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Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.

ABSTRACT: While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin ?5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and ?-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

SUBMITTER: Stubb A

PROVIDER: S-EPMC6802214 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.

Stubb Aki A Guzmán Camilo C Närvä Elisa E Aaron Jesse J Chew Teng-Leong TL Saari Markku M Miihkinen Mitro M Jacquemet Guillaume G Ivaska Johanna J

Nature communications 20191018 1

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamina ...[more]

PMID: 31628312

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Project description:BackgroundGeneration of insulin-producing cells from human pluripotent stem cells (hPSCs) in vitro would be useful for drug discovery and cell therapy in diabetes. Three-dimensional (3D) culture is important for the acquisition of mature insulin-producing cells from hPSCs, but the mechanism by which it promotes β cell maturation is poorly understood.MethodsWe established a stepwise method to induce high-efficiency differentiation of human embryonic stem cells (hESCs) into mature monohormonal pancreatic endocrine cells (PECs), with the last maturation stage in 3D culture. To comprehensively compare two-dimensional (2D) and 3D cultures, we examined gene expression, pancreas-specific markers, and functional characteristics in 2D culture-induced PECs and 3D culture-induced PECs. The mechanisms were considered from the perspectives of cell-cell and cell-extracellular matrix interactions which are fundamentally different between 2D and 3D cultures.ResultsThe expression of the pancreatic endocrine-specific transcription factors PDX1, NKX6.1, NGN3, ISL1, and PAX6 and the hormones INS, GCG, and SST was significantly increased in 3D culture-induced PECs. 3D culture yielded monohormonal endocrine cells, while 2D culture-induced PECs co-expressed INS and GCG or INS and SST or even expressed all three hormones. We found that focal adhesion kinase (FAK) phosphorylation was significantly downregulated in 3D culture-induced PECs, and treatment with the selective FAK inhibitor PF-228 improved the expression of β cell-specific transcription factors in 2D culture-induced PECs. We further demonstrated that 3D culture may promote endocrine commitment by limiting FAK-dependent activation of the SMAD2/3 pathway. Moreover, the expression of the gap junction protein Connexin 36 was much higher in 3D culture-induced PECs than in 2D culture-induced PECs, and inhibition of the FAK pathway in 2D culture increased Connexin 36 expression.ConclusionWe developed a strategy to induce differentiation of monohormonal mature PECs from hPSCs and found limited FAK-dependent activation of the SMAD2/3 pathway and unregulated expression of Connexin 36 in 3D culture-induced PECs. This study has important implications for the generation of mature, functional β cells for drug discovery and cell transplantation therapy for diabetes and sheds new light on the signaling events that regulate endocrine specification.

Project description:Stem cells are adherent cells whose multipotency and differentiation can be regulated by numerous microenvironmental signals including soluble growth factors and surface topography. This study describes a simple method for creating distinct micropatterns via microphase separation resulting from polymer demixing of poly(desaminotyrosyl-tyrosine carbonate) (PDTEC) and polystyrene (PS). Substrates with co-continuous (ribbons) or discontinuous (islands and pits) PDTEC regions were obtained by varying the ratio of PDTEC and sacrificial PS. Human mesenchymal stem cells (MSCs) cultured on co-continuous PDTEC substrates for 3 days in bipotential adipogenic/osteogenic (AD/OS) induction medium showed no change in cell morphology but exhibited increased anisotropic cytoskeletal organization and larger focal adhesions when compared to MSCs cultured on discontinuous micropatterns. After 14 days in bipotential AD/OS induction medium, MSCs cultured on co-continuous micropatterns exhibited increased expression of osteogenic markers, whereas MSCs on discontinuous PDTEC substrates showed a low expression of adipogenic and osteogenic differentiation markers. Substrates with graded micropatterns were able to reproduce the influence of local underlying topography on MSC differentiation, thus demonstrating their potential for high throughput analysis. This work presents polymer demixing as a simple, non-lithographic technique to produce a wide range of micropatterns on surfaces with complex geometries to influence cellular and tissue regenerative responses.Statement of significanceA better understanding of how engineered microenvironments influence stem cell differentiation is integral to increasing the use of stem cells and materials in a wide range of tissue engineering applications. In this study, we show the range of topography obtained by polymer demixing is sufficient for investigating how surface topography affects stem cell morphology and differentiation. Our findings show that co-continuous topographies favor early (3-day) cytoskeletal anisotropy and focal adhesion maturation as well as long-term (14-day) expression of osteogenic differentiation markers. Taken together, this study presents a simple approach to pattern topographies that induce divergent responses in stem cell morphology and differentiation.

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Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.

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Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells.

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