Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profile of normal human mammary epithelial stem/progenitor and myoepithelial cells

ABSTRACT: There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in DFCI-1 medium retain a fraction with progenitor cell properties. These cells co-express basal, luminal and stem/progenitor cell markers. Clonal derivatives of progenitors co-expressing these markers fall into two distinct types: K5+/K19- (Type I) and K5+/K19+ (Type II). We show that both types of progenitor cells have self-renewal and differentiation ability. Through microarray analysis, we want to identify genes and pathways linked to human mammary epithelial stem/progenitor cell self-renewal and differentiation. Normal human mammary epithelial cells (hMECs) were isolated from Reduction Mammoplasty and immortalized by hTERT. Type I K5+/K19- and Type II K5+/K19+ cell colonies were isolated from hTERT-immortalized hMECs and cultured in MEGM medium for self-renewal and differentiation. Total RNA isolated from Type I, Type II, and differentiated Myoepithelial (Myo) cells were used on Affymetrix microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Gautam Malhotra

PROVIDER: E-GEOD-22580 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:TSV40ER and hTERT immortalized mammospheres undergo transformation in culture to generate breast cancer stem cells The stem cell hypothesis of cancer predicts that cancers arise from the transformation of normal stem/progenitor cells present within adult tissues. However, the molecular mechanisms that lead to the transformation of these cells remain largely unknown, mainly owing to the lack of suitable model systems. In this study, we have generated an immortalized breast epithelial cell line, NBLE, by over-expressing the SV40 Early Region and the catalytic domain of human telomerase into mammosphere-derived cells that are enriched in breast stem/progenitor cells. While primary mammospheres fail to grow beyond four passages in suspension culture, NBLE cells propagated continuously generating spheres at a high frequency of 35%. These cells also expressed stemness-related genes, and differentiated into both luminal and myoepithelial lineages. Interestingly, after around 135 population doublings in culture, the late passage cells exhibited a mesenchymal morphology and contained >90% of CD44+/CD24Low/- cells resembling breast cancer stem cells. When injected subcutaneously into nude mice, the late passage NBLEs formed invasive tumors closely resembling breast adenocarcinomas, the most common type of breast cancer. The transformed NBLE cells showed hyperactivation of Wnt, Hh and TGFbeta pathways, suggesting that the deregulation of these self-renewal pathways may have in part contributed to tumorigenesis. Gene expression analysis further revealed a high degree of similarity with breast cancer stem cells. Thus, our study provides a unique model system to study signaling pathways involved in self-renewal, differentiation, and transformation of breast stem/progenitor cells. Additionally, the NBLE cells can be utilized for screening drugs specifically targeting breast cancer stem cells. NBLE cells were generated by over-expressing SV40ER and hTERT into mammosphere-derived cells. These cells harnored CD44+/CD24- fraction that resembled breast cancer stem cells. To understand the unique gene expression of this fraction we performed microarray on CD44+/CD24- versus rest fractions from NBLE cells (Rep 1 and Rep 2). To see if this gene profile has any similarity with breast cancer stem cells, we performed CD44+/CD24- versus rest microarray on two primary breast tumors (CB272 and CB258-Rep1 andRep2).

Project description:Immortalized colonic epithelial progenitor cells derived from normal human colon biopsies express stem cell markers and differentiate in vitro The sustained growth of normal non-transformed human colonic epithelial cells has historically been exceptionally challenging. We here report the successful long-term propagation of colon biopsy derived cells expressing both epithelial and mesenchymal features, intestinal stem cell markers, and demonstrating multilineage epithelial differentiation capability. Cells isolated from the biopsies of two patients undergoing routine screening colonoscopy have been successfully passaged in vitro, subsequently immortalized with non-oncogenic proteins (cyclin dependent kinase 4 [CDK4] and the catalytic component of human telomerase [hTERT]), and maintained for well over a year in continuous cell culture. Immunofluorescence experiments reveal the immortalized populations of cells significantly express the progenitor epithelial cell marker mucin-1 and the colon epithelial specific marker A33. In addition, staining shows subsets of cells express chromogranin A, mucin-2, and dipeptyl peptidase 4, corresponding to neuroendocrine, mucus secreting, and absorptive colonic cell lineages, respectively. The cells also possess some mesenchymal features, including vimentin and α- smooth muscle actin expression when proliferating actively. Finally, immortalized cells express various stem cell markers including LGR5, BMI1, CD29, and CD44. When cells are seeded at low density in Matrigel® in the absence of a mesenchymal feeder layer individual cells are able to divide and form self-organizing, cyst-like structures with a subset of cells exhibiting either mucin-2 or polarized villin staining. These findings demonstrate that the immortalized human colonic cells are capable of self renewal and multilineage differentiation. These cells should serve as valuable cellular reagents for studying normal colon stem cell biology, differentiation, and disease development.

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Gene expression profile of normal human mammary epithelial stem/progenitor and myoepithelial cells

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