable
pluripotency of ES cells. Gail
Martin is credited with coining
the term “embryonic stem cell”
(64). In her article, she refers to
permissive culture conditions
that promote stem cell proper-
ties, and she further describes
the implications for patho-
physiological discoveries re-
lated to her technique: “. . .
Embryonic stem cells were iso-
lated from . . . blastocysts cul-
tured in medium . . . [which]
might contain a growth factor
that stimulates the proliferation
or inhibits the differentiation of
. . . embryonic cells. . . . This
method . . . makes feasible the
FIGURE 4. Comparison of mouse, monkey, and human pluripotent stem cells. ES cell, embryonic stem cell; EG, isolation of pluripotent cell lines
embryonic germ cell; EC, embryonic carcinoma cell; SSEA, stage-specific embryonic antigen; TRA-1, tumor rejec-
from . . . embryo . . . carrying
tion antigen-1; LIF, leukemia-inhibitory factor; bFGF, basic fibroblast growth factor; EB, embryoid bodies; gp, glyco-
protein (adapted from, NIH Stem Cell Information. http://stemcells.nih.gov. Accessed May 1, 2008 [79]).
mutant genes . . . [and] should
make possible new approaches
to the study of early mam-
blood is the blood remaining in the placenta and umbilical cord, malian development” (64, p 7634).
which can be accessed during birth. Since 1988, scientists have Two years later, Elizabeth J. Robertson demonstrated that stem
used cord blood stem cells to treat leukemia, thalassemia, and cells isolated from parthenogenetic mouse embryos could form
anaplastic anemia. The unique genetic signature of cord blood a variety of tissues, including nerve and muscle (44, 102).
stem cells is partly responsible for higher success rates in patients
who receive a transplant of cord blood stem cells from a family 1992: Rat Neural Stem Cells Cultured
member as compared with cells from a nonrelative or even bone in Vitro as Neurospheres
marrow-derived HSCs. Cord blood collection is relatively easy Investigators at the California Institute of Technology first
and free of risk to the donor. Families with histories of hemato- isolated multipotent neural stem cells from the mammalian neu-
logical disorders are encouraged to consider banking their baby’s ral crest in 1992. These neural stem cells generated both multi-
umbilical cord blood for potential future therapy. It is vital that potent progeny, illustrating the self-renewal definition of stem
cord blood stem cells are collected within 15 minutes after birth cells, as well as committed clonal progeny that differentiated
and processed within 48 minutes at the cord blood bank’s labo- into neurons and glia (116). Progeny fate depended on substrate
ratory to preserve cell viability. manipulation. The authors wrote, “These findings have implica-
tions for models of neural crest development in vivo, and estab-
1981: Mouse ES Cells Derived from the Inner Cell Mass lish a system for studying the generation of cellular diversity by
Before 1981, HSCs were a common research focus, whereas a multipotent stem cell in vitro” (116, p 973).
the potential of ES cells was merely theoretical. Then, in a land- Concurrently, cloning technology was being developed at an
mark article, scientists Martin Evans, Matthew Kaufman, and alarming rate, far faster than most of the lay public could com-

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 FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY

specific cell surface markers for

undifferentiated cells that were
distinct from murine markers.
The latter observation implies
that basic species differences
exist between early mouse and
human development. Even
after months of undifferenti-
ated in vitro proliferation, the
cells maintained pluripotency.
The cell line is still in use today.
Thomson aptly wrote, “Em-
bryonic stem cells provide a
powerful approach for intro-
ducing specific genetic chang-
es” (125, p 1145), a fact that has
since figured prominently in
biomedical research through
the generation of knockout
mice and today can be ack-
nowledged as a feature of para-
mount benefit to mankind. In
his article, Thomson elucidates
the potential utility of his find-
ing in advancing mammalian
FIGURE 5. Comparison of mouse and human embryonic stem cells. SSEA, stage-specific embryonic antigen; TRA-1, developmental biology and
tumor rejection antigen-1; N/A, not applicable (adapted from, Klimanskaya I, Rosenthal N, Lanza R: Derive and con- drug discovery: “Screens based
quer: Sourcing and differentiating stem cells for therapeutic applications. Nat Rev Drug Discov 7:131–142, 2008 [47]).
on the in vitro differentiation of
human ES cells to specific line-
prehend. Achievements in stem cell science for the next few ages could identify gene targets for new drugs, genes that could
years alternated between impressive technical feats credited to be used for tissue regeneration therapies, and teratogenic or toxic
those advancing the field of cloning and those working with compounds” (125, p 1146). He correctly predicted particular ben-
pluripotent stem cells. efit with regard to developmental events that cannot be studied
directly in the intact human embryo, including birth defects,
1996: First Mammal Cloned from Adult Somatic Cell infertility, pregnancy loss, and early postimplantation develop-
In 1996, the ewe Dolly was the first mammal to be cloned ment. Thomson foresaw the value of human ES cells in studying
from an adult somatic cell, using the process of nuclear transfer the development and function of tissues that differ between mice
(53, 54, 105, 138), which will be elaborated on later. She was and humans, including placenta and extraembryonic mem-
cloned by Ian Wilmut, Keith Campbell, and colleagues at the branes. Finally, he alluded to future aims in transplantation med-
Roslin Institute in Edinburgh, Scotland. This pioneering research icine, articulating that clarifying the mechanisms controlling dif-
helped create the basis for therapeutic and reproductive cloning. ferentiation was a prerequisite to the proficient, directed
Dolly lived until the age of 6 years. She was documented to have differentiation of ES cells to specialized types of functional cells
telomeres almost the same length as her mother’s, much shorter
with physiological relevance. He outlined that the consistent
than those of other sheep that were physiologically her age. Most
manufacturing of large quantities of human cells such as neu-
sheep live until about 13 years. Being cloned from an older sheep
rons would potentially permit cell replacement capacities in neu-
was implicated as a potential cause for her shortened telomeres
rodegenerative diseases. Challenges included “. . . strategies to
and early death; others think that her autopsy-proven pul-
prevent immune rejection of the transplanted cells . . . [these]
monary disease explains her early death.
include banking ES cells with defined major histocompatibility
1998: First Human ES Cell Line Derived complex backgrounds or genetically manipulating ES cells to . . .
In 1998, James Thomson derived the first human pluripotent combat immune rejection. . . . Substantial advances in basic
ES cell line at the University of Wisconsin-Madison (125) from developmental biology are required to direct ES cells efficiently
human blastocysts, 18 years after Gail Martin isolated mouse ES to lineages of human clinical importance” (125, p 1147).
cells. This landmark feat is widely cited as one of the most crit-
ical breakthroughs in modern stem cell science. 2001: Scientists Clone First Human Embryos
The human ES cells exhibited normal karyotypes, formed deriv- On October 31, 2001, 4 years after Dolly was cloned, scientists at
ative tissues of all 3 embryonic layers, and expressed primate- Advanced Cell Technology in Worcester, MA, cloned the first 4- to

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FARIN ET AL.

Women between the ages of 24

A to 32 who had at least 1 child
and were willing to contribute
eggs anonymously were re-
cruited while skin biopsies
were collected from individu-
als to be cloned (the donors).
The skin biopsies were used to
isolate fibroblasts, and their
donors included patients with
diabetes or spinal cord injury
B because of the potential benefit
to them from therapeutic
cloning.
The technique is described
and illustrated in basic terms
(Fig. 7). First, an extremely fine
needle was used to aspirate the
C genetic material from a mature
egg; the donor cell nucleus or
entire cell was then injected
into the enucleated egg. The
transplanted egg was incubated
under conditions prompting
FIGURE 6. Avenues to generating pluripotent cell derivatives. A, somatic cell nuclear transfer (SCNT). The nucleus division and growth. The tim-
of the fertilized oocyte is removed, and the nucleus of a recipient’s somatic cell is transferred into the enucleated oocyte. ing of each cloning attempt
The somatic nucleus is reprogrammed by the competent cytoplasm, and the oocyte with the donor nucleus starts cleav- was dependent upon the men-
ing and forms a morula that continues to develop into a blastocyst. Pluripotent embryonic stem cells (ESC) with the strual cycles of the egg contrib-
recipient’s genome are derived from either the morula or the inner cell mass of the blastocyst. Their differentiated deriv- utors, who were injected with
atives are immunologically identical to the recipient’s own. B, parthenote pluripotent cells. An unfertilized oocyte is hormones so that they would
activated and starts to cleave as a normal embryo. Pluripotent cells are derived from a morula or the inner cell mass ovulate many eggs at once.
of a blastocyst. The absence of the paternal contribution greatly diminishes the human leukocyte antigen (HLA) vari- Initial failure led to adoption of
ability and thus allows a bank of various HLA types of pluripotent cells and/or their derivatives to be built. C, trans-
a modified technique, devel-
differentiation. The recipient’s somatic cell is turned into a pluripotent cell via cellular reprogramming using trans-
oped by Teruhiko Wakayama,
fection of a defined set of transcription factors or loading the target cell with the cellular lysate of a pluripotent cell.
The new pluripotent cell and its derivatives are immunologically identical to the recipient’s and so can be used for who created the first cloned
transplantation (from, Klimanskaya I, Rosenthal N, Lanza R: Derive and conquer: Sourcing and differentiating stem mice in 1998. Some eggs were
cells for therapeutic applications. Nat Rev Drug Discov 7:131–142, 2008 [47]). injected with nuclei from skin
fibroblasts, while others were
injected with ovarian cells
6-cell human embryos (14, 53, 54). The nuclear transplantation called cumulus cells that usually nurture developing eggs in the
experiments were reportedly conducted to develop blastocysts to ovary and can be found still attached to eggs after ovulation.
isolate human ES cells and develop replacement cells and tissues Cumulus cells are small enough to be injected whole. Ultimately,
for therapeutic purposes. Only 1 embryo progressed to the 6-cell 71 eggs from 7 volunteers were required before the first cloned
stage, at which point it stopped dividing (the blastocyst stage embryo was generated.
would have required 50–100 cells). The scientists did successfully The scientists then turned their attention to parthenogenesis
develop blastocysts parthenogenetically by prompting human eggs (asexual reproduction), whereby they induced human eggs to
alone, without sperm or other additional nuclear material, to divide into early embryos without being fertilized by a sperm or
repeatedly divide. The authors noted that their accomplishment without being enucleated and injected with a donor cell. Because
“[represented] the dawn of a new age in medicine by demonstrat- eggs proceed from the diploid state to the haploid state late in
ing that the goal of therapeutic cloning is within reach” (15, p 44). their maturation cycle, they can be isolated while they still retain
They defended their objective of therapeutic cloning as distinct a full set of genes. This technique was felt to be advantageous
from reproductive cloning, a practice to which they are opposed. because stem cells derived from such parthenogenetically acti-
Whereas therapeutic cloning attempts to use a patient’s own vated cells would be unlikely to be rejected after transplantation
genetic material to generate cells or tissue to treat disease or dys- because of their extreme similarity to a patient’s own cells. On the
function, reproductive cloning seeks to implant a cloned embryo other hand, they would not be identical to the individual’s cells
into a woman’s uterus with hopes of developing a cloned baby. because of the obligatory gene shuffling that occurs during ooge-

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FIGURE 7. Therapeutic cloning (from, Cibelli JB, Lanza RP, West
MD, Ezzell C: The first human cloned embryo. Sci Am 286:44–51, 2002, at
nesis. Cells derived parthenogenetically might also be more
acceptable to those who are opposed to derivation of stem cells
from cloned early embryos. The scientists described a scenario in
which a woman with heart disease has her own eggs collected
and activated in the laboratory to yield blastocysts. Stem cells iso-
lated from the blastocyst could be chemically induced to become
cardiac muscle cells, which are then implanted back into the
patient to repair the diseased heart. Androgenesis, in which a
zygote containing only paternal chromosomes is developed,
could conceivably create autologous stem cells to treat a man. The
scientists exposed 22 eggs to a chemical environment that
changed the concentration of intracellular ions. After 5 days of
growing in culture, 6 eggs had developed into what appeared to
be blastocysts, but none clearly contained the inner cell mass
required to isolate stem cells.
The next few years witnessed astounding technical feats with
the aim of deriving multipotent, and preferably pluripotent,
stem cells without requiring embryos, given the controversy
generated by the traditional technique of deriving pluripotent
stem cells at the cost of embryonal destruction. Stem cells were
discovered in dental pulp, umbilical cord blood, and amniotic
fluid. Eventually, pluripotent stem cells were derived in mice
without embryos at all, and, subsequently, human hepatocytes
were generated from umbilical cord stem cells, not ES cells. Not
surprisingly, human induced pluripotent stem (iPS) cells soon
followed, and subsequently, human ES cell lines were generated
without embryonal destruction.
2003: Multipotent Adult Stem/Postnatal Cells
Discovered in Children’s Primary Teeth
Dr. Songtao Shi of the National Institutes of Health’s National
Institute of Dental and Craniofacial Research (74, 107) discov-
ered a unique and rich supply of adult stem or postnatal cells in
a naturally exfoliated human organ, the dental pulp of chil-
dren’s primary, or deciduous, teeth. Shi, a pediatric dentist, was
helping his daughter pull out a loose baby tooth when he
noticed residual pulp inside the tooth. The discovery that the
stem cells remained viable inside the tooth for hours after it
was de-rooted prompted him to consider harvestation. Shi
NEUROSURGERY
FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY
Scientific American Online. http://cmbi.bjmu.edu.cn/news/0111/76.htm.
Accessed May 1, 2008 [15]).
extracted and cultured the pulp, isolated viable multipotent
stem cells, and discovered that about 12 to 20 stem cells from
each tooth could reliably grow in culture. The cells were named
stem cells from human exfoliated deciduous teeth (SHED).
Children normally develop a set of 20 deciduous teeth, which
appear after 6 months of life and are replaced between ages 6
and 12. These relatively accessible adult multipotent stem cells
were found to be highly proliferative; compared with dental
pulp stem cells from permanent teeth, SHED divide much more
rapidly, suggesting that SHED may be in a more immature state
than adult stem cells. Furthermore, these clonogenic cells could
be induced to express surface proteins, reflecting their transi-
tion from stem cells to neural cells, adipocytes, and odontoblasts.
After in vivo transplantation, SHED could induce bone forma-
tion, generate dentin, and survive in mouse brain while express-
ing neural markers (Fig. 8). Deciduous teeth are believed to be
capable of providing enough stem cells for potential clinical
application, including autologous stem cell transplantation to
aid in the repair of damaged teeth, regenerate bone, and treat
neurological diseases. Future efforts involve comprehensively
detailing all the cell types that can be generated from SHED.
2005: Human Umbilical Cord Blood-derived Embryonic-
like Stem Cells Discovered
Researchers at Kingston University in England claimed to have
discovered a third category of stem cells, cord blood-derived
embryonic-like stem cells (CBE). The group claimed that these
cells are more potent than adult stem cells (69). After elective
Caesarean section, umbilical cord blood components are sepa-
rated immunomagnetically. Cells are then cultured, and CBE
colonies are isolated. These colonies exhibit the human stem cell
restricted markers TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, and
Oct4. CBEs cultured with hepatocyte growth medium resulted in
expression of hepatic markers α-fetoprotein and albumin. The
authors described the potentially considerable ramifications of
this discovery: “To produce . . . tissue for transplantation, without
feeder layers, and with the . . . recipient’s immunological pheno-
type, is a . . . scientific hindrance. . . . The annual global 100 million
human birth rate proposes umbilical cord blood as the largest
VOLUME 64 | NUMBER 1 | JANUARY 2009 | 23
FARIN ET AL.

potential for clinical applica-

tions” (69, p 245).

2005: President Bush

Approves Federal Funds
for Restricted Stem Cell
Research
On December 20, 2005,
President George W. Bush for-
mally approv ed the use of
federal funds for research on
existing stem cell lines by
signing into law H.R. 2520,
the Stem Cell Therapeutic and
Research Act of 2005. The Act
created a new federal pro-
gram that promotes stem cell
research by re quiring cord
blood banks to donate to re-
searchers units not suitable
for transplantation because of
disease or size. The Act also
ex panded the current bone
marrow registry program to
also include cord blood (36).
Pres ident Bush’s decision
reflects widespread and high-
level recognition of the thera-
peutic potential of stem cells.
However, the decision to fund
only research on existing stem
cell lines and to severely
restrict federal funding for
embryonal research is viewed
by many as a significant neg-
ative position that reflects the
President’s personal religious
beliefs, and it led many scien-
tists to search for alternative
FIGURE 8. A–H, neural differentiation of stem cells from human exfoliated deciduous teeth (SHED). Immunocytochemical
pluripotent stem cell sources.
staining depicts SHED expressing the neural cell markers nestin, glial fibrillary acidic protein (GFAP), neurofilament-M
Partly in response to signifi-
(NFM), 2ⴕ,3ⴕ-cyclic nucleotide-3ⴕ-phosphodiesterase (CNPase), βIII-tubulin, glutamic acid decarboxylase (GAD), and neu-
ronal nuclei (NeuN). I, Western blot analysis confirmed that SHED expressed neural markers as described above. After 4 weeks cant global legislative and
of culture in the presence of B27 supplement, basic fibroblast growth factor, and epidermal growth factor, expression levels of ethical concerns complicating
βIII-tubulin, GAD, and NeuN were up-regulated when compared with regular culture conditions. However, expression lev- ES cell research, investigators
els of nestin, GFAP, CNPase, and NFM remained the same after the treatment. J–O, SHED may coexpress neuronal mark- more intensely sought out
ers including βIII-tubulin (green)/GAD (red) and βIII-tubulin (green)/NFM (red). The morphology of SHED showed elon- alternative means to derive
gated cell-cytoplasmic processes that sometimes coexpress neural markers (triangles) or only express individual neural marker pluripotent stem cells.
(arrows). P–S, toluidine blue (0.1%) staining depicting the altered morphology of SHED after induction with neural culture
medium (P and Q, arrows). Immunopositive staining for anti-MAP2 and anti-Tau antibodies on dendrites and axon (R and August 2006: Murine
S, arrows), respectively. Double-staining experiments showed that βIII-tubulin-positive cells were also detected in the same Pluripotent Stem Cells
field (R, triangle, green). T–W, SHED continued to express glial cell markers including nestin (red), CNPase (red), GFAP Generated from Fibroblasts
(red), and NFM (green) by immunocytostaining (from, Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S: with Specific Factors
SHED: Stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 100:5807–5812, 2003 [74]).
Accordingly, in 2006, Taka-
hashi and Yamanaka (121)
untouched stem cell source. . . . CBEs are a viable . . . alternative demonstrated that differentiated adult fibroblasts can be “repro-
from embryonic stem cells . . . without ethical constraint and with grammed” backward or induced to become embryonic-like

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pluripotent stem cells. Transference of nuclear contents into
oocytes resulted in cells engineered to express 4 defined factors,
Oct3/4, Sox2, c-Myc, and Klf4, which are known to be impor-
tant for maintaining the “stemness” of ES cells. Induction also
required ES cell culture conditions. These iPS cells revealed the
morphology and growth properties of true ES cells and
expressed ES cell marker genes. Subcutaneous transplantation
of the induced cells into nude mice resulted in teratomas. After
injection into blastocysts, the induced cells contributed to mouse
embryonic development.
November 2006: Mouse ES Cells Preserve
Life in Mice with Liver Failure
Japanese scientists collaborating with the National Institutes
of Health successfully induced mouse ES cells to differentiate
into hepatocytes, forming an implantable bioartificial liver that
replaced aspects of physiologically normal liver function. The
ES cell-derived hepatocytes expressed liver-specific genes,
secreted albumin, and metabolized ammonia, lidocaine, and
diazepam. Treatment of 90% hepatectomized mice with an
implanted bioartificial liver seeded with ES cell-derived hepa-
tocytes improved liver function and prolonged survival,
whereas treatment with a bioartificial liver seeded with control
cells did not. The hope is to carry out the same results with
human stem cells (111).
January 2007: Discovery of Human Amniotic
Stem Cell Lines
Scientists at Wake Forest University, led by Dr. Anthony
Atala, and at Harvard University reported discovery of amni-
otic fluid-derived stem cells expressing both ES and adult stem
cell markers; embryonal destruction is precluded with this
methodology (20). Undifferentiated cells doubled in 36 hours
and did not become tumorigenic, although they could give rise
to tissues of all 3 embryonic germ layers. After dozens of pop-
ulation doublings, long telomeres and normal karyotypes were
retained. Differentiated cells derived from amniotic fluid stem
cells included neuronal lineage cells, hepatic lineage cells, and
osteogenic lineage cells.
Dr. Robert Lanza, chief scientist at the stem cell company
Advanced Cell Technology, which later cloned the first human
embryo, stated that “ . . . this work represents a giant step for-
ward for stem cell research” (22a). Dr. George Daley, a Harvard
University stem cell researcher, indicated that expectant par-
ents may someday be able to store amniotic stem cells for
future tissue replacement in a sick child without fear of
immune rejection. However, he warned, “While they are fasci-
nating subjects of study in their own right, they are not a sub-
stitute for human ES cells, which allow scientists to address a
host of other interesting questions in early human develop-
ment” (74a). Interestingly, some of the DNA retrieved from
the amniotic stem cells contained Y chromosomes, indicating
that the cells came from male babies rather than the pregnant
mothers. Stem cells were reportedly easily extracted without
significant risk to mother or fetus; brain, liver, and bone tissue
types were subsequently developed (Figs. 9–12).
NEUROSURGERY
FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY
June 2007: Mouse Fibroblasts Reprogrammed
to Embryonic State
As a prelude to the same phenomenon soon thereafter
observed in humans, research reported by 3 different groups
showed that skin cells, or fibroblasts, can be reprogrammed to
an embryonic state in mice (18). Shinya Yamanaka of Kyoto
University, who pioneered the technique, had induced pluripo-
tent cells from mouse fibroblasts in 2006 via retroviral transfer
of 4 genes; resultant transcription factors facilitated the expres-
sion of additional genes that generated pluripotency. Although
the induced pluripotent cells had some characteristics of
embryonic cells in that they could propagate continuously and
could form teratomas, they failed to produce a chimera mouse
when injected into a developing embryo. (A chimera would be
characterized by DNA from both the original embryo and the
induced pluripotent cells.) The 4 transcription factors used by
Yamanaka in 2006 reprogrammed less than 0.1% of the million
cells in a skin biopsy.
A
B
FIGURE 9. Clonal human amniotic fluid-derived stem (AFS) cells are
broadly multipotent. The clonality of AFS cells and differentiated cells was
verified. A, reverse transcription-polymerase chain reaction (RT-PCR)
analysis of messenger ribonucleic acids for lineages indicated. U, control
undifferentiated cells; D, cells maintained under conditions to promote
osteogenic, myogenic, adipogenic, endothelial, hepatic, and neurogenic dif-
ferentiation. B, Southern blot analysis of inserted green fluorescent protein
(GFP) retroviral deoxyribonucleic acid (DNA) in differentiated cells from
A; arrow indicates 4-kilobase pair (kbp) junction fragment in BamHI-
digested DNA from undifferentiated cells of a GFP-positive subclone of AFS
cells (Lane 1) and the second round subclone (Lane 2) used for transcript
analysis after differentiation under adipogenic (Lane 3), endothelial (Lane
4), hepatic (Lane 5), osteogenic (Lane 6), myogenic (Lane 7), and neuro-
genic (Lane 8) conditions (from, De Coppi P, Bartsch G Jr, Siddiqui MM,
Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ,
Furth ME, Soker S, Atala A: Isolation of amniotic stem cell lines with
potential for therapy. Nat Biotechnol 25:100–106, 2007 [20]).
VOLUME 64 | NUMBER 1 | JANUARY 2009 | 25
FARIN ET AL.
A B
C C
D
FIGURE 10. Neurogenic differentiation of human AFS cells in culture. A,
immunofluorescence staining for nestin (a neural marker) after 8 days in
the first stage of neurogenic differentiation. B, phase contrast micrograph
showing cells after the second stage of neurogenic differentiation under
conditions biased for production of dopaminergic neurons; individual cells
with pyramidal morphology were assessed for potassium channels by volt-
age clamping. C, RT-PCR showing expression of the housekeeping enzyme
glyceraldehyde-3-phosphate dehydrogenase (Lane 1) and the neuronal G
protein-coupled, inwardly rectifying K+ channel (GIRK2, Lane 2) after sec-
ond stage of neurogenic differentiation. M, size markers. Sizes of PCR
products are shown. bp, base pairs. D, secretion of neurotransmitter glu-
tamic acid in response to potassium ions in cells after neurogenic differen-
tiation in the presence of nerve growth factor (NGF) (open bars) and after
maintenance of undifferentiated cells in standard growth medium (closed
bars) (from, De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC,
Perin L, Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ, Furth ME, Soker
S, Atala A: Isolation of amniotic stem cell lines with potential for therapy.
Nat Biotechnol 25:100–106, 2007 [20]).
Isolating cells that were successfully reprogrammed was
more efficiently accomplished in 2007 by inserting a gene for
antibiotic resistance that is activated only when proteins charac-
teristic of stem cells are expressed. Antibiotics are used to elim-
inate nonreprogrammed cells (83). A group led by Rudolf
Jaenisch (136) at the Whitehead Institute for Biomedical
Research in Cambridge, MA, and a collaborative effort between
26 | VOLUME 64 | NUMBER 1 | JANUARY 2009
A B
D
E F
FIGURE 11. Engraftment of neurogenically differentiated human AFS
cells in mouse brain. Differentiation was induced by incubation with NGF,
and cells were injected into brains of newborn mice. Samples shown were
obtained 1 month after injection. Red staining with antibody to a 65-kDa
mitochondrial protein (perinuclear localization) reveals human cells. Blue
staining (4ⴕ,6-diamidino-2-phenylindole, DAPI) is a fluorescent stain that
binds strongly to DNA; superimposed image in D and insets of A, B, E, F
show cell nuclei. A, lateral ventricle; B, higher-magnification view of lat-
eral ventricle; C, periventricular area and hippocampus; D, same field as C,
with DAPI staining superimposed; E, third ventricle; F, olfactory bulb
(from, De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L,
Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ, Furth ME, Soker S, Atala
A: Isolation of amniotic stem cell lines with potential for therapy. Nat
Biotechnol 25:100–106, 2007 [20]).
Konrad Hochedlinger of the Harvard Stem Cell Institute and
Kathrin Plath (63) of the University of California, Los Angeles,
used the same factors as Yamanaka did in 2007 and obtained
similar results. These groups produced chimeric mice using
induced pluripotent cells that were isolated in this manner, and
the mice passed on induced pluripotent DNA to their offspring.
Jaenisch also produced embryos and fetuses whose cells were
derived entirely from induced pluripotent cells.
This June 2007 discovery was laudable for providing pluri-
potent stem cells that were genetically matched to individuals
while minimizing ethical and technical dilemmas involved in
embryonal destruction or human cloning to otherwise obtain a
genetic match. Whereas human cloning is limited by the num-
ber of available eggs and technical difficulties, Yamanaka’s
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 FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY

structed blastocysts expressed

A B C markers similar to those ex-
pressed by fertilized embryos.
The authors summarized, “Our
results represent a significant
breakthrough in elucidating
the role of nuclear remodeling
events in reprogramming fol-
lowing SCNT” (71, p 2232).
In 2004, cell nuclear transfer,
or cloning, was reportedly used
by Hwang Woo-Suk in Korea
D E F to produce a line of human ES
cells from fertilized human
oocytes. His work was sub-
sequently shown to be fabri-
cated.

October 2007: Nobel

Prize Awarded for
Engineering Knockout
Mice with ES Cells
The 2007 Nobel Prize for
Physiology or Medicine was
awarded to Mario Capecchi,
Martin Evans, and Oliver
FIGURE 12. Tissue engineered bone from human AFS cells. A, calcium deposition of human AFS cells maintained Smithies for their discoveries
in osteogenic differentiation medium in vitro was quantified by measuring calcium-cresolophthalein complex levels
of “principles for introducing
(solid line) and compared with that of undifferentiated (broken line) AFS cells. B, staining of control alginate/collagen
scaffold recovered 8 weeks after implantation in nu/nu mouse. C, staining of scaffold of AFS cells in alginate/collagen
specific gene modifications in
scaffold recovered 8 weeks after implantation; black staining indicates strong mineralization. D–F, micro-computed mice by the use of embryonic
tomographic scan of mouse 18 weeks after implantation of constructs. 䉴, region of implantation of control scaffold with- stem cells” and generating
out AFS cells; * and 䉫, scaffolds seeded with AFS cells. Close-up views of seeded scaffolds are indicated by symbols knockout mice (Fig. 13) (124).
(E, F) (from, De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC, Snyder The magnitude of their enor-
EY, Yoo JJ, Furth ME, Soker S, Atala A: Isolation of amniotic stem cell lines with potential for therapy. Nat mous contribution to medi-
Biotechnol 25:100–106, 2007 [20]). cine is challenging to summa-
rize adequately. Their efforts
method utilizes the most basic cells and simple laboratory tech- emphasize, in this most crucial context, the irreplaceable value
niques. Conceivably, if the same techniques were applied to of pluripotent ES cells.
humans, researchers could produce iPS cells from patients with Capecchi and Smithies initially noted that homologous recom-
Parkinson’s disease or diabetes for therapeutic cell replace- bination, or the natural exchange of DNA sequences within chro-
ment. However, the induced pluripotent cells cannot, as of mosome pairs to increase genetic variety, could be instigated
now, be used safely to generate genetically matched cells for between introduced DNA and endogenous chromosomes, result-
transplantation, as the technique carries a 20% risk of onco- ing in repair of defective genes. It appeared that any mammalian
genic potential in mice. gene could be targeted for specific modification by homologous
recombination. Evans then provided the crucial conduit to the
June 2007: First Successful Primate Stem Cell mouse germline so that DNA modifications would be inherited
Line Created via the ES cell. He observed that ES cells with normal karyotypes
Scientist Shoukhrat Mitalipov of Oregon Health & Science could be established from early mouse embryos. Embryos from
University reported the first successful creation of a primate one mouse strain were injected with ES cells from another mouse
stem cell line through somatic cell nuclear transfer (SCNT) (10, strain, resulting in mosaic embryos that were then carried to term
71). This technique requires cytoplast-mediated reprogramming by surrogate mothers. The mosaic offspring was subsequently
of the donor nucleus through nuclear remodeling. This triumph mated. ES cell-derived genes were then identified in pups, reflect-
had proven challenging during previous attempts at SCNT in ing Mendelian inheritance of genetic modifications. Evans began
primates; in this case, more than 20% of SCNT embryos recon- to modify the ES cells with retroviruses, which integrated their
structed with fetal fibroblasts progressed to blastocysts. Suc- genes into the embryonic chromosomes, generating the first gene-
cessful reprogramming was reflected in the fact that recon- targeted ES cells. Evans’ findings, along with Capecchi and

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FARIN ET AL.

produce almost any variety of

DNA modification in the
mouse genome by gene target-
ing and thereby investigate
almost every feature of mam-
malian physiology. This potent
technical feat of gene targeting
in mice has already produced
more than 500 different mouse
models of human disease,
enabling investigators to con-
firm the roles of hundreds of
genes in mammalian embry-
onic and fetal development
including organogenesis and
body plan establishment, adult
physiology, aging, and disease
mechanisms. The models also
enable testing of the effects of
gene therapy. Today, gene tar-
geting has evolved so far as to
permit the introduction of tem-
porally regulated mutations as
well as organ-specific muta-
tions during development and
in the adult animal. As the
Nobel Committee aptly noted,
the birth of the knockout
mouse marked the beginning
of a new era in genetics.

November 2007: iPS Cells

Developed from
Adult Human Fibroblasts
Only a few months after
Yamanaka’s 2007 publication
(83), 2 similar articles by Taka-
hashi et al. (122) and Yu et al.
(143) showed that human iPS
cells could be generated from
mature human dermal fibro-
blasts. Through SCNT, trans-
acting factors of the mamma-
FIGURE 13. General strategy for gene targeting in mice (from, The Nobel Prize in Physiology or Medicine 2007. lian oocyte reprogram somatic
http://nobelprize.org/nobel_prizes/medicine/laureates/2007/press.html. Accessed May 1, 2008 [124]). nuclei to an undifferentiated
state. Human iPS cells were
Smithies’ demonstration that genes could be targeted by homolo- similar to human ES cells in morphology, karyotypes, prolifer-
gous recombination in cultured cells, led to the concept of homol- ation, surface antigens, and gene expression. In addition, iPS
ogous recombination in ES cells. Initial experiments were per- cells could differentiate into teratomas in vivo. This momen-
formed with the hprt gene for Lesch-Nyhan syndrome. Capecchi tous achievement demonstrated the feasibility of producing a
had developed a more advantageous tactic for targeting genes, pluripotent stem cell from almost any other human cell rather
termed positive-negative selection. The original documentation than necessitating an embryo or egg. As Takahashi et al. (122)
of homologous recombination in ES cells used to generate gene- pointed out, “Successful reprogramming of differentiated
targeted mice was published in 1989. human somatic cells into a pluripotent state would allow cre-
To date, more than 10 000 murine genes, or half the genes in the ation of patient- and disease-specific stem cells” (122, p 861)
mammalian genome, have been knocked out, with the capacity to with tremendous clinical applications in disease models, drug

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development, and transplantation medicine. However, the risk
of tumorigenesis resulting from retroviral gene transfer (Oct3/4,
Sox2, Klf4, and c-Myc in Yamanaka’s work and Oct4, Sox2,
NANOG, and Lin28 in Thomson’s work) requires more analysis.
January 2008: Human ES Cell Lines Generated without
Embryo Destruction
Advanced Cell Technology announced the development of 5
human ES cell lines generated without embryonal destruction
(13). All previous human ES cell lines involved destruction of
embryos. These results have the potential to definitively end
the ethical debate surrounding the use of embryos to derive
stem cells.
Single cells were removed from embryos using a technique
similar to preimplantation genetic diagnosis. The biopsied
embryos continued to develop normally to the blastocyst stage
and were then frozen. The isolated cells were cultured with the
use of a proprietary methodology recreating the optimal inner
cell mass developmental environment. This substantially
improved the efficiency of pluripotent stem cell derivation,
achieving a rate comparable to the traditional approach of
deriving stem cells from a blastocyst’s inner cell mass. The 5
lines of stem cells maintained normal karyotypes and markers
of pluripotency for up to 50 passages. They also differentiated
into cell types of all 3 germ layers, including blood cells, neu-
rons, myocytes, cartilage, and other cell types of therapeutic
significance. Human ES cell coculture was not essential to the
derivation. The stem cell lines that were generated appear to
have the same characteristics as other human ES cell lines,
including expression of the same markers of pluripotency, self-
renewing capacity, and genetic stability. Robert Lanza, M.D.,
Chief Scientific Officer at Advanced Cell Technology and senior
author of the article, commented, “If the White House approves
this new methodology, researchers could effectively double or
triple the number of stem cell lines available within a few
months. Too many needless deaths continue to occur while this
research is being held up. I hope the President will act now and
approve these stem cell lines quickly” (1a).
January 2008: Development of Human Cloned Blastocysts
Scientists at Stemagen Corp. in La Jolla, CA, claimed develop-
ment of human cloned blastocysts after SCNT with adult fibrob-
lasts (25). First, oocytes obtained from egg donors who were 20
to 24 years of age were enucleated. Then, SCNT embryos were
constructed using skin cells from 2 men. The cells were incu-
bated; cleavage and blastocyst development were observed.
Unfortunately, no stem cells were extracted, but DNA was con-
firmed to match the male donors. Blastocysts were also obtained
from parthenogenetically activated oocytes. In 2005, British sci-
entists reported using human ES cells to produce a cloned
embryo, but no stem cells were extracted then, either. This study
demonstrates, for the first time, that SCNT can be used to gen-
erate cloned human blastocysts using differentiated adult donor
nuclei that are reprogrammed by human oocytes. Dr. Samuel
Wood, coauthor of the article and Chief Executive of Stemagen,
reports that he is now attempting to produce stem cell lines
NEUROSURGERY
FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY
from the embryos. Richard Doerflinger, spokesman for the
United States Conference of Catholic Bishops, criticized that the
process still “involves creating human lives in the laboratory
solely to destroy them for alleged benefit to others” (25).
Although there was no progress beyond the blastocyst stage, the
process did enable all intended parents to successfully achieve
ongoing pregnancies with the oocytes retained for reproduc-
tive purposes.
February 2008: Improved Generation of Mouse iPS Cells
In February 2008, Yamanaka developed murine iPS cells that
appeared to be more similar to ES cells than the previously
developed induced pluripotent cells. Previously, iPS cells gener-
ated from mouse and human fibroblasts required retroviral
transduction of 4 transcription factors. In their recent article,
Aoi et al. (2) describe generation of iPS cells from adult mouse
hepatocytes and gastric epithelial cells without the need for
retroviral integration. Specifically, 4 transcription factors
(Oct3/4, Sox2, Klf4, and c-Myc) were introduced by retroviral
vectors into hepatocytes or gastric epithelial cells. iPS cells were
generated by germline transmission of induction genes, as
judged from the presence of transgenes, without retroviral inte-
gration into specific sites. Few retroviral integration sites were
noted at all, and no common retroviral integration sites were
found among multiple clones. Instead, integration sites were
randomly distributed in multiple chromosomes. Each of the 4
factors were individually omitted to assess the effect on the gen-
eration of iPS cells. When Oct3/4, Sox2, or Klf4 were omitted,
no iPS cell colonies emerged. c-Myc omission decreased colony
numbers 20 to 40%, reflecting its minor role in the generation of
cells, compared with its role in generating iPS fibroblast cells.
Generation of iPS cells with gene transfer methods, or direct
reprogramming of lineage-committed somatic cells, instead of
by retroviral integration of induction genes into specific sites,
diminishes tumorigenicity after transplantation into patients
and is a major technical advance (2).
An abridged timeline of the aforementioned significant
events in stem cell science is illustrated in Figure 14.
CONCLUSIONS
Although stem cell research has been applied to an extensive
list of disorders (19, 42, 43, 50, 82, 84–86, 88, 96, 119), its true
long-term potential extends beyond any narrow discipline.
Investigators are optimistic that, in the coming decades, stem
cells may be used successfully to battle medicine’s toughest
foes: malignant cancer, cardiovascular disease, and central nerv-
ous system injuries and deficits. Those in need of transplants
hope newly engineered organs will one day obviate the need to
wait for organs. Perhaps most interestingly, the embryogenetic
mishaps leading to birth defects will be better characterized by
studying stem cells. Immediate future efforts must include as
their focus safeguarding humans against any tumorigenic
potential from stem cell transplantation.
Although stem cell research is still in its infancy, its imagina-
ble clinical applications seem almost limitless. These potentially
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FARIN ET AL.

FIGURE 14. Timeline of

significant events in stem
cell science. SCID, severe
combined immunodefi-
ciency; NIH, National
Institutes of Health.

30 | VOLUME 64 | NUMBER 1 | JANUARY 2009 www.neurosurgery-online.com


immortal cells are believed to be the key to understanding the
mysterious processes of human embryogenesis, development,
aging, and disease. The ability to generate any type of cell in the
body theoretically allows stem cells to repair any damaged or
inadequate tissue and conceivably to cure disease. Patients and
physicians alike eagerly anticipate the safe, effective, and ethi-
cal clinical use of stem cells for therapeutic applications.
Disclosure
The authors have no personal financial or institutional interest in any of the
drugs, materials, or devices described in this article.
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Acknowledgment
We thank James C. Wald, Esq., for his assistance in produing the timeline for
this article.
COMMENTS
I n this article, Farin et al. provide the first of a 5-part review of stem
cell biology. This component tells the history, from the early work in
the 1960s to dramatic developments in the past few months regarding
the production of stem cells from adult mammalian cells. The series is
background for neurosurgeons on what, we hope, will be the next
wave in medicine—and especially in central nervous system (CNS)
regenerative medicine—for battles with cancer, cardiovascular disease,
CNS regeneration injuries, or congenital deficits. The authors correctly
34 | VOLUME 64 | NUMBER 1 | JANUARY 2009
point out that the thorough evaluation of this topic will include the his-
tory, scientific proofs of CNS neurogenesis, as well as the application of
this technology not only to stem cells but also to the derivation and
control of function of other adult cells; finally, the article presents a dis-
cussion of the philosophical and ethical issues that surround this topic.
The work is still in its very early stages, but this review suggests that
exciting work in associated areas, such as cloning and knockout mice,
has already completely changed the way we assess the pathophysiol-
ogy of disorders. Also, it highlights the use of stem cell techniques to
delineate causes of neoplasia in the CNS. We look forward to partici-
pating as a discipline in the development of this potential evolution in
diagnosis, pathophysiology, and therapeutic advances.
Robert J. Dempsey
Madison, Wisconsin
T he identification of stem cells in the CNS is the single most impor-
tant scientific discovery in our lifetime. Let me repeat that: the sin-
gle most important scientific discovery in our lifetime!
This report provides a chronology of the major events in the evolu-
tion of stem cell work in the CNS. It serves as a reference for all of us
to appreciate how our discipline has evolved. The residents that we
train today will look back at this era as a turning point in the treatment
of neurological disease. The future of stem cell work will alter their per-
ceptions of CNS tumors, vascular disease, degenerative disease, and
trauma. This is a wonderful time to enter the field of neurosurgery.
Current residents will someday use the tools described here to cure the
diseases and injuries that we can only manage. This report is an intro-
duction into how we arrived at this place. Subsequent reports on stem
cell biology and research will show our readers where we are headed.
Joseph M. Piepmeier
New Haven, Connecticut
I n this comprehensive treatise, the authors review some of the histor-
ical developments that have led to the current excitement in the
discipline of stem cell biology. From the earliest days, the study of
embryology and the discovery that stem cells exist have provided
tremendous promise in terms of potential regeneration/repair of tis-
sues damaged by age and/or disease. The scientists of this discipline
have been very cautious in ensuring that over-interpretation of results
does not lead to therapeutic adventures that could have disastrous
repercussions in terms of unfulfilled outcomes in human patients.
This steady and cautious progress is now beginning to bear fruit: the
most recent discoveries of reprogramming of adult differentiated cells
back into a stem cell state through re-expression of a set of only 4 genes
(Oct 3/4, Sox2, KLf4, and c-Myc or Oct4, Sox2, NANOG, and Lin28)
provide evidence that only a few master pathways will require re-
instruction to achieve the desired effect of “stemness.” This type of
finding thus opens up avenues of research related to drug or ligand dis-
coveries that may lead one to turn these sets of genes back on, in any
cell, to repair a tissue or organ.
The authors provide us with a valuable timeline of the key discover-
ies that have led us to the current state of scientific fervor. The reader will
find this a valuable review to bring himself or herself up to date on the
nomenclature and key figures of this discipline over the past 50 years.
E. Antonio Chiocca
Columbus, Ohio
www.neurosurgery-online.com
 FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY

TABLE A1. Glossarya

Adult (or somatic) stem cell An undifferentiated cell found in a differentiated tissue that can renew itself and differentiate (with certain limitations) to give
rise to all the specialized cell types of the tissue from which it originated; i.e., possessing multipotency and unipotency but not
pluripotency in its native state. Pluripotency has been achieved recently through environmental and genetic manipulations.
Allogeneic transplantation Cell, tissue, or organ transplants from 1 member of a species to a genetically other member of the same species.
Autologous transplantation Cell, tissue, or organ transplants from 1 individual back to the same individual. Such transplants from self do not induce an
immune response and are not rejected. For example, a cancer patient may have her hematopoietic stem cells or bone marrow
removed and stored during treatment with radiation or chemotherapy to kill all blood-forming cells (and, perhaps, all cancer);
her blood-forming capacity is then rescued with autologous hematopoietic stem cells or bone marrow.
Blastocoel The fluid-filled cavity inside the blastocyst of the developing embryo.
Blastocyst A thin-walled hollow sphere made up of an outer layer of cells (trophoblast) that forms the placenta, a fluid filled cavity
(blastocoel), and an inner cell mass containing pluripotent embryonic stem cells. Also called the blastula, the blastocyst is a
preimplantation embryo of about 150 cells produced by cleavage of the zygote after fertilization at approximately 5 days. Further
reproductive development occurs only if the blastocyst is successfully implanted in the uterus.
Blastomere A cell produced during cleavage of a fertilized egg.
Bone marrow stromal cells Also known as mesenchymal stem cells; a mixed population of stem cells derived from the non-blood-forming fraction of bone
marrow that does not give rise to blood cells but instead generates bone, cartilage, fat, and fibrous connective tissue.
Cell culture A technique for growing or maintaining cells on an artificial medium (substance) under laboratory conditions (e.g., Petri dish or
test tube).
Cell division Method by which a single cell divides to create 2 cells. There are 2 main types of cell division: mitosis and meiosis.
Cell line Cells that can be maintained and grown in culture and display an immortal or indefinite life span.
Cell type A specific subset of cells within the body, defined by their appearance, location, and function.
Cell-based therapies Treatment in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cells
or tissues, alter normal cell response, stimulate native signaling cascades, perform missing metabolic functions, or changing
the normal course of repair into true regeneration.
Cellular reprogramming Transformation of a somatic cell into a pluripotent one via manipulation of regulatory genes and gene products that govern the
state of pluripotency with the purpose of efficiently generating human isogenic pluripotent stem cells and improving the efficiency
of somatic cell nuclear transfer.
Chondrocyte The functional cell type that makes cartilage for joints, ear canals, trachea, epiglottis, larynx, the discs between vertebrae, and
the ends of ribs.
Cleavage The early divisions of the fertilized egg.
Clone To generate identical copies of a molecule, cell, or organism. When it is used to refer to cells grown in a tissue culture dish, a
clone is a line of cells that is genetically identical to the originating cell. This cloned line is produced by cell division (mitosis)
of the originating cell. The term clone may also be used to refer to an animal produced by somatic cell nuclear transfer.
Cloning The process in which an organism, molecule, or cell produces 1 or more genetically alike copies of itself by asexual means.
Cloning may occur by propagation of cuttings, as in the case of plants; continual budding, as in the case of hydra; fission, as in
the case of bacteria and protozoa; parthenogenic asexual reproduction, as in the case of aphids; or somatic cell nuclear transfer,
as in the case of higher-order animals such as mammals. The term cloning can also be applied to a group of cells undergoing replication
by repetitive mitoses (cell divisions). “Therapeutic cloning” creates a line of stem cells genetically identical to the originating cell
for use in research. “Reproductive cloning” creates an organism genetically identical to the organism providing the originating cell.
Culture medium The liquid that covers cells in a culture dish and contains nutrients to feed the cells. Medium may also include other growth factors
added to produce desired changes in the cells.
Deoxyribonucleic acid (DNA) A chemical found primarily in the nucleus of cells; the genetic code.
Differentiation Progressive restriction of the developmental potential and increasing specialization of function which takes place during the
development of the embryo and leads to the formation of specialized cells, tissues, and organs.
Directed differentiation Manipulating stem cell culture conditions to induce differentiation into a particular cell type.
Ectoderm The outermost of 3 germ layers derived from the inner cell mass of the blastocyst that gives rise in later development to the skin,
cells of the amnion and chorion, nervous system, enamel of the teeth, lens of the eye, and neural crest.
Embryo The product of a fertilized egg, from the zygote until the fetal stage.
Embryoid bodies Spheroid colonies seen in culture, produced by the growth of embryonic stem cells in suspension. Embryoid bodies contain
cell types derived from all 3 germ layers, and the distribution and timing of the appearance of specific cell types corresponds to
that observed within the embryo.

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TABLE A1. continued.

Embryonic germ cells Pluripotent stem cells that are derived from early germ cells that exist between the blastocyst stage of development until their
conversion within gonads to egg or sperm stem cells. Embryonic germ cells are thought to have properties similar to those of embryonic
stem cells.
Embryonic stem cells Pluripotent undifferentiated cell lines established usually from the inner cell mass of the blastocyst stage (5-day-old embryo) of
development. Within the population of cultured embryonic stem cells are cells that can produce more embryonic stem cells or,
under conditions of differentiation, give rise to collections of cells that include most, if not all, cell types that can be found in a
postimplantation embryo, fetus, or developed organism, but not trophoblast or placenta. To date, no embryonic stem cells
cultured in vitro can give rise to developed organisms or even developed organs. A test of pluripotency of mouse embryonic stem
cells is to inject them into blastocysts before implantation; the progeny of these cells can participate in all germline and somatic
tissues along with host blastocyst-derived cells. By definition, an embryonic stem cell is self-renewing (can replicate itself), is pluripotent
(can form all cell types found in the body), and theoretically is immortal.
Endoderm The inner of 3 germ layers of the early embryo that gives rise in later development to tissues such as the lungs, the intestine, the
liver, and the pancreas.
Enucleated A cell with its nucleus removed.
Fertilization The joining of the male gamete (sperm) and the female gamete (egg).
Fetus The stage in development from the end of the embryonic stage, 7–8 weeks after fertilization, to developed organism that ends
at birth.
Fibroblast A connective or support cell found within most tissues of the body, such as skin. Fibroblasts provide an instructive support
scaffold to help the functional cell types of a specific organ perform correctly.
Gamete A reproductive cell containing half of the genetic material necessary to form a complete human organism. During fertilization,
male and female gametes (sperm and ovum, respectively) fuse, producing a zygote.
Gene A functional unit of heredity that is a segment of DNA found on chromosomes in the nucleus of a cell. Genes direct the formation
of an enzyme or other protein.
Germ layers Fertilization of an egg stimulates cell division, and the resulting cells are organized into 3 different layers, called germ layers. The
3 germ layers are the endoderm, mesoderm, and ectoderm and are the 3 precursory tissue layers of the early, primitive embryo
(which form at approximately 2 weeks in the human) that give rise to all tissues of the body.
Germline cells Cells that arise from the inner cell mass and are irreversibly committed to give rise to eggs or sperm. Primitive germline cells (which
are germline stem cells) migrate from the posterior of the early postimplantation embryo to the developing gonads, known as genital
ridges, where they commit to spermatogenesis (in testes) and oogenesis (in ovaries).
Hematopoiesis The development and formation of various types of blood cells.
Hematopoietic cell Transplantation of cells with blood-forming potential, usually bone marrow. Hematopoietic stem cells
transplantation provide rapid and sustained reconstitution of blood formation and are found in adult bone marrow, umbilical cord blood,
mobilized peripheral blood (the nucleated cell fraction of blood after treatment of the donor with agents that increase the passage
of hematopoietic stem cells from bone marrow to blood), and fetal liver.
Hematopoietic Progenitor cells from which all blood cells derive (red blood cells, white blood cells, platelets).
stem cells Hematopoietic stem cells give rise to 2 distinct cells: a replica of the stem cell and a cell that will further proliferate and
differentiate into a mature blood cell. Hematopoietic cells are found within the bone marrow of adults. In the fetus, hematopoietic
cells are found within the liver, spleen, bone marrow, and amniotic fluid surrounding the fetus in the womb. These precursors
of mature blood cells are defined by their ability to replace the bone marrow system after its obliteration (for example, by
[gamma]-irradiation) and can continue to produce mature blood cells.
Hepatocyte The functional cell type of the liver that makes enzymes for detoxifying metabolic waste, destroying red blood cells and reclaiming
their constituents, and synthesis of proteins for blood plasma.
Histocompatible A tissue or organ from a donor (the person giving the organ or tissue) that will not be rejected by the recipient (the patient in whom
the tissue or organ is transplanted). Rejection is caused because the immune system of the recipient sees the transplanted organ
or tissue as foreign and attempts to destroy it. Tissues from 2 random people are rarely histocompatible. In siblings, the probability
of histocompatibility is higher, while identical twins are almost always histocompatible.
Homologous Similar or uniform, often used in the context of genes and DNA sequences. In the context of stem cells, the term homologous
recombination is a technique used to disable a gene in embryonic stem cells.
Homologous A technique used to inactivate a gene and determine its function in a living animal. The process of
recombination homologous recombination is more efficient in embryonic stem cells than in other cell types. It is achieved by introducing a stretch
of DNA that is similar or identical (homologous) to part of a gene and to some of the DNA surrounding the gene, but different
(not homologous) to a specific section of the gene. The DNA is then introduced into the stem cells, and the stretch of homologous
DNA will recognize the similar sequences of the gene within the cell and replace it. The cell is then left with a segment of DNA
in the gene that has the incorrect sequence, which interrupts gene function. The gene is then said to be knocked out. From these
embryonic stem cells, an entire mouse can be made by injecting the altered stem cells into a blastocyst, and implanting the
blastocyst into a female mouse. This is a way to make genetically engineered mice with altered gene function. These experiments
are crucial to understand how specific genes work and interact in living animals.

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 FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY

TABLE A1. continued.

Human embryonic A pluripotent stem cell that is derived from the inner cell mass of a blastocyst and can differentiate into several tissue types in a
stem cell dish. They are similar to embryonic stem cells from the mouse; however, human embryonic stem cells are harder to grow than
mouse embryonic stem cells.
In vitro Latin for “in glass”; in a laboratory dish or test tube; an artificial environment.
In vitro fertilization A technique in which an egg is fertilized by sperm outside the body. For use in assisted reproduction, the fertilized egg is
implanted in the uterus at approximately 3 to 4 days of cell division for the purpose of development into a baby. For use in
research, the fertilized egg is maintained in cell culture until it develops into the blastocyst stage at approximately 5 days of
cell division and stem cells can be removed.
Inner cell mass A small group of cells attached to the wall of the blastocyst (the embryo at a very early stage of development). Embryonic stem
cells are made by isolating and culturing the cells that make up the inner cell mass. In development, it is the inner cell mass that
will eventually give rise to all the organs and tissues of the future embryo and fetus, but it does not give rise to the extraembryonic
tissues, such as the placenta.
Long-term self-renewal The ability of stem cells to renew themselves by dividing into the same nonspecialized cell type over long periods (many months
to years) depending on the specific type of stem cell.
Meiosis Cell division of a gamete to reduce the chromosomes within it to half the normal number. This is to ensure that fertilization
restores the full number of chromosomes rather than causing aneuploidy, or an abnormal number of chromosomes.
Mesenchymal stem cells Also known as bone marrow stromal cells; cells from the immature embryonic connective tissue mainly found in bone marrow
that can develop into distinct mesenchymal tissue such as bone, fat, tendons, lymphatic tissue, muscles, adipose tissue, cartilage,
nervous tissue, and blood and blood vessels.
Mesoderm Middle germ layer of a group of cells derived from the inner cell mass of the blastocyst; it gives rise to bone, muscle, connective
tissue, kidneys, and related structures.
Microenvironment The molecules and compounds, such as nutrients and growth factors in the fluid surrounding a cell in an organism or in the laboratory,
that play an important role in determining the characteristics of the cell.
Mitosis Cell division that allows a population of cells to increase its numbers or to maintain its numbers.
Morphology Study of the shape and visual appearance of cells, tissues, and organs.
Morula A globular solid mass of cells (called blastomeres) formed by cleavage of a zygote.
Multipotency Ability of a single stem cell to develop into more than 1 cell type of the body.
Multipotent stem cells Stem cells whose progeny are of multiple differentiated cell types, but all within a particular tissue, organ, or physiological
system. These relatively undifferentiated cells of the same lineage retain the ability to divide and cycle throughout postnatal life
to provide cells that can become specialized and take the place of those that die or are lost. For example, blood-forming
(hematopoietic) stem cells are single multipotent cells that can produce progeny that include hematopoietic stem cells, blood
cell-restricted oligopotent progenitors, and all cell types and elements (e.g., platelets) that are normal components of the blood.
Other examples include myoblasts, myeloid progenitor cells, fibroblasts, tumor stem cells, and skin stem cells.
Myocyte The functional cell type of muscles.
Neural stem cell A type of stem cell that resides in the brain and can make new neurons and glia (oligodendrocytes and astrocytes).
Oligodendrocyte A supporting cell that provides insulation to nerve cells by forming a myelin sheath (a fatty layer) around axons.
Oligopotent progenitor cells Progenitor cells that can produce more than 1 type of mature cells. For example, the clonal common myeloid progenitor is a progenitor
cell which can give rise to blood granulocytes, monocytes, red blood cells, platelets, basophils, eosinophils, and dendritic cells,
but not T lymphocytes, B lymphocytes, or natural killer cells.
Oocyte An egg before maturation; a female gametocyte; also called an ovocyte.
Parthenogenesis A form of reproduction in which an egg is activated without the fusion of sperm with the egg cell. The egg behaves as if it has
been fertilized. Parthenogenesis occurs commonly among insects and other arthropods. Artificially inducing parthenogenesis with
human eggs may be a means to isolate stem cells from an embryo without fertilization.
Passage A round of cell growth and proliferation in cell culture.
Phenotype The description of the characteristics of a cell, a tissue, or an animal. For example, black and white fur of a mouse are 2
phenotypes that can be found. The phenotype is determined by the genes (or the genotype) and by the environment. For example,
short stature is a phenotype that can be genetically determined (and therefore inherited from the parents), but it can also be
caused by malnourishment during childhood (and therefore be caused by the environment).
Placenta The oval spongy structure in the uterus from which the fetus derives its nourishment and oxygen. The placenta develops from
the outer cell layer of the blastocyst, called the trophoblast.
Plasticity The ability of stem cells from an adult tissue to generate the differentiated cell types of another tissue.
Pluripotency Ability of a single stem cell to give rise to all of the various cell types that make up the body. Pluripotent cells cannot make so-
called “extraembryonic” tissues, such as the amnion, chorion, and other components of the placenta. Scientists demonstrate
pluripotency by providing evidence of stable developmental potential, even after prolonged culture, to form derivatives of all 3
embryonic germ layers from the progeny of a single cell and to generate a teratoma after injection into an immunosuppressed
mouse.

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FARIN ET AL.

TABLE A1. continued.

Pluripotent stem cells Stem cells that include in their progeny all cell types that can be found in a postimplantation embryo, fetus, or developed
organism, but not embryonic components of the trophoblast and placenta (these are usually called extraembryonic). Pluripotent
stem cells give rise to multipotent and unipotent stem cells as the embryo develops.
Polar body Structure produced when an early egg cell, or oogonium, undergoes meiosis. In the first meiosis, the oogonium divides its
chromosomes evenly between the 2 cells but divides its cytoplasm unequally. One cell retains most of the cytoplasm, while the
other gets almost none, leaving it very small. This smaller cell is called the first polar body. The first polar body usually degenerates.
The ovum, or larger cell, then divides again, producing a second polar body with half the amount of chromosomes but almost no
cytoplasm. The second polar body splits off and remains adjacent to the large cell, or oocyte, until it (the second polar body)
degenerates. Only 1 large functional oocyte, or egg, is produced at the end of meiosis.
Postimplantation embryos Implanted embryos in all early stages of development until the establishment of the body plan of a developed organism with identifiable
tissues and organs.
Pre-embryos Eggs that are fertilized and develop to approximately 2–3 days old in a laboratory (in vitro fertilization) and are ready for
implantation for the purposes of human reproduction or removal of stem cells for the purposes of research.
Preimplantation With regard to an embryo, preimplantation indicates that the embryo has not yet implanted in the wall of the uterus. Human embryonic
stem cells are derived from preimplantation stage embryos fertilized outside a woman’s body (in vitro).
Preimplantation embryos Fertilized eggs (zygotes) and all of the developmental stages up to, but not beyond, the blastocyst stage.
Primitive streak The beginning of the vertebral column in the human embryo that develops at approximately 14 days after conception.
Progenitor cell Derived from stem cells and intermediate to the production of mature cells. Often incorrectly confused with stem cell; an early
descendant of a stem cell that can only differentiate, but not renew itself. In contrast, a stem cell exhibits potency and self-
renewal. A progenitor cell is often more limited in the types of cells it may develop into, compared with a stem cell, i.e., it
exhibits increasing differentiation.
Proliferation Expansion of cells by the continuous division of single cells into 2 identical daughter cells.
Regenerative medicine A treatment in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed
cell populations or tissues. (See also Cell-based therapies.)
Reproductive cloning Creation of an animal being identical to the animal that donated the somatic cell nucleus. The embryo is implanted in a uterus
and develops into a live being. The first animal to be created by reproductive cloning was Dolly, the sheep born at the Roslin
Institute in Scotland in 1996. (See also Somatic cell nuclear transfer).
Signals Internal and external factors that control changes in cell structure and function.
Somatic cell nuclear transfer A technique that combines an enucleated egg (nucleus removed) and the nucleus of a somatic cell to make an embryo. Somatic
cell nuclear transfer can be used for therapeutic or reproductive purposes, but the initial stage that combines an enucleated egg
and a somatic cell nucleus is the same. With regard to reproductive cloning, somatic cell nuclear transfer attempts to produce
a fetus that is implanted into a woman’s uterus; the live offspring is genetically identical to the donor of the somatic cell DNA.
Somatic cells All cells within the developing or developed organism with the exception of germline cells.
Somatic stem cells Nonembryonic stem cells that are not derived from gametes (egg or sperm cells).
Stem cell line Stem cells that have been growing in cell culture for 6 or more months without becoming specialized and appear genetically normal.
Stem cell transplantation The transfer of stem cells from 1 individual to another within the same species (homologous transplantation) or between species
(xenotransplantation), or transfer within the same individual (autologous transplantation). The source and location of the stem
cells determines their potency or pluripotency to differentiate into various cell types.
Stem cells Cells with the ability to divide for indefinite periods in culture (self-renewal) and to give rise to specialized cells (potency).
Stromal cells Non-blood cells derived from blood organs, such as bone marrow or fetal liver, which are capable of supporting growth of
blood cells in vitro. Stromal cells that make the matrix within the bone marrow are derived from mesenchymal stem cells.
Subculturing Transferring cultured cells, with or without dilution, from 1 culture vessel to another.
Surface markers Proteins on the outside surface of a cell that are unique to certain cell types, which are visualized using antibodies or other
detection methods.
Teratoma Scientists verify that they have established a human embryonic stem cell line by injecting putative stem cells into mice with a
dysfunctional immune system. Since the injected cells cannot be destroyed by the mouse’s immune system, they survive and form
a multilayered benign tumor called a teratoma. Teratomas serve to establish the ability of a stem cell to give rise to all cell types
in the body, as teratomas contain cells derived from each of the 3 embryonic germ layers.
Therapeutic cloning Somatic cell nuclear transfer, or cloning, for the isolation of embryonic stem cells that exactly match an individual. The embryonic
stem cells are derived from the blastocyst (before it becomes a fetus) and can be instructed to form particular cell types (e.g., heart
muscle) to be implanted into damaged tissue (e.g., heart) to restore its function. If the stem cells are placed back into the
individual who donated the DNA for the somatic cell nuclear transfer, the embryonic stem cells and their derivatives are
genetically identical and thus immunocompatible (they will not be rejected).

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 FOUNDATIONS AND LANDMARKS IN STEM CELL BIOLOGY

TABLE A1. continued.

Totipotent stem cells Stem cells that include in their progeny all cell types that can be found in an embryo, fetus, or developed organism, as well as
the cells that form the embryonic components of the trophoblast and placenta required to support development and birth.
Considered the “master cells of the body.” The zygote and the cells at the very early stages after fertilization (i.e., the 2-cell
stage) are considered totipotent.
Transdifferentiation The ability of a particular cell of 1 tissue, organ, or system, including stem or progenitor cells, to be caused to differentiate into
a cell type characteristic of another tissue, organ, or system; e.g., hematopoietic stem cells to liver hepatocytes.
Transplantation biology The science that studies the transplantation of organs and cells, include graft rejection.
Trophoblast The extraembryonic tissue (outer layer of cells of the blastocyst) responsible for uterine implantation, developing into the placenta,
and controlling the exchange of oxygen and metabolites between mother and embryo. In contrast to embryonic stem cells, the
trophoblast does not come from the inner cell mass, but from cells surrounding it.
Umbilical cord blood Hematopoietic stem cells collected from the umbilical cord during and shortly after delivery. These stem cells are in the
stem cells blood at the time of delivery because they transition from the liver, where blood formation takes place during fetal life, to the
bone marrow, where blood is made postnatally. Umbilical cord blood stem cells are similar to stem cells that reside in bone marrow,
and they can be used for the treatment of leukemia and other diseases of the blood. Cord blood is currently used to rescue
patients who have undergone chemotherapy to destroy their bone marrow. Efforts are now being undertaken to consistently
harvest and store these cells for future potential use.
Undifferentiated cell A cell that has not yet generated structures or manufactured proteins characteristic of a specialized cell type.
Unipotent stem cells Stem cells that self-renew as well as give rise to a single mature cell type; e.g., spermatogenic stem cells.
Wharton’s jelly A gelatinous substance within the umbilical cord; it has recently been shown to be a source of potentially pluripotent stem cells.
Zygote The cell formed by the union of 2 gametes; the result of sperm-egg interactions leading to the fusion of sperm and egg nuclei.
The zygote usually begins cell division to give rise to the stages of the preimplantation embryo, from the 2-cell stage to the
blastocyst; however, only the fertilized egg is the zygote.

a
Sources: National Institutes of Health, International Society for Stem Cell Research, Cambridge Healthtech Institute, and the University of Kansas.

Mario R. Capecchi (left), Sir Martin J. Evans (center), and Oliver Smithies (right), recipients of the Nobel Prize in Physiology or Medicine in 2007 “for
their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells.” See Apuzzo, p 1, and Farin et al.,
pp 15–39.

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