Results: 5

1.
Fig. 3.

Fig. 3. From: Rare somatic cells from human breast tissue exhibit extensive lineage plasticity.

Expression of pluripotency markers NANOG, OCT3/4, and SOX2 in single-cell–derived R1 colonies. (A) Phase contrast images of hESCs H7 (Top row), EpCAM+-R1–derived colonies (two Middle rows) and R4 cells (Bottom row) cultured on feeders and immunofluorescence analysis documenting expression of NANOG, OCT3/4, and SOX2. Scatterplot shows EpCAM distribution in R1 sorted cells (third row). R4 cells lack expression of NANOG and OCT3/4 but express the epithelial marker EpCAM. (B) Coincident expression of NANOG, OCT3/4, and SOX2 in individual cells assessed by FACS after 14 d of culture on feeder layer. Left: R1 single cell subclones; Center: hESCs H7; Right: R1 unstained (isotype control). Upper three panels: coanalysis of NANOG and OCT3/4. Lower three panels: coanalysis of SOX2 and OCT3/4. Far Right Upper panel: 93% of cells from R1 single cell subclones coexpressed EpCAM and OCT3/4. (C) Western blot analysis of NANOG, OCT3/4, and SOX2 in single-cell–derived R1 colonies on feeders after 14 d. H7, positive control; reduction mammary fibroblasts (RMFs) and R4 cells, negative controls. Loading control: γ-tubulin. Results were obtained in five of five analyses.

Somdutta Roy, et al. Proc Natl Acad Sci U S A. 2013 March 19;110(12):4598-4603.
2.
Fig. 4.

Fig. 4. From: Rare somatic cells from human breast tissue exhibit extensive lineage plasticity.

R1 cells form teratomas in immunocompromised mice. R1 cells directly isolated from reduction mammoplasty (A) or a single-cell–derived R1 clone expanded in culture (B) were grafted under the renal capsule of 7- to 8-wk-old female SCID/beige mice. Teratomas, harvested 16 wk after injection, were paraffin embedded, serial sectioned, and stained for the panhuman-specific marker lamin A/C to document the human origin of these structures and for lineage-specific markers. Neuroepithelial (ectodermal) marker, GFAP; pancreatic (endodermal) marker, PDX1; or cartilage (mesodermal) marker, HAPLN1 validated human-specific tissue formation in teratomas. Insets: magnified GFAP+ cells. Representative views show bone stained with Alizarin Red and a gastrointestinal structure with goblet cells expressing TFF3 (A, Lower). Additional views of structures expressing the endodermal marker AFP and corresponding staining for human lamin A/C are shown (B, Lower). (Scale bars, 150 µm.) Directly sorted R1 cells generated teratomas in two of five analyses, failed to generate any mass or structures in two of five analyses and gave rise to a mesoderm derivative (cartilage) in one of five analyses. In one of one attempt, a single-cell–derived R1 subclone formed a teratoma and gave rise to representative three germ layers.

Somdutta Roy, et al. Proc Natl Acad Sci U S A. 2013 March 19;110(12):4598-4603.
3.
Fig. 1.

Fig. 1. From: Rare somatic cells from human breast tissue exhibit extensive lineage plasticity.

Assessment of R1 for ectodermal multilineage potential. (A) Expression of multi- and pluripotency-associated genes in directly sorted R1–R4 subpopulations (n = 4) and hESC H7 (n = 3) assayed by qPCR array. Results are expressed as fold changes compared with hESC H9 (n = 3) and normalized to the housekeeping gene GAPDH. Blue shading highlights the similarities in gene expression levels for H7 and R1. (B) Distribution of R1–R4 subsets within the total epithelial population and ability to form mammospheres from either 1 cell or 1,000 cells. Results are expressed as averaged mean and percentages ±SEM (n = 10). Individual values are shown in Dataset S1. (C) Mammosphere initiating capacity for R1–R4 subpopulations was assessed using 10,000 cells (first passage) and 1,000 cells (subsequent passages) (n = 5). Actual values are shown in the legend of Fig. S3A. (D) Distribution of myoepithelial (α-6-integrin/CD49f+), luminal (MUC-1+), and bipotent (α-6-integrin/CD49f+ and MUC-1+) cells assessed by FACS after dissociation of colonies derived from successive passages of mammospheres. P, passage. Quantitative data are shown in the SI Methods. (E) Human β-casein expression in R1–R3 mammosphere-derived cells by Western blot analysis. Loading control: actin. Positive control: BT-20 cell line. (F) Ducts consisting of a luminal layer expressing CK8/18 (green) and a myoepithelial layer expressing α-SMA (red) stained with human-specific antibodies documenting the human origin of R1-derived structures formed in mouse fat pads. (Scale bar, 100 µm.)

Somdutta Roy, et al. Proc Natl Acad Sci U S A. 2013 March 19;110(12):4598-4603.
4.
Fig. 2.

Fig. 2. From: Rare somatic cells from human breast tissue exhibit extensive lineage plasticity.

Assessment of R1 for endodermal and mesodermal lineage potential. (A) Immunostaining of R1 cells for PDX1, SOX17, and NKX6.1 after 12 d of pancreatic differentiation: 40% cells were SOX17+, almost all SOX17+ cells also expressed PDX1 and NKX6.1. (B) Oil Red O staining of R1 cells and MSCs (experimental control) after 9 d of adipogenic differentiation. Transcript levels of fatty acid binding protein 4 (FABP4), LEPTIN, and PPARγ (normalized to glucuronidase B) (GUSB) in directly sorted R1, R1 in expansion medium (R1-ctrl), R1 in adipogenic differentiation medium (R1-diff), MSC in expansion medium (MSC-ctrl), MSC in adipogenic differentiation medium (MSC-diff), and directly sorted R4. R4 cells fail to grow under R1 expansion conditions. Expression differences were validated at the protein level for FABP4 by immunostaining. (C) Analysis of expression of the endothelial marker CD31 in R1 cells after 7 d of endothelial differentiation by FACS. Positive control: human umbilical vein endothelial cells (HUVEC) cells. (D) Cord formation capacity evaluated after 24 h in endothelial Matrigel differentiation assay by phase-contrast microscopy for (a) CD31+ R1 cells from C, (b) CD31+ HUVEC from C, (c) primary human mammary epithelial cells, or (d) primary mammary epithelial cells after 24 h of culture in mammary epithelial cell growth medium (MEGM) as a negative control. (Scale bars, 100 μm.) These results were obtained in five of five analyses.

Somdutta Roy, et al. Proc Natl Acad Sci U S A. 2013 March 19;110(12):4598-4603.
5.
Fig. 5.

Fig. 5. From: Rare somatic cells from human breast tissue exhibit extensive lineage plasticity.

R1-derived clones are mortal and distinct from hESCs, iPSCs, and MSCs. (A) Expression of selected genes was compared between breast-derived R1 cells [either uncultured (n = 4) or expanded on feeder layers (n = 3) or in media (n = 3)], hESCs (n = 2), human iPSCs (n = 3), and human MSCs (n = 4) by qPCR-array analysis. Gene expression values in R1 cells, under the three conditions described above, are similar to each other and share several key expression characteristics with pluripotent hESCs and human iPSCs (OCT3/4, NANOG, SOX2, CD24 and EpCAM) (green shaded values) easily distinguishable from MSCs. Likewise, these cells share other expression characteristics with MSCs (DNMT3B and NT5E/CD73) (red shaded values) easily distinguishable from hESC and human iPSCs. Several genes (GPC4 and THY1/CD90) (gray shaded values) exhibit unique expression levels in the three breast-derived R1 cells compared with hESC, iPSCs, and MSCs verifying their unique identity. Transcript expression levels, normalized to GAPDH expression, are relative to H9 human ES cells. Full details of analysis are found in Dataset S2. (B) Growth curve of a single R1 cell expanded in culture demonstrating that cells eventually enter senescence. Black arrows indicate passage doublings (PDs) 43, 50, and 56 at which karyotypes were confirmed to be diploid 46, XX (normal). (C) Representative karyotype at PD 50. (D) Red dots (on curve in B) indicate PDs [mid (PD 51) and late (PD 58)] at which cell cycle was analyzed. FACS analysis using propidium iodide (PI) staining demonstrated a G1 arrest at late passage. (E) Expression of hTERT normalized to GUSB and telomerase activity evaluated using the TRAPeze XL Telomerase Detection kit were measured in the indicated cell lines. TPG, total products generated. Cells from five of five individual patient samples arrested at late passage and maintained diploid 46, XX karyotypes.

Somdutta Roy, et al. Proc Natl Acad Sci U S A. 2013 March 19;110(12):4598-4603.

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