Dataset Information

TGF-?1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural in vitro Models.

ABSTRACT: Persistent neural stem cell (NSC) proliferation is, among others, a hallmark of immaturity in human induced pluripotent stem cell (hiPSC)-based neural models. TGF-?1 is known to regulate NSCs in vivo during embryonic development in rodents. Here we examined the role of TGF-?1 as a potential candidate to promote in vitro differentiation of hiPSCs-derived NSCs and maturation of neuronal progenies. We present that TGF-?1 is specifically present in early phases of human fetal brain development. We applied confocal imaging and electrophysiological assessment in hiPSC-NSC and 3D neural in vitro models and demonstrate that TGF-?1 is a signaling protein, which specifically suppresses proliferation, enhances neuronal and glial differentiation, without effecting neuronal maturation. Moreover, we demonstrate that TGF-?1 is equally efficient in enhancing neuronal differentiation of human NSCs as an artificial synthetic small molecule. The presented approach provides a proof-of-concept to replace artificial small molecules with more physiological signaling factors, which paves the way to improve the physiological relevance of human neural developmental in vitro models.

SUBMITTER: Izsak J

PROVIDER: S-EPMC7655796 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

TGF-β1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural in vitro Models.

Izsak Julia J Vizlin-Hodzic Dzeneta D Iljin Margarita M Strandberg Joakim J Jadasz Janusz J Olsson Bontell Thomas T Theiss Stephan S Hanse Eric E Ågren Hans H Funa Keiko K Illes Sebastian S

Frontiers in cell and developmental biology 20201028

Persistent neural stem cell (NSC) proliferation is, among others, a hallmark of immaturity in human induced pluripotent stem cell (hiPSC)-based neural models. TGF-β1 is known to regulate NSCs in vivo during embryonic development in rodents. Here we examined the role of TGF-β1 as a potential candidate to promote in vitro differentiation of hiPSCs-derived NSCs and maturation of neuronal progenies. We present that TGF-β1 is specifically present in early phases of human fetal brain dev ...[more]

PMID: 33195202

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Project description:Hydroxysafflor yellow A (HSYA) is an active ingredient of Carthamus tinctorius L.. This study aimed to evaluate the effects of HSYA on transforming growth factor-β1 (TGF-β1)-induced changes in proliferation, migration, differentiation, and extracellular matrix accumulation and degradation in human fetal lung fibroblasts (MRC-5), to explore the mechanisms whereby HSYA may alleviate pulmonary fibrosis. MRC-5 cells were incubated with various doses of HSYA and/or the TGF-β receptor type I kinase inhibitor SB431542 and then stimulated with TGF-β1. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt assay. Cell migration was detected by wound-healing assay. Protein levels of α-smooth muscle actin (α-SMA), collagen I α 1 (COL1A1), and fibronectin (FN) were measured by immunofluorescence. Protein levels of matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-2, TGF-β type II receptor (TβRII), and TGF-β type I receptor were detected by western blotting. TβRII knockdown with siRNA interfered with the inhibitory effect of HSYA on α-SMA, COL1A1, and FN expression, and TGF-β1-induced Sma and Mad protein (Smad), and extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway activation. The antagonistic effect of HSYA on the binding of fluorescein isothiocyanate-TGF-β1 to MRC-5 cell cytoplasmic receptors was measured by flow cytometry. HSYA significantly suppressed TGF-β1-induced cell proliferation and migration. HSYA could antagonize the binding of FITC-TGF-β1 to MRC-5 cell cytoplasmic receptors. Also HSYA inhibited TGF-β1-activated cell expression of α-SMA, COL1A1, and FN and phosphorylation level of Smad2, Smad3, and ERK by targeting TβRII in MRC-5 cells. These findings suggest that TβRII might be the target responsible for the inhibitory effects of HSYA on TGF-β1-induced pathological changes in pulmonary fibrosis.

Project description:Experimental models of the central nervous system (CNS) are imperative for developmental and pathophysiological studies of neurological diseases. Among these models, three-dimensional (3D) induced pluripotent stem cell (iPSC)-derived brain organoid models have been successful in mitigating some of the drawbacks of 2D models; however, they are plagued by high organoid-to-organoid variability, making it difficult to compare specific gene regulatory pathways across 3D organoids with those of the native brain. Single-cell RNA sequencing (scRNA-seq) transcriptome datasets have recently emerged as powerful tools to perform integrative analyses and compare variability across organoids. However, transcriptome studies focusing on late-stage neural functionality development have been underexplored. Here, we combine and analyze 8 brain organoid transcriptome databases to study the correlation between differentiation protocols and their resulting cellular functionality across various 3D organoid and exogenous brain models. We utilize dimensionality reduction methods including principal component analysis (PCA) and uniform manifold approximation projection (UMAP) to identify and visualize cellular diversity among 3D models and subsequently use gene set enrichment analysis (GSEA) and developmental trajectory inference to quantify neuronal behaviors such as axon guidance, synapse transmission and action potential. We showed high similarity in cellular composition, cellular differentiation pathways and expression of functional genes in human brain organoids during induction and differentiation phases, i.e., up to 3 months in culture. However, during the maturation phase, i.e., 6-month timepoint, we observed significant developmental deficits and depletion of neuronal and astrocytes functional genes as indicated by our GSEA results. Our results caution against use of organoids to model pathophysiology and drug response at this advanced time point and provide insights to tune in vitro iPSC differentiation protocols to achieve desired neuronal functionality and improve current protocols.

Dataset Information

TGF-?1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural in vitro Models.

Publications

TGF-β1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural <i>in vitro</i> Models.

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