Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions

Background

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway.

Methods

PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2.

Results

PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs.

Conclusions

Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation.

General significance

These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway.     

Extracellular matrix: A dynamic microenvironment for stem cell niche

Background

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche.

Scope of review

We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions.

Major conclusions

ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior.

General significance

ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Major signaling pathways in intestinal stem cells

Background

The discovery of markers to identify the intestinal stem cell population and the generation of powerful transgenic mouse models to study stem cell physiology have led to seminal discoveries in stem cell biology.

Scope of review

In this review we give an overview of the current knowledge in the field of intestinal stem cells (ISCs) highlighting the most recent progress on markers defining the ISC population and pathways governing intestinal stem cell maintenance and differentiation. Furthermore we review their interaction with other stem cell related pathways. Finally we give an overview of alteration of these pathways in human inflammatory gastrointestinal diseases.

Major conclusions

We highlight the complex network of interactions occurring among different pathways and put in perspective the many layers of regulation that occur in maintaining the intestinal homeostasis.

General significance

Understanding the involvement of ISCs in inflammatory diseases can potentially lead to new therapeutic approaches to treat inflammatory GI pathologies such as IBD and celiac disease and could reveal the molecular mechanisms leading to the pathogenesis of dysplasia and cancer in inflammatory chronic conditions. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin

Background

Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.

Methods

NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.

Results

Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.

Conclusions

These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.

General Significance

Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.     

On the role of Wnt/β-catenin signaling in stem cells

Background

Stem cells are mainly characterized by two properties: self-renewal and the potency to differentiate into diverse cell types. These processes are regulated by different growth factors including members of the Wnt protein family. Wnt proteins are secreted glycoproteins that can activate different intracellular signaling pathways.

Scope of review

Here we summarize our current knowledge on the role of Wnt/β-catenin signaling with respect to these two main features of stem cells.

Major conclusions

A particular focus is given on the function of Wnt signaling in embryonic stem cells. Wnt signaling can also improve reprogramming of somatic cells towards iPS cells highlighting the importance of this pathway for self-renewal and pluripotency. As an example for the role of Wnt signaling in adult stem cell behavior, we furthermore focus on intestinal stem cells located in the crypts of the small intestine.

General significance

A broad knowledge about stem cell properties and the influence of intrinsic and extrinsic factors on these processes is a requirement for the use of these cells in regenerative medicine in the future or to understand cancer development in the adult. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Matrix regulators in neural stem cell functions

Background

Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism.

Scope of review

In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior.

Major conclusions

The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors.

General significance

The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Developmental signaling pathways in cancer stem cells of solid tumors

Background

The intricate regulation of several signaling pathways is essential for embryonic development and adult tissue homeostasis. Cancers commonly display aberrant activity within these pathways. A population of cells identified in several cancers, termed cancer stem cells (CSCs) show similar properties to normal stem cells and evidence suggests that altered developmental signaling pathways play an important role in maintaining CSCs and thereby the tumor itself.

Scope of review

This review will focus on the roles of the Notch, Wnt and Hedgehog pathways in the brain, breast and colon cancers. We describe the roles these pathways play in normal tissue homeostasis through the regulation of stem cell fate in these three tissues, and the experimental evidence indicating that the role of these pathways in cancers of these is directly linked to CSCs.

Major conclusions

A large body of evidence is accumulating to indicate that the deregulation of Notch, Wnt and Hedgehog pathways play important roles in both normal and cancer stem cells. We are only beginning to understand how these pathways interact, how they are coordinated during normal development and adult tissue homeostasis, and how they are deregulated during cancer. However, it is becoming increasingly clear that if we are to target CSCs therapeutically, it will likely be necessary to develop combination therapies.

General significance

If CSCs are the driving force behind tumor maintenance and growth then understanding the molecular mechanisms regulating CSCs is essential. Such knowledge will contribute to better targeted therapies that could significantly enhance cancer treatments and patient survival. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Cartilage tissue engineering: Molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction

Background

Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes.

Scope of review

This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions.

Major conclusions

Current research involves the use of chondrocytes or progenitor stem cells, associated with “smart” biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process.

General significance

This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Bioreactors to influence stem cell fate: Augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems

Background

Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro.

Scope of review

This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems.

Major conclusions

The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased.

General significance

Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

PKC activation contributes to caspase-mediated eIF2α phosphorylation and cell death

Back ground

Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α), involved in translation, promotes cell suicide or survival. Since multiple signaling pathways are implicated in cell death, the present study has analyzed the importance of PKC activation in the stress-induced eIF2α phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda (Sf9) and in their extracts.

Methods

Cell death is analyzed by flow cytometry. Caspase activation is measured by Ac-DEVD-AFC hydrolysis and also by the cleavage of purified recombinant PERK, an endoplasmic reticulum-resident eIF2α kinase. Status of eIF2α phosphorylation and cytochrome c levels are analyzed by western blots.

Results

PMA, an activator of PKC, does not promote cell death or affect eIF2α phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2α phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2α phosphorylation in cell extracts. These changes are mitigated more efficiently by caspase inhibitor, z-VAD-fmk, than by calphostin, an inhibitor of PKC. In contrast, tunicamycin-induced eIF2α phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.

Conclusions

While caspase activation is a cause and consequence of eIF2α phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2α phosphorylation, and cell death in Sf9 cells.

General significance

Caspases can activate multiple signaling pathways to enhance cell death.     

COMP-Ang1 inhibits apoptosis as well as improves the attenuated osteogenic differentiation of mesenchymal stem cells induced by advanced glycation end products

Background

In the present study, we have investigated the possibility that cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), important factor in angiogenesis, osteogenesis and the survival of mesenchymal stem cells (MSCs) through the Ang1/Tie2 pathway has beneficial effects on osteogenic differentiated cells (ODCs) from MSCs treated by advanced glycation end products (AGE), which are pathological factors of diabetes.

Methods

Primary culture of MSCs was used. For comparison analysis of AGE and COMP-Ang1 effects, we performed cell viability assay with each treated variety concentration for 24 h. Apoptosis rate and Caspase-3 activity were measured by each ELISA assay. To make sure with Ang1/Tie2 pathway, we performed small interfering RNA transfected to MSCs. Real-time RT-PCR was performed to identify ODCs marker genes. Immunoblotting was used to evaluate the expression of Tie2, AKT, p38 and ERK.

Results

Our results clearly demonstrate that COMP-Ang1 upregulates the phosphorylation of AKT and p38 by activating the Ang1/Tie2 signaling pathway, indicating that COMP-Ang1 affects both AGE-induced apoptosis and the attenuated osteogenic differentiation of MSCs through the p38/MAPK and PI3K/AKT pathways.

Conclusions

COMP-Ang1 improves cell viability and differentiation function of ODCs against AGE via Ang/Tie2 signaling pathway.

General significance

Our results suggest the potential importance of COMP-Ang1 as a new therapy for impaired bone formation that is associated with diabetes and advanced age.     

Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt/GSK3β pathways

Background

Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established.

Scope of Review

EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering (RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells.

Major Conclusions

Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediated Akt/GSK3β signaling pathways.

General significance

These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinical relevance in EC.