Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro.
OBJECTIVE
Long-term propagation of human colonic epithelial cells (HCEC) of adult origin has been a challenge; currently used HCEC lines are of malignant origin and/or contain multiple cytogenetic changes. We sought to immortalize human colon biopsy-derived cells expressing stem cell markers and retaining multilineage epithelial differentiation capability.


METHODS
We isolated and cultured cells from biopsy samples of 2 patients undergoing routine screening colonoscopy. Cells were immortalized by expression of the nononcogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of human telomerase (hTERT) and maintained for more than 1 year in culture.


RESULTS
The actively proliferating HCECs expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. Upon growth arrest, cells assumed a cuboidal shape, decreased their mesenchymal features, and expressed markers of colonic epithelial cells such as cytokeratin 18, zonula occludens-1, mucins-1 and -2, antigen A33, and dipeptidyl peptidase 4. Immortalized cells expressed stem cell markers that included LGR5, BMI1, CD29, and CD44. When placed in Matrigel in the absence of a mesenchymal feeder layer, individual cells divided and formed self-organizing, cyst-like structures; a subset of cells exhibited mucin-2 or polarized villin staining.


CONCLUSIONS
We established immortalized HCECs that are capable of self-renewal and multilineage differentiation. These cells should serve as valuable reagents for studying colon stem cell biology, differentiation, and pathogenesis.
Roig
Andres I
AI
Department of Cell Biology, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Eskiocak
Ugur
U
Hight
Suzie K
SK
Kim
Sang Bum
SB
Delgado
Oliver
O
Souza
Rhonda F
RF
Spechler
Stuart J
SJ
Wright
Woodring E
WE
Shay
Jerry W
JW
D015415:Biomarkers / Q000378:metabolism*; D001706:Biopsy; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D049109:Cell Proliferation; D048430:Cell Shape; D003094:Collagen / Q000378:metabolism; D003106:Colon / Q000378:metabolism* / Q000648:ultrastructure; D003113:Colonoscopy; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics; D004338:Drug Combinations; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007621:Karyotyping; D007797:Laminin / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism* / Q000648:ultrastructure; D019098:Telomerase / Q000235:genetics; D013997:Time Factors; D014162:Transfection
D015415:Biomarkers; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; C509186:TERT protein, human; D019098:Telomerase