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Proliferation of Adult Human Small Intestinal Epithelial Stem Cells by a GSK-3β Inhibitor, CHIR99021 Encapsulated in Poly Lactic-co-Glycolic Acid Nanoparticles

  • Author(s): Francis, Rubina Maria Jenova
  • Advisor(s): Dunn, James
  • et al.
Abstract

Successful identification of `Lgr5' as a bona fide stem cell marker of small intestinal crypt base columnar (CBC) adult epithelial stem cells is a major breakthrough in the realm of intestinal stem cell biology and regenerative medicine. Although various culture conditions exist to support the growth of intestinal epithelial cells in vitro, no exogenous conditioned media or growth factor is sufficient enough to provide the essential `niche' signal to sustain growth and proliferation of Lgr5+ stem cells in cultures. More recently, a small molecule inhibitor which is a glycogen synthase kinase 3β (GSK-3β) inhibitor, CHIR99021 was identified as an exogenous factor that contributes toward long-term culture and expansion of mouse small intestinal epithelial stem cells. This small molecule, by specifically inhibiting the activity of GSK-3β, activates the Wnt/β-catenin (canonical Wnt) pathway which is quintessential for maintaining self-renewal and proliferation of CBC stem cells, simultaneously regulating Notch, BMP, and other cell survival pathways. In an attempt to translate this effect in vivo, this report offers an efficient way to encapsulate this small molecule using a nanoparticulate drug delivery vehicular system (PLGA nanoparticles) aimed for a tissue localized delivery. This work focuses on studying the stemness-retaining effect induced by this inhibitor on adult human small intestinal epithelial cells in vitro. To elucidate the efficacy of PLGA encapsulated CHIR99021, various cell culture experiments were conducted on: (i) lentiviral TCF/LEF reporter transduced spheroids to justify the mechanism that CHIR99021triggers canonical Wnt pathway in adult small intestinal epithelial cells, (ii) non-transduced spheroids to determine the morphological and structural changes (conversion of spheroids to enteroids) induced as a result by activation of Wnt/ β-catenin signaling, (iii) small intestinal crypts to evaluate the proliferation of Lgr5+ stem cells and also their survival in the absence of an apoptotic inhibitor but in the presence of CHIR99021. Overall, this work demonstrates that the small molecule inhibitor encapsulated in PLGA nanoparticles is pharmacologically active and its efficacy is demonstrated by various in vitro experiments on adult human small intestinal epithelial cells.

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