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FIG. 1. Stem cell hierarchy. Zygote and early cell division stages (blastomeres) to the morula stage are defined as totipotent, because they can generate a complex organism. At the blastocyst stage, only the cells of the inner cell mass (ICM) retain the capacity to build up all three primary germ layers, the endoderm, mesoderm, and ectoderm as well as the primordial germ cells (PGC), the founder cells of male and female gametes. In adult tissues, multipotent stem and progenitor cells exist in tissues and organs to replace lost or injured cells. At present, it is not known to what extent adult stem cells may also develop (transdifferentiate) into cells of other lineages or what factors could enhance their differentiation capability (dashed lines). Embryonic stem (ES) cells, derived from the ICM, have the developmental capacity to differentiate in vitro into cells of all somatic cell lineages as well as into male and female germ cells.
FIG. 2. Developmental origin of pluripotent embryonic stem cell lines of the mouse. The scheme demonstrates the derivation of embryonic stem cells (ESC), embryonic carcinoma cells (ECC), and embryonic germ cells (EGC) from different embryonic stages of the mouse. ECC are derived from malignant teratocarcinomas that originate from embryos (blastocysts or egg cylinder stages) transplanted to extrauterine sites. EGC are cultured from primordial germ cells (PGC) isolated from the genital ridges between embryonic day 9 to 12.5. Bar = 100 µm. [From Boheler et al. (40).]
FIG. 3. Human pluripotent embryonic stem (ES) and embryonic germ (EG) cells have been derived from in vitro cultured ICM cells of blastocysts (after in vitro fertilization) and from primordial germ cells (PGC) isolated from aborted fetuses, respectively.
FIG. 4. Regulation of self-renewal in mouse ES cells by Oct3/4, Nanog, BMP-dependent SMAD, and LIF-dependent JAK/STAT3 signaling pathways. A: transcription factors, such as Oct3/4, Nanog, Sox2, and FoxD3, control early developmental stages from totipotent to pluripotent developmental stages. B: self-renewal (proliferation) of undifferentiated mouse ES cells is regulated by Nanog, Oct-3/4, and tightly regulated interactions between LIF-dependent JAK/STAT3 pathway(s) and BMP-dependent activation of Id target genes. A MEK-ERK signaling mechanism prevents ES cell self-renewal. Oct-3/4 and Nanog expression prevents differentiation into trophectoderm, primitive endoderm, and mesoderm cells. C: the relative expression level of Oct-3/4 determines the fate of ES cells. [Adapted from Cavaleri and Schöler (71), Ying et al. (410), and Niwa et al. (251).]
FIG. 5. Gene targeting, conditional expression, and ES cell-derived models in vivo and in vitro. A: site-specific insertion and excision events in ES cells can be mediated by Cre recombinase-loxP recombination. In this example, a gene locus in ES cells has been targeted by homologous recombination to insert a PGK-neoR cassette flanked by two loxP sites. Following selection with G418, a clonal ES cell line containing one wild-type (WT) allele and one targeted allele (TA) was isolated and transiently transfected with pBS185 (CMV promoter-driven Cre recombinase) and pPPP (PGK-PacR cassette flanked by two loxP sites). After puromycin selection, the ES cells were clonally expanded to identify independent and stable integration events. Possible Cre recombinase-mediated insertion or deletion events are indicated in the diagram. B: genotyping by PCR was performed to identify clonal ES cell lines that had lost the neomycin resistance cassette. An internal control (-globin, -Glo) was included for each DNA preparation to ensure against false negatives. Similar protocols are employed to genotype transgenic mice. C: clonal ES cell lines can be tested by Southern analysis to identify which cell clones had undergone deletion or insertion events. In this example, four distinct bands could be identified: 1) an 8.9-kb band corresponding to the WT allele; 2) a 9.4-kb band of the original targeted allele containing the neomycin resistance cassette; 3) a 7.9-kb band where the neomycin resistance cassette has been lost and the flanking loxP sites have recombined (deletion); and 4) a 6.6-kb band generated by digestion of the newly inserted Cre recombinase targeted allele. D: targeted ES cell lines can be injected into blastocysts and used to generate chimeric mice that can be bred to generate homozygous animal models. E: in some instances, gene targeting can lead to embryonic lethality, but targeted chromosomal pairs coupled with in vitro differentiation can be used to elucidate the underlying mechanisms of embryonic lethality in mice. Loss of functional ryanodine receptor (RyR2), for example, leads to embryonic lethality at E10.5, but following in vitro differentiation of ES cells, we found that RyR2 regulated the spontaneous rate of beating (beats per minute, bpm) in ES cell-derived cardiomyocytes (408), and this effect on rate resulted in inadequate blood perfusion and embryonic lethality in mice.
FIG. 6. In vitro differentiation of ES cells. Undifferentiated mouse ES cells (A) develop in vitro via three-dimensional aggregates (embryoid body, B) into differentiated cell types of all three primary germ layers. Shown are differentiated cell types labeled by tissue-specific antibodies (in parentheses). C: cardiomyocytes (titin Z-band epitope). D: skeletal muscle (titin Z-band epitope). E: smooth muscle (smooth muscle -actin). F: neuronal (III tubulin). G: glial (glial fibrillary acidic protein, GFAP). H: epithelial cells (cytokeratin 8). I: pancreatic endocrine cells [insulin (red), C-peptide (green), insulin and C-peptide colabeling (yellow)]. K and L: hepatocytes (K, albumin; L, 1-antitrypsin). Bars = 0.5 µm (H), 20 µm (I), 25 µm (C, D, E), 30 µm (K, L), 50 µm (B, G), and 100 µm (A, F).
FIG. 7. Schematic overview of gene trapping. A: endogenous wild-type genes usually produce heterogeneous nuclear RNA transcripts that are spliced to form mature mRNAs. One approach to gene trapping employs constructs that contain a reporter gene sequence between a splice acceptor (SA) and a polyadenylation signal (pA). When inserted into a functional gene, the endogenous splice donor (SD) and gene trap splice acceptor are processed to form a fusion transcript to activate the reporter gene contained in the gene trap construct. The transgene is only activated when it integrates correctly within an active transcriptional unit. Some translational fusions (frame shifts) may inactivate the reporter activity or may target the translated proteins into subcellular locations where reporter activity is not easily detectable. Gene trapping therefore selects for integration events in functional genes, and it is especially useful for the analysis of mammalian cells that have complex genomic organizations that consist of promoters and exons that are separated by introns. B: expression of the gene trap is assayed for reporter gene expression (e.g., -galactosidase activity), and staining is indicative of an insertion event. In this figure, we show a gene trap construct incorporated within jumonji and expressing LacZ. Embryos (E9.5 and E11.5) were stained with X-gal. (Figure kindly provided by G. Lyons.)
FIG. 8. Principal steps of serial analysis of gene expression (SAGE). Two assumptions are critical for SAGE analyses: 1) short DNA sequences (10–14 bp) are sufficient to identify individual gene products and 2) concatenation (linking together) of short DNA sequences or tags increases the efficiency of identifying expressed mRNAs in a sequence-based assay. To generate the sequences, purified mRNA from ES cells (or any other cell line) is used to generate double-stranded cDNA. With the use of streptavidin-coated magnetic beads, double-stranded cDNA is purified, followed by digestion with a type I restriction enzyme or anchoring enzyme that recognizes specific sites located in the double-stranded DNA recognition sequence (CATG for NlaIII). The fragments, located closest to the biotinylated primer, are purified by binding to magnetic beads, divided in half and ligated to two linker/primer sets. SAGE tags are generated by digestion of the cDNA molecules with a type II restriction enzyme or tagging enzyme, which cleaves DNA several bases away from the recognition sites. The SAGE tags are joined to form ditags and amplified by PCR with a set of primers that recognize linkers A and B. The ditags are separated from the linkers and ligated together to form concatemers of purified ditags. These are then subcloned into a plasmid vector, amplified, and sequenced. The individual tags can then be extracted by identifying the CATG anchoring enzyme sequences. Each individual tag sequence is then run against GenBank databases to identify the corresponding gene product, and comparisons among SAGE libraries (http://www.ncbi.nlm.nih.gov/sage/) facilitate the identification of factors implicated in ES cell identity. [Adapted from Boheler and Wobus (43).]
FIG. 9. Proposed strategies of cell therapy using human ES cells for the treatment of heart and central nervous system diseases and diabetes. Pluripotent human ES cells must first be propagated in vitro. ES cells may then be selectively differentiated into cardiac, neural, or pancreatic progenitor cells, which have the capacity for terminal differentiation in vitro. Defined progenitor cells are selected and purified followed by further differentiation/maturation and transplantation into the injured or damaged tissue to integrate and develop into functional cardiomyocytes, neurons, and pancreatic endocrine cells, respectively. For the treatment of cardiac infarcts or diabetes, mature cells may be necessary, whereas for the treatment of neurodegenerative diseases, neuronal progenitor cells could be applicable. [Adapted from Boheler and Wobus (43).]
FIG. 10. Strategy of human "therapeutic cloning" to generate autologous tissue grafts. Somatic donor cell nuclei are fused to enucleated oocytes. In the context of the oocyte cytoplasm, the genome of adult cells is reprogrammed to an embryonic status. From this embryo, blastocysts are developed that are used to establish human ES cells. These ES cells are subsequently differentiated in vitro into the desired cell type to generate an autologous tissue graft for transplantation. [Adapted from Lanza et al. (201).]
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