Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2+ cells generating gastric spheroids differed from adult glandular Lgr5+ stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5+ stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2+ cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2+ cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

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Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical subcloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell−derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications.

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