High‐throughput combinatorial screening reveals interactions between signaling molecules that regulate adult neural stem cell fate

Abstract

Advancing our knowledge of how neural stem cell (NSC) behavior in the adult hippocampus is regulated has implications for elucidating basic mechanisms of learning and memory as well as for neurodegenerative disease therapy. To date, numerous biochemical cues from the endogenous hippocampal NSC niche have been identified as modulators of NSC quiescence, proliferation, and differentiation; however, the complex repertoire of signaling factors within stem cell niches raises the question of how cues act in combination with one another to influence NSC physiology. To help overcome experimental bottlenecks in studying this question, we adapted a high-throughput microculture system, with over 500 distinct microenvironments, to conduct a systematic combinatorial screen of key signaling cues and collect high-content phenotype data on endpoint NSC populations. This novel application of the platform consumed only 0.2% of reagent volumes used in conventional 96-well plates, and resulted in the discovery of numerous statistically significant interactions among key endogenous signals. Antagonistic relationships between fibroblast growth factor 2, transforming growth factor β (TGF-β), and Wnt-3a were found to impact NSC proliferation and differentiation, whereas a synergistic relationship between Wnt-3a and Ephrin-B2 on neuronal differentiation and maturation was found. Furthermore, TGF-β and bone morphogenetic protein 4 combined with Wnt-3a and Ephrin-B2 resulted in a coordinated effect on neuronal differentiation and maturation. Overall, this study offers candidates for further elucidation of significant mechanisms guiding NSC fate choice and contributes strategies for enhancing control over stem cell-based therapies for neurodegenerative diseases.

Pressure regulated basis for gene transcription by delta-cell micro-compliance modeled in silico: Biphenyl, bisphenol and small molecule ligand models of cell contraction-expansion

“…Thus, based on study determinations, the gene expression pattern for small molecule hormone receptor interaction between the 2.6 x 10 5 to 2.1 x 10 6 min · count range results in a negative Δ C micro response and an initial downward shift in the contraction with unilateral expansion as compared to positive Δ C micro response with contraction and bidirectional expansion, and irrespective of the specific steriod axis receptor class (ER, AR). Furthermore, it appears that an initial negative (-) Δ C micro response within the 1.86 x 10 6 to 1.94 x 10 6 (IGF-II · IGF-IIR) min · count range is coupled to a positive Δ C micro response, as Dht or R1881 · CM AR (1.79E + 05–2.45E + 05, + P eff ) results in the transcription of pro-proliferative genes [ 118 , 119 ] , that is proposed to be by an initial (-) P eff Χ then an (+) P eff Υ intermediate step coupled to resultant transcriptional activation of MKI67 ( P eff 0.329) [Part I, not cited; 38 ] ( Table 9 ); thus, expression of a focal adhesion or endocytic component may be involved, which would apply to calvarial osteoblasts that overexpress the IGF-IIR/M6P receptor [ 120 ] , and similarly to transformed cells with P eff shift to IGF1R expression [ 121 ] , and could result in altered cell phenotype such as mononucleated: multinucleated [ 5 ] , or lineage commitment such as glial: non-glial during the pre: post exposure period without interaction [ 122 ] .…”

Application and prospects of high-throughput screening for in vitro neurogenesis

“…Nakajima et al [ 163 ] used a cell-based assay to coimmobilize growth factors and natural or synthetic matrices, and they found that EGF promoted the maintenance of NSCs and that two nerve growth factor (NGF) family members, NGF and NT-3, could facilitate NSCs toward neuronal differentiation. In addition to for cell-based microarrays requiring immobilization of biomolecules, Muckom et al [ 177 ] applied a high-throughput microculture system consisting of complementary micropillars and microwells that could hold 532 independent microenvironments for cell culture. They seeded adult rodent NSCs and provided 6 soluble factors, BMP4, TGF-β, FGF-2, shh, Wnt-3a and Ephin-B2, and evaluated the extent to which their individual signals and double, tertiary and quaternary signal combinations could influence neural differentiation[ 177 ].…”

Exploring landscapes of brain morphogenesis with organoids

“…Indeed, presentations from Linda Griffith, Fan Yang and Cole DeForest focused on extracellular matrix components and standardization of the 3D microenvironment (Cambria et al, 2015;DeForest and Tirrell, 2015;Wang et al, 2014), and work presented by Guo-li Ming demonstrated the ability to produce largely identical organoids on a bigger scale (Qian et al, 2016). Patterning was also an area of focus, with Randolph Ashton, Matthias Lutolf, Madeline Lancaster and David Schaffer presenting work on intrinsic and extrinsic cues that control cell fate determination (Knight et al, 2018;Muckom et al, 2018;Ranga et al, 2016).…”