Single cell transcriptomics of fetal kidney and Wilms tumor nephrogenic progenitors

Purpose: Bulk transcriptomics analysis of Wilms tumor SIX2+CITED1+ cells to compare and identify unique nephron progenitor transcriptome profiling (RNA-seq) signature between unfavorable and favorable Wilms Tumors and against human fetal kidney Methods: Wilms tumor and human fetal kidney samples were collected and transported on ice at 4°C in RPMI-1640 and a single cell suspensions were prepared following mechanical and enzymatic dissociation as previously publication. Enzymatic dissociation was performed with 125?U/ml collagenase I in RPMI-1640 at 37?°C for 35?min. The digested cells were then passed through a 100-µm cell strainer and a 40-µm cell strainer with washes of 1x PBS. The cell suspension was than centrifuged at 1500?rpm for 5?min and erythrocytes were eliminated using a red blood cell lysis kit. WT SIX2+CITED1+ cells were isolated using SIX2-Cy5 and CITED1-Cy3 Smartflare RNA probes following manufacturer's instructions and previous publication. Briefly, cells were incubated over night at 37?°C with both RNA probes diluted at 1:20 in PBS and 25ul/ml in RPMI-1640 supplemented with 5% FBS, and 0.2% antimicrobial agent Primocin. After 16-18 h, cells were dissociation using TrypLE 1x for 5 min, cells were centrifuged at 1500 rpm for 5 min and prepared for FACS. Freshly isolated SIX2+CITED1+ cells from a 16 WGA hFK, freshly isolated SIX2+CITED1+ cells from WT8 (favorable stage II Wilms' Tumor), freshly digested xenograft derived from cultured SIX2+CITED1+ cells (Passage 6) isolated from WT8, freshly digested xenograft generated from freshly isolated SIX2+CITED1+ cells from WT8, and total cell population of WT8. Approximately 3,000 cells were captured on a 10x Chromium device using a 10X V2 Single Cell 3' Solution kit. All protocols were performed following manufacturer's instructions. Final library concentrations were determined using a Qubit high Sensitivity DNA assay Kit. Final sequencing libraries were analyzed on a Sequencing: HiSeq 4000, 2x150bp PE, and ~625M total reads/lane (QuickBiology) to determine the library size; Approximately, 300 million reads per sample were sequenced Results: Transcriptomics analysis of Wilms Tumor SIX2+CITED1+ cells in comparison to human fetal kidneys confirmed the nephrogenic signature of hFK-SIX2+CITED1+ and WT-SIX2+CITED1+ cells but highlighted differences in expression of pluripotency and self renewal-related genes.Trajectory inference analysis generated from sc-RNAseq data of SIX2+CITED1+ cells and cells dissociated from total WT (from where the SIX2+CITED1+ cells were isolated), suggests that SIX2+CITED1+ cells are the root cells that give rise to all the tumor cell types. Conclusion: Our study represents the first single cell transcriptomic characterization of Wilms Tumor cancer stem cells (SIX2+CITED1+) against human fetal kidney SIX2+CITED1+ cells and against the total tumor and xenografts from cultured and fresh isolated SIX2+CITED1+ WT cells. Identifying that WT SIX2+CITED1+ cells arise early in nephrogenesis and have an expression pattern favoring proliferation over differentiation that overlaps considerably with the expression pattern of both the tumor of origin and with the expression pattern of WT they generate after grafting. Overall design: Single cell transcriptomics of SIX2+CITED1+ cells from hFK and WT against total WT and xenografts generated from cultured and freshly isolated SIX2+CITED1+ cells.

### 研究目的
对肾母细胞瘤（Wilms Tumor, WT）SIX2+CITED1+细胞开展批量转录组分析，旨在对比并鉴定不良预后与良好预后肾母细胞瘤之间，以及与人类胎儿肾脏之间独特的肾祖细胞转录组测序（RNA-seq）特征。

### 实验方法
收集肾母细胞瘤及人类胎儿肾脏样本，置于添加有RPMI-1640培养基的冰浴中4℃转运；参照既往发表研究，通过机械联合酶解法制备单细胞悬液：于37℃下使用含125 U/ml I型胶原酶的RPMI-1640培养基消化35 min，将消化后的细胞依次通过100 μm及40 μm细胞筛，并用1×PBS冲洗细胞悬液；随后以1500 rpm离心5 min，采用红细胞裂解试剂盒去除红细胞。
按照制造商说明书及既往发表研究，使用SIX2-Cy5与CITED1-Cy3 Smartflare荧光RNA探针分离肾母细胞瘤的SIX2+CITED1+细胞：将探针以1:20的比例稀释于添加有5%胎牛血清、25 μl/ml Primocin抗菌剂的RPMI-1640培养基中，与细胞于37℃共孵育过夜；孵育16~18 h后，使用1×TrypLE解离液消化细胞5 min，以1500 rpm离心5 min，制备后用于荧光激活细胞分选（FACS）。
本次实验涉及的样本包括：16周孕龄人类胎儿肾脏（hFK）新鲜分离的SIX2+CITED1+细胞、良好预后II期肾母细胞瘤WT8新鲜分离的SIX2+CITED1+细胞、从WT8分离的第6代培养SIX2+CITED1+细胞构建的异种移植瘤新鲜消化细胞、从WT8新鲜分离的SIX2+CITED1+细胞构建的异种移植瘤新鲜消化细胞，以及WT8的总细胞群。
使用10X V2单细胞3'试剂盒搭配10x Chromium系统，每个样本捕获约3000个细胞；采用Qubit高灵敏度DNA检测试剂盒测定最终文库浓度；使用HiSeq 4000平台进行2×150 bp双端测序，每个泳道约6.25亿条reads（QuickBiology公司），每个样本测序量约3亿条reads。

### 实验结果
对比人类胎儿肾脏的肾母细胞瘤SIX2+CITED1+细胞转录组分析结果，证实了hFK-SIX2+CITED1+与WT-SIX2+CITED1+细胞均具有肾发生特征，但凸显了二者在多能性及自我更新相关基因表达上的差异。基于SIX2+CITED1+细胞及从总肾母细胞瘤（分离SIX2+CITED1+细胞的来源组织）解离细胞的单细胞RNA测序（sc-RNAseq）数据开展的轨迹推断分析显示，SIX2+CITED1+细胞是产生所有肿瘤细胞类型的起始细胞群。

### 研究结论
本研究首次完成了肾母细胞瘤癌干细胞（SIX2+CITED1+）的单细胞转录组表征，并以人类胎儿肾脏SIX2+CITED1+细胞、WT8总肿瘤细胞群，以及从培养及新鲜分离的SIX2+CITED1+ WT细胞构建的异种移植瘤作为对照。研究发现，WT SIX2+CITED1+细胞起源于肾发生早期，其表达模式倾向于增殖而非分化，且与起源肿瘤及移植后生成的肿瘤的表达模式高度重合。

### 实验整体设计
本研究的单细胞转录组分析样本包括：来自人类胎儿肾脏的SIX2+CITED1+细胞、来自肾母细胞瘤的SIX2+CITED1+细胞，以及肾母细胞瘤总细胞群和从培养及新鲜分离的SIX2+CITED1+细胞构建的异种移植瘤。