Modulation of Oligodendrocyte Differentiation by Mechanotransduction

Oligodendrocytes (OLs) are responsible for the myelination of axons in the central nervous system (CNS). The differentiation of OLs encompasses several stages, through which cells undergo dramatic biochemical and morphological changes. OL differentiation is modulated by soluble factors (SFs)—such as growth factors and hormones—, known to be essential for each maturation stage. Besides SFs, insoluble factors such as extracellular matrix (ECM) proteins and other microenvironmental elements also play a pivotal role during OL differentiation. Recently, a growing number of studies were published concerning the effect of biophysical properties of the extracellular milieu on OL differentiation and myelination, showing the importance of ECM stiffness and topography, strain forces and spatial constraints. For instance, it was shown in vitro that OL differentiation and maturation is enhanced by substrates within the reported range of stiffness of the brain and that this effect is potentiated by the presence of merosin, whereas the myelination process is influenced by the diameter of axonal-like fibers. In this mini review article, we will discuss the effect of mechanical cues during OL differentiation and the possible molecular mechanisms involved in such regulation

The secretome of stem cells isolated from the adipose tissue and wharton jelly acts differently on central nervous system derived cell populations

Introduction: It is hypothesized that administration of stromal/stem cells isolated from the adipose tissue (ASCs) and umbilical cord (HUCPVCs) can ameliorate the inured CNS. However it is still not clear whether they have similar or opposite effects on primary cultures of neuronal populations. The objective of the present work was to determine if ASCs and HUCPVCs preferentially act, or not, on specific cell populations within the CNS. Methods: Primary cultures of hippocampal neurons were exposed to ASCs and HUCPVCs conditioned media (CM) (obtained 24, 48, 72 and 96 hours after 3 days of culture) for 1 week. Results: Cell viability experiments (MTS test) revealed that CM obtained from both cell populations at all time points did not cause any deleterious effects on neuronal cells. In fact, it was determined that whenever the ASCs CM were supplemented with bFGF and B27, there was a significant increase on the metabolic viability and neuronal cell density of the cultures. On the other hand in the absence of CM supplementation, it was the HUCPVCs secretome that had the highest impact on the metabolic viability and cell density. In an attempt to unveil which factors could be involved in the observed effects, a screening for the presence of basic fibroblast growth factor (bFGF), nerve growth factor (NGF), stem cell factor (SCF), hepatocyte growth factors (HGF) and vascular endothelial growth factor (VEGF) in the CM was performed. Results revealed the presence of all these factors in ASCs CM, except bFGF; in contrast, in HUCPVCs CM it was only possible to detect robust NGF expression. Conclusions: Overall the results herein confirm important differences on the secretome of ASCs and HUCPVCs, which leads to distinct effects on the metabolic viability and neuronal cell densities in primary cultures of hippocampal neurons; however, the factor(s) that promote the stronger effect of the HUCPVCs CM in neuronal survival is (are) still to be identified.Pennington Biomedical Research FoundationFoundation Calouste de Gulbenkian - The Gulbenkian Programme to Support Research in the Life Sciences and Ciência 2007 ProgramFundação para a Ciência e a Tecnologia (FCT

Soft culture substrates favor stem-like cellular phenotype and facilitate reprogramming of human mesenchymal stem/stromal cells (hMSCs) through mechanotransduction

Fundação para a Ciência e a Tecnologia) - FCT - grant FCT-UID/NEU/04539/2019. European Regional Development Fund (ERDF/FEDER) through the Operational Program Competitiveness Factors (Programa Operacional Factores de Competitividade) - COMPETE - funding through Project 'Stem cell based platforms for Regenerative and Therapeutic Medicine', Centro-07-ST24-FEDER-002008. M.G. acknowledges funding by the ERDF/FEDER through COMPETE and by national funds by FCT through grant FCOMP-01-0124-FEDER-021150 - PTDC/SAU-889 ENB/119292/2010 and grant POCI-01-0145-FEDER-029516, co-financed by the ERDF/FEDER under the framework Competitiveness and Internationalization Operational Program (Programa Operacional Competitividade e Internacionalizacao -POCI), national funds through FCT/'Ministerio da Ciencia, Tecnologia e Ensino Superior' (FCT/MCTES) through the Portuguese State Budget. Grant PTDC/SAU-ENB/113696/2009 was attributed to R.P.N. R.D.M.T. and J.C. thank the support of FEDER funds through COMPETE and by national funds by FCT under the strategic project UID/FIS/04564/2016 and under POCI-01-0145-FEDER-031743 - PTDC/BIA-CEL/31743/2017. R.D.M.T. acknowledges FCT's support through the FCT Researcher Program.Biophysical cues influence many aspects of cell behavior. Stiffness of the extracellular matrix is probed by cells and transduced into biochemical signals through mechanotransduction protein networks, strongly influencing stem cell behavior. Cellular stemness is intimately related with mechanical properties of the cell, like intracellular contractility and stiffness, which in turn are influenced by the microenvironment. Pluripotency is associated with soft and low-contractility cells. Hence, we postulated that soft cell culture substrates, presumably inducing low cellular contractility and stiffness, increase the reprogramming efficiency of mesenchymal stem/stromal cells (MSCs) into induced pluripotent stem cells (iPSCs). We demonstrate that soft substrates (1.5 or 15 kPa polydimethylsiloxane – PDMS) caused modulation of several cellular features of MSCs into a phenotype closer to pluripotent stem cells (PSCs). MSCs cultured on soft substrates presented more relaxed nuclei, lower maturation of focal adhesions and F-actin assembling, more euchromatic and less heterochromatic nuclear DNA regions, and increased expression of pluripotency-related genes. These changes correlate with the reprogramming of MSCs, with a positive impact on the kinetics, robustness of colony formation and reprogramming efficiency. Additionally, substrate stiffness influences several phenotypic features of iPS cells and colonies, and data indicates that soft substrates favor full iPSC reprogramming.publishersversionpublishe

Cofilin-1 is a mechanosensitive regulator of transcription

The mechanical properties of the extracellular environment are interrogated by cells and integrated through mechanotransduction. Many cellular processes depend on actomyosin-dependent contractility, which is influenced by the microenvironment's stiffness. Here, we explored the influence of substrate stiffness on the proteome of proliferating undifferentiated human umbilical cord-matrix mesenchymal stem/stromal cells. The relative abundance of several proteins changed significantly by expanding cells on soft (∼3 kPa) or stiff substrates (GPa). Many such proteins are associated with the regulation of the actin cytoskeleton, a major player of mechanotransduction and cell physiology in response to mechanical cues. Specifically, Cofilin-1 levels were elevated in cells cultured on soft comparing with stiff substrates. Furthermore, Cofilin-1 was de-phosphorylated (active) and present in the nuclei of cells kept on soft substrates, in contrast with phosphorylated (inactive) and widespread distribution in cells on stiff. Soft substrates promoted Cofilin-1-dependent increased RNA transcription and faster RNA polymerase II-mediated transcription elongation. Cofilin-1 is part of a novel mechanism linking mechanotransduction and transcription.publishe

Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein — MBP — and proteolipid protein — PLP — (respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation

Modulation of oligodendrocyte differentiation by mechanotransduction

Oligodendrocytes (OLs) are responsible for the myelination of axons in the central nervous system. The differentiation of OLs encompasses several stages, through which cells undergo dramatic biochemical and morphological changes. Oligodendrocyte differentiation is modulated by soluble factors (SFs) — such as growth factors and hormones —, known to be essential for each maturation stage. Besides SFs, insoluble factors such as extracellular matrix (ECM) proteins and other microenvironmental elements also play a pivotal role during OL differentiation. Recently, a growing number of studies were published concerning the effect of biophysical properties of the extracellular milieu on oligodendrocyte differentiation and myelination, showing the importance of ECM stiffness and topography, strain forces and spatial constraints. For instance, it was shown in vitro that OL differentiation and maturation is enhanced by substrates within the reported range of stiffness of the brain and that this effect is potentiated by the presence of merosin, whereas the myelination process is influenced by the diameter of axonal-like fibers. In this mini review, we will discuss the effect of mechanical cues during oligodendrocyte differentiation and the possible molecular mechanisms involved in such regulation

Modulation of signaling pathways by DJ-1: An updated overview

Efforts have been made to understand the physiological and pathological role of DJ-1, a Parkinson's disease (PD)-associated protein, to provide new insights into PD pathophysiology. Such studies have revealed several neuroprotective roles of DJ-1, from which its ability to modulate signaling pathways seems to be of utmost importance for cell death regulation by DJ-1. Indeed, research on these topics has led to a higher number of publications disclosing a variety of mechanisms through which DJ-1 is able to modulate signaling pathways in distinct disease-related contexts. Thus, this graphical review presents the most relevant findings concerning the mechanisms through which DJ-1 exerts its regulatory activity on signaling cascades relevant for DJ-1 neuroprotective action, namely ERK1/2, PI3K/Akt, and ASK1 pathways, and Nrf2 and p53 transcription factors-related signaling. A greater focus was given to perform an overview of the research interests over the last years, especially in the most recent works, to highlight the current research lines in this topic, and point out future directions in the field. In addition, the impact of DJ-1 mutations causative of PD and the importance of the redox status of DJ-1's cysteine residues for the action of DJ-1 on signaling modulation was also addressed to uncover the potential pathological mechanisms associated with loss of DJ-1 native function

Towards the Maturation and Characterization of Smooth Muscle Cells Derived from Human Embryonic Stem Cells

In this study we demonstrate that CD34+ cells derived from human embryonic stem cells (hESCs) have higher smooth muscle cell (SMC) potential than CD34− cells. We report that from all inductive signals tested, retinoic acid (RA) and platelet derived growth factor (PDGFBB) are the most effective agents in guiding the differentiation of CD34+ cells into smooth muscle progenitor cells (SMPCs) characterized by the expression of SMC genes and proteins, secretion of SMC-related cytokines, contraction in response to depolarization agents and vasoactive peptides and expression of SMC-related genes in a 3D environment. These cells are also characterized by a low organization of the contractile proteins and the contractility response is mediated by Ca2+, which involves the activation of Rho A/Rho kinase- and Ca2+/calmodulin (CaM)/myosin light chain kinase (MLCK)-dependent pathways. We further show that SMPCs obtained from the differentiation of CD34+ cells with RA, but not with PDGFBB, can be maturated in medium supplemented with endothelin-1 showing at the end individualized contractile filaments. Overall the hESC-derived SMCs presented in this work might be an unlimited source of SMCs for tissue engineering and regenerative medicine.Marie Curie-Reintegration GrantMIT-Portugal programCrioestaminalAssociação Viver a CiênciaFundação para a Ciência e a Tecnologia (PTDC/SA-BEB/098468/2008 and PTDC/CTM/099659/2008 to L.F.; SFRH/BD/40077/2007 to H.V.

Hypothermia Does Not Boost the Neuroprotection Promoted by Umbilical Cord Blood Cells in a Neonatal Hypoxia-Ischemia Rat Model

Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of death and long-term disability in the perinatal period. Currently, therapeutic hypothermia is the standard of care for this condition with modest efficacy and strict enrollment criteria. Therapy with umbilical cord blood cells (UCBC) has come forward as a strong candidate for the treatment of neonatal HIE, but no preclinical studies have yet compared the action of UCBC combined with hypothermia (HT) with the action of each therapy by itself. Thus, to evaluate the potential of each therapeutic approach, a hypoxic-ischemic brain lesion was induced in postnatal day ten rat pups; two hours later, HT was applied for 4 h; and 24, 48, and 72 h post-injury, UCBC were administered intravenously. The neonatal hypoxic-ischemic injury led to a brain lesion involving about 48% of the left hemisphere that was not improved by HT (36%) or UCBC alone (28%), but only with the combined therapies (25%; p = 0.0294). Moreover, a decrease in glial reactivity and improved functional outcomes were observed in both groups treated with UCBC. Overall, these results support UCBC as a successful therapeutic approach for HIE, even when treatment with therapeutic hypothermia is not possible