This study aimed to assess the changes of small intestinal morphology, progenitors, differentiated epithelial cells, and potential mechanisms in neonatal piglets. Hematoxylin and eosin staining of samples from 36 piglets suggested that dramatic changes were observed in the jejunum crypts depth and crypt fission index of neonatal piglets (P < 0.001). The number of intestinal stem cells (ISC) tended to increase (P < 0.10), and a decreased number of enteroendocrine cells appeared in the jejunal crypt on d 7 (P < 0.05). Furthermore, the mRNA expression of jejunal chromogranin A (ChgA) was down-regulated in d 7 piglets (P < 0.05). There was an up-regulation of the adult ISC marker gene of SPARC related modular calcium binding 2 (Smoc2), and Wnt/β-catenin target genes on d 7 (P < 0.05). These results were further verified in vitro enteroid culture experiments. A mass of hollow spheroids was cultured from the fetal intestine of 0-d-old piglets (P < 0.001), whereas substantial organoids with budding and branching structures were cultured from the intestine of 7-d-old piglets (P < 0.001). The difference was reflected by the organoid budding efficiency, crypt domains per organoid, and the surface area of the organoid. Furthermore, spheroids on d 0 had more Ki67-positive cells and enteroendocrine cells (P < 0.05) and showed a decreasing trend in the ISC and goblet cells (P < 0.10). Moreover, the mRNA expression of spheroids differed markedly from that of organoids, with low expression of intestinal differentiation gene (Lysozyme; P < 0.05), epithelial-specific markers (Villin, E-cadherin; P < 0.05), and adult ISC markers (leucine-rich repeat-containing G protein-coupled receptor 5 [Lgr5], Smoc2; P < 0.001), and up-regulation of fetal marker (connexin 43 [Cnx43]; P < 0.05). The mRNA expression of relevant genes was up-regulated, and involved in Wnt/β-catenin, epidermal growth factor (EGF), Notch, and bone morphogenetic protein (BMP) signaling on d 7 organoids (P < 0.05). Spheroids displayed low differentiated phenotype and high proliferation, while organoids exhibited strong differentiation potential. These results indicated that the conversion from the fetal progenitors (spheroids) to adult ISC (normal organoids) might largely be responsible for the fast development of intestinal epithelial cells in neonatal piglets.

本研究旨在评估新生仔猪小肠形态、祖细胞、分化上皮细胞和潜在机制的变化。36头仔猪样品的苏木精和伊红染色表明新生仔猪空肠隐窝深度和隐窝裂变指数发生了显着变化（P  <0.001）。肠道干细胞（ISC）数量呈增加趋势（P  < 0.10），第7天空肠隐窝出现肠内分泌细胞数量减少（P  < 0.05）。此外，在 d 7 仔猪中空肠嗜铬粒蛋白 A ( ChgA )的 mRNA 表达下调 ( P < 0.05)。SPARC相关模块钙结合2（Smoc2）和Wnt/β-catenin靶基因的成人ISC标记基因在第7天上调（P  < 0.05）。这些结果在体外小肠培养实验中得到了进一步验证。从 0 日龄仔猪的胎肠中培养出大量空心球体（P  < 0.001），而从 7 日龄仔猪的肠中培养出大量具有出芽和分枝结构的类器官（P  < 0.001）。这种差异反映在类器官出芽效率、每个类器官的隐窝域和类器官的表面积上。此外，第 0 天的球体有更多的 Ki67 阳性细胞和肠内分泌细胞（P < 0.05)，ISC 和杯状细胞呈下降趋势( P  < 0.10)。此外，球体的 mRNA 表达与类器官存在显着差异，肠分化基因（溶菌酶；P  < 0.05）、上皮特异性标志物（Villin、E-cadherin；P  < 0.05）和成人 ISC 标志物（富含亮氨酸重复序列的 G 蛋白偶联受体 5 [ Lgr5 ]、Smoc2；P  < 0.001）和胎儿标志物（连接蛋白 43 [ Cnx43 ]；P < 0.05)。相关基因的mRNA表达上调，并参与了d 7 类器官的Wnt/β-catenin、表皮生长因子（EGF）、Notch和骨形态发生蛋白（BMP）信号通路（P  < 0.05）。球体表现出低分化表型和高增殖，而类器官表现出很强的分化潜力。这些结果表明，从胎儿祖细胞（球状体）到成年 ISC（正常类器官）的转化可能是新生仔猪肠上皮细胞快速发育的主要原因。