Holtzer Laurent, Wesseling-Rozendaal Yvonne, Verhaegh Wim, van de Stolpe Anja
Molecular Pathway Diagnostics, Philips, Eindhoven, The Netherlands.
Stem Cell Res. 2022 May;61:102748. doi: 10.1016/j.scr.2022.102748. Epub 2022 Mar 14.
Important challenges in stem cell research and regenerative medicine are reliable assessment of pluripotency state and purity of differentiated cell populations. Pluripotency and differentiation are regulated and determined by activity of developmental signal transduction pathways (STPs). To date activity of these STPs could not be directly measured on a cell sample. Here we validate a novel assay platform for measurement of activity of developmental STPs (STP) for use in stem cells and stem cell derivatives. In addition to previously developed STP assays, we report development of an additional STP assay for the MAPK-AP1 pathway. Subsequently, activity of Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB signaling pathways was calculated from Affymetrix transcriptome data of human pluripotent embryonic (hES) and iPS cell lines under different culture conditions, organ-derived multipotent stem cells, and differentiated cell types, to generate quantitative STP activity profiles. Results show that the STP assay technology enables reliable and quantitative measurement of multiple STP activities simultaneously on any individual cell sample. Using the technology, we found that culture conditions dominantly influence the pluripotent stem cell STP activity profile, while the origin of the stem cell line was a minor variable. A pluripotency STP activity profile (Pluripotency qPAP) was defined (active PI3K, MAPK, Hedgehog, Notch, TGFβ, and NFκB pathway, inactive Wnt pathway). Differentiation of hES cells to intestinal progenitor cells resulted in an STP activity profile characterized by active PI3K, Wnt and Notch pathways, comparable to the STP activity profile measured on primary intestinal crypt stem cells. Quantitative STP activity measurement is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture/differentiation, and enable controlled manipulation of pluripotency/differentiation state using pathway targeting compounds.
干细胞研究和再生医学中的重要挑战是对多能性状态和分化细胞群体纯度进行可靠评估。多能性和分化由发育信号转导通路（STP）的活性调节和决定。迄今为止，这些STP的活性无法在细胞样本上直接测量。在此，我们验证了一种用于测量发育性STP（STP）活性的新型检测平台，以用于干细胞和干细胞衍生物。除了先前开发的STP检测方法外，我们还报告了针对MAPK-AP1通路的另一种STP检测方法的开发。随后，根据不同培养条件下人类多能胚胎（hES）和诱导多能干细胞系、器官来源的多能干细胞以及分化细胞类型的Affymetrix转录组数据，计算Notch、Hedgehog、TGFβ、Wnt、PI3K、MAPK-AP1和NFκB信号通路的活性，以生成定量的STP活性图谱。结果表明，STP检测技术能够在任何单个细胞样本上同时可靠且定量地测量多种STP活性。使用该技术，我们发现培养条件对多能干细胞STP活性图谱有主要影响，而干细胞系的来源是一个较小的变量。定义了一种多能性STP活性图谱（多能性qPAP）（PI3K、MAPK、Hedgehog、Notch、TGFβ和NFκB通路活跃，Wnt通路不活跃）。hES细胞向肠祖细胞的分化导致了一种以PI3K、Wnt和Notch通路活跃为特征的STP活性图谱，与在原代肠隐窝干细胞上测量的STP活性图谱相当。定量STP活性测量有望提高多能和多能干细胞培养/分化的实验可重复性和标准化，并能够使用通路靶向化合物对多能性/分化状态进行可控操作。
