Unlimited expansion of intestinal stem cells from a wide range of ages

Abstract

The recent technical advance in cloning and culturing ground-state intestinal stem cells (ISC) provides us an opportunity of accurate assessment of age-related impact on the function of highly proliferative intestinal stem cells. Our ability of indefinitely and robustly expanding single-stem-cell derived pedigrees in vitro allows us to study intestinal stem cells at the clonal level. Interestingly, comparable number of ISC clones was yielded from 1mm endoscopic biopsy of all donors despite the age. They were passaged in vitro as pedigrees and expanded to 1 billion cells in approximately sixty days without changes in stemness demonstrated by clonogenicity and multipotency. Therefore, our study shows that ISCs from a wide range of ages can be cloned and expanded to unlimited number in vitro with similar efficiency and stability. These patient-derived ISCs harbor intrinsic immortality and are ideal for autologous transplantation, supporting the promise of adult-stem-cell based personalized medicine.

Figure 1.

Cloning ISC^GS from patients of wide-range ages A. left, Histogram depicting ages of the patients included in this study. right, Histogram depicting days needed for single ISC^GS from each patient dividing to 1 billion ISCGS. B. Phase contrast images of typical colonies derived from biopsies of 16yr, 42yr and 76yr old patients. Scale bar, 100μm. C. Surface view of ALI cultures. D. Histological sections through ALI cultures via hematoxylin-eosin staining. E. Immunofluorescence on sections of ALI cultures with indicated antibodies to secretory cell markers Mucin 2, Chromogranin A and Defensin A6. Scale bar, 100μm.

Figure 2.

Long-term stability of ISC^GS A. upper, Clonogenicity assay revealing Rhodamine red-stained colonies grown 20days following seeding 1000 ISC^GS cells. Scale bar, 10mm. lower, Quantification of clonogenicity of ground state stem cells from endoscopic biopsy at indicated age range. Error bars, s.d. B. Individual colonies are sampled from the pool and grown as separate lines in isolation. C. upper, Phase contrast images of typical clones at early and late passage of wild-type ISC^GS. middle, Histological sections through ALI cultures via hematoxylin-eosin staining. lower, Immunofluorescence on sections of ALI cultures with indicated antibodies to secretory cell markers Mucin 2. D. Clonogenicity assay of early and late passages of ISC^GS revealing nearly unchanged number of Rhodamine red-stained colonies grown 10 days following seeding 2,000 passaged ISC^GS.