Scaffold-free 3D cell culture of primary skin fibroblasts induces profound changes of the matrisome

Vu, Bich; Souza, Glauco R.; Dengjel, Jörn

doi:10.1016/j.mbplus.2021.100066

Cited by 29 publications

(27 citation statements)

References 36 publications

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“…As it has been shown that 3D cell culture better recapitulates in-vivo observations and as 3D cell culture has a profound effect on the composition of the proteome [ 42 , 43 ], additional new insights can be generated by the use of 3D cell culture systems. To better understand potential mechanisms of action, separate 3D cell cultures might still be the method of choice.…”

Section: Discussionmentioning

confidence: 99%

A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Activates Autophagy in Primary Skin Fibroblasts

Martínez-Martínez

Atzei

Vionnet

et al. 2022

IJMS

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Wound healing pathologies are an increasing problem in ageing societies. Chronic, non-healing wounds, which cause high morbidity and severely reduce the quality of life of affected individuals, are frequently observed in aged individuals and people suffering from diseases affected by the Western lifestyle, such as diabetes. Causal treatments that support proper wound healing are still scarce. Here, we performed expression proteomics to study the effects of the small molecule TOP-N53 on primary human skin fibroblasts and keratinocytes. TOP-N53 is a dual-acting nitric oxide donor and phosphodiesterase-5 inhibitor increasing cGMP levels to support proper wound healing. In contrast to keratinocytes, which did not exhibit global proteome alterations, TOP-N53 had profound effects on the proteome of skin fibroblasts. In fibroblasts, TOP-N53 activated the cytoprotective, lysosomal degradation pathway autophagy and induced the expression of the selective autophagy receptor p62/SQSTM1. Thus, activation of autophagy might in part be responsible for beneficial effects of TOP-N53.

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Section: Discussionmentioning

confidence: 99%

A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Activates Autophagy in Primary Skin Fibroblasts

Martínez-Martínez

Atzei

Vionnet

et al. 2022

IJMS

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“…Samples were concentrated with a SpeedVac vacuum concentrator (Thermo Fisher Scientific), and the final volume was adjusted to 20 μL using buffer A*/A (30% buffer A* with 3% acetonitrile and 0.3% TFA in water, and 70% buffer A). Samples were analysed on a Q Exactive Plus mass spectrometer, coupled to an EasyLC 1000 (Thermo Fisher Scientific), as described [ 34 ].…”

Section: Methodsmentioning

confidence: 99%

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions

et al. 2022

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The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.

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“…Several studies have been devised to culture 3D cell spheroids of ASCs ( Di Stefano et al, 2020 ; Hoefner et al, 2020 ; Sung et al, 2020 ; Yin et al, 2020 ). In these studies, ULA-3D culture has been more widely used, and in addition to its simplicity and inexpensive nature, this method has the following advantages: it is compatible with many cell lines, initiates by self-assembly, and consists of natural cells and their deposited extracellular matrix (ECM) ( Vu et al, 2021 ). These methods are also more suitable for biological analysis methods that solve basic scientific issues, such as the regulation of protein abundance due to changes in the 3D environment.…”

Section: Introductionmentioning

confidence: 99%

A Novel 3D Culture Model of Human ASCs Reduces Cell Death in Spheroid Cores and Maintains Inner Cell Proliferation Compared With a Nonadherent 3D Culture

Luo

Zhang

Wang

et al. 2021

Front. Cell Dev. Biol.

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3D cell culture technologies have recently shown very valuable promise for applications in regenerative medicine, but the most common 3D culture methods for mesenchymal stem cells still have limitations for clinical application, mainly due to the slowdown of inner cell proliferation and increase in cell death rate. We previously developed a new 3D culture of adipose-derived mesenchymal stem cells (ASCs) based on its self-feeder layer, which solves the two issues of ASC 3D cell culture on ultra-low attachment (ULA) surface. In this study, we compared the 3D spheroids formed on the self-feeder layer (SLF-3D ASCs) with the spheroids formed by using ULA plates (ULA-3D ASCs). We discovered that the cells of SLF-3D spheroids still have a greater proliferation ability than ULA-3D ASCs, and the volume of these spheroids increases rather than shrinks, with more viable cells in 3D spheroids compared with the ULA-3D ASCs. Furthermore, it was discovered that the SLF-3D ASCs are likely to exhibit the abovementioned unique properties due to change in the expression level of ECM-related genes, like COL3A1, MMP3, HAS1, and FN1. These results indicate that the SLF-3D spheroid is a promising way forward for clinical application.

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Scaffold-free 3D cell culture of primary skin fibroblasts induces profound changes of the matrisome

Cited by 29 publications

References 36 publications

A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Activates Autophagy in Primary Skin Fibroblasts

A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Activates Autophagy in Primary Skin Fibroblasts

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions

A Novel 3D Culture Model of Human ASCs Reduces Cell Death in Spheroid Cores and Maintains Inner Cell Proliferation Compared With a Nonadherent 3D Culture

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