Date of Completion

8-22-2013

Embargo Period

8-22-2013

Keywords

Stem Cell, Intestinal Organoid, Lgr5, Dclk1, Tert

Major Advisor

Charles Giardina

Associate Advisor

Craig E. Nelson

Associate Advisor

Ping Zhang

Field of Study

Biochemistry

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Embryonic stem cells, with the ability of self-renewal and differentiation to generate cells of all three germ layers, are promising sources to address developmental questions and also for use in clinical regenerative medicine. Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. Intestinal organoids are multicellular crypt-like structures that can be derived from isolated intestinal stem cells or from embryonic stem cells (ESCs) following described differentiation procedures. In addition to their potential usefulness in tissue repair, intestinal organoids may also be useful as tissue surrogates to study basic aspects of intestinal cell biology and for the identification of novel compounds for intestinal diseases and disorders.

In this work, we first generated a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A to generate intestinal organoids. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.

We next analyzed their cellular compositions of ESC-derived intestinal organoids under different conditions. We found that the resulting organoids were enriched for intestinal stem cells expressing intestinal stem cell markers Lgr5, Msi1, Tert and Dclk1, relative to cells stripped from colonic mucosa. Similar to intestinal crypt cells, the Tert and Lgr5 expressing cells resolved into two distinct populations. Under two different differentiation procedures, the intestinal stem cell population was found to be largely unaffected whereas the expression of secretory vs. absorptive cell genes could be altered. Finally, intestinal organoids derived from cancer-prone ApcMin/+ mice showed reduced Atoh1 expression, as reported for native intestinal tissue, indicating that aberrant differentiation signaling is reproduced in the organoid system. Our data support the use of ESC-derived intestinal organoids as tissue surrogates for the study of early events in intestinal stem cell differentiation. We propose that these organoids may be particularly useful for modeling colonic mucosa at risk for cancer development.

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