The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids

Abstract

Intestinal organoids are innovative in vitro tools to study the digestive epithelium. The objective of this study was to generate jejunum and colon organoids from suckling and weaned piglets in order to determine the extent to which organoids retain a location-specific and a developmental stage-specific phenotype. Organoids were studied at three time points by gene expression profiling for comparison with the transcriptomic patterns observed in crypts in vivo. In addition, the gut microbiota and the metabolome were analyzed to characterize the luminal environment of epithelial cells at the origin of organoids. The location-specific expression of 60 genes differentially expressed between jejunum and colon crypts from suckling piglets was partially retained (48%) in the derived organoids at all time point. The regional expression of these genes was independent of luminal signals since the major differences in microbiota and metabolome observed in vivo between the jejunum and the colon were not reproduced in vitro. In contrast, the regional expression of other genes was erased in organoids. Moreover, the developmental stage-specific expression of 30 genes differentially expressed between the jejunum crypts of suckling and weaned piglets was not stably retained in the derived organoids. Differentiation of organoids was necessary to observe the regional expression of certain genes while it was not sufficient to reproduce developmental stage-specific expression patterns. In conclusion, piglet intestinal organoids retained a location-specific phenotype while the characteristics of developmental stage were erased in vitro. Reproducing more closely the luminal environment might help to increase the physiological relevance of intestinal organoids.

Disruption of the primocolonizing microbiota alters epithelial homeostasis and imprints stem cells in the colon of neonatal piglets

“…Indeed, the expression of LYZ and TLR4 remained reduced in organoids derived from piglets treated with colistin, even after several passages that ensured the elimination of residual microbiota-derived compounds. These results are in line with previous studies showing that tissue-derived intestinal organoids retained some transcriptomic signatures specific to the gut region 38,[64][65][66] and to the disease state. 67,68 In contrast, other studies showed that organoids derived from germ-free or conventional mice did not retain the transcriptomic differences observed in vivo.…”

The Early Life Microbiota Is Not a Major Factor Underlying the Susceptibility to Postweaning Diarrhea in Piglets

“…Previous experiments in the Caco-2 human cell line showed that TMA reduced cell growth but did not alter the epithelial barrier function ( 35 , 36 ). To circumvent the lack of cellular diversity and genomic abnormalities of cell lines, we tested the effects of TMA in suckling piglet colon organoids that contain the main cell types and reproduce the 3D architecture of the intestinal epithelium ( 37 , 38 ). Our results showed that, contrary to our hypothesis, TMA did not alter epithelial homeostasis when considering cell morphology, TEER, and the gene expression of proteins involved in tight-junctions, epithelium renewal, innate immunity, redox enzymes, or ion transport.…”

“…We used colon organoids derived from a 21-day-old suckling piglet and cryopreserved at passage 1, as described previously ( 37 ). A cryovial containing colon organoids was thawed at 37°C and centrifuged (at 300 g, 5 min, and room temperature).…”

“…Experiments were repeated five times (passages 3 to 8). A total of 500 ng RNA purified with Direct-zol RNA MiniPrep Plus kit (Zymo Research) were retrotranscribed into cDNA with the kit GoScript Reverse Transcription Mix, Random primer (Promega) as described previously ( 37 ). Gene expression was analyzed by real-time qPCR using Biomark microfluidic system using a Dynamic Array IFC for gene expression (Fluidigm) according to the manufacturer recommendations and with specific primers (Table S2).…”

Initial pig developmental stage influences intestinal organoid growth but not phenotype

“…These changes are thought to be largely driven by changes in the luminal environment occurring at weaning, including both gut microbiota composition and nutrients modifications [11]. Mussard et al revealed that porcine organoids derived from pre-(21-day old) and post-weaning (35-day old) piglets harbored very close phenotypes, suggesting that the weaning-induced changes in luminal environment do not imprint piglet ISC [12]. Beside weaning, the neonatal period (from birth to post-natal day 28 in pigs), is also a key period in terms of intestinal development, with changes in epithelium morphology and gene expression with post-natal age [13], as well as ISC dynamics depending on gut location [14].…”