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1.
FIG. 5.

FIG. 5. From: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors.

Detection of a population of WT1+/PAX2+ cells. (A–D) Four fields of view showing the presence of a rare population of cells that co-localize WT1 and PAX2 in GCTM2 cell fractions (arrow). (E) Magnified image of a cell co-expressing WT1 and PAX2. (F) Corresponding nuclear stain for the isotype control.

S. Adelia Lin, et al. Stem Cells Dev. 2010 October;19(10):1637-1648.
2.
FIG. 1.

FIG. 1. From: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors.

Immunohistochemical analyses of human embryo sections (A–F) and kidney-like structures within teratomas (G–J) formed from hESC injected into SCID mice. a. Staining of 8-week embryonic kidney for WT1 revealed cytoplasmic staining of podocytes (arrow). (B) Isotype-matched negative control for A. (C and D) Staining of 7-week embryonic kidney with cadherin 16 showed immunoreactivity within nephric duct (black arrow) and MM-derived (white arrow) structures. (E) Isotype-matched control for C and D. (F) Podocalyxin was localized to nephric duct (black arrow) and MM-derived (white arrow) structures within the 9.5-week human embryonic kidney. (G) Cadherin 16 staining of a duct-like structure within a teratoma. (H) WT1 immunoreactivity on rare cells within a teratoma. (I) Podocalyxin staining of a glomerular-like structure found in a teratoma and the epithelial tubule, possibly a proximal tubule (white arrow). (J) Isotype-matched negative control for I. Scale bar = 100 μm.

S. Adelia Lin, et al. Stem Cells Dev. 2010 October;19(10):1637-1648.
3.
FIG. 2.

FIG. 2. From: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors.

Cell-sorting strategy. (A) Representative FACS plots showing viable, differentiated human embryonic stem (ES) cells fractionated based on co-expression of CD24, podocalyxin, and varying levels of GCTM2 (denoted ++Hi, ++Low, and ++−). (a) Cells were first distinguished based on forward and side scatter and (b) dead cells were excluded based on the uptake of the viability marker propidium iodide. (d) Cells that co-expressed CD24 and podocalyxin were further gated based on GCTM2 expression resulting in 3 populations. (B) Quantitative gene expression analysis indicates that when compared with unfractionated cells, the ++Low and ++− fractions showed a trend of increased expression of kidney development genes LHX1, PAX2, and WT1 and reduced expression of pluripotent genes OCT4 and GDF3. * indicates a statistically significant difference. Values are presented as means ± standard error of the mean (SEM), n = 5.

S. Adelia Lin, et al. Stem Cells Dev. 2010 October;19(10):1637-1648.
4.
FIG. 4.

FIG. 4. From: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors.

Validation of microarray results by quantitative polymerase chain reaction (PCR). Results of the microarray were validated by quantitative PCR screen of genes involved in the development of each compartment of the mesoderm where expression levels were first normalized against 18S rRNA and calibrated using a CD30-expressing human embryonic stem (ES) cell line as an internal standard. The QPCR results demonstrated a slight up-regulation in transcript levels of all 3 intermediate mesoderm and 2 lateral mesoderm genes, CD34 and CDH5, in the ++− fraction compared with the other 3 fractions, indicating that cells expressing these transcripts are enriched from the unfractionated cell population by sorting based on CD24+/Podo+/GCTM2. Conversely, an overall lower level of gene expression was observed for paraxial mesoderm genes, which also showed little variation in transcript abundance across the cellular fractions, indicating that the cells that express these genes are not selectively isolated by sorting with the above markers. Values are presented as means ± SEM, n = 3.

S. Adelia Lin, et al. Stem Cells Dev. 2010 October;19(10):1637-1648.
5.
FIG. 3.

FIG. 3. From: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors.

Microarray analyses of undifferentiated, unfractionated, and sorted fractions. (A) Heat maps of undifferentiated, unfractionated, and sorted fractions. Combined heat map of 3 biological replicates shows down-regulation of stem cell genes (yellow) and up-regulation of developmental genes (blue) across the undifferentiated (UD), unfractionated (UF), +Low, and ++− fractions. All 3 replicates show similar expression patterns of pluripotent and developmental genes confirming the reproducibility of the array results. (B) Gene ontology analyses performed on the top 1,000 up-regulated genes (B-stat > 0) in the ++− compared with the undifferentiated fraction show a slight increase in percentage of kidney and vascular development-associated genes in the ++− fraction relative to either spontaneously differentiated embryonic stem (ES) cells or cell populations derived from varying stem cell marker expression-based fractionation (red arrowheads). Ontology analysis also indicated a reduced proportion of genes involved in neural and skeletal muscle development in the ++− cell fraction (black arrowheads). (C) Cluster analyses. (a) Cluster analysis of the top 200 up-regulated genes in the ++− fraction compared with undifferentiated ES cells cross-matched with a genitourinary development-specific gene expression database (GUDMAP) shows an overrepresentation of transcripts associated with E11.5 murine metanephric interstitium (green box) and E15.5 nephrogenic and cortical interstitium (yellow box). (b) This overrepresentation was not observed when a randomly generated list of genes was subjected to the same cross-match. (c) Cluster analysis performed on all genes up-regulated only in ++− compared with undifferentiated, spontaneously differentiated, and ++Low fractions shows these genes are associated with the MM at E11.5 (green box), podocytes at E13.5 and E15.5 (yellow box), and the collecting duct and proximal tubules (purple box) when cross-matched with the GUDMAP database.

S. Adelia Lin, et al. Stem Cells Dev. 2010 October;19(10):1637-1648.

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