Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

Abstract

Stem cell research is emerging both as a scientifically and clinically relevant area. One of the current challenges in stem cell research and regenerative medicine is assessment of the pluripotency state of induced pluripotent stem (iPS) cells. Once a stem cell differentiation process is initiated the challenge is how to assess the state of differentiation, and the purity of the differentiated cell population. Stem cell potency and differentiation states are determined by tightly coordinated activity of developmental signaling pathways, such as the Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB pathways. Source of the stem cells and culture protocols may influence stem cell phenotype, with potential consequences for pluripotency and in general for experimental reproducibility. Human pluripotent embryonic (hES) and iPS stem cell lines under different culture conditions, organ derived multipotent stem cells, and differentiated cell types, were phenotyped with respect to functional activity of developmental signaling pathways. Methods: We previously reported on the development and validation of a novel assay platform for quantitative measurement of activity of multiple signal transduction pathways (STP) simultaneously in a single sample, based on interpreting a preselected set of target mRNA expression levels. Assays were used to calculate Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB signal transduction pathway activity scores for individual cell samples, using publicly available Affymetrix expression microarray data. Results: Culture conditions (e.g. mouse versus human feeder) influenced pluripotent stem cell pathway activity profiles. hES and iPS stem cell lines cultured in the same lab under similar conditions showed minimal variation in pathway activity profile despite different genetic backgrounds, while across different labs larger variations were measured, even for the same stem cell line. Pathway activity scores for PI3K, MAPK, Hedgehog, Notch, TGFβ, and NFκB pathways rapidly decreased upon pluripotent stem cell differentiation, while increasing for the Wnt pathway. Further differentiation to intestinal progenitor cells resulted in higher PI3K, Wnt and Notch pathway activity. In multipotent intestinal crypt stem cells obtained from intestinal mucosa samples, similar Notch and even higher Wnt pathway activity were measured, which disappeared upon differentiation to mucosal cells. Conclusion: Results support the validity of using these STP assays for quantitative phenotyping of stem cells and differentiated derivatives, and enabled definition of a pluripotency profile with high PI3K, MAPK, Hedgehog, TGFβ, Notch, and NFκB, and low Wnt pathway activity scores. Measurement of combined signaling pathway activity scores is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture and differentiation. It enables controlled manipulation of signaling pathway activity using pathway targeting compounds. An envisioned additional utility may lie in quality control for regenerative medicine purposes.

Improved diagnosis of inflammatory bowel disease and prediction and monitoring of response to anti-TNF alpha treatment based on measurement of signal transduction pathway activity

“…We previously reported a completely new method to quantitatively measure activity of clinically relevant signal transduction pathways (STP) on transcriptome data of individual cell-containing samples 7 , 8 , 9 , 10 , 11 , 12 , 7 , 13 , 14 . Using this technology to analyze RNA expression data of colon mucosa biopsy samples, signaling pathway activity was investigated in CD and UC, and related to pathophysiology of IBD and the clinical response to anti-TNFα treatment.…”

“…Development and validation of computational models to quantitatively measure activity of the androgen receptor (AR), MAPK, NFκB, WNT, TGFβ, JAK-STAT1/2, and JAK-STAT3 pathways have been described before, and can in principle be used to analyze pathway activity on any cell type, based on analysis of transcriptome data 7 , 8 , 9 , 10 , 11 , 12 , 7 , 13 , 14 . For the current study, pathway activity scores (PAS) were measured on publicly available Affymetrix expression microarray data from preclinical and clinical IBD studies.…”

Measuring host immune response status by simultaneous quantitative measurement of activity of signal transduction pathways that coordinate functional activity of immune cells from innate and adaptive immune system

“…Development and validation of Affymetrix-based assays for measuring activity of the estrogen (ER) and androgen receptor (AR), PI3K-FOXO, MAPK, NFκB, TGFβ, Notch, JAK-STAT1/2, and JAK-STAT3 pathways has been described before, and can in principle be used to analyze pathway activity on any cell type (Bouwman et al, 2020), (Verhaegh et al, 2014), (Stolpe et al, 2019), (Canté-Barrett et al, 2020), (Wesseling-Rozendaal et al, 2021), (van Ooijen et al, 2018) including blood cells (Bouwman et al, 2020), (Wesseling-Rozendaal et al, 2020),(van de , (Bouwman et al, 2021). For the current study, quantitative pathway activity scores (PAS) were measured on Affymetrix expression microarray data.…”