A hierarchical, data-driven approach to modeling single-cell populations predicts latent causes of cell-to-cell variability. Loos et al

We hypothesized that nerve growth factor (NGF)-mediated Erk1/2 phosphorylation is differently modulated in subpopulations of primary sensory neurons when neurons are cultured on poly-D-lysine (PDL) versus collagen type I (Col I). For this, dissociated sensory neurons from dorsal root ganglia of male Sprague Dawley rats were cultured overnight on PDL or Col I and were stimulated acutely with NGF (kinetic: 0, 1, 5, 15, 30, 60, 120 min and 20 ng/ml; dose response: 1h and 0, 0.16, 0.8, 4, 20, 100, 500 ng/ml). NGF-induced signaling activity was monitored by detecting phosphorylated Erk1/2 via immunocytochemistry with phospho-specific antibody labelling (NGF kinetic (file: KM14_KM28KM31KM34_NGFkinetic_pErk_ResultsFinal_Cells) and NGF dose response (file: KM14_KM60KM62KM67KM68_NGFdoseresponse_pErk_ResultsFinal_Cells)). Additionally, co-labelling with antibodies against TrkA (receptor of NGF, file: KM14_KM100KM102KM104KM106_NGFdoseresponse_TrkApERK_ResultsFinal_Cells) and Erk1/2 (file: KM14_KM101KM103KM105KM107_NGFdoseresponse_ERKpERK_ResultsFinal_Cells) should determine differences in these pathway components between PDL/NGF and Col I/NGF treated sensory neurons. Cultures were fully digitalized by the Cellomics ArrayScan XTi HCS microscope and analyzed on a single-cell level by automated image analysis algorithms and R-script processing. NGF kinetic and dose response data show a significant amplitude increase of Erk1/2 phosphorylation in Col I treated cultures. pErk/TrkA and pErk/Erk data present an increase in TrkA and Erk1/2 expression in Col I treated cultures. This experimental single-cell data was used to demonstrate the capacity of our novel hierarchical, data-driven approach to modeling single-cell populations.

File description (header):

PlateID: Plate IDs corresponding to labbook entries of 3-4 replicate experiments
Well: corresponding wells of 96-well plate
Stain: Staining of wells with corresponding antibody combinations for spill over compensation of fluorescent channels (Ch1, Ch1Ch2, Ch1Ch3, Ch1Ch2Ch3)
Cond: treatment condition (PDL: poly-D-lysine; Col I: collagen type I, SpillOver: for data compensation)
Comp: applied compound (Ctrl: solvent, NGF: nerve growth factor, Fsk: forskolin (positive control))
Conc: used concentration (ng/ml) or if not applicable tested wells
Time: stimulation time (min)
Area: Cell size in µm2
Ch1-Ch4: fluorescent intensities of indicated antibody stainings (ch-UCHL1: chicken polyclonal antibody against UCHL1; rb-pErk: rabbit monoclonal antibody against phospho-Erk1/2; mm-UCHL1: mouse monoclonal antibody against UCHL1; go-TrkA: goat polyclonal antibody against TrkA; mo-Erk1/2: mouse monoclonal antibody against ERK1/2)

我们提出假设：当原代感觉神经元分别培养于聚-D-赖氨酸（poly-D-lysine, PDL）与I型胶原蛋白（collagen type I, Col I）底物时，神经生长因子（nerve growth factor, NGF）介导的Erk1/2磷酸化过程在原代感觉神经元的不同亚群中会受到差异化调控。为此，我们从雄性斯普拉格-道利（Sprague Dawley）大鼠背根神经节中解离感觉神经元，将其分别接种于PDL或Col I底物上过夜培养，随后以NGF进行急性刺激：动力学实验设置时间点为0、1、5、15、30、60、120分钟，NGF浓度统一为20 ng/ml；剂量反应实验设置刺激时长为1小时，NGF浓度分别为0、0.16、0.8、4、20、100、500 ng/ml。通过磷酸化特异性抗体标记结合免疫细胞化学法检测磷酸化Erk1/2，以此监测NGF诱导的信号通路活性，其中动力学实验相关文件为KM14_KM28KM31KM34_NGFkinetic_pErk_ResultsFinal_Cells，剂量反应实验相关文件为KM14_KM60KM62KM67KM68_NGFdoseresponse_pErk_ResultsFinal_Cells。此外，我们还通过分别靶向TrkA（NGF的受体）与Erk1/2的抗体进行共标记，相关文件分别为KM14_KM100KM102KM104KM106_NGFdoseresponse_TrkApERK_ResultsFinal_Cells与KM14_KM101KM103KM105KM107_NGFdoseresponse_ERKpERK_ResultsFinal_Cells，以此分析PDL/NGF与Col I/NGF处理组感觉神经元中上述通路组分的表达差异。培养物通过Cellomics ArrayScan XTi高内涵筛选（High Content Screening, HCS）显微镜完成全数字化成像，并通过自动化图像分析算法与R脚本处理实现单细胞水平的数据分析。NGF动力学与剂量反应实验数据显示，Col I处理组的Erk1/2磷酸化幅度显著升高。pErk/TrkA与pErk/Erk数据表明，Col I处理组的TrkA与Erk1/2表达量有所提升。本实验产生的单细胞数据用于验证我们提出的新型分层数据驱动建模方法在单细胞群体建模中的应用潜力。

文件描述（表头说明）：
PlateID：对应3-4次重复实验的实验记录编号
Well：96孔板对应的孔位
Stain：用于荧光通道串扰补偿的孔位染色组合（Ch1、Ch1Ch2、Ch1Ch3、Ch1Ch2Ch3）
Cond：处理条件（PDL：聚-D-赖氨酸（poly-D-lysine）；Col I：I型胶原蛋白（collagen type I）；SpillOver：用于数据串扰补偿）
Comp：施加的化合物（Ctrl：溶剂对照；NGF：神经生长因子（nerve growth factor）；Fsk：福司柯林（forskolin，阳性对照））
Conc：使用的浓度（单位：ng/ml），若无适用浓度则标注检测孔位
Time：刺激时长（单位：分钟）
Area：细胞面积（单位：µm²）
Ch1-Ch4：对应抗体染色的荧光强度（ch-UCHL1：靶向UCHL1的鸡多克隆抗体；rb-pErk：靶向磷酸化Erk1/2的兔单克隆抗体；mm-UCHL1：靶向UCHL1的小鼠单克隆抗体；go-TrkA：靶向TrkA的山羊多克隆抗体；mo-Erk1/2：靶向ERK1/2的小鼠单克隆抗体）