Culturing and transcriptome profiling of progenitor-like colonies derived from adult mouse pancreas

Abstract Background Transplantation of insulin-producing cells is considered an important diabetes therapy. Many research studies have shown that insulin-producing cells can be derived from the in-vitro cultured pancreatic colonies with self-renewal ability and multilineage potential. Even though these progenitor-like colonies have been prepared from adult pancreas cells, the efficient culture method is hardly established and regulation of the colonies is rarely known. We confirmed previously that single cells acquired from adult mouse pancreas could form cyst-like colonies in a 3D semi-solid system containing Matrigel and methylcellulose. These colonies could be passaged continuously without losing progenitor-like capacity. In the previous culturing system, however, conditioned medium from murine embryonic-stem-cell-derived pancreatic-like cells was used. This unregulated ingredient may reduce repeatability and affect following study. Thus, a new culturing system with certain components needs to be developed. Methods Single cell suspension was acquired from adult mouse pancreas and cultured in a Matrigel-based 3D system with epidermal growth factor, Nicotinamide, B27, and Noggin to form ring colonies. Serial-passage assay was performed to evaluate self-renewal ability. Real-time polymerase chain reaction and immunostaining were used to detect the expression of progenitor-related genes. A 2D differentiation method was used to testify the multilineage potency of the colonies. High-throughput sequencing (HTS) of the colonies was performed to profile the differentially expressed genes. Results We developed a 3D culturing system deprived of conditioned medium to propagate those colonies with high proliferative efficiency. HTS of the transcriptome of mRNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) showed differentially expressed genes compared to the whole pancreas (as control). In mRNAs, several surface marker genes were identified in the colonies. Moreover in noncoding RNAs, miR-21a, miR-31 and miR-155 were upregulated and miR-217, miR-802 and miR-375 were downregulated in colonies along with a number of other miRNAs and lncRNAs. Conclusions Our results offer an efficient culture system for pancreatic progenitor-like colonies and HTS of the colonies serves as a target resource for following study of in-vitro cultured pancreatic progenitors. These findings should also contribute to our understanding of the transcriptional regulation of these progenitor-like colonies and the mechanisms behind their functions

Data_Sheet_1_The microbiome types of colorectal tissue are potentially associated with the prognosis of patients with colorectal cancer.docx

As the second leading cause of cancer worldwide, colorectal cancer (CRC) is associated with a poor prognosis. Although recent studies have explored prognostic markers in patients with CRC, whether tissue microbes carry prognostic information remains unknown. Here, by assessing the colorectal tissue microbes of 533 CRC patients, we found that Proteobacteria (43.5%), Firmicutes (25.3%), and Actinobacteria (23.0%) dominated the colorectal tissue microbiota, which was different from the gut microbiota. Moreover, two clear clusters were obtained by clustering based on the tissue microbes across all samples. By comparison, the relative abundances of Proteobacteria and Bacteroidetes in cluster 1 were significantly higher than those in cluster 2; while compared with cluster 1, Firmicutes and Actinobacteria were more abundant in cluster 2. In addition, the Firmicutes/Bacteroidetes ratios in cluster 1 were significantly lower than those in cluster 2. Further, compared with cluster 2, patients in cluster 1 had relatively poor survival (Log-rank test, p = 0.0067). By correlating tissue microbes with patient survival, we found that the relative abundance of dominant phyla, including Proteobacteria, Firmicutes, and Bacteroidetes, was significantly associated with survival in CRC patients. Besides, the co-occurrence network of tissue microbes at the phylum level of cluster 2 was more complicated than that of cluster 1. Lastly, we detected some pathogenic bacteria enriched in cluster 1 that promote the development of CRC, thus leading to poor survival. In contrast, cluster 2 showed significant increases in the abundance of some probiotics and genera that resist cancer development. Altogether, this study provides the first evidence that the tissue microbiome of CRC patients carries prognostic information and can help design approaches for clinically evaluating the survival of CRC patients.</p

α-Ketoglutarate Promotes Pancreatic Progenitor-Like Cell Proliferation

A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG), a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133+ and CD133− cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133+ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet). αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet

Riemann solvers and numerical methods for fluid dynamics: a practical introduction

High resolution upwind and centred methods are a mature generation of computational techniques applicable to a range of disciplines, Computational Fluid Dynamics being the most prominent. This book gives a practical presentation of this class of techniques