Culturing and transcriptome profiling of progenitor-like colonies derived from adult mouse pancreas.
Culturing and transcriptome profiling of progenitor-like colonies derived from adult mouse pancreas.
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来自成年小鼠胰腺的祖细胞样集落的培养和转录组分析
DOI：
10.1186/s13287-017-0626-y
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发表时间：
2017-07-26
影响因子：
7.5
通讯作者：
Jin L
中科院分区：
文献类型：
--
作者：
Ma D;Tang S;Song J;Wu Q;Zhang F;Xing Y;Pan Y;Zhang Y;Jiang J;Zhang Y;Jin L
胰岛素产生细胞的移植被认为是一种重要的糖尿病治疗方法。许多研究表明，体外培养的胰腺集落可以分化为具有自我更新能力和多向分化潜能的胰岛素分泌细胞。即使这些祖细胞样集落已经从成年胰腺细胞制备，有效的培养方法几乎没有建立，并且集落的调控很少为人所知。我们以前证实，从成年小鼠胰腺获得的单细胞可以在含有Matrigel和甲基纤维素的3D半固体系统中形成囊肿样集落。这些集落可以连续传代而不丧失祖细胞样能力。然而，在先前的培养系统中，使用来自鼠胚胎干细胞衍生的胰腺样细胞的条件培养基。这种不受管制的成分可能会降低重复性并影响后续研究。因此，需要开发具有某些组件的新培养系统。从成年小鼠胰腺获得单细胞悬浮液，并在具有表皮生长因子、烟酰胺、B27和头蛋白的基于Matrigel的3D系统中培养以形成环状集落。采用连续传代实验评价细胞的自我更新能力。采用实时荧光定量聚合酶链反应和免疫组化方法检测其祖细胞相关基因的表达。采用二维分化法验证了克隆的多向分化能力。对菌落进行高通量测序（HTS）以分析差异表达的基因。我们开发了一个三维培养系统剥夺了条件培养基繁殖这些集落具有高增殖效率。mRNA、microRNA（miRNA）和长链非编码RNA（lncRNA）的转录组的HTS显示与整个胰腺（作为对照）相比差异表达的基因。在mRNA中，在菌落中鉴定了几种表面标记基因。此外，在非编码RNA中，miR-21 a、miR-31和miR-155在集落中上调，而miR-217、miR-802和miR-375与许多其他miRNA和lncRNA一起沿着下调。我们的结果提供了一个有效的培养系统，胰腺祖细胞样集落和HTS的集落作为一个目标资源，在体外培养的胰腺祖细胞的研究。这些发现也将有助于我们理解这些祖细胞样集落的转录调控及其功能背后的机制。本文的在线版本（doi：10.1186/s13287-017-0626-y）包含补充材料，可供授权用户使用。
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. 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. 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. 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. The online version of this article (doi:10.1186/s13287-017-0626-y) contains supplementary material, which is available to authorized users.
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影响因子：
2.9
作者：
Azevedo-Pouly AC;Sutaria DS;Jiang J;Elgamal OA;Amari F;Allard D;Grippo PJ;Coppola V;Schmittgen TD
通讯作者：
Schmittgen TD
影响因子：
10.5
作者：
Arnes L;Akerman I;Balderes DA;Ferrer J;Sussel L
通讯作者：
Sussel L
影响因子：
7.7
作者：
Filios SR;Shalev A
通讯作者：
Shalev A
影响因子：
1.2
作者：
Jin L;Gao D;Feng T;Tremblay JR;Ghazalli N;Luo A;Rawson J;Quijano JC;Chai J;Wedeken L;Hsu J;LeBon J;Walker S;Shih HP;Mahdavi A;Tirrell DA;Riggs AD;Ku HT
通讯作者：
Ku HT
影响因子：
9.2
作者：
Boo L;Ho WY;Ali NM;Yeap SK;Ky H;Chan KG;Yin WF;Satharasinghe DA;Liew WC;Tan SW;Ong HK;Cheong SK
通讯作者：
Cheong SK