Role of IGF1 and IGF1/VEGF on Human Mesenchymal Stromal Cells in Bone Healing: Two Sources and Two Fates.

In the repair of skeletal defects one of the major obstacles still remains an efficient vascularization of engineered scaffolds. We have examined the ability of insulin growth factor-1, alone or in association with vascular endothelial growth factor, to modulate the osteoblastic or endothelial commitment of periosteum-derived progenitor cells (PDPCs) and skin-derived multipotent stromal cells (S-MSCs). A selected gene panel for endothelial and osteoblastic differentiation as well as genes that can affect MAPK and PI3K/AKT signaling pathways were investigated. Moreover, gene expression profile of Sox2, Oct4, and Nanog transcription factors was assessed. Our results showed that under growth factor stimulation PDPCs are induced toward an osteoblastic differentiation, while S-MSCs seem to move along an endothelial phenotype. This different commitment seems to be linked to a diverse MAPK or PI3K/AKT signaling pathway activation. The analysis of genes for stemness evidenced that at least in PDPCs multipotency and differentiation could coexist. These results open interesting perspective for the development of innovative bone tissue engineering approaches based on a good network of angiogenesis and osteogenesis processes

Cognitive and brain cytokine profile of non-demented individuals with cerebral amyloid-beta deposition

Brain inflammation has been increasingly associated with early amyloid accumulation in Alzheimer's disease models; however, evidence of its occurrence in humans remains scarce. To elucidate whether amyloid deposition is associated with neuroinflammation and cognitive deficits, we studied brain inflammatory cytokine expression and cognitive decline in non-demented elderly individuals with and without cerebral amyloid-beta deposition. Global cognition, episodic, working, and semantic memory, perceptual speed, visuospatial ability, and longitudinal decline (5.7 ± 3.6 years) in each cognitive domain were compared between elderly individuals (66-79 years) with and without cerebral amyloid-beta deposition. The expression of 20 inflammatory cytokines was analyzed in frozen temporal, parietal, and frontal cortices and compared between older individuals with and without amyloid-beta deposition in each brain region. Correlation analyses were performed to analyze associations between amyloid-beta load, cytokine expression, and cognitive decline. Individuals with cortical amyloid-beta deposition displayed deficits and a faster rate of cognitive decline in perceptual speed as compared with those individuals without amyloid-beta. This decline was positively associated with cortical amyloid-beta levels. Elderly individuals with amyloid-beta deposition had higher levels of IL-1β, IL-6, and eotaxin-3 in the temporal cortex accompanied by an increase in MCP-1 and IL-1β in the parietal cortex and a trend towards higher levels of IL-1β and MCP-1 in the frontal cortex as compared with age-matched amyloid-free individuals. Brain IL-1β levels displayed a positive association with cortical amyloid burden in each brain region. Finally, differential cytokine expression in each cortical region was associated with cognitive decline. Elderly individuals with amyloid-beta neuropathology but no symptomatic manifestation of dementia, exhibit cognitive decline and increased brain cytokine expression. Such observations suggest that increased cytokine expression might be an early event in the Alzheimer's continuum. The online version contains supplementary material available at 10.1186/s12974-021-02169-0

Expression of neural markers by undifferentiated mesenchymal-like stem cells from different sources

The spontaneous expression of neural markers, already demonstrated in bone marrow (BM) mesenchymal stem cells (MSCs), has been considered as evidence of the MSCs' predisposition to differentiate toward neural lineages, supporting their use in stem cell-based therapy for neural repair. In this study we have evaluated, by immunocytochemistry, immunoblotting, and flow cytometry experiments, the expression of neural markers in undifferentiated MSCs from different sources: human adipose stem cells (hASCs), human skin-derived mesenchymal stem cells (hS-MSCs), human periodontal ligament stem cells (hPDLSCs,) and human dental pulp stem cells (hDPSCs). Our results demonstrate that the neuronal markers \u3b2III-tubulin and NeuN, unlike other evaluated markers, are spontaneously expressed by a very high percentage of undifferentiated hASCs, hS-MSCs, hPDLSCs, and hDPSCs. Conversely, the neural progenitor marker nestin is expressed only by a high percentage of undifferentiated hPDLSCs and hDPSCs. Our results suggest that the expression of \u3b2III-tubulin and NeuN could be a common feature of stem cells and not exclusive to neuronal cells. This could result in a reassessment of the use of \u3b2III-tubulin and NeuN as the only evidence proving neuronal differentiation. Further studies will be necessary to elucidate the relevance of the spontaneous expression of these markers in stem cells

Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation

Amniotic fluid-derived stem cells for cardiovascular tissue engineering applications

Recent research has demonstrated that a population of stem cells can be isolated from amniotic fluid removed by amniocentesis that are broadly multipotent and non-tumorogenic. These amniotic fluid-derived stem cells (AFSC) could potentially provide an autologous cell source for treatment of congenital defects identified during gestation, particularly cardiovascular defects. In this review, the various methods of isolating, sorting and culturing AFSC are compared, along with techniques for inducing differentiation into cardiac myocytes and endothelial cells. Though research has not demonstrated complete and high yield cardiac differentiation, AFSC have been shown to effectively differentiate into endothelial cells and can effectively support cardiac tissue. Additionally, several tissue engineering and regenerative therapeutic approaches for the use of these cells in heart patches, injection after myocardial infarction, heart valves, vascularized scaffolds and blood vessels are summarized. These applications show great promise in the treatment of congenital cardiovascular defects, and further studies of isolation, culture, and differentiation of AFSC will help to develop their use for tissue engineering, regenerative medicine, and cardiovascular therapies

Detection of phosphatidylserine with a modified polar head group in human keratinocytes exposed to the radical generator AAPH

Phosphatidylserine (PS) is preferentially located in the inner leaflet of the cell membrane, and translocation of PS oxidized in fatty acyl chains to the outside of membrane has been reported as signaling to macrophage receptors to clear apoptotic cells. It was recently shown that PS can be oxidized in serine moiety of polar head-group. In the present work, a targeted lipidomic approach was applied to detecting OxPS modified at the polar head-group in keratinocytes that were exposed to the radical generator AAPH. Glycerophosphoacetic acid derivatives (GPAA) were found to be the major oxidation products of OxPS modified at the polar head-group during oxidation induced by AAPH-generated radicals, similarly to previous observations for the oxidation induced by OH radical. The neutral loss scan of 58Da and a novel precursor ion scan of m/z 137.1 (HOPO3CH2COOH) allowed the recognition of GPAA derivatives in the total lipid extracts obtained from HaCaT cells treated with AAPH. The positive identification of serine head group oxidation products in cells under controlled oxidative conditions opens new perspectives and justifies further studies in other cellular environments in order to understand fully the role of PS polar head-group oxidation in cell homeostasis and disease

DNA Methyltransferase Controls Stem Cell Aging by Regulating BMI1 and EZH2 through MicroRNAs

Epigenetic regulation of gene expression is well known mechanism that regulates cellular senescence of cancer cells. Here we show that inhibition of DNA methyltransferases (DNMTs) with 5-azacytidine (5-AzaC) or with specific small interfering RNA (siRNA) against DNMT1 and 3b induced the cellular senescence of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) and increased p16INK4A and p21CIP1/WAF1 expression. DNMT inhibition changed histone marks into the active forms and decreased the methylation of CpG islands in the p16INK4A and p21CIP1/WAF1 promoter regions. Enrichment of EZH2, the key factor that methylates histone H3 lysine 9 and 27 residues, was decreased on the p16INK4A and p21CIP1/WAF1 promoter regions. We found that DNMT inhibition decreased expression levels of Polycomb-group (PcG) proteins and increased expression of microRNAs (miRNAs), which target PcG proteins. Decreased CpG island methylation and increased levels of active histone marks at genomic regions encoding miRNAs were observed after 5-AzaC treatment. Taken together, DNMTs have a critical role in regulating the cellular senescence of hUCB-MSCs through controlling not only the DNA methylation status but also active/inactive histone marks at genomic regions of PcG-targeting miRNAs and p16INK4A and p21CIP1/WAF1 promoter regions

Characterization of tumor-derived mesenchymal stem cells potentially differentiating into cancer-associated fibroblasts in lung cancer

PurposeThe goal of this study was to understand if mesenchymal stem cells isolated from lung tumor tissue (T-MSCs) may differentiate into cancer associated fibroblasts (CAFs), that promote neoplastic progression, angiogenesis and metastasis in the epithelial solid tumors, mimicking the tumor microenvironmental influence.MethodsMSCs were been obtained from healthy (Control, C-MSCs) and tumor (T-MSCs) tissue of one patient who underwent a lobectomy for a lung adenocarcinoma pT1bN0. Isolated cells were characterized for the presence of molecular markers (identified by routine diagnostic characterization in differentiated tumoral cells), stemness properties, and CAF-related markers expression. Subsequently, cells were co-cultured with a lung adenocarcinoma cell line (A549 cells) to evaluate the effects on proliferation, oncogene expression and IL6 secretion.ResultsC- and T-MSCs did not present EGFR mutations unlike tumor tissue and showed a stem-like immunophenotype, characterized by the ability to differentiate towards osteo-, chondro- and adipogenic lineages. The expression of markers referred to CAFs (-SMA, HI-1, MMP11, VEGF, CXCL12, TGF-1, TGF-RII, IL6, TNF) was significantly higher in T-MSCs than in C-MSCs. The co-cultures with A549 cells led to the over-expression of selected oncogenes and to the increase of IL6 secretion in T-MSCs but not in C-MSCs.ConclusionsMSCs isolated from tumor tissue displayed distinct properties compared to MSCs isolated from healthy tissue, suggesting T-MSCs differentiation towards a CAF-related phenotype under the influence of the tumoral microenvironment