Table of contents :

Timing :

• Kern plasma relation theory : the theory that for each cell there exists a definite size relation of nuclear mass to cell mass. The question of whether cells need an active size-sensing mechanism or whether they stochastically revert back to a mean size after several divisions following either chemical or signaling perturbation has been debated for about 40 years. A size threshold adjusts cell cycle length in the next cycle to ensure maintenance of a proper balance between growth and proliferation rates in various vertebrate cell types including human, mouse, and chicken erythroblasts and fibroblasts in vitro^ref : concept had been demonstrated previously in yeast^{ref1, ref2}. There is a 'sizer' and 'timer' in animal cells. The main question was what happens in vivo, where cells are growing with abundant nutrients and an array of different growth and mitogenic factors together with cell adhesion effects
• interphase / resting or vegetative phase or stage : the stage of a cell or its nucleus when no mitotic changes are going on; the interval between two successive cell divisions, during which the chromosomes are not individually distinguishable and the normal physiological processes proceed
• G₀ : MKI67 / antigen Ki-67 identified by monoclonal antibody (MIB-1) protein is a proliferation antigen, which is present in G₁, S, G₂ and M phases of the cell cycle. Quiescent or resting cells in the G₀ phase of the cell cycle do not express the Ki-67 antigen.
• G₁ (for gap 1) or pre-synthethic phase
• S (for synthesis) phase. Each chromosome is copied to generate 2 identical sister chromatids and cohesion is established between them. Cohesion is thought to be mediated both by DNA connections (topological concatenations) that are generated during DNA replication and by a proteinaceous linkage (probably the cohesin complex) that hold sister chromatids together until anaphase. Cohesin is a 4-member protein complex that is required to hold together the sister chromatids of newly replicated chromosomes. Cohesin contains a heterodimer of the SMC proteins SMC1 and SMC3, which is associated with the non-SMC proteins SCC1 and SCC3. Cohesin proteins were identified by 2 Saccharomyces cerevisiae laboratories in genetic screens that were designed to identify the glue that is required for sister-chromatid attachemtn. Both screens correctly presumed that mutational disruption of the glue would cause precocious sister-chromatid sepration in metaphase arrested cells. Cytological proof of sister-chromatid separation defects relied on clever approaches to mark a single locus on each sister chromatid. Similar proteins have now been identified as members of a cohesin complex in several organisms. Cohesin has the cell-cycle-dependent chromatin-localization patern that would be predicted for a cohesion factor. In yeast, cohesin loads onto chromatids at replication, and remains boud until its abrupt dissociation at anaphase, when cohesion must be released. In higher organisms, the bulk of cohesin dissociates at prophase, although a smaller amount persists at centromeres and dissociates at anaphase. This prophase release of cohesin is necessary for sister-chromatid resolution. If cohesin is not removed during anaphase, sister chromatids fail to separate. Chromatin immunoprecipitation and immunofluorescence experiments show cohesin along chromsome arms and concentrated at the centromere, which is a site of tight cohesion, as would be expected for a complex that connects sister chromatids. The localized pattern of cohesin during meiosis reveals the most about its role in cohesion. During meiosis I when homologues are paired, cohesin localizes along chromosome arms, but when sister chromatids are paired during meiosis II, cohesin is only found between centromeres. Most organisms contain a meiosis-specific cohesin complex, with a meiosis-specific SCC1 variant called REC8 in place of SCC1. Meiotic chromatids that lack REC8 canot maintain cohesion. Reciprocally, meiotic chromatids on which cohesin inappropriately persists cannot lose cohesion and fail to separate at meiosis I and meiosis II. Dissociation of cohesin at anaphase is triggered by the proteolytic cleavage of its SCC1 or REC8 subunit by a protease called separase. Cleavage of SCC1 is necessary for sistert chromatid separation, and is an irreversible entry into anaphase. Separase is inhibited by a protein called securin until the anaphase-promoting complex (APC) ubiquitylates and destroys securin. The APC, in turn, is unable to perform its role until the spindle checkpoint has been cleared. These and other mechanisms ensure that cohesin remains between sister chromatids until the cell is ready to entry anaphase. Light dictates the cell cycle in zebrafish by controlling entry into S-phase. Exposure of larvae to light-dark (LD) cycles causes a range of different cell types to enter S phase predominantly at the end of the day. When larvae are raised in constant darkness (DD), a low level of arrhythmic S phase is observed. In addition, light-entrained cell cycle rhythms persist for several days after transfer to DD, both observations pointing to the involvement of the circadian clock The number of LD cycles experienced is essential for establishing this rhythm during larval development^ref.
• G₂ (for gap 2) or post-synthethic phase
• karyostasis : the so-called resting stage of the nucleus between mitotic divisions.
• M (for mitosis) or D (for division) phase
• mitosis / equational division : a method of indirect division of a cell, consisting of a complex of various processes, by means of which the 2 daughter nuclei normally receive identical complements of the number of chromosomes characteristic of the somatic cells of the species : in centriola-less Protozoa and Fungi it occurs within the nucleus. NOTE: The term mitosis is used interchangeably with cell division, but strictly speaking it refers to nuclear division, whereas cytokinesis refers to division of the cytoplasm. In some cells, as in many fungi and the fertilized eggs of many insects, nuclear division occurs within the cell unaccompanied by division of the cytoplasm and formation of daughter cells.
• karyokinesis : the phenomena involved in division of the nucleus, usually an early stage in the process of cell division, or mitosis.
• asymmetrical karyokinesis : mitosis in which the chromosomes divide unequally and into dissimilar masses.
• hyperchromatic karyokinesis : mitosis in which the number of chromosomes is abnormally large.
• hypochromatic karyokinesis : mitosis in which the number of chromosomes is abnormally small.
• karyomitosis : division of the nucleus of a cell preceding mitosis.
It is the process by which the body grows and replaces cells and is divided into 4 phases:
• prophase :
• formation of paired chromosomes : after replication and cohesin loading, chromosomes are in an extended configuration and appear as amorphous mass in the nucleus. As cells enter prophase, chromosomes begin the process of condensation, undergoing a marked change in structure that continues until they are fully compacted at metaphase. During prophase condensation, the sister chromatids begin to dissociate along their arms and organize along their individual axes - a process that is called resolution. In many organisms this can be viewed cytologically as the transition from indistibguishable chromosomes to 2 rod-shaped arms that are attached at the centromere. Condensin is a 5-member protein complex that is required for chromosome organization and segregation. It contains a heterodimer of SMC proteins (SMC2 and SMC4) and 3 associated non-SMC proteins (CAP-D2, CAP-G and CAP-H). In most organisms, condensin only associates with chromosomes at times of the cell cycle when they are condensed (prophase to anaphase). Our understanding of condensin comes from both biochemical and genetic studies. The bacterial condensin MukBEF compacts DNA into a repetitive, stable structure^ref. Chicken SCII, later determined to be SMC2, was identified as one of the proteins that remained after chrosomes were stripped down to an insoluble "chromosome scaffold", on which looped chromatin domains are thought to be organized. The complete known complex, containing CAP-E (SMC2), CAP-C (SMC4), CAP-D2, CAP-G and CAP-H, was identified by a sedimentation method for purifying Xenopus mitotic chromosome associated proteins (CAPs) from egg extracts. CAP-D2 was also identified independently. The 5-member protein complex was named condensin because sperm chromosomes introduced into an egg extract that was depleted of any subunit formed a diffuse mass rather than a condensed structure. Genetic studies also uncovered condensin subunits. Mutations in Schizosacchromyces pombe cut3 (SMC2) and cut14 (SMC4) cause a cell untimely torn ("cut") phenotype when the division septum cuts through unsegregated chromosomes at the cell centre. These mutants were proposed to affect chromosome condensation rather than the separation of centromeres on sister chromatids, becaue the distrance between  2 fluorescent in situ hybridization (FISH) probes on a chromosome arm increased the mutants, but centromere probes separated normally. The 3 S.pombe non-SMC condensin subunits were later biochemically identified and shown to associate with the SMC proteins. In Saccharomyces cerevisiae, condensin subunit mutations were created by a similar FISH assay to have defects in chromosome segregation and condensation. In Drosophila and Caenorhabditis elegans, condensin subunits were identified fortuitously in genetic screens for unrelated processes, and were later shown to affect mitotic chromosome morphology and segregation. Observations of chromosomes in theise condensin subunit mutants indicated that condensin might have an important role in resolving sister chromatids at prophase, but might not be the only metaphase condensation factor. Mutation of a Drosophila condensin subunit caused wider less-distinct prophase sister chromatids, yet these chromatids had shortened along their longitudinal axis by metaphase. Similarly, depletion of C.elegans condensin components caused wispy instead of rigid prophase chromosomes that nevertheless align into a relatively compact metaphase plate. A common and notable phenotype of condensin mutants in many organisms is the failure to completely separate connections between sister chromatids as they pull apart at anaphase during mitosis. A similar anaphase segregation defect has also been observed during meiosis II. Condensin binds to DNA and has ATPase activity. When incubated with relaxed circular DNA in the presence of a type I topoisomerase, condensin causes ATP-dependent positive supercoiling. In the presence of a type II topoisomerase, condensin converts nicked circular DNA into positive knots. These results led to the model that condensin introduces global writhe into the DNA, promoting condensation by stabilizing large positively supercoiled DNA loops. Conversely, cohesin catenates nicked circular DNA when incubated with topoisomerase II, and causes DNA-protein aggregates in gel-shift experiments. So, condensin, swhich compresses one sister into itself, has intramolecular activities, whereas cohesin, which adheres 2 different sister chromatids, has intermolecular activities. Electron microscopy (EM) shows that the arms of condensin are close together, where those of cohesin are spread apart in a "V" shape. These activities and structures support the model that condensin acts as an intramolecular crosslinker by grabbing sites on a single DNA strand and bringing them together, whereas cohesin acts as an intermolecular crosslinker by grabbing and holding 2 different sister chromatids. New microscopic views of cohesin and condensin have inspired a revised view of their mechanism of action. Atomic-force microscopy shows that the SMCs of condensin form a globular head onto which the non-SMCs assemble, and a coiled tail the end of which touches DNA. These results suggest a loop fastener model in which the condensin hinge binds one region of DNA, then non-SMC proteins mediate an ATP-dependent opening and closing of the SMC "V" to enclose a loop of DNA. Another study using electron spectroscopic imaging of condensin suggests an "orientated gyre" model rather than a "global writhe" model, because a single condensin molecule seems to introduce 2 stacked supercoils into closed plasmid DNA. On the basis of this finding, it has been proposed that an ATP-hydrolysis cycle changes the conformation of condensin and allws it to trap 2 orientated positive supercoils in its coiled-coil arms. A related "embrace model" has been proposed for cohesin. EM and interaction data indicate that SMC1 and SMC3 are linked at one end by hinge interactions and at the other by interaction with SCC1. Cohesin was, therefore, proposed to form a large loop that encircles both sister chromatids, fastened by SCC1 at one end. Whereas earlier models envisioned cohesin grabbing sister chromatids, this model indicates that the arms of cohesin might hold 2 sister chromatds in an "embrace" until proteolytic cleavage of SCC1 disrupts the cohesin loop.
• disappearance of nuclear membrane
• appearance of the achromatic spindle
• spindle fibers : the microtubules radiating from the centrioles during mitosis and forming a spindle-shaped configuration
• karyoplastin : the substance of a mitotic spindle; the parachromatin
• formation of polar or pole plates: platelike bodies at the end of the spindle in certain forms of mitosis)
• prometaphase generally begins with the disintegration of the nuclear membrane. When this has occurred, a more fluid zone is noted in the center of the cell, in which the chromosomes move freely and in apparent disorder, making their way toward the equator. A specialized chromosome region, the centromere, assembles a proteinaceous structure - the kinetochore - that mediates attachment to the microtubule spindle. Sister chromatids are arranged so that each centromere interacts with microtubules from only one pole, a phenomenon that is called bi-orientation

metaphase : chromosomes become fully condensed by metaphase. Condensation provides mechanical strength and reduces volume so that chromosomes can withstand spindle forces as they are pulled to opposite sides of the cell. At metaphse, the chromosomes are completely aligned at the centre of the cell, along what is called the metaphase plate. A spindle-checkpoint mechanism monitors chromosome-spindle attachment and delays anaphase until the chromosomes are attached and under tension^ref. Arrangement of chromosomes in the equatorial plane (equatorial plate : the platelike collection of chromosomes at the equator of the spindle in karyokinesis) of the central spindle to form the monaster. Chromosomes separate into exactly similar halves.
• anaphase : at anaphase, the sister chromatids separate in a coordinated burst of movement. Once the spindle is relieved, the anaphase-promoting complex (APC) triggers the proteolysis of target proteins and chromosome segregation follows^ref. One target of the APC is a protein called securin that prevents the protease separase from cleaving a cohesin subunit untile the commitment is made to anaphase. This mechanism ensures that the cohesin glue is not dissolved until the cell is ready for division^ref. The 2 groups of daughter chromosomes separate and move along the fibers of the central spindle, each toward one of the asters, forming the diaster.
• anaphase A
• anaphase B
• telophase : in telophase, the newly segregated chromosomes decondense themselves into a reticulum and the daughter nuclei are formed; susbsequently, cytokinesis divides the cytoplasm, forming 2 complete daughter cells
• dispireme : the stage of cell division which follows the diaster; so called because the cytoplasm is divided into 2 parts, in each of which the chromatin appears to assume the form of a coil

Kinases involved in the regulation of mitosis :
• Aurora kinases :
• aurora-A / serine/threonine kinase 6 (STK6) (spindle poles) : regulates centrosome maturation and spindle assembly
• aurora-B (kinetochore; midzone) : regulates chromosomal segregation, spindle orientation, essential for cytokinesis
• aurora-C (centrosomes) : unknown
• Polo kinases (PLK) :
• PLK1 (centrosomes; kinetochores) : regulates mitotic entry and exit, and cytokinesis
• PLK2 (kinetochores; mitotic spindle) : regulates mitotic entry and exit, and cytokinesis; downstream target of p53
• PLK3 (midbody; centrosome; mitotic spindle; Golgi) : regulates mitotic dynamics and centrosomal function; involved in DNA-damage stress response and Golgi dynamics
• cyclin-dependent kinases (CDKs) :
• CDK1 / CDC2 regulates mitotic entry and exit; associates with cyclin A and cyclin B
• cyclins
• cyclin A
• cyclin A1
• cyclin B
• cyclin C
• cyclin D
• cyclin D1 (CCND1) / B-cell lymphoma 1 (BCL1) / Prad1
• cyclin D2 (CCND2)
• cyclin D3 (CCND3)
Both triple knockout mutant mice lacking all 3 D-type cyclins—cyclin D1, D2, and D3^ref- and complementary deletions of cdk6 and cdk4 single and double mutants^ref develop normally until the later stages of gestation, after which they died due to severe anemia, among other abnormalities. These observations suggest that CDK6 is required for expansion of certain differentiated compartments, rather than for proliferation of early hematopoietic precursors. Previous evidence had shown a critical need for the D-type cyclins by proliferating cancer cells : for this reason, it was suggested that D-type cyclins might be a good target for therapeutic strategies in human cancer. The inhibitors p16, p21, and p27 are much lower in the Spanish double knockouts, but levels of p21 and p27 are unchanged in the Sicinski triple knockouts, suggesting contradictory results.
• cyclin E
• never in mitosis, gene A (NIMA) kinases :
• NIMA-related kinase (NEKs)
• NEK1
• NEK2 regulates centrosomal organization
• NEK3
• NEK4
• NEK5
• NEK6
• NEK7
• NEK8
• NEK9
• NEK10
• NEK11
• spindle-checkpoint kinases (kinetochore; centrosome (TTK and ESK only)) : regulate mitotic checkpoint to ensure accurate chromosomal segregation; activated by signals from unattached kinetochores to prevent premature entry into anaphase
• budding uninhibited by benzimidazole 1 (BUB1)
• BUB-related kinase 1 (BUBR1) / BUB1B
• TTK / EC STY kinase (ESK)
Germline stem cells use intracellular mechanisms involving centrosome function and cortically localized adenomatous polyposis coli (APC) to orient spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews and the other, displaced away, initiates differentiation
• mitapsis : the fusion of the chromatin granules in the final stage of cell conjugation.
• spindle elongation theory : the theory which suggests that the spindle and asters have a decisive role in cell division. The theory is based on the observation that the elongation of the cell at anaphase is accompanied by a shrinkage at the equator. The centers are believed to be pushed apart by the spindle tubules, since the spindles and asters appear to be rigid structures.
• aster / astrosphere / cytaster / kinosphere : a structure seen in a cell during the prophase of mitosis, composed of a system of microtubules arranged in astral rays around the centrosome
• central body : the structures at the center of the aster during mitosis
• central apparatus : the dynamic organ of the cell that participates in mitosis; it consists of centrosome and astrosphere (the central mass of an aster, exclusive of the rays)
• mitosome : a body formed from the spindle fibers of the preceding mitosis; a spindle remnant
• homeotypic mitosis : the ordinary type of cell division in mitosis, as occurs also in the second, or equational, division of meiosis
• heterotypic mitosis : mitosis in which the halves of bivalent chromosomes move away from each other toward the poles, as occurs in the first, or reductional, division of meiosis
• pathologic mitosis : atypical, asymmetrical mitosis indicative of malignancy
• tetraster : a figure in abnormal mitosis characterized by 4 centrosomal centers or asters
• multicentric or pluripolar mitosis : cell division that results in the formation of > 2 daughter cells
Web resources : Mitosis World by Ted Salmon at UNC Chapel Hill
• meiosis / reductive division : a special method of cell division, occurring in maturation of the sex cells (hence only in those Eukarya with sexual reproduction), by means of which each daughter nucleus receives half the number of chromosomes characteristic of the somatic cells of the species
• meiosis I
• prophase I consists of 5 stages:
• leptotene : the stage of meiosis in which the chromosomes are slender, like threads
• zygotene / amphitene : the synaptic stage of the first meiotic prophase in which the 2 leptotene chromosomes undergo pairing by the formation of synaptonemal complexes to form a bivalent structure
• synaptonemal complex : a thick, threadlike structure formed during the zygotene (synaptic) stage of meiosis by the intertwining of 2 leptotene chromosomes so that they are indistinguishable separately.
• pachytene : synaptonemal complexes shorten, thicken, and continue to intertwine, and each of the conjoined (bivalent) chromosomes separate into 2 sister chromatids, which are held together by a centromere, to form a tetrad. During this phase the chromatids break up and corresponding regions of the nonsister chromatids of the paired chromosomes are exchanged in a process known as crossing over
• diplotene (in Amphibiadictyotene : the protracted stage resembling suspended prophase in which the primary oocyte persists from late fetal life until discharged from the ovary at or after puberty; lampbrush chromosomes): the 2 chromosomes in each bivalent begin to repel one another and a split occurs between the chromosomes, which are then held together by regions where exchanges have taken place (chiasmata) during crossing over
• meiotic prophase arrest requires the presence of the G_sPCR GPR3 in the oocyte that elevates cAMP^ref, which has been previously shown to be critical for preventing completion of meiosis^ref. 2 signals must control the receptor—one from the follicle cells to maintain arrest and one from the pituitary gland to release the arrest. The previous observation that removing an oocyte in meiotic arrest from its follicle results in spontaneous resumption of meiosis would be explained by the removal of the signal from the follicle. In addition, then, somehow the gonadotropin would result in the cessation of the production of that ligand
• diakinesis : the stage of first meiotic prophase in which the nucleolus and nuclear envelope disappear and the spindle fibers form.
• prometaphase I
• metaphase I
• anaphase I
• anaphase IA
• independent assortment : the random distribution of different combinations of the parental chromosomes to the gametes. As a result, each gamete normally possesses one genome (set of 23 chromosomes) that includes one chromosome of each type. The genes on the chromosome also assort independently unless they are linked
• anaphase IB
• telophase I
• interkinesis : a period intervening between the first and second divisions in meiosis, similar to the interphase in mitosis
• G₁
• G₂
• meiosis II
• prophase II resembles that in mitotic division
• prometaphase II
• metaphase II
• anaphase II
• anaphase IIA
• anaphase IIB
• telophase II
• cytodieresis / cytokinesis is achieved by the constriction of an actomyosin-based contractile ring and its associated plasma membrane, partitioning the cytoplasm into the daughter cells within a few minutes. Blebbistatin specifically blocks all nonmuscle myosin II-dependent processes in vertebrate cells, including cytokinesis and cell blebbing.
• contractile ring theory : a theory advanced to explain the formation of a furrow in a dividing cell. According to this theory, the gelated ring in the cortex of the dividing cell contracts (cortical gel contraction) like the nonmotile portion of an amoeba, and, therefore, decreases the surface area. Actually, however, before division the surface increases by about 26%
• expanding surface theory : a theory of cell division which postulates that a nuclear substance is liberated, probably from chromosomes, which causes expansion of the cellular membrane at the poles; as the polar areas expand, the equator contracts, leading to division.
• intermediate body of Flemming : a small bridge of acidophil material connecting the two daughter cells for a time at the end of mitosis
Web resources : Mitosis and cytokinesis
• plasmodium : a dinucleated cell resulting from mitosis not followed by cytodieresis
• mammalian cell cycle control and DNA repair systems

genotoxic stress (CPT and related anticancer drugs^ref) => ATR phosphorylation of Chk1 at Ser-345 both activates Chk1 and marks this protein for proteolytic degradation => checkpoint termination under normal conditions^ref
Web resources :
• G1/S checkpoint
• G2/M DNA damage checkpoint
• Kohn's maps, at NCI
• Introduction
• Molecular species index
• Map A: cyclins & E2F
• Map B: p53 & DNA repair
• Annotation list
• References
• Ser and Thr phosphorylation events regulate cell cycle progression
• p110^Rb and cell cycle control
• p53 and cell cycle control
• p53 story
• Cdc42 and cell cycle control

• epithelia : the covering of internal and external surfaces of the body, including the lining of vessels and other small cavities. It consists of cells joined by small amounts of cementing substances. Epithelium is classified into types on the basis of the number of layers deep and the shape of the superficial cells


• lining epithelia
• absorbing epithelium
• protective epithelium : epithelium that forms a protective covering, as the epidermis.
• ciliated epithelium :  any type bearing vibratile cilia on the free surface.
• pigmentary epithelium / pigmented epithelium : epithelium containing granules of pigment.
• pavement epithelium / tessellated epithelium : simple squamous epithelium
• stratified epithelium / laminated epithelium : epithelium in which the cells are arranged in several layers
• pseudostratified epithelium : a type of epithelium which occurs in the large excretory ducts of the parotid and several other glands and in the male urethra. The nuclei are spaced at different levels and the cells are quite variable in shape, giving the appearance of a stratified epithelium.
• columnar epithelium : a type composed of tall prismatic cells
• pyramidal epithelium : columnar epithelium whose cells have been modified by pressure into truncated pyramids.
• rod epithelium : epithelium the cells of which are rod-shaped
• cubical epithelium / cuboidal epithelium : a type composed of cells which have a cubical shape
• transitional epithelium : epithelium that was originally thought to represent a transitional form between stratified squamous and columnar epithelium, found characteristically in the mucous membrane of the excretory passages of the urinary system; in the contracted condition it consists of many cell layers, whereas in the stretched condition usually only 2 layers can be distinguished.
• secretion epithelium
• glandular epithelium : epithelium made up of glandular or secreting cells
• crescents of Giannuzzi / demilunes of Heidenhain / Giannuzzi's bodies, cells, or demilunes / crescent, demilune, or marginal cells / semilunar bodies : crescent-shaped patches of serous cells surrounding the mucous tubules in seromucous glands, and formed by the outnumbered albuminous cells pushed to the blind ends of the terminal portions or into saccular outpocketings
• merocrine gland : one in which the secretory cells maintain their integrity throughout the secretory cycle (see eccrine sweat-gland)
• apocrine gland : a gland whose discharged secretion contains part of the secreting cells (see apocrine sweat glands and mammary gland)
• holocrine gland : a gland whose discharged secretion contains entire secreting cells
• sense or sensory epithelium / neuroepithelium
• olfactory epithelium : pseudostratified epithelium lining the olfactory region of the nasal cavity, and containing the receptors for the sense of smell
• nervous epithelium(some authors consider it as another kind of tissue)
• connective tissues
• properly said connective tissue
• mucous connective tissue
• weary fibrillar connective tissue
• thick fibrillar connective tissue
• blood
• fatty tissue
• cartilage
• bone tissue
• lymphoid tissue
• muscular tissue
• smooth muscle
• skeletal muscle
• myocardium
• stem cells (focus on human stem cells) (see also cell therapy with stem cells)
• not differentiated yet
• can undergo multilineage differentiation

Lineage restriction or commitment =>
• totipotent cell : zygote and the following 4 cells can give rise to a fully differentiated adult organism, ie any type in the adult organism body and any cell of the extraembryonic membranes (placenta)
• pluripotent cell : after few divisions into development, cells can give rise to cells of all 3 germ layers, but cannot contribute to extraembryonic membranes, and require particular culture conditions
• ESCs
• EGCs
• ECCs
• multipotent or oligopotent cells : germ layer-specific cells emerge later in development and are present (quiescent) in adult tissues activating to repopulate and regenerate at a level related to the degree of environmental cues.
• unipotent, progenitor or precursor cells : organ-specific
• terminally differentiated cell
• able to  indefinite self-renewal (see tissue regeneration), apart from differentiation, apoptosis or migration
• supports development, tissue homeostasis and repair
• from embryo tissues
• embyonic stem cell (ESC) : from fertilized egg or parthenogenesis (to circumvent some of the ethical concerns; see Dividing, unfertilized eggs could offer new therapeutic option from Nature Science Update).
• before 16-cells blastocyst stage (i.e before E5)
• located in embryonic node / inner cell mass
• long-term self-renewal depending on expression of TFs :
• Oct-3/4 promotes self-renewal during differentiation from morula to early blastocyst to late blastocyst to postimplantation egg cylinder, while inhibits differentiation to trophectoderm
• Sox-2 promotes self-renewal during differentiation from late blastocyst to postimplantation egg cylinder
• FoxD3 promotes self-renewal during differentiation from late blastocyst to postimplantation egg cylinder
• STAT3 inhibits differentiation to mesoderm
• NANOG promotes self-renewal during differentiation from early to late blastocyst, while inhibits differentiation to visceral/parietal endoderm. p53 binds to the promoter of Nanog and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53S315A knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53S315A ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis^ref. Through cell fusion, ESCs can erase the developmental programming of differentiated cell nuclei and impose pluripotency. Molecules that mediate this conversion should be identifiable in ESCs. One candidate is the variant homeodomain protein Nanog, which has the capacity to entrain undifferentiated ES cell propagation. In fusions between ESCs and NSCs, increased levels of Nanog stimulate pluripotent gene activation from the somatic cell genome and enable an up to 200-fold increase in the recovery of hybrid colonies, all of which show ESC characteristics. Nanog also improves hybrid yield when thymocytes or fibroblasts are fused to ESCs; however, fewer colonies are obtained than from ESC x NSC fusions, consistent with a hierarchical susceptibility to reprogramming among somatic cell types. Notably, for NCS x ESC fusions elevated Nanog enables primary hybrids to develop into ESC colonies with identical frequency to homotypic ESC x ESC fusion products. This means that in hybrids, increased Nanog is sufficient for the NSC epigenome to be reset completely to a state of pluripotency^ref
• stable diploid karyotype : anyway ESCs that are cultured in the lab accumulate an alarming array of genetic changes, including mutations known to be linked to cancer. The finding throws into question whether such cells could eventually be used for therapy, unless they can be kept fresh and checked for mutations before use. The longer the cells are kept, and the more they divide, the more errors they build up in their genetic code. But previous, smaller studies of stem cells had not found problematic levels of mutations. Chakravarti and his colleagues decided to take a closer look, examining 9 of the hESC lines that have federal approval. They compared frozen, archived cells with 'daughter' generations that had been created from these. Many of the archived cells seemed normal, although some had already divided tens of times to build up cell numbers into the billions. Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10^-9 per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, 8 of 9 late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes^ref. The finding undermines a general assumption that stem cells remain unblemished until they are programmed to become a certain type of cell. It suggests that the biological properties of the cells before and after replicating could be different. It remains unclear what would happen if these stem cells were transplanted into a patient. But the results should encourage the use of fresher stem cells or, preferably, genetic screens of stem-cell lines before they are used for therapy. Some take a "glass half full" view of the findings, because the billions of archived cells seemed normal. This shows that the replications needed to boost stem-cell numbers to usable levels do not necessarily cause problems. The study supports the idea that more, fresh stem-cell lines would be useful for the scientific community: US federal research currently relies on a very limited number of lines.
• NIH Stem Cell Registry
• lack G₁ checkpoint
• lack of X inactivation if it has a XX genome
• growth on ...
• irradiated quiescent fibroblasts feeder layer (but probably FGF alone is sufficient)
• conditioned medium (CM) / cell-free culture supernatant (CFCS)
• Hayflick number = 450
• totipotent in generation of differentiated cell types from all the germ layers (3 in Trilateria), including, e.g., cochlear hairy cells. It can be demonstrated :
• in vivo
• injection of ESC from the inner cell mass of one blastocyst into the cavity of another blastocyst results in chimera formation
• ectopic injection of ESC into adult mice results in teratoma formation
• in vitro : this is the only ethically acceptable way for Homo sapiens. Please note nowadays long-term ES (LT-ES) cultures have been obtained only from Mus musculus, Homo sapiens and Macaca mulatta.
• inducing formation of complex cystoid embryoid bodies (EB) in which endodermal, mesodermal, and ectodermal formations can be detected
• by subtracting leukemia inhibiting factor (LIF) / differentiation inhibiting activity (DIA)
• by giving :
• retinoic acid (RA)
• 3-methoxybenzamide
• clonogenicity
• integration in all fetal tissues (chimera formation)
• germ line colonization
• inducible proliferative/differentiative state
Sources of ESC lines :
• aborted fetuses
• destruction of preimplantation human embryos created during FIVET procedures, whose ethical status is undefined yet. Some religions and many people who view the human preimplantation embryo as a person or subject with rights and interests believe that the intentional destruction of an embryo is equivalent to murmer : then the beneficial goal of saving or prolonging life would not matter, for no life may be taken to preserve the life of one or even many persons (except in cases of self-defence, war and dire necessity). On the other side widely held philosophical and moral views hold that status as a person or as an entity with interests requires, at the very least, a nervous system capable of sentience, if not also of cognition and consciousness, which is not present in the undifferentiated cells of preimplantation human embryos, although it is different from ordinary human tissue because of the unique potential it has to develop into a new human being ("special respect"). The distinction between use and derivation reflects the basic distinction in the ethics of complicity between causing an immoral or wrongful act to occur and benefiting from it once it has occurred.
• parthenogenetic eggs
• human parthenotes^ref
• nonhuman primate parthenogenetic stem cells^ref : parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy as sperm is required for implantation and placenta formation and cannot survive longer than several days. Monkey (Macaca fascicularis) eggs can be parthenogenetically developed in vitro to the blastocyst stage, harvested and cultured to create a pluripotent line of monkey stem cells (Cyno-1 cells) TERT⁺, AlkP⁺, Oct-3/4⁺, stage-specific embryonic antigen 4 (SSEA-4)⁺, tumor rejection antigen 1-60 (TRA 1-60)⁺, and tumor rejection antigen 1-81 (TRA 1-81)⁺ (traditional markers of human ES cells). They have a normal chromosome karyotype (40 + 2) and can be maintained in vitro in an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiated in vitro into dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells are injected into SCID mice, teratomas with derivatives from all 3 embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (nestin⁺). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-gated sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.
Cell culture : early-stage mammalian embryos develop in a low O₂ environment (hypoxia). hESCs, however, are generally cultured under an atmosphere of 21% O₂ (normoxia), under which conditions they tend to differentiate spontaneously. Such conditions may not be the most suitable, therefore, for hESC propagation. hESCs grow as well under hypoxic (3% and 5% O₂) as under normoxic conditions (21% O₂), and growth was only slightly reduced at 1% O₂. Hypoxic culture reduces the amount of spontaneous cell differentiation that occurs in hES colonies, as assessed morphologically, biochemically (by the production of hCG and progesterone), and immunohistochemically (by the loss of stage-specific embryonic antigen-4 and Oct-3/4 and gain of stage-specific embryonic antigen-1 marker expression). In addition, hESC growth under hypoxia provided enhanced formation of embryoid bodies. Hypoxic culture would appear to be necessary to maintain full pluripotency of hES cells^ref.
In vitro differentiations :
• germ cells differentiated after meiosis :
• egg cells differentiated from female mouse ESCs^ref
• sperm  (FE-J1⁺) without tail differentiated from male mouse embryoid bodies ESC (EBs : SSEA1⁺) (rather like an early embryo)^ref. They can successfully fertilize and develop diploid blastocysts in only 20% of challenged eggs (probably due to awry DNA remethylation)^ref: whether the embryos would have produced live births is not known, as the experiment was halted 5 days after fertilization (if reprogramming were affected, the embryos may have aborted later). It could lead to alternative ways to help infertile couples conceive by in vitro fertilization : human sperm will be more difficult to make than mouse sperm : reprogramming problems aside, researchers will first need to make their stem cells using therapeutic cloning.
• in mice, primordial germ cells (PGCs) appear in gastrulating embryos at embryonic day 7.25 and have been generated in vitro from the outer germinal membrane of an ovum (epiblast), provided the epiblast is cocultured with cells expressing BMP4 and BMP8b. How the founder population of PGCs is separated from the rest of the pluripotent epiblast has been unclear because of technical difficulties in distinguishing PGCs from ES cells, since standard PGC markers (Oct-3/4 and alkaline phosphatase) are also positive in ES cells. By using the mouse vasa homolog (Mvh) helicase as a molecular marker specific for differentiating PGCs it has been shown that male ES cells can form PGCs equivalent to those found in the fetal gonad (expressing also GCNA1 and SYCP3) in vitro when co-cultured in presence of BMP4-producing cells. After coculture with gonadal cells and transplantation into the male gonad, ES-derived PGCs differentiated into spermatogenic cells and, ultimately, sperm.
• immature immune cells differentiated from mouse ESCs whose Notch receptors are engaged by Delta-like 1 ligand (DL1) (a protein essential in T-cell production) expressed on the OP9-DL1 stromal cell line. ESC-derived T cell progenitors effectively reconstituted the T cell compartment of immunodeficient mice, enabling an effective response to a viral infection^ref. The technique would work best if the immature immune cells could somehow be delivered into the thymus. They might one day be used in cancer and HIV patients whose own supply has been wiped out : HIV patients could be given cells genetically enhanced to fight the virus. Grafts of bone marrow stem cells must be matched to the patient to avoid rejection, take months to produce T cells, and the small number they make leaves patients vulnerable to infections. In theory, the lab-made immune cells could be used for any patient, because they lack surface molecules that trigger rejection. And because embryonic stem cells keep dividing indefinitely, they might sprout an unlimited supply.
• specific neuronal subtypes :
• Pax-6⁺radial glial cells are generated by selection of highly proliferative ESCs followed by treatment with retinoic acid. As they do in vivo, these cells went on to generate neurons with remarkably uniform biochemical and electrophysiological characteristics^ref.
• hESCs generate early neuroectodermal cells, which organize into rosettes and expressed Pax6 but not Sox1, and then late neuroectodermal cells, which formed neural tube-like structures and expressed both Pax6 and Sox1. Only the early, but not the late, neuroectodermal cells were efficiently posteriorized by retinoic acid and, in the presence of sonic hedgehog, differentiated into spinal motoneurons. The in vitro-generated motoneurons expressed HB9, HoxC8, choline acetyltransferase and SLC18A3 / vesicular acetylcholine transporter, induced clustering of acetylcholine receptors in myotubes, and were electrophysiologically active. These findings indicate that retinoic acid action is required during neuroectoderm induction for motoneuron specification and suggest that stem cells have restricted capacity to generate region-specific projection neurons even at an early developmental stage^ref.
• type II pneumocytes can be derived in vitro from murine ES cells. After withdrawal of leukemia inhibitory factor (LIF) and formation of embryoid bodies in maintenance medium for 10, 20, and 30 days, differentiating ES cells were kept in the same medium or transferred to serum-free small airway growth medium (SAGM) for a further 3 or 14 days of culture. The presence of type II pneumocytes in the resulting mixed cultures was demonstrated by RT-PCR of surfactant protein C (SPC) mRNA, immunostaining of SPC, and electron microscopy of osmiophilic lamellar bodies only at 30 days sampling time. SAGM appeared to be more favorable for type II cell formation than ES medium. No SPC transcripts were found in differentiating cells grown under the same conditions without formation of embryoid bodies. These findings could form the basis for the enrichment of ES cell-derived cultures with type II pneumocytes, and provide an in vitro system for investigating mechanisms of lung repair and regeneration^ref.
• small airway pneumocytes were differentiated from hESC in 2003 (Polak J, Tissue Engineering)
• definitive endoderm, such as the pancreas. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation^ref. Definitive and visceral endoderm were distinguished by using a mouse ES cell line that bears the gfp and human IL2R marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc⁺Sox17⁺ definitive endoderm and Gsc^-Sox17⁺ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, 7 surface molecules were identified that are expressed differentially in the two populations. One of the 7 markers, Cxcr4, to which a mAb is available allowed us to monitor and purify the Gsc⁺ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm^ref
Transplantation of ESC-derived, differentiated cells could prove useful in :
• drug development and toxicity tests
• tissue/cells for therapy
• experiments to study development and gene control
Culture methods : your stem cells have just arrived. Now, how do you convert those frozen vials into a thriving, hESC research program?
• 1. don't expect to jump right into hESCs research. Even if your lab is ready to go, it is still going to take you about 16 weeks minimum, to go from deriving your MEFs to actually growing up a lot of hESCs and being able to freeze them down. So even with a complete lab, you're looking at 4 to 6 months before you can even experiment with these cells
• 2. don't buy commercial murine embryo fibroblast (MEFs) : though it is possible to buy MEFs, WiCell recommends making them in-house. It only costs $26 for 1 pregnant mouse, which can yield 12 to 15 vials of MEFs. It costs about $50 to get 2 to 4 vials of MEFs from a commercial vendor, so you're paying twice the cost for one-sixth the MEFs. ... In addition, you never know what you're going to get from a commercial vendor. WiCell only uses MEFs to passage 5, and after that, they really start dying off. A lot of commercial vendors will sell MEFs at passage 3 or 4, which makes them useless for embryonic stem cell culture.
• 3. MEFs must be irradiated to halt their growth. WiCell's protocol calls for a dose of 8,000 rads, but this number is variable. With a cesium irradiator, 8,000 rads seems about right, but with an x-ray machine the required dose is more like 12,000 rads. You can also inactivate chemically with mitomycin C, but irradiation is recommended if possible. ESCs are extremely sensitive to chemicals : when you're chemically inactivating your MEFs, you need to ensure there are no traces of the chemical on your cells before you add the embryonic stem cells
• 4. thaw cells as quickly as you can possibly do it without cutting corners. I've found a few cases where I've thawed a vial and a new person at WiCell thawed out a duplicate, and she got about 50% of the colonies I got even though they were the exact same cells... A difference of 30 to 45 seconds makes a big difference in the amount of cells you recover. Practice the protocol several times before actually thawing your hESCs, to minimize delays.
• 5. don't expect a completely dense culture right after a thaw ; only 0.1% to 1% of cells survive the procedure. Feed every single day because something might pop up in 3 days, 5 days, maybe even 7 or 8 days, all of a sudden a little colony will pop up. A thaw isn't deemed a failure until 14 days have passed. And don't neglect the plate's edges. Frequently you find a lot of colonies around these edges. (Visualize these by placing your finger along the rim of the plate to block the diffraction of light.)
• 6. feed and assess them every day and never let them overgrow : they generally need to be split about once a week. hESC form tight, homogeneous, flat colonies with sharp borders, while differentiating colonies have ragged, uneven edges or transparent centers. Under low magnification, healthy colonies appear white, while colonies that have "balled up" in solution and then settled appear yellow; these latter colonies mimic a developing embryo and will likely differentiate.
• 7. an experienced hESC veteran will worry about his or her cells. If you worry, you'll look at them under the microscope a lot, get a real sense of what they look like, what they look like before they differentiate. You learn to anticipate when the cells are going to do something tomorrow. And that's important, because hESC work really is more art than science. There's much the class cannot teach, but we teach you how to make the correct judgement.
• 8. though it makes culture maintenance difficult, ESC differentiation is a good thing, because it indicates a healthy culture. A trouble-free culture should raise flags. If it looks too good to be true, it probably is : when your ESCs are all of a sudden growing really well, and really fast, and they always look great, and they are never differentiating, that's when you should be suspicious that something is wrong. At that point, karyotype.
• 9. we have an informal rule for deciding what we want to do with our plate of hESCs. Under 10% differentiation, pass the cells as-is. Between 10% and 50% differentiation, remove those cells by a process called "pick-to-remove." Above 50% differentiation, mechanically remove the undifferentiated colonies to a fresh plate of MEFs, using a process called "pick-to-keep."
• 10. if you're picking-to-keep often, you're probably doing something wrong. You should find yourself picking to remove about once every other passage, give or take. So therefore, you should never hit over half being differentiated. ... If you find yourself picking to keep, say more than once every other month, maybe once every 3 months ... you're neglecting your cells. You should be doing pick-to-remove, not pick-to-keep.
• 11. picking too frequently can cause abnormal cell outgrowth, so it's important not to be a perfectionist. If you see a few patches of differentiation, maybe 2% to 3% of the culture, that's not that much and you can be sure those colonies are probably normal. But if, month after month, you constantly pick to produce a pure, undifferentiated culture, you can promote an abnormality – trisomy 12 or 17, for instance. Plus, there's no better way to contaminate your cells than picking.
• 12. if you have perfect cell culture technique and perfect sterile technique, there's no need for antibiotics. Besides, they could adversely affect your culture. You never know when you're adding another element to your media how it's going to affect your ESCs. Those antibiotics could select for abnormal cells for all you know.
• 13. of the 22 federally approved hESC lines, 5 come from the Wisconsin Alumni Research Foundation, which established WiCell in 1999 to, among other things, distribute those lines to researchers around the world. The Institute has fulfilled 300 requests to date, at $5,000 each. That represents some 40% of all hESCs shipped, according to NIH statistics. WiCell distributes cells between passage 20 and 25 that have been karyotyped to ensure they are genetically normal. But you should have them karyotyped, too. We recommend you get them karyotyped before you start experiments, just to make sure they are normal. Then, when you start getting really great data, it's good to check before you finish, and again before you publish. You should karyotype every 10 passages after passage 50
• 14. build up an emergency supply of frozen cells as soon as possible after thawing. Once you're at the point of expanding, start freezing, a few vials at a time. You want to start freezing as soon as possible, because you want the lowest passage cells. Don't wait until you have lots of cells to start freezing. Cells are ready to freeze about 2 days before they are ready to split, at about day 5. That's when the cells are growing most rapidly and will be thaw most robustly
• 15. hESC culture cannot realistically be a side project. Between culturing MEFs and splitting and maintaining the hESCs, the culture work consumes > 20 hours per week. As a result, it's critical that you train another person to cover your culture needs in the event you are sick or just need a vacation. It's too much of a commitment for one person to do alone, so always have a backup."^ref
• embryonic germinal cells (EGC)
• coming from cultures of PGCs from gonadal ridge of 5-9 weeks fetal tissue that results from elective abortions
• as for ESCs, they :
• can form EBs
• retain a normal karyotype
• can generate both XX and XY cultures
• express a set of markers characteristic of pluripotent cells
• spontaneously differentiate into derivatives of all 3 primary germ layers
• but ...
• Hayflick number = 80
• not able to induce teratoma in vivo in mice
• differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries : germline stem cells begin to differentiate by forming interconnected germ cell cysts (cystocytes), and under certain conditions male mouse cystocytes have been postulated to revert into functional progenitors. 4- and 8-cell Drosophila germline cystocytes generated either in second instar larval ovaries or in adults over-producing the BMP4-like stem cell signal Decapentaplegic efficiently convert into single stem-like cells. These de-differentiated cells can develop into functional germline stem cells and support normal fertility. Cystocytes represent a relatively abundant source of regenerative precursors that might help replenish germ cells after depletion by genotoxic chemicals, radiation or normal aging^ref.
• embryonic carcinoma (EC) cells are PGCs, able to induce teratoma when implanted into differentiated tissues.
• amniotic epithelial cells develop from the epiblast by 8 days after fertilization and prior to gastrulation opening the possibility that they might maintain the plasticity of pre-gastrulation embryo cells. Amniotic epithelial cells isolated from human term placenta express surface makers normally present on embryonic stem and germ cells. In addition, amniotic epithelial cells express the pluripotent stem cell specific transcription factors Oct-3/4, and Nanog. Under certain culture conditions, amniotic epithelial cells form spheroid structures which retained stem cell characteristics. Amniotic epithelial cells do not require other cell derived feeder layers to maintain Oct-4 expression, do not express telomerase and are non-tumorigenic upon transplantation. Based on immunohistochemical and genetic analysis, amniotic epithelial cells have the potential to differentiate to all three germ layers-endoderm (liver, pancreas), mesoderm (cardiomyocyte), and ectoderm (neural cells) in vitro. Amnion derived from term placenta following live birth may be a useful and non-controversial source of stem cells for cell transplantation and regenerative medicine^ref. According to US census figures, there are > 4 million live births each year. For each discarded placenta, there are about 300 million amniotic epithelial cells that could be expanded to between 10 and 60 billion cells relatively easily.
National laws on ESCs :
• USA laws allow federal funding on human ESC research only for those 22 lines that already existed before President Bush Aug 9, 2001 policy, ie those listed in the NIH Human ESC Registry (ESCR) :
• BresaGen, Inc., Athens, Georgia, USA (4 different stem cell lines)
• CyThera, Inc., San Diego, California, USA (9)
• ES Cell International, Melbourne, Australia (6)
• Geron Corporation, Menlo Park, California, USA (7)
• Göteborg University, Göteborg, Sweden (19)
• Karolinska Institute, Stockholm, Sweden (6)
• Maria Biotech Co.Ltd, Maria Infertility Hospital Medical Institute, Seoul, South Korea (3)
• MizMedi Hospital, Seoul National University, Seoul, South Korea (1)
• National Centre for Biological Sciences / Tata Institute of Fundamental Research, Bangalore, India (3)
• Pochon CHA University, Seoul, Korea (2)
• Reliance Life Sciences, Mumbai, India (7)
• Technion-Israel Institute of Technology, Haifa, Israel (4)
• University of California, San Francisco (UCSF), California, USA (2)
• Wisconsin Alumni Research Foundation [other site], Madison, Wisconsin, USA (5)
All 22 are grown in Petri dishes containing nutrient broth (usually containing animal serum) and mouse feeder cells (typically mouse embryonic fibroblasts (MEF)). Embryonic mouse cells have on their surface N-glycolylneuraminic acid (Neu5Gc). Humans are genetically unable to produce Neu5Gc, because of a mutation that occurred after our last common ancestor with great apes. Although Neu5Gc is presumed absent from normal humans, small amounts have been claimed to exist in human tumors and fetal meconium. Both human ESCs (HESC) and derived embryoid bodies metabolically incorporate substantial amounts of Neu5Gc under standard conditions. Exogenous Neu5Gc reaches lysosomes of HESCs via pinocytic/endocytic pathways, and is exported in free form into the cytosol, becoming available for activation and transfer to glycoconjugates^ref. Neu5Gc has never been reported in plants or microbes: Neu5Gc is rare in poultry and fish, common in milk products, and enriched in red meats. Eating these foods has sensitized people to the molecule and normal humans have variable amounts of circulating IgA, IgM, and IgG antibodies against Neu5Gc, with the highest levels comparable to those of the previously known anti-a-galactose xenoreactive antibodies. This finding represents an instance wherein humans absorb and metabolically incorporate a nonhuman dietary component enriched in foods of mammalian origin, even while generating xenoreactive, and potentially autoreactive, antibodies against the same molecule^ref. Exposure to human sera with antibodies specific for Neu5Gc resulted in binding of immunoglobulin and deposition of complement, which would lead to cell killing in vivo. This suggests the cells are seen as foreign, and that transplanting them into the body would trigger the immune system to reject them. This does not mean that all available lines should be thrown out, but we need to take caution when using these cells as therapeutics. We eat animal products and drink milk all the time and get this acid into our cells, and yet we are not always suffering from raging autoimmune disease. Varki's team did not test all of the 22 federally funded human ESC lines that were created in the USA before the ban came down, but the cellular mechanism for absorbing Neu5Gc is universal, and all US stem-cell cultures have probably been exposed to animal material. The current stem-cell lines have little clinical value, but that is not an issue for pursuing basic research. In fact, these lines will help to develop animal-free conditions for growing and maintaining human ESCs and minimizing safety concerns. Until better growth conditions are established, a group in Sweden with stem cells that have never been exposed to materials from animals will keep the cells frozen and unavailable for use. In summer 2004, more than half the members of the Senate urged easing limits on new cell lines, noting that potential contamination could make available lines use for humans uncertain. Existing cells might be isolated from animal products for a time, allowing the acid to be diluted. Levels of Neu5Gc on HESC and embryoid bodies dropped after culture in heat-inactivated anti-Neu5Gc antibody-negative human serum, reducing (but not completely eliminating) binding of antibodies and complement from high-titer sera, while allowing maintenance of the undifferentiated state. Complete elimination of Neu5Gc would be likely to require using human serum with human feeder layers, ideally starting with fresh HESC that have never been exposed to animal products^ref.
Mouse blastocysts with and without zonae pellucidae are susceptible to complement-dependent antibody cytotoxicity. Exposure of blastocysts to rabbit anti-mouse serum together with complement results in the death of all cells; however, when blastocysts are exposed to antiserum alone and then transferred to guinea pig complement, only the trophoblastic cells are killed. These results suggest that the mouse blastocyst is not permeable for certain antibodies. The inner cell masses can easily be separated from the remnants of trophoblastic cells and are then able to grow and differentiate in vitro. This method of immunosurgery can be used to obtain large quantities of pure inner cell masses (ICMs) in a relatively short period of time^ref. It was shown to work for human blastocyts, too^ref
An ESC line was derived without exposure to cells or serum : frozen cleavage-stage embryos were thawed and cultured to the blastocyst stage. Inner cell masses were isolated by immunosurgery and plated onto ECM-coated plates that can be easily sterilised. 6 established human ESC lines were also maintained with this serum and feeder free culture system. The cells maintained normal karyotype and markers of pluripotency, including Oct-3/4, stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumour-rejection antigen (TRA)-1-60, TRA-1-81, and alkaline phosphatase. After > 6 months of undifferentiated proliferation, these cells retained the potential to form derivatives of all 3 embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for > 30 passages) with the established stem-cell lines. This system eliminates exposure of human ESC and their progeny to animal and human feeder layers, and thus the risk of contamination with pathogenic agents capable of transmitting diseases to patients^ref.
Harvard University researcher Doug Melton announced in March 2004 that he had created 17 new stem cell lines with private funding. Melton has been pegged to codirect the new Harvard Stem Cell Institute, a privately funded venture banding together nearly 100 researchers.
• California : a ballot initiative to raise $3 billion dollars for stem-cell research passed with the endorsement of its Republican governor, Arnold Schwarzenegger. By 59% to 41% of votes, Californians said "yes" to Proposition 71, the California Stem Cell Research and Cures Initiative, which will raise around $300 million a year for a decade through bond sales. The money will pay for research that has not been eligible for government money since 9 Aug 2001, when President George W. Bush limited federal spending on human embryonic stem-cell research to cell lines in existence as of that date. The creation of new cell lines involves the destruction of a days-old human embryo. Bush's opponent in the presidential race, Senator John Kerry, had promised to reverse the ruling, a move that would have freed up an indeterminate amount of additional funds for stem-cell work. Conceived 3 years ago by a group of wealthy Californians whose families include diabetes sufferers, it will create a new research entity, the Institute for Regenerative Medicine, to distribute the funds and establish research guidelines. It will amend the state constitution to guarantee biologists' right to do embryonic stem-cell research, and protect the institute from interference or supervision by the legislature. A Field Poll taken 2 days before the election showed that the 'yes' vote was driven largely by a belief in medical research and its ability to find treatments for disease. That has been the message of the campaign, which raised nearly $20 million over 6 months to promote the measure. Opponents, who raised only a few hundred thousand dollars, included moral objectors to embryo research and a larger share who worried about the money involved. According to the same poll, 45% of probable 'no' voters said either that California couldn't afford to borrow the money or that the state should leave it to federal government and industry to come up with the cash. Several opposition groups are also worried about the lack of clear ethical guidelines in the measure, given the moral concerns surrounding the work. Now that the Institute for Regenerative Medicine has the green light, those groups plan to be vigilant. The discrepancy between federal and state rules may create confusion among researchers over how to set up collaborations between states and could lead to an exodus of stem-cell scientists to the state. The projected cost of the California's stem cell initiative is US$ 6 billions : the Governor Schwarzenegger rescindment of vehicle licence fees caused lost of 4 billions of US$, while the Calfornia's deficit is estimated to be 10 billions of US$
• New Jersey
• Connecticut legislature may pass in 2005 legislation allowing both adult and embryonic stem cell researchwith strong bipartisan support. In 2004, the state Senate passed a bill to allow stem cell research with overwhelming support, but the bill was subsequently defeated by 4 votes in the House of Representatives. Governor would take between $10 and $20 million from the current budget surplus to promote stem cell research in the state. The proposed legislation was written with the help of CURE, a nonprofit group of biotech companies and educational institutions that promotes the biotech industry and biological research in Connecticut. Connecticut is a leading employer in the biotech pharmaceutical field, but now that proposition 71 has passed in California and there are funds to recruit stem cell biologists, they are going to be able to start to recruit people away from existing institutions in Connecticut.
• Nebraska : senator Joel Johnson, a Republican and former surgeon decided to fight back and introduce another bill, LB580, to ban reproductive cloning but permitting research on embryonic stem cells and let cells divide for 14 days. In the past two years, Nebraska has toyed with a bill banning all forms of cloning. Last year, Johnson and like-minded legislators used filibustering to delay a vote, forcing the other side to produce 33 votes (out of 49) required to stop the debate. This left supporters of the ban a few votes shy of victory before the session ended. A bill that bans embryonic stem cell research would be problematic for Nebraska. Medical research across the state has increased dramatically, and that momentum could come to a grinding halt if legislators began limiting researchers. The University of Nebraska Medical Center recently opened its new $77 million Durham Research Center of Excellence in Omaha, and in 2004, UNMC researchers attracted $80 million in private and public funding. The bill was modeled after one that Sen. Orrin Hatch (R-Utah) introduced. Both Democrats and Republicans have cosigned his bill, although the Nebraska Senate is officially nonpartisan, and no member runs for office under a political party. Legally, the Senate cannot pass, nor can the governor sign, contradictory bills.
• Italy : a law approved in December 2003 : bans any testing of embryos for research and experimental purposes, freezing embryos or embryo suppression, and forbids preimplantation diagnosis for preventing genetically transmitted diseases. It also prohibits donor insemination, limits fertility treatment to stable, heterosexual couples, and states that no more than 3 cells may be fertilized in vitro and that they must be transferred into the womb simultaneously. Such tight restrictions, however, have not prevented a series of "test-tube mix-ups" at fertility centers in recent months. In one case, 2 women had to be prescribed the morning-after pill because each had been inseminated with the wrong man's sperm. But Sirchia's real problems began this week, after he announced the outcome of an innovative stem cell transplant, carried out at the San Matteo Hospital in Pavia, which cured a five-year-old boy of thalassemia. The new therapy involved using cells from the placenta of both of the boy's recently born twin brothers. While reporting on the "historic outcome" at a press conference, Sirchia did not mention that the twin brothers were designer babies, born healthy thanks to preimplantation screening and assisted reproduction carried out at a fertility center in Istanbul, Turkey. The stem cell transplant can be done, with lower probabilities, with donors born following a natural birth. Sirchia allocated 400,000 euros for 2004 to the transplant transfusion and immunology centre of the Ospedale Maggiore in Milan–an organization with which the minister has had very close bounds- for the conservation of 31,000 frozen "orphan" embryos. The center in question opened when Sirchia worked with transplants, he was head of the department for about 28 years. The amount of money given to the center is huge. The main cost for embryo conservation is that one of liquid azote, which costs only 50 cents per litre. In a statement, Sirchia explained that the money would cover not only the cost of liquid azote, but also "the complex organization of such a center and embryo transport from centers across the country to Milan
• 29 countries allow stem cell work (in 2004 China, Japan, Singapore, France and Spain entered this list)
• Singapore parliament on Thursday, September 2, 2004 passed a new law drawn up by the health ministry, which prohibits reproductive cloning^ref. Crucially for the country's life sciences community, the law does not forbid cell nuclear transfer for the purposes of developing stem cells. The health ministry introduced the draft law in May this year, saying that it was taking a step-by-step approach to regulating biomedical research. The first step is the "Human cloning and other prohibited practices bill," which imposes a fine of up to SGD $100,000 (USD $58,700) or 10 years in prison, or both. The bill prohibits the placing of any cloned human embryos in bodies of humans or animals. There are also prohibitions on the import or export of any cloned embryos and the commercial trading of human eggs, human sperm and human embryos. The legislation also forbids the developing of human embryos created by means other than fertilization for more than 14 days, and forbids researchers from developing human embryos outside the body of a woman for more than 14 days. The 14-day cut-off leaves plenty of time for the derivation of stem cells. Beyond 14 days you see the start of the primitive streak development : for harvesting stem cells you only need 4, 5, or 6 days after fertilization or nuclear transfer.
• Spain : embryo research has been permitted in Spain since July 2003, when the previous government approved legislative reforms related to human assisted reproduction. The changes allowed research on embryos leftover from in vitro fertilization (IVF) treatments, but did not include specific mechanisms for permitting scientists to apply to undertake projects. Under the October 29 2004 Royal Decree approved by the new Socialist government, embryos created by IVF will only be available for research use if they have been frozen for more than 5 years and if the couple involved explicitly authorizes their use for this purpose. Couples who chose to allow their embryos to be used in this way will sign an informed consent form and grant permission for a specific research project. They will not be eligible for remuneration and will have not rights to subsequent patents.
• Asia : the United Kingdom considers itself a leader in the stem cell research field, but some of the country's scientists fear that lead could be squandered due to inadequate funding. In recent days, a group of financial figures and researchers announced plans to establish a £100 million (USD $187.3 million) public–private foundation with the aim of shoring up funding in the area^ref. In September 2004, the UK Department of Trade and Industry (DTI) sent a group of leading figures from the field of stem cell science on a 2-week trip to the China (Beijing, Tianjin, and Shanghai), South Korea (Woo Suk Hwang's lab at the National University and the Stem Cell Research Centre), and Singapore (Biopolis) to assess the quality of science being done and to evaluate possible commercial opportunities : stem cell research was impressive^ref.
• new human embryonic stem cell (hESC) lines, SNUhES1, SNUhES2, and SNUhES3, established from the inner cell mass using an STO feeder layer, satisfy the criteria that characterize pluripotent hESCs: The cell lines express high levels of alkaline phosphatase, cell surface markers (such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), transcription factor Oct-4, and telomerase. When grafted into severe combined immunodeficient mice after prolonged proliferation, these cells maintained the developmental potentials to form derivatives of all three embryonic germ layers. The cell lines have normal karyotypes and distinct identities, revealed from DNA fingerprinting. Interestingly, analysis by electron microscopy clearly shows the morphological difference between undifferentiated and differentiated hESCs. Undifferentiated hESCs have a high ratio of nucleus to cytoplasm, prominent nucleoli, indistinct cell membranes, free ribosomes, and small mitochondria with a few crista, whereas differentiated cells retain irregular nuclear morphology, desmosomes, extensive cytoplasmic membranes, tonofilaments, and highly developed cellular organelles such as Golgi complex with secretory vesicles, endoplasmic reticulum studded with ribosomes, and large mitochondria. Existence of desmosomes and tonofilaments indicates that these cells differentiated into epithelial cells. When in vitro differentiation potentials of these cell lines into cardiomyocytes were examined, SNUhES3 was found to differentiate into cardiomyocytes most effectively^ref.
• from foetal tissues
• fetal liver hematopoietic stem cell (FL HSC) : CD11b / Mac-1⁺AA4.2⁺, most are long-term reconstituting HSCs (LT-HSC), but they cycle more rapidly than adult LT-HSCs. They can generate B-1a B cells, Vg3⁺ and Vg4⁺gd T cells, too. Unlike adult common myeloid progenitor (CMP), FL CMP, but not FL granulocyte-monocyte progenitor (GMP) or FL megakaryocyte-erythrocyte progenitor (MEP), give rise to B cells (but never T cells) both in vivo and in vitro. Likewise, while adult common lymphoid progenitor (CLP) have no macrophage activity in vivo or in vitro, FL CLP form macrophage (but never granulocyte, erythrocyte or megakaryocyte) colonies in methylcellulose and stromal cell culture.
• fetal liver mesenchymal stem cell (MSC) differentiate into osteogenic, chondrogenic and adipogenic lineages when induced in vitro, which means that they have the capability to mature into bone, cartilage and fat tissue. Fetal MSCs are immunologically privileged cells and have potentials for allogeneic transplantation
• human umbilical cord perivascular (HUCPV) cells (CD45^–, CD34^–, SH2⁺, SH3⁺, Thy-1⁺, CD44⁺) occur at a frequency of 1 in 300 in Wharton's jelly. They were often discarded because previous research has concentrated only on the cord blood, where the frequency of MSCs is only 1 in 200 million. Once isolated, it only takes 21 days to generate enough stem cells for up to 1000 therapeutic cell doses. Each HUCPV cell harvest (2–5 x 10⁶, depending on the length of cord available) gave rise to a morphologically homogeneous fibroblastic cell population, which expressed -actin, desmin, vimentin, and 3G5 (a pericyte marker) in culture. We determined the colony-forming unit-fibro-blast (CFU-F) frequency of primary HUCPV cells to be 1:333 and the doubling time, which was 60 hours at passage 0 (P0), decreased to 20 hours at P2. This resulted in a significant cell expansion, producing over 1010 HUCPV cells within 30 days of culture. Furthermore, HUCPV cells cultured in nonosteogenic conditions contained a subpopulation that exhibited a functional osteogenic phenotype and elaborated bone nodules. The frequency of this CFU-osteogenic subpopulation at P1 was 2.6/10⁵ CFU-F, which increased to 7.5/10⁵ CFU-F at P2. Addition of osteogenic supplements to the culture medium resulted in these frequencies increasing to 1.2/10⁴ and 1.3/104 CFU-F, respectively, for P1 and P2. CFU-O were not seen at P0 in either osteogenic or non-osteogenic culture conditions, but P0 HUCPV cells did contain a 20% subpopulation that presented neither class I nor class II cell-surface major histocompatibility complexes (MHC^–/–). This population increased to 95% following passage and cryopreservation (P5). Due to their rapid doubling time, high frequencies of CFU-F and CFU-O, and high MHC^–/– phenotype, HUCPV cells represent a significant source of cells for allogeneic mesenchymal cell-based therapies^ref.
• olfactory ensheathing glial cells (OECs) extracted from the olfactory bulbs of fetuses aborted during the second trimester of pregnancy are thought to have the capacity to provide an environment in which damaged neuron cells recover
Sources : fetal stem cells enters the bloodstream of pregnant women and can be isolated before they differentiated into specific cell types within mum's tissues
• from adult tissues
• adult stem cells (ASC) : pluripotent or multipotent. Their number decreases with increasing age. Anyway their accessibility for autologous therapies in adult patients offers them a distinct advantage over ESCs. When cultured in vitro they differentiate into various cell types losing their stemness. They can be recovered from the following tissues :
• deciduous teeth : stem cells from the pulp of human exfoliated deciduous teeth (SHED) not only became a range of cells, but also could trigger bone formation in mice. They also found the cells multiply 2-3 times faster than stem cells from adult bone marrow and adult teeth. Incisors and canines only yield roughly 20 stem cells each, with molars yielding fewer still. Nevertheless, each stem cell from a baby tooth can reproduce itself billions of times. Someday, he added, we can ask parents to put the baby tooth that comes out in milk, put it in the refrigerator and give a call the next day, and we can get stem cells out. You can freeze them in liquid nitrogen and save them for years and years.
• blood vessels
• bone marrow : bone marrow stem cells (BMSC)
• hematopoietic stem cell (HSC) / hematopoietic progenitor stem cell (HPSC) / hematopoietic stem/progenitor cells (HSC/Ps) / colony-forming unit–spleen (CFU-S) (a name for the HSC, based on the fact that in mice whose marrow has been ablated by irradiation this cell gives rise to colonies of marrow cells in the spleen) : there is < 1 HSC every 50,000 (< 0.05%) bone marrow hematopoietic cells. A single stem cell can restore the entire lymphohematopoietic system of a lethally irradiated animal^ref. CD7⁺33^-38^-44⁺48^-ref90 / Thy-1⁺117 / c-kit⁺133 / AC133⁺150^+ref201 / endothelial protein C receptor (EPCR)^+ref202b / Tie-2⁺244^-ref, Rho^lo, Stro-1⁺, Flt1 / VEGFR1⁺, Runx1⁺ & CBFb⁺, neurofilament⁺, CXCR4, suspension growth⁺, growth factor independent 1 (GFI1)⁺. Deficiency of Rac1 inhibits the ability of HSC to reconstitute hematopoiesis in an engraftment model (which requires adhesion and proliferation in the bone marrow), and the absence of both Rac1 and Rac2 led to the movement of progenitor cells out of he bone marrow and into the peripheral circulation, as a result of decreased adhesion to fibronectin. Rac1 and Rac2 also have roles in regulating the cytoskeletal changes that are required for engraftment and mobilization. Rac1^-/-, and to a greater extent Rac1^-/-Rac2^-/-, HSCs have reduced proliferation in response to growth factors in vitro, correlating with decreased levels of cyclin D1 and phosphorylation of ERK, and increased level of KIP1. By contrast, Rac2^-/- cells are more susceptible to apoptosis after growth-factor stimulation, associated with reduced Akt activation. Therefore, it seems that Rac1 regulates entry into the cell cycle, whereas Rac2 mainly regulates cell survival^ref. Self-renewal is increased by intrinsic (overexpression of HoxB4, Wnt, Notch, and Sonic hedgehog (Shh) signaling pathways) and extrinsic signals (SCF / SLF, Flt3L, thrombopoietin, IL-3 and IL-6) : symmetric and asymmetric mitoses can occur (in the mouse they produce ~ 2.4 x 10⁸ blood cells daily). In the bone marrow, HSC specialized niches are likely formed from a subset of bone marrow stromal cells, which may play a further role in "translating" external signals to support or inhibit (in a contact-dependent or independente fashion) HSC developmental decisions. Migration through the bloodstream to distinct bone marrow niches or to peripheral tissues (eventually contributing to regeneration of non-hematopoietic tissues by transdifferentiation ?) is an additional developmental alternative for HSC. They can no longer generate B-1a B cells, Vg3⁺ and Vg4⁺gd T cells. GFI1^{ref1, ref2} is a negative regulator of HSC proliferation, and the excessive proliferation of GFI1-deficient HSCs results in exhaustion and the observed loss of self-renewal function. This role as a factor restricting HSC proliferation is in contrast to the observation that GFI1 promotes T-cell proliferation, highlighting that transcription-factor function is context dependent and can be cell-type specific.
• CD34⁺Sca-1 / LY-6A⁺Lin^-Hoechst 33342 stained^lorhodamine stained^lo
• Hoechst 33342-stained side population (SP) (ABCB1 / P-glycoprotein (P-GP) / MDR1-mediated stain efflux, blocked by verapamil) : CD31 / PECAM-1 / ENDOCAM⁺34^-, Flk1 / VEGFR2 / KDR^-, VEGF-A⁺, Flt1 / VEGFR1^-, VE-cadherin^-, von Willebrand factor (vWF)^-, factor VIII^-, SM-a-actin^-, calponin^-, SM22a^-, a-actinin^-, desmin^-, Tal-1⁺.
• adult long-term reconstituting (or repopulating) HSC (LT-HSC) CD11b / Mac-1^-13⁺33⁺123 / IL-3Ra⁺AA4.2^-, have the greatest self-renewal capacity and give rise to all hematopoietic lineages throughout life. They are rare in the adult HSC pool

‡
• adult short-term reconstituting (or repopulating) HSC (ST-HSC) generate all hematopoietic lineages for only 8-10 weeks. They are common in the adult HSC pool
The transcriptional repressor Slug / snail-homolog 2 (SNAI2) specifically rescues hematopoietic progenitor cells from lethal doses of g radiation : it is transcriptionally induced by p53 upon irradiation and then protects the damaged cell from apoptosis by directly repressing p53-mediated transcription of puma / BBC3, a key BH3-only antagonist of the antiapoptotic Bcl-2 proteins. The physiologic significance of Slug-mediated repression of puma was established by demonstrating that mice deficient in both genes survive doses of total-body irradiation that lethally deplete hematopoietic progenitor populations in mice lacking only slug. Thus, Slug functions downstream of p53 in developing blood cells as a critical switch that prevents their apoptosis by antagonizing the trans-activation of puma by p53^ref.
Normal tissue injured by ischaemia or trauma and tumor tissue produce VEGF-A₁₆₅, CXCL12 / SDF1 and G-CSF, which in turn up-regulate MMP-9 / gelatinase B in hematopoietic and stromal bone marrow cells. MMPs then activate cytokines promoting cell motility and adhesion. The HSC subset termed circulating endothelial progenitors (CEP) / endothelial progenitor cells (EPC) undergo chemokinesis from bone marrow and are recruited into neo-angiogeneic sites by VEGF-A---Flt1 / VEGFR1 and VEGF-A---Flk1 / VEGFR2 / KDR interactions. Interactions that regulate CEP trafficking through bone marrow microvascular endothelial cells (BMEC) are :

on BMEC	on CEP	function
p110CD62E / E-selectin / ELAM-1 / LECAM-2 (IL-1 / TNF-a stimulation)	CLA-1  other syalyl-Lewis A and B molecules	rolling / growth regulation
CD106 / VCAM-1 / INCAM-110 (IL-1 / TNF-a stimulation)	CD49d / VLA-4 / a4-integrin	firm adhesion / mobilization
CD54 / ICAM-1 (present on resting BMEC)	CD11aCD18 / LFA-1 / aLb2 integrin	firm adhesion / growth regulation
CD31 / PECAM-1 / ENDOCAM	CD31 / PECAM-1 / ENDOCAM	transmigration
delta-like-1 (DLL1)	Notch homolog 1 (TAN-1)	adhesion / growing regulation

EPCs have been recently found to exist circulating in peripheral blood of adults, and home to sites of neovascularization in peripheral tissues. They can also be differentiated from peripheral blood mononuclear cells (PBMNCs). In tumor tissues, EPCs are found in highly vascularized lesions. Early-outgrowth of EPCs (EO-EPCs) were generated from PBMNCs, by culture in the presence of angiogenic factors. These EO-EPCs had the morphological and functional features of endothelial cells and, additionally, they shared antigen-presenting ability. They induced the proliferation of allogeneic lymphocytes in a MLR, and could generate CTLs, with the ability to lyze tumor cells in an antigen-specific manner. The antigen-presenting ability of EO-EPCs, however, was weaker than that of monocyte-derived dendritic cells, but stronger than peripheral blood monocytes. Since EO-EPCs play an important role in the development of tumor angiogenesis, targeting EPCs would be an effective anti-angiogenic strategy. Alternatively, due to their antigen-presenting ability, EO-EPCs can be used as the effectors of anti-tumor immunotherapy. Since they share endothelial antigens, the activation of a cellular immunity against angiogenic vessels can be expected^ref.
HSCs are capable of shifting from a state of relative quiescence under homeostatic conditions to rapid proliferation under conditions of stress. The mechanisms that regulate the relative quiescence of stem cells and its association with self-renewal are unclear, as is the contribution of molecular regulators of the cell cycle to these decisions. Mad1^-/-p27^Kip1-/- bone marrow has a 5.7-fold increase in the frequency of HSCs, and surprisingly, an expanded pool of quiescent HSCs. However, Mad1^-/-p27^Kip1-/- stem cells exhibit an enhanced proliferative response under conditions of stress, such as cytokine stimulation in vitro and regeneration of the haematopoietic system after ablation in vivo. Together these data demonstrate that the MYC-antagonist MAD1 and cyclin-dependent kinase inhibitor p27^Kip1 cooperate to regulate the self-renewal and differentiation of HSCs in a context-dependent manner^ref.
Until recently, stem cells were thought to be endowed with unlimited self-renewal capacity and, thus, assumed exempt from aging. But accumulating evidence over the past decade compellingly argues that a measurable and progressive replicative impairment in the hematopoietic, intestinal, and muscle stem cell activity exists from adulthood to old age, resulting in a decline in stem cell function and rendering stem cell aging as the possible link between cellular aging and organismal aging. By using a previously uncharacterized congenic animal model to study genetic regulation of HSC aging, it was demonstrated definitively that a locus on murine chromosome 2 regulates hematopoietic stem cell aging. In addition to demonstrating that hematopoietic stem cell aging is regulated by a distinct genetic element, experimental evidence links the response of HSCs to DNA DSBs to cellular aging, suggesting DNA integrity influences stem cell aging^ref.
Testing for HSCs expansion in vitro :
• severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay has proven the most reliable method thus far^{ref1, ref2}. When this assay was used, the maximal expansion that was achieved was 2- to 4-fold expansion^{ref1, ref2, ref3, ref4}. However, it does not mean a 2- to 4-fold increase of SRC numbers because it is difficult to distinguish between a truly increased SRC number or increased SRC proliferative potential following culture (and therefore increased human engraftment detection) by this assay^ref. On the other hand, recent data suggest that surface expression of CXCR4 and CD26 in CD34⁺ cells by ex vivo manipulation have a positive and negative effect, respectively, on their homing and engraftment in NOD/SCID mice^{ref1, ref2}. Thus, the true self-renewal division of SRCs in vitro could not be assessed by the quantitative SRC assay. While quantitative SRC assay using limiting dilution analysis (LDA) is the gold standard for measuring expansion, it is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method
• retrovirus-mediated gene marking and direct intra-bone marrow transplantation (iBMT) of CD34⁺ cord blood (CB) cells^ref cultured in serum-free and stroma-dependent (SFSD) conditions using the murine bone marrow stromal cell line HESS-5 and human cytokines FLT3L, thrombopoietin, and stem cell factor (SCF)^{ref1, ref2}. Since these vectors essentially integrate at random, each genomic integration site serves as a distinct clonal marker that can be used to trace the progeny of individual stem cells after transplantation^{ref1, ref2}. Gene-marking studies in SRC assays have successfully elucidated the in vivo kinetics of human HSCs such as proliferation, self-renewal, and multilineage differentiation^{ref1, ref2}. iBMT is 15 times more sensitive than conventional intravenous injection for the detection of SRCs^ref. In an attempt to improve the efficiency of homing, seeding, and repopulation, iBMT has been applied to detect clones used to repopulate different individuals^ref. If the number of SRCs amplified 5-fold during culture, we should ideally find every clone in 5 different mice when cultured cells are divided and transplanted by this method into 10 mice. However, 74 clones were detected in 110 mice that underwent transplantation. Therefore, the net amplification of SRCs was calculated to be at least 1.5-fold. The discrepancy between 1.5-fold amplification from clonal analysis and 5.03-fold expansion from the quantitative SRC assay may be due to the increased SRC proliferative potential and/or homing and engraftment efficiency following culture, which affects the quantitative SRC assay using intravenous injection^{ref1, ref2, ref3}. To further dissect these 2 factors, comparing cultured cells by limiting dilution using iBMT and clonal analysis would be useful. Further study is required to elucidate their relative contribution. To elucidate the in vivo kinetics of human HSCs, CD34⁺38^- cells were infected with lentivirus vector and transplanted into immunodeficient mice. The multilineage differentiation and self-renewal abilities of individual thymus-repopulating clones in primary recipients, and their descending clones paired secondary recipients, were analyzed by tracing lentivirus gene integration sites in each lymphomyeloid progeny using a linear amplification-mediated PCR strategy. This clonal analysis revealed that a single human thymus-repopulating cell had the ability to produce lymphoid and myeloid lineage cells in primary recipient and each secondary recipient, indicating that individual human HSCs expand clonally by self-renewal division. Furthermore, the proportion of HSC clones present in the CD34⁺ cell population decreased as HSCs replicated during extensive repopulation and also as the differentiation capacity of the HSC clones became limited. This indicates the restriction of the ability of individual HSCs despite the expansion of total HSC population. The extensive self-renewal potential was confined in the relatively small proportion of HSC clones. This clonal tracking studies clearly demonstrated that heterogeneity in the self-renewal capacity of HSC clones underlies the differences in clonal longevity in the CD34⁺ stem cell pool^ref
New classes of SRCs have been recently identified^{ref1, ref2, ref3, ref4}
• long-term repopulating cells (LTRCs)
• short-term repopulating cells (STRCs) with lymphomyeloid differentiation potential were identified by transplanting CD34⁺CD38⁺ cells into NOD/SCID-b₂m-null mice^ref. Rapid-SRCs (R-SRCs) identified in Lin^-CD34⁺CD38^lowCD36^- cells repopulate the myeloid and erythroid lineage within 2 weeks of iBMT^ref
Transdifferentiation of HSCs : HSCs have been proposed as a potential source of neural cells for use in repairing brain lesions, but previous studies indicate a low rate of neuronal differentiation and have not provided definite evidence of neuronal phenotype. To test the neurogenic potential of human HSCs, CD34⁺ HSCs from adult human bone marrow were implanted into lesions of the developing spinal cord in the chicken embryo and followed their differentiation by using IHC, retrograde labeling, and electrophysiology. Human cells derived from the implanted population express the neuronal markers NeuN and MAP2 at substantially higher rates than previously reported. These cells exhibit neuronal cytoarchitecture, extend axons into the ventral roots or several segments in length within the spinal white matter, are decorated with synaptotagmin⁺ and GABA⁺ synaptic terminals, and exhibit active membrane properties and spontaneous synaptic potentials characteristic of functionally integrated neurons. Neuronal differentiation is accompanied by loss of CD34 expression. Careful examination with confocal microscopy reveals no signs of heterokaryons, and human cells never express a chicken-specific antigen, suggesting that fusion with host chicken cells is unlikely. The microenvironment in the regenerating spinal cord of the chicken embryo stimulates substantial proportions of adult human HSCs to differentiate into full-fledged neurons. This may open new possibilities for a high-yield production of neurons from a patient's own bone marrow^ref.
although optimal dwell time of TCR-pMHC interaction is a requirement at low cognate pMHC density on the APC surface, at high epitope density there is no impairment of T cell activation by extended TCR-pMHC dwell times^ref.
The expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34^- cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, CDKs, CKIs, and growth-arrest genes. According to the expression of their receptors and transducers, IL-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34^- cells. Conversely, CD34⁺ cells were sensitive to the mitogenic stimulus of TPO. Furthermore, CD34^- cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34⁺ cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34⁺ cells. Thus, the different kinetic status of CD34^- and CD34⁺ cells, detailed by molecular and functional analysis, significantly influences their biological behavior^ref.
Web resources : The human haematopoietic stem cell (HSC) database (SCDb).
• mesenchymal stem cell (MSC) : CD29⁺34^-44⁺71⁺90 / Thy-1⁺106 / VCAM-1⁺120a / TNFRI⁺124 / IL-4R⁺, SH2⁺, SH3⁺, Stro-1⁺, grow as adherent cells in culture.
• multipotent adult progenitor cell (MAPC) / mesodermal progenitor cells (MPC)are MHC class I^- DR^- cells multipotent in generation of differentiated cell types from all the germ layers and do not induce teratoma when implanted into differentiated tissues. Anyway they could arise over time in culture from a fusion between distinct tissue-specific cell-types in the starting population. They fail to stimulate CD4⁺ and CD8⁺ T-cell alloresponses, and were targets of NK cytolysis^ref
• they can differentiate into new fat, cartilage, muscle and bone but stretched or RhoA-tranduced MSCs are more likely to become bone cells. Walking and jogging, which help to strengthen bones, might physiologically place MSCs under stress and trigger the production of bone cells
• bone marrow cells enter and turn into endometrial cells : these findings could explain why some women with endometriosis sometimes have endometrial cells in faraway places, such as the lung or brain
Noninvasive radionuclide imaging (combined SPECT/CT scanner) is well suited to dynamically track the biodistribution and trafficking of MSCs to both target (infarcted) and nontarget organs^ref
They can be mobilized into bloodstream by CXCR4 inhibitors.
‡
• peripheral blood
• HSC (peripheral blood progenitor cells (PBPC)) : stem cells found in the peripheral blood rather than the bone marrow; their numbers can be artificially increased by exposure to hematopoietic growth factors so that they can be extracted before myeloablative chemotherapy and later infused as an autologous bone marrow transplantation. In response to the inclusion of purified CR3/43 anti-MHC monoclonal antibody to well-established culture conditions, mononuclear cells obtained from a single blood donor are converted into pluripotent haematopoietic, neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells
• hemangioblast
‡
• liver
• hepatocytes are the major contributors to liver regeneration
• oval cells (small cells with little cytoplasma and oval nuclei. When liver damage is very severe (paracetamol, CCl₄), or when carcinogens or toxins inhibit hepatocyte expansion (2-acetylaminofluorene), oval cells emerge from the canal of Hering - the point where the terminal bile ducts meet the periportal hepatocytes - and migrate into the hepatic lobule  where they can differentiate into hepatocytres or biliary epithelial cells)
• from dedifferentiation of biliary epithelial cells
• from bone marrow stem cells (BMSCs) : CD34⁺90 / Thy-1⁺117 / c-kit⁺Sca-1 / LY-6A⁺Lin^lo/-. They can generate ...
• hepatocytes (cytokeratin 8⁺; with the same life cycle, ie undergoing polyploidization)
• biliary epithelial cells (CK-19⁺)
• hepatic endothelial cells (factor VIII-associated antigen / protein⁺)
Please note during foetal life pluripotential hepatoblasts reside in the aorta-gonads-mesonephros (AGM) region that are able to differentiate into both hepatocytes (thanks to OsM produced by CD45⁺ hematopoietic cells in fetal liver) and biliary epithelial cells.
• breast: stem cells discovered in material from cosmetic breast reduction surgery. They are tucked into ducts, between epithelial and muscular layers.
• exocrine pancreas : pancreatic islet and ductal populations of the adult mouse pancreas contain precursor cells that proliferate in vitro to form clonal colonies that coexpress neural and pancreatic precursor markers. Upon differentiation, individual clonal colonies produce distinct populations of neurons and glial cells, pancreatic endocrine b-, a- and d-cells, and pancreatic exocrine and stellate cells. Moreover, the newly generated b-like cells demonstrate glucose-dependent Ca²⁺ responsiveness and insulin release. Pancreas colonies do not express markers of ESCs, nor genes suggestive of mesodermal or neural crest origins^ref.
• endocrine pancreas : the nonendocrine fraction of cells that remain after islet isolation consists of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. There is no evidence of b-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas^ref.
• gut
• ventrally emigrating neural tube (VENT) cells within myenteric and submucosal plexus of the enteric nervous system, circular smooth muscle cell layer, and mucosal lining of the stomach and small intestine. Some differentiate into neurons and glial cells of the enteric nervous system, once believed to be derived solely from the neural crest cells. VENT cells originate in the ventral part of the hindbrain, emigrate through the site of attachment of the cranial nerves, and colonize a variety of developing structures including the gastrointestinal tract.
• myocardium

	islet-1 (isl1)+ cardioblasts	cardiac Sca-1 / LY-6A+ cells	cardiac SP cells
Hoeschst 33342 dye efflux	4.5% of cells	3.6% of cells	100% of cells
marker expression	Sca-1 / LY-6A- CD31- CD117 / c-kit- Nkx2.5+ GATA4+ myocytic marker-	Sca-1 / LY-6A+ CD31+ CD117 / c-kit- Nkx2.5- GATA4+ myocytic marker-	Sca-1 / LY-6A+ CD31- CD117 / c-kitlo Nkx2.5- GATA4- myocytic marker-
in vivo localization
number	500-600
progenitor identity determined by lineage tracing
myocytic differentiation in vitro
myocytic differentiation in vivo after cell transplantation
functional evaluation of in vitro differentiated cells
differentiation method	co-culture with neonatal cardiocytes	5-azacytidine	differentiation medium (MEM + 10% FCS + 10-8 M dexamethasone)
differentiated phenotype	+ GATA4, Nkx2.5 + sarcomeric proteins + striations, contractions + E-M coupling + Ca2+ potentials + action potential	+ GATA4, Nkx2.5 + sarcomeric proteins + striations, contractions + sarcomeres	+ GATA4, Nkx2.5, MEF2 + sarcomeric proteins, smooth muscle proteins, endothelial proteins + striations, contractions + sarcomeres
clonogenic	ND	ND	yes
self-renewal	yes	yes ?	yes
multi-potent	no ?	ND	yes (cardiocytes, smooth muscle, endothelial cells)
regenerative capacity	TBD	TBD	yes

• cardiac stem cell (CSC) : ABCB1 / P-glycoprotein (P-GP) / MDR1⁺Sca-1 / LY-6A⁺CD34⁺117 / c-kit⁺Lin^-. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing approximately 70% of the ventricle.
• cardioblasts / cardiac progenitors : despite little evidence for native cardiac precursor cells in the postnatal heart, islet-1 (isl1)⁺ cardiac progenitors have been identified in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1⁺ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca²⁺-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease^ref. Only a few hundred progenitor cells remain in the heart after birth, and this number decreases with age. In the lab, several hundred progenitor cells taken from animals after birth produced millions of cardiac-muscle cells. The progenitor cells are found mainly in ventricles. Unlike cardiac stem cells (CSC), which have a seemingly unlimited capacity for self-renewal, progenitor cells undergo a finite number of divisions. But both types of cells have potential for use in repairing heart damage. And cardiac progenitor cells have one considerable advantage over stem cells: scientists can easily coax them into becoming fully specialized heart-muscle cells, without chemical or hormonal stimuli. In the future, doctors might be able to capture and grow progenitor cells and then transplant them back into the patient's heart. For now, however, isolating substantial numbers of these special cells remains a technical challenge
• skeletal muscle
• muscle-derived stem cells (MDSC) : CD34^+/-43^-45^-56⁺117 / c-kit^-Sca-1 / LY-6A⁺Bcl-2⁺Pax7^-, all myogenic regulatory factor (MRF)^-. They lie between the sarcolemma and the basal lamina of each myofiber. They can be isolated by exclusion of Hoechst 33342 exclusion (side population (SP)) at FACS or slow adhesion characteristics in pre-plating from freshly dissociated muscle. Some subpopulations exist :
• mesenchymal stem cells (MSC) (? overlapping with bone marrow MSCs ?) : CD10^lo13⁺45^-56^lo.
‡
• adipogenic (when stimulated in vitro with dexamethasone) : saturated neutral lipids⁺, PPARg⁺, C/EBPa⁺.
• osteogenic (when stimulated in vitro with dexamethasone or overexpressing BMP-2) : osteocalcin⁺, calcium phosphates⁺, AlkP⁺.
• hematopoietic : CD45⁺.
• chondrogenic (when stimulated in vitro with dexamethasone) : sulfonated GAGs⁺, collagen type II⁺.
• other myogenic lineages (when stimulated in vitro with dexamethasone)
• side population (SP) cells have been isolated based on their ability to efflux the DNA-binding dye Hoechst
‡
• quiescent satellite cells (elongated cells that are closely associated with a muscle fiber; they either are flattened against the fiber or occupy shallow depressions in its surface; wrongly referred to by many as muscle stem cells, they are committed myogenic precursors) : M-cadherin^+/-, c-Met⁺, CD34(truncated isoform)⁺106 / VCAM-1 / INCAM-110⁺, Myf5^+/-, MyoD^+/-, desmin^+/-, Pax7⁺. A decrease in Notch signaling in old muscle causes age-related decline of muscle regeneration

‡ myotrauma
• activated satellite cell / proliferating myogenic precursor / premyoblast : M-cadherin⁺, c-Met⁺, CD34⁺ (full length), Myf5⁺, MyoD⁺, desmin⁺, myogenin^+/-, Pax3.

‡
• myoblasts (terminally differentiated cell) : myogenin⁺, MRF4 and other MRFs⁺, CD34^-, Pax7^-. They undergo fusion to form new multinucleated skeletal myofibers
Over-expression of the MSX1 gene drives a proportion of differentiated myotube nuclei to de-differentiate, re-enter the cell cyle, and subsequently differentiate into other cell types^ref.
Treatment with the 2,6-disubstituted purine reversine induces regression of C2C12 mouse myoblasts to multipotent progenitor cells which can redifferentiate into osteoblasts and adipocytes^ref

Satellite-like cells, sharing both myogenic and endothelial markers, can be obtained from the dorsal aorta of murine embryos : such findings suggest that at least some portion of the satellite cell population may develop indepndently of embryonic myogenesis and may be rooted in vascular development. Anyway the circulating stem cells that can enter muscle via the circulation play no significant role in maintenance or repair of muscle under normal circumstances.
• bone
• CNS
• white-matter progenitor cells (WMPCs)^ref from subcortical white matter of the adult human brain constitute an abundant pool of mitotically competent neurogenic and glial progenitor cells that can be isolated by FACS after either transfection with GFP under the control of the PDE2A promoter, or A2B5 (mAb specific for GQ ganglioside)-targeted immunotagging. Although these cells give rise largely to oligodendrocytes, in low-density culture some also can be passaged as neurospheres in vitro and generate functionally competent neurons and glia both in vitro and after xenograft to the fetal rat brain
• oligodendrocyte progenitor cells (OPCs) (A2B5⁺polysialylated neural cell adhesion molecule (PSA-NCAM)^-) are found in large numbers in both late-gestation and adult human forebrain. PDGF, NT3, and neuregulin 1 / glial growth factor 2 (GGF2) promote proliferation, while IGF-1, exert a maturational effect.
• neural stem cell (NSC) from brain cancer patients are more accurately termed committed progenitor cells because their fate is restricted. Due to similarity to bone marrow (e.g. ECM and strome role, neurofilament⁺ and CD133 / AC133⁺BMI1⁺), these regions are called brain marrow.
• subventricular zone (SVZ) (more commonly known as the periventricular subependymal zone) : in adult mammals, including humans, SVZ contains cells that have the characteristics of glial cells and that can generate neuronal cells in culture^{ref1, ref2, ref3}. In adult humans these glial progenitor cells form a prominent layer, or ribbon, that is restricted to a specific region in the brain that lines the lateral cerebral ventricle^ref. This region is also present in non-human primates, but it is thinner and less well delineated than in humans^{ref1, ref2, ref3, ref4}. Based on the knowledge that about 0.7% of cells in the human SVZ contain a specific nuclear protein, Ki-67, that is associated with DNA synthesis and hence cell division, it is inferred that these cells can proliferate^ref. However, the number of cells that are actually dividing is probably smaller than this, as Ki-67 can also be present in dying cells^{ref1, ref2}. Importantly, although the cells that were Ki-67⁺ also contained marker molecues for astrocytes, they did not contain markers for immature neurons. This observation is similar to that seen with subependymal cells in other parts of the adult mammalian brain, such as the spinal cord or the brain stem, which also do not generate neurons in vivo. However, if these cells are cultured or are transplanted to acceptable cellular environments such as the hippocampus, they can show some neuronal features^ref. A pathway known as the rostral migratory stream (RMS) - which in mammals with a highly developed sense of smell (e.g. rodents) contains neurons that migrate in thousands every day from the SVZ to the olfactory bulb^{ref1, ref2} - is absent in humans : this may be explained by the long distance that separates the olfactory bulbs from the cerebrum in humans and/or our relatively macro-osmatic capabilities. Data from rodents indicate that SVZ NSCs are astrocytes^{ref1, ref2, ref3, ref4}. In the presence of exogenous mitogens, SVZ-derived NSCs self-renew and can differentiate into astrocytes, oligodendrocytes and neurons in vitro^{ref1, ref2, ref3}. Explants of intraoperative and postmortem adult human brain also contain progenitors that can give rise to new neurons and glia^{ref1, ref2, ref3, ref4, ref5, ref6}, but the organization and function of the human germinal zones that contain these progenitors remain poorly understood. It seems that these migratory neuronal precursors are present in humans during infancy but disappear during childhood^ref. This is not unexpected, considering the dramatic loss of olfactory receptor genes in the relatively small human olfactory bulb and the substantial reduction in the size of the rostral migratory stream in non-human primates^ref. However, it highlights the danger of extrapolating results from rodents to humans. Back to the potential glial progenitors, then - what might be their function, given that they do not give rise to neurons? Apart from serving as a major source for the turnover of astrocytes and oligodendrocytes, which occurs normally in the adult primate brain, they also provide a reservoir for a certain type of astrocyte that migrates to sites of traumatic, infectious or degenerative brain damage. In addition, their uncontrolled proliferation is considered to be a main cause of certain types of invasive glial brain tumour. However, it is instructive that these dormant glial progenitors never generate malignant tumours of any neuronal cell type. This indicates that they are probably formidable suppressors that block the production of neuronal cells in the adult human brain^ref. The lack of neuronal turnover and/or replacement of injkured beurons in the adult human brain is not due to the absence of potential neural stem cells. Rather, it is more likely to be due to a remarkable resistance to accept such cells into a mature neuronal network. The systematic decrease in the extent of adult neurogenesis during vertebrate evolution, culminating in primates, may be the result of an adaptation to keep neuronal populations with their accumulated experience for an entire lifespan^ref. Although the therapeutic transplantation of new neurons to regions of the human brain that are responsible for more advanced brain functions may be counterproductive, their transplantation to other regions, such as the sensory or motor systems of the brain, could have enormous clinical significance. So the identification of the cellular and molecular mechanisms that prevent adult NSCs from becoming inttegrated into functional neuronal networks would be a major achievement. Without this, the stimulation of dormant NSCs, and even their survival, may not be sufficient to accomplish brain repair. In the postnatal SVZ, local cues or signaling molecules released from neuroblasts limit the proliferation of GFAP-expressing progenitors thought to be stem cells. However, signals between SVZ cells have not been identified. Depolarization of neuroblasts induces nonsynaptic SNARE-independentGABA_A receptor currents in GFAP-expressing cells, the time course of which depends on GABA uptake in acute mouse slices. GABA_A receptors are tonically activated in GFAP-expressing cells, consistent with the presence of spontaneous depolarizations in neuroblasts that are sufficient to induce GABA release. These data demonstrate the existence of nonsynaptic GABAergic signaling between neuroblasts and GFAP-expressing cells. Furthermore, GABA_A receptor activation in GFAP-expressing cells limits their progression through the cell cycle. Thus, as GFAP-expressing cells generate neuroblasts, GABA released from neuroblasts provides a feedback mechanism to control the proliferation of GFAP-expressing progenitors by activating GABA_A receptors^ref. The neural stem cell niches in the subventricular zone and dentate gyrus are established sequentially and not until late embryonic stages^ref
• hippocampus : with the exceptions of a specific cell type in the dentate gyrus of the hippocampus^ref, the human brain shows no sign of spontaneous neuronal turnover^{ref1, ref2}
When cultured in vitro on laminin layer, FGF-2 and EGF can induce progression to transit amplifying cells (TAC) (PSA-NCAM⁺, a-nestin⁺, a-vimentin⁺, a-GFAP⁺, abIII tubulin⁺ and NSE⁺) arranged into neurospheres (which contain some NSCs but a majority of more restricted neural progenitor cells (NPC)) and then differentiation to all glial and neuronal lineages, populating developing or degenerating (apoptotic) regions of the CNS. Li⁺ increase their expansion by 30%. They constitutively produce a broad range of peptide neurotrophic factors (including BDNF, GDNF, NGF, NT-3, NT-4/5), adhesion molecules (eg L1), ECMs (eg reelin) and lysosomal enzymes.
• retinal stem cell : adult urodeles have the amazing capacity to regenerate an entire retina after complete retinectomy. The CMZ of vertebrates contains cells that are labeled by BrdU (retinal progenitors? neural progenitors?). Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Muller glia.
• cornea
• corneal epithelial stem cells : ABCG2⁺p63⁺TERT⁺14-3-3s^-. Located in epithelial crypts of corneoscleral limbus^ref.
• inner ear
• utricular sensory epithelial stem cells are capable of differentiating into cells representative of ectodermal, endodermal and mesodermal lineages, including hairy cell–like cells
• vestibular organs stem cells
• fatty tissue : leftovers of liposuction of human subcutaneous adipose tissue harbor adipose stromal cells (ASCs) / adipose-derived adult stem (ADAS) cells (CD10⁺11b / a_M integrin^-13⁺29⁺44⁺45^-49e⁺59⁺90⁺HLA-ABC⁺HLA-DR⁺). The absence of the panhematopoietic marker, CD45, indicates that the ADAS cells do not derive from circulating HSCs^ref. They secrete angiogenic and antiapoptotic factors :
• 1203±254 pg of VEGF per 10⁶ cells
• 12 280±2944 pg of HGF per 10⁶ cells
• 1247±346 pg of TGF-ß per 10⁶ cells
• when ASCs are cultured in hypoxic conditions, VEGF secretion increases 5-fold to 5980±1066 pg/106 cells (P=0.0016)
• the secretion of VEGF can also be augmented 200-fold by transfection of ASCs with a plasmid encoding VEGF(P<0.05)
• conditioned media obtained from hypoxic ASCs significantly increases endothelial cell growth (P<0.001) and reduces endothelial cell apoptosis (P<0.05).
Source : isolated from human subcutaneous adipose tissue liposuction waste by collagenase digestion and differential centrifugation. Linear regression analysis of the cells derived from female donors demonstrates a significant negative correlation between the number of ADAS cells/mL_lipoaspirate with the BMI but not the age of the donor^ref.
When puckled from a processed lipoaspirate, primary culture of the cells differentiate in vitro into chondrocytes, skeletal muscle fibers, neurons, osteoblasts, and of course adipocytes^ref.
Web resources : International Fat Applied Technology Society (IFATS)
• skin
• keratinocyte stem cells have high proliferative potential. Epidermal ste-cell progeny give rise to the differentiated cells of the
• interfollicular epidermis (IFE) (long known from observations on healing from burns) : a_2B integrin^hia₃ integrin^hib₁ integrin^hidesmoplakin^lodesmoglein 3^lo
• holoclones : product of a true stem cell which has undergone > 140 doublings. Under standard conditions, < 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate
• paraclones : a population of senescent or differerentiating progenitors which contains exclusively cells with a short replicative lifespan (< 15 cell generations), after which they uniformly abort and terminally differentiate
• meroclones : a population of transient amplifying cells whose growth potential is between the holoclone and the paraclone
The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones. The tips of the rete ridges in human palm are a major repository of epidermal stem cells.
• hair follicles
• bulge cells in the bulge region : connexin 43^-, keratin 15⁺
• sebaceous glands
Telomere length, as well as the catalytic component of telomerase, Tert, are critical determinants in the mobilization of epidermal stem cells. Telomere shortening inhibited mobilization of stem cells out of their niche, impaired hair growth, and resulted in suppression of stem cell proliferative capacity in vitro. In contrast, Tert overexpression in the absence of changes in telomere length promoted stem cell mobilization, hair growth, and stem cell proliferation in vitro. The effects of telomeres and telomerase on stem cell biology anticipate their role in cancer and aging^ref.
• dermis :
• skin-derived precursors (SKP)^ref derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells and generate both neural and mesodermal progeny. SKPs can first be isolated from skin during embryogenesis and persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood^ref.
• isolated multipotential mesenchymal stem cells (MSC) from human postnatal dermal tissues were expanded and maintained for over 100 population doublings with retention of their chromosomal complement and potential for multilineage differentiation. Progeny of cell lines established from a single dermal MSC could be differentiated into adipogenic, osteogenic, and myogenic lineages, consistent with the conclusion that we established a clonal, multipotential, somatic MSC cell line. This study is the first to show potential growth of multipotential human MSC cell lines from a single cell that can be used for the engineering of tissues in vivo. Clonal growth of MSCs presents profound implications in our understanding of differentiation and development, and should provide a valuable resource for tissue repair^ref.
• cell types whose regenerative capacity can be enhanced by coculture with minimally passaged dermal cells or exogenous supply of the ECM protein laminin-10/11 :
• transit-amplifying cells (TAC) are destined to undergo terminal differentiation after a few rounds of division
• early differentiating keratinocytes, which are more committed progenitor cells
Transplantation of all classes of keratinocyte progenitors into an in vivo setting demonstrated that tissue regeneration can be elicited from stem cells, TACs, and early differentiating keratinocytes for up to 10 weeks^ref.
• adult mouse skin contains stem cells that can generate multiple types of tissue when grafted (patches of fur, including skin, follicles, hair, and oil-producing glands)^{ref1, ref2}. If humans are like mice, researchers could use a similar method to extract the stem cells, grow them up and re-implant them into a balding scalp or wound
• both hair and feather follicles show robust regenerative powers that episodically renew the epithelial organ. However, the evolution of feathers (from reptiles to birds) and hairs (from reptiles to mammals) are independent events and their follicular structures result from convergent evolution. Because feathers do not have the anatomical equivalent of a hair follicle bulge, we are interested in determining where their stem cells are localized. By applying long-term label retention, transplantation and DiI tracing to map stem cell activities, feather follicles contain slow-cycling long-term label-retaining cells (LRCs), transient amplifying cells and differentiating keratinocytes. Each population, located in anatomically distinct regions, undergoes dynamic homeostasis during the feather cycle. In the growing follicle, LRCs are enriched in a 'collar bulge' niche. In the moulting follicle, LRCs shift to populate a papillar ectoderm niche near the dermal papilla. On transplantation, LRCs show multipotentiality. In a 3D view, LRCs are configured as a ring that is horizontally placed in radially symmetric feathers but tilted in bilaterally symmetric feathers. The changing topology of stem cell activities may contribute to the construction of complex feather forms^ref.
• placenta (isolated in Jan 2004 from researchers from the National Health Research Institute (NHRI), National Taiwan University Hospital and Cathay General Hospital) : human term placenta is a temporary organ with fetal contributions that is discarded postpartum, so it does not involve the ethical concerns surrounding ESC research. In addition, the placenta, which weighs 700 grams to 800 grams, is a high-yield source of stem cells compared with cord blood, which is only 80 grams to 90 grams in a newborn. These placenta-derived multipotent cells (PDMCs) exhibit many markers common to mesenchymal stem cells—including CD105/endoglin/SH-2, SH-3, and SH-4—and they lack hematopoietic-, endothelial-, and trophoblastic-specific cell markers. In addition, PDMCs exhibit ESC surface markers of SSEA-4, TRA-1-61, and TRA-1-80. Adipogenic, osteogenic, and neurogenic differentiation were achieved after culturing under the appropriate conditions. PDMCs could provide an ethically uncontroversial and easily accessible source of multipotent cells for future experimental and clinical applications^ref.
• germline stem cells (GSCs) :
• for spermatogenesis
• for oogenesis : although males retain GSCs for spermatogenesis throughout adult life, oocyte production in females of most mammalian species is believed to cease before birth^{ref1, ref2, ref3, ref4}. Accordingly, a central dogma of mammalian reproductive biology is that females are born with a finite, non-renewing pool of germ cells, all of which are arrested in meiosis I (oocytes) and are enclosed by somatic cells in structures referred to as follicles. Oocyte numbers decline throughout postnatal life^{ref1, ref2, ref3} through mechanisms involving apoptosis^{ref1, ref2}, eventually leaving the ovaries barren of germ cells^ref. In humans, exhaustion of the oocyte reserve occurs around the fifth decade of life, driving the menopause^ref. The process that is believed to occur in female mammals with respect to germ-cell development differs from that of several invertebrate organisms, including Drosophila melanogaster, in which GSCs maintain oocyte production in adult ovaries^{ref1, ref2, ref3}. This apparent evolutionary disparity is female-specific, as the role of GSCs in maintaining spermatogenesis has been documented in essentially all metazoan species^{ref1, ref2, ref3}. But Jonathan Tilly from Harvard Medical School, Boston, first reported in early 2004 that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance (up to 33% of ovarian follicles), are needed to continuously replenish the follicle pool, which does not dry up. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia : cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries have been shown. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary^ref. It may be that, with age, fewer or more defective eggs are produced because the stem cells are dying off or growing old. In future women might freeze their youthful ovarian stem cells for re-implantation and production of fresh eggs at a later age. Alternatively, researchers might find drugs that can revive flagging stem cells. It is not yet clear whether such stem cells exist in human ovaries or how prolific they might be. Anyway the researchers were unable to isolate the cells, so critics were not convinced. Now, Tilly has isolated the cells and shown that they display genetic markers that are characteristic of stem cells with the ability to develop into eggs. Although he will not disclose his method, he says he obtained 150 to 200 such cells from a single mouse ovary, but he cautions that the cells need to be characterized further before the case is proven. He has identified a gene in the mice that appears to regulate the stem cells' activity : when he knocked out this gene, the resulting mice had 40% more follicles in their ovaries than normal. And he has even identified a molecule, which he calls GSA8, that has a similar effect. Tilly will not reveal the identity of GSA8, but he told that when he injected it into female mice just before puberty, they ended up with almost double the normal number of follicles. If it works in humans, such a chemical could provide a revolutionary treatment for women with a low egg-count, such as cancer survivors or those nearing menopause. The finding supports an earlier suggestion that mammal ovaries could produce eggs throughout life, and may shatter the dogma that women are born with a finite supply. Why would Mother Nature put all her eggs in one basket when they would just sit there and accumulate DNA-related damage? Female flies, fish, birds and now mice all appear to make new eggs throughout life, so it is unlikely that humans would be any different. Ovarian surface epithelial (OSE) cells grown in a lab for 5-6 days differentiated into immature small cells of various different types, while in presence of phenol red they completed the first stage of the division needed to become mature human eggs, capable of being fertilised and developing into an embryo. The OSE cells can be easily harvested from the surface of the ovaries using a laparoscope. The discovery could help those women with poor fertility or who are set to go through an early menopause, who lack follicles in their ovaries, to have a better chance of conceiving through IVF. Development of numerous mature oocytes (eggs) from adult ovarian stem cells in vitro (in the laboratory) offers new strategies for the egg preservation, IVF utilisation, and treatment of female infertility. To test directly the physiological relevance of circulating cells for female fertility, transplantation and parabiotic mouse models were estab35shed to assess the capacity of circulating bone marrow cells to generate ovulated oocytes, both in the steady state and after induced damage. These studies showed no evidence that bone marrow cells, or any other normally circulating cells, contribute to the formation of mature, ovulated oocytes. Instead, cells that travelled to the ovary through the bloodstream exhibited properties characteristic of committed blood leukocytes^ref.
Click here for a scheme of ASCs transdifferentiations / stem-cell plasticity (also across germ layer boundaries !) : by clicking on arrows you will be directed to PubMed abstracts for main reference(s).
Criteria to prove ASC plasticity / transdifferentiation :
• in vitro (GF administration)
• show that the cell is phenotipically like the resident cells of the intended organ
• to prove that the cell is functionally like the resident cells of the intended organ
• gene expression profile specific for the new cell type (FACS, RNA-FISH, IHC, ...)
• in vivo (ASC transplantation)=> in vitro
• identify the differentiated cell as being donor derived
• transgenic donor cells into wild-type recipients
• sex-mismatched (cross-gender) allografts
• in situ hybridization (ISH) looking for Y chromosome after male cells transplantation into immunosuppressed female recipient
• looking for presence of Barr body after female somatic cells trasplantation into immunosuppressed male recipient
• to prove that the cell is functionally like the resident cells of the organ
• rescue from an enzymatic deficiency in the liver
• sinchronous contraction in the heart
• action potential generation in the brain
Recent experiments suggest that the idea that one cell type can dedifferentiate and then redifferentiate into another is an oversimplification :
• BM as a source of germ cells that could sustain oocyte production in adulthood : it has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ATM-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes^ref.
Several explanations, not necessarily mutually exclusive, have emerged :
• stem cells from the bone marrow can be mobilized by i.v. administration of SCF+ G-CSF, travel via the bloodstream to various sites and then differentiate into nonblood cell types: supporting evidence is that SCs are much more likely to be located near a blood vessel than not.
• stem cells from a variety of tissues (in various states of quiescence) can be traced at some point to
• blood
• bone marrow
• cell fusion between 1 or more ASCs and a differentiated cell (which then becomes tetraploid or polyploid) it is not yet know whether cell fusion is an artifact of cell culture, or a fleeting and rare in vivo event that may be an intermediate in one cell type's reinvention as another (both pro-fusion^{1,2,3, and 4}and against fusion references exist¹). The ASCs may reprogramme host cells' genetic material : as the cells contain genetic information from various origins, it's possible they could be unstable and lead to tumor formation. In some experiments HSCs did not engraft and repopulate the liver directly, but hematopoietic engraftment occurred first, followed by liver repopulation starting about 2 months later : maybe a differentiated progeny of HSC (macrophages, lymphocytes, ...) is the actual fusion partner. A reduction division, perhaps by multipolar mitosis is a further confounding factor to the investigation of tissue conversion, potentially masking the fusion history of these cells. Bone-marrow–derived cells (BMDCs) can fuse with with Purkinje neurons, cardiomyocytes and hepatocytes.
• culturing cells may also permit mutations to occur that can change developmental fate
Unanswered questions
• how many tissues contain ASCs ?
• why is there such a lack of ASC markers ?
• is the ASC state actively maintained ?
• what kind of signals can prevent differentiation ?
• is there a relation between ASC and cancer cells ?
• do ASC exhibit plasticity as a normal event in vivo ?
• can ASC transit between tissues via circulation ?
• what are the homing signals for ASCs ?
• is there a universal ASC ?
Scientists have created a "miracle mouse" that can regenerate amputated limbs or badly damaged organs, making it able to recover from injuries that would kill or permanently disable normal animals. The experimental animal is unique among mammals in its ability to regrow its heart, toes, joints and tail. When cells from the test mouse are injected into ordinary mice, they too acquire the ability to regenerate. The discoveries raise the prospect that humans could one day be given the ability to regenerate lost or damaged organs, opening up a new era in medicine. The ability of mice to regenerate appears to be controlled by about a dozen genes : it seems almost certain that humans have comparable genes. We have experimented with amputating or damaging several different organs, such as the heart, toes, tail and ears, and just watched them regrow. The only organ that did not grow back was the brain. When we injected foetal liver cells taken from those animals into ordinary mice, they too gained the power of regeneration : this persisted even 6 months after the injection. Heber-Katz made her discovery when she noticed that the identification holes that scientists punch in the ears of experimental mice healed without any signs of scarring. The self-healing mice, from a strain known as MRL, were then subjected to a series of surgical procedures. In one the mice had their toes amputated - but the digits grew back, complete with joints. In another test some of the tail was cut off but also regenerated. Then the researchers used a cryoprobe to freeze parts of the animals' hearts, only to see these grow back again. A similar phenomenon was observed when the optic nerve was severed and the liver partially destroyed. The MRL mouse seems to have a higher rate of cell division : its cells live and die faster and get replaced faster. The same genes could confer greater longevity and researchers are measuring the animals' survival rate. The mice are, however, only 18 months old and the normal lifespan is 2 years so it is too early to reach conclusions. Scientists have long known that less complex creatures have an impressive ability to regenerate. Many fish and amphibians can regrow internal organs or even whole limbs.
Adult human nuclei injected into immature frog egg cytoplasm are coaxed back to an adaptable, stem-cell-like state : the technique is different from the process used to create Dolly the sheep as this approach leaves the egg's nucleus in place. When the researchers injected the adult human nuclei into immature frog egg nuclei, rather than into the surrounding cytoplasm, Oct-3/4 levels shot up by a factor of 10 : molecules in the frog nucleus may be responsible for the eggs' revitalizing abilities.
The UK Stem Cell Bank, the world's first stem cell bank, has opened its doors in May 2004, funded by the Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC) : the first 2 stem-cell lines (derived from early human embryos, from tissue donated by patients undergoing fertility treatments) to be banked are from King's College London (KCL) and the Centre for Life, a research facility in Newcastle upon Tyne. The centre is also unique as it plans to store the full gamut of stem-cell lines: embryonic, fetal and adult.
Web resources :
• Stem Cell Brief by Royal Society
• Newcastle Centre for Life
• Stem cell information at NIH
• Harvard Stem Cell Institute (HSCI)
• Journals : Stem Cells
Bibliography : Nature insight Stem cells in Nature Vol.414, 1 November 2001

Web resources :

• Cell cycle
• Cell cycle at COPE

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