The Life and Death of Differentiated and Dedifferentiated Intestinal Absorptive and Secretory Cells
Authors: Pearce Sarah C. , Kishida Kunihiro , Yu Shiyan , Gao Nan , Ferraris Ronaldo P. 
Published: June 2021

Intestinal stem cells (ISC) differentiate into many types of secretory and absorptive progenies which migrate up the villi and are exfoliated after 3–5 d. Because epithelial homeostasis requires balanced rates of proliferation, migration, differentiation and apoptosis, studies increasing our understanding of cell turnover are critical. We used murine primary intestinal organoids to determine lifespan of different intestinal cell types. We tested the hypotheses that organoids directed to differentiate into distinct cell types would exhibit lifespans similar to those previously estimated in vivo, and that forced dedifferentiation of organoids containing mature cells would result in reacquisition of ISC characteristics and extension of lifespan. We directed, via Wnt and Notch signaling, strict ISC differentiation into specialized organoids comprised primarily of stem cells (ISC), enterocytes (ENT), goblet cells (GOB), or Paneth cells (PAN). Cell type enrichment was confirmed via biomarker mRNA expression and immunofluorescence, as follows: stem cells (~95%), enterocytes (~90%), goblet (~85%) or Paneth cells (~65%) which contain relative proportions much higher than in situ.We found that continuously proliferating ISC organoids can be passaged several times; in contrast, ENT and GOB organoids do not live past 5 or 4 d, respectively, and PAN organoids live ~11 d. These findings are similar to known in vivo lifespans. During differentiation, ENT and GOB organoids exhibited a gradual increase in expression of enterocyte biomarker sucrase isomaltase and goblet cell biomarker mucin‐2, respectively, while levels of stem cell markers Leucine Rich Repeat Containing G Protein‐Coupled Receptor 5 (Lgr5) and SRY‐Box 9 (Sox9) decreased dramatically. Forced dedifferentiation of ENT and GOB organoids was characterized by a dramatic decrease in expression of differentiated cell markers paralleled by pronounced increases in stem cell characteristics.Differentiation was accompanied by sharp decreases over time in anti‐apoptotic B‐cell lymphoma 2 (Bcl2) in both ENT and GOB organoids (>90%). Changes in expression of pro‐apoptotic caspase‐3 (Casp3) and Fas Cell Surface Death Receptor (Fas) were insignificant. Dedifferentiated ENT organoids (dENT) expressed altered apoptotic markers compared to their normal ENT and ISC counterparts. Casp3 and Fas levels decreased 50 and 70% respectively in dENT organoids compared to ENT controls while Bcl2 and Tumor protein p53 (P53) increased significantly (1.6 and 5‐fold respectively). In dedifferentiated goblet organoids (dGOB), Bcl2 and P53 also increased 2–3‐fold. Phenotypically, life‐span increased markedly in both dENT and dGOB organoids by more than 2‐fold due to dedifferentiation, but cell morphology was not altered.In conclusion, we were able to differentiate organoids enriched in distinct cell lineages, then to induce these specialized organoids to undergo in vitro dedifferentiation which markedly extended their life span. In vitro lifespan of specific cell type organoids was similar to that in vivo, and was strongly correlated with mRNA levels of Bcl2. Additionally, we were able to induce specialized organoids to undergo in vitro dedifferentiation and markedly extend their lifespan. This study furthers our understanding of organoid physiology, intestinal cell turnover, and plasticity of cell fate.Support or Funding InformationSupported by NSF Grants No. IOS‐1121049 and 1456673 (RPF). RPF also received support from NIH R01‐DK‐102934 (NG).