Expression analysis of BMP7 responsive P2RY1/ALK3 expressing progenitor like cells within the human exocrine pancreas

Purpose: Here we demonstrate ALK3Bright/PDX1+ cells residing within the human pancreatic ducts have progenitor like characteristics. Using flow cytometery, live-cell sorting of ALK3bright/PDX1+ cells is possible using a surrogate surface marker for PDX1 (P2RY1). Treating ALK3bright/P2RY1+ cells with BMP7 results in their expansion. Later removal of BMP7 results in the differentiation of these cells to ß-like cells. Here we compare the mRNA expression profiles of these three different cell types (in triplicate). Methods: mRNA profiles of ALK3Bright/P2RY1+ cells isolated from human non-endocrine pancreatic tissue, ALK3Bright/P2RY1+ cells treated with BMP7 and ALK3Bright/P2RY1+ cells differentiated to ß-like cells after BMP7 removal were generated by deep sequencing, in triplicate, using Illumina HiSeq PE Cluster Kit v4 and Illumina HiSeq Flow Cell v4 with 50 nt paired end reads plus dual index reads using the Illumina HiSeq SBS kit v4. Sequence reads that passed quality filters were analyzed at the transcript isoform level following alignment using TopHat v2.1.0 followed by exon and gene level counting using Bioconductor easyRNASeq v 2.4.7. Conclusions: Our study represents the first detailed analysis of ALK3Bright/P2RY1+ sorted cells with biological replicates. We demonstrate ALK3Bright/P2RY1+ cells were shown to form progenitor-like epithelial colonies characterized by NKX6.1 and PDX1 expression. Unlike the negative fraction controls, these colonies responded to BMP-7 by generating new ß-like cells as well as cells from other pancreatic lineages. The transcriptional profile of these cells and their BMP7 treated counterparts suggest a mitotic and progenitor like state. Our studies confirm the progenitor-like nature of ALK3Bright/PDX1+ cells within the human pancreas and suggest a specific anatomical location within the ductal network. Overall design: Comparison of transcriptional expression in Alk3Bright/P2RY1+ cells, Alk3Bright/P2RY1+ cells treated with BMP7 and Alk3Bright/P2RY1+ cells allowed to differentiate after BMP7 removal. Human islets, isolated from the same donors were included as a control.研究目的：本研究证实，存在于人类胰腺导管内的ALK3Bright/PDX1+ 细胞（ALK3Bright/PDX1+ cells）具备祖细胞样特性。以P2RY1（P2RY1）作为PDX1（PDX1）的替代表面标志物，可通过流式细胞术（flow cytometry）完成ALK3Bright/PDX1+ 细胞的活细胞分选（live-cell sorting）。使用骨形态发生蛋白7（BMP7）处理ALK3Bright/P2RY1+ 细胞可实现其扩增，移除BMP7后则可诱导这些细胞分化为β样细胞。本研究对这三类细胞的mRNA表达谱（mRNA expression profiles）进行了三次重复比较分析。 实验方法：本研究通过深度测序（deep sequencing）生成了三类细胞的mRNA表达谱，均设置三次生物学重复：从人类非内分泌胰腺组织中分离的ALK3Bright/P2RY1+ 细胞、经BMP7处理的ALK3Bright/P2RY1+ 细胞，以及移除BMP7后诱导分化为β样细胞的ALK3Bright/P2RY1+ 细胞。测序使用Illumina HiSeq PE Cluster Kit v4、Illumina HiSeq Flow Cell v4，采用50 nt双端测序读段及双索引读段，并搭配Illumina HiSeq SBS Kit v4完成测序。对通过质量过滤（quality filters）的序列读段，先使用TopHat v2.1.0进行序列比对（alignment），随后在转录本亚型（transcript isoform）水平开展分析，并通过Bioconductor easyRNASeq v2.4.7完成外显子与基因水平的计数。 研究结论：本研究首次针对分选出的ALK3Bright/P2RY1+ 细胞开展了带有生物学重复（biological replicates）的详细转录组分析。研究证实，ALK3Bright/P2RY1+ 细胞可形成以上皮细胞标志物NKX6.1（NKX6.1）与PDX1表达为特征的祖细胞样上皮集落。与阴性组分对照组不同，此类集落可响应BMP7刺激，生成新的β样细胞及其他胰腺谱系细胞。上述细胞及其经BMP7处理的对应细胞的转录谱（transcriptional profile），均提示其处于有丝分裂活跃且具备祖细胞特性的状态。本研究证实了人类胰腺导管内ALK3Bright/PDX1+ 细胞的祖细胞样属性，并提示其在导管网络（ductal network）中存在特定的解剖学定位。 总体实验设计：对比三类细胞的转录表达谱：ALK3Bright/P2RY1+ 细胞、经BMP7处理的ALK3Bright/P2RY1+ 细胞，以及移除BMP7后完成分化的ALK3Bright/P2RY1+ 细胞。同时纳入从相同供体中分离的人类胰岛（human islets）作为对照。