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Short Peptides Protect Oral Stem Cells from Ageing

Authors:
Bruna Sinjari at Università degli Studi G. d'Annunzio Chieti e Pescara
Bruna Sinjari
Francesca Diomede at Università degli Studi G. d'Annunzio Chieti e Pescara
Francesca Diomede
Vladimir Khavinson
Vladimir Khavinson
Vladimir Khavinson
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Ekaterina S Mironova at Saint Petersburg Institute of Bioregulation and Gerontology
Ekaterina S Mironova
  • Saint Petersburg Institute of Bioregulation and Gerontology
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Abstract and Figures

Primary stem cells, after several cell divisions, enter into a senescence state, that is characterized by alterations to spindle-shape typical morphology. This concern is one of the main problems in the use of human mesenchymal stem cells (hMSCs) in clinical applications which demand cells in large numbers. Short peptides had geroprotective properties and stimulated stem cell differentiation. The aim of the study is to demonstrate the role of AEDG and KED peptides in maintaining oral hMSCs morphology and functions over long-term expansion. 2 types of hMSCs were investigated: human periodontal ligament stem cells (hPLSCs) and human gingival mesenchymal stem cells (hGMSCs). Cells at the 25th passage were divided into 3 groups: 1 – control (without adding peptide), 2 – treated with AEDG peptide, 3 – treated with KED peptide. Cell cultures were analyzed by an immunofluorescence method and RT-PCR on the p16 and p21 senescence markers expression. AEDG peptide decreased p16 and p21 mRNA expression by 1.56–2.44 times in comparison with the control group. KED peptide decreased p16 and p21 mRNA expression by 1.82–3.23 times in comparison with the control group. These results were confirmed by immunofluorescent visualization. AEDG and KED peptides could be used as supplementary substances in a culture medium to delay the expression of senescence markers in long term stem cell cultivation in order to promote the large-scale in vitro expansion necessarily required for stem cell therapy clinical application. The data obtained confirm the geroprotective effect of AEDG and KED peptide, which was shown early in animal and cells models.
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Short Peptides Protect Oral Stem Cells from Ageing
Bruna Sinjari
1
&Francesca Diomede
1
&Vladimir Khavinson
2,3,4
&Ekaterina Mironova
2
&Natalia Linkova
2,5
&
Svetlana Trofimova
2,5
&Oriana Trubiani
1
&Sergio Caputi
1
#Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Primary stem cells, after several cell divisions, enter into a senescence state, that is characterized by alterations to spindle-shape
typical morphology. This concern is one of the main problems in the use of human mesenchymal stem cells (hMSCs) in clinical
applications which demand cells in large numbers. Short peptides had geroprotective properties and stimulated stem cell
differentiation. The aim of the study is to demonstrate the role of AEDG and KED peptides in maintaining oral hMSCs
morphology and functions over long-term expansion. 2 types of hMSCs were investigated: human periodontal ligament stem
cells (hPLSCs) and human gingival mesenchymal stem cells (hGMSCs).Cells at the 25th passage were divided into 3 groups: 1 –
control (without adding peptide), 2 –treated with AEDG peptide, 3 –treated with KED peptide. Cell cultures were analyzed by
an immunofluorescence method and RT-PCR on the p16 and p21 senescence markers expression. AEDG peptide decreased p16
and p21 mRNA expression by 1.56–2.44 times in comparison with the control group. KED peptide decreased p16 and p21
mRNA expression by 1.82–3.23 times in comparison with the control group. These results were confirmed by immunofluores-
cent visualization. AEDG and KED peptides could be used as supplementary substances in a culture medium to delay the
expression of senescence markers in long term stem cell cultivation in order to promote the large-scale in vitro expansion
necessarily required for stem cell therapy clinical application. The data obtained confirm the geroprotective effect of AEDG
and KED peptide, which was shown early in animal and cells models.
Keywords AEDG peptide .KED peptide .Stem cells .Cell senescence .p16 .p21 .Geroprotection
Introduction
In vitro cell growth is a mechanism that gradually induces the
loss of cellular function. Human mesenchymal stem cells
(hMSCs) cultured for long-term passage lose self-renewal
capacity, multi-lineage differentiation potential and go into a
replicative senescence state. This concern is one of the main
problems in the use of hMSCs in clinical applications which
demand cells in large numbers. Primary somatic cells, after
several cell divisions, enter into a senescence state, that is
characterized by alterations to spindle-shape typical morphol-
ogy [1,2]. Otherwise, they lose their multilineage differentia-
tion potential and the capacity to migrate, becoming un-
suitable for clinical use [3–5]. For stem cell transplanta-
tion, low grade of cell senescence plays a key role in the
large-scale expansion of hMSCs [6,7]. hMSCs are widely
used in regenerative medicine as well as in age associated
diseases [8]. They should have multi-differentiation poten-
tial, be easily accessible and be obtainable by minimally-
invasive or non-invasive collection procedures. Bone
marrow-derived mesenchymal stem cells were the first
MSCs obtained in vitro, however they require an invasive
technique to obtain [9]. Dental tissue serves as an alterna-
tive source for MSCs showing a higher colony forming
unit and proliferation rate efficiency, along with a similar
gene expression profile for genes related to mineralization
and to their neural crest origin [10]. Periodontal ligament,
Bruna Sinjari and Francesca Diomede contributed equally to this work.
*Natalia Linkova
miayy@yandex.ru
1
Department of Medical, Oral and Biotechnological Sciences,
University “G. d’Annunzio”Chieti-Pescara, Chieti, Italy
2
Department of Biogerontology, Saint Petersburg Institute of
Bioregulation and Gerontology, St. Petersburg, Russia
3
Department of Geriatrics, Propaedeutics and Nursing Management,
Mechnikov North-Western State Medical University, St.
Petersburg, Russia
4
Pavlov Institute of Physiology, Russian Academy of Sciences, St.
Petersburg, Russia
5
Department of Therapy, Geriatrics, and Anti-Aging Medicine,
Academy of Postgraduate Education, Moscow, Russia
https://doi.org/10.1007/s12015-019-09921-3
Stem Cell Reviews and Reports (2020) 16:159–166
Published online: 1 November 2019
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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... This means that di-, tri-and some tetrapeptides have both enough free space in the active center and the ability to saturate their hydrogen bonds, and find partners in electrostatic and hydrophobic interactions, which is indicative of the possibility for USP data to be transported across the membrane by LAT1 transporter. Geroprotector [12] Regulator of the epiphysis functions [172] Neuroprotector [173] Crystagen (EDP) −20.22 ...
... Geroprotector [12] Regulator of the epiphysis functions [172] Neuroprotector [173] ...
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Full-text available
  • Apr 2018
Background: The role of bone tissue engineering in the field of regenerative medicine has been a main research topic over the past few years. There has been much interest in the use of three-dimensional (3D) engineered scaffolds (PLA) complexed with human gingival mesenchymal stem cells (hGMSCs) as a new therapeutic strategy to improve bone tissue regeneration. These devices can mimic a more favorable endogenous microenvironment for cells in vivo by providing 3D substrates which are able to support cell survival, proliferation and differentiation. The present study evaluated the in vitro and in vivo capability of bone defect regeneration of 3D PLA, hGMSCs, extracellular vesicles (EVs), or polyethyleneimine (PEI)-engineered EVs (PEI-EVs) in the following experimental groups: 3D-PLA, 3D-PLA + hGMSCs, 3D-PLA + EVs, 3D-PLA + EVs + hGMSCs, 3D-PLA + PEI-EVs, 3D-PLA + PEI-EVs + hGMSCs. Methods: The structural parameters of the scaffold were evaluated using both scanning electron microscopy and nondestructive microcomputed tomography. Nanotopographic surface features were investigated by means of atomic force microscopy. Scaffolds showed a statistically significant mass loss along the 112-day evaluation. Results: Our in vitro results revealed that both 3D-PLA + EVs + hGMSCs and 3D-PLA + PEI-EVs + hGMSCs showed no cytotoxicity. However, 3D-PLA + PEI-EVs + hGMSCs exhibited greater osteogenic inductivity as revealed by morphological evaluation and transcriptomic analysis performed by next-generation sequencing (NGS). In addition, in vivo results showed that 3D-PLA + PEI-EVs + hGMSCs and 3D-PLA + PEI-EVs scaffolds implanted in rats subjected to cortical calvaria bone tissue damage were able to improve bone healing by showing better osteogenic properties. These results were supported also by computed tomography evaluation that revealed the repair of bone calvaria damage. Conclusion: The re-establishing of the integrity of the bone lesions could be a promising strategy in the treatment of accidental or surgery trauma, especially for cranial bones.
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Mesenchymal stem cells provide paracrine neuroprotective resources that delay degeneration of co-cultured organotypic neuroretinal cultures
Article
  • May 2019
Through the paracrine effects of stem cells, including the secretion of neurotrophic, immunomodulatory, and anti-apoptotic factors, cell-based therapies offer a new all-encompassing approach to treatment of neurodegenerative diseases. In this study, we used physically separated co-cultures of porcine neuroretina (NR) and human mesenchymal stem cells (MSC) to evaluate the MSC paracrine neuroprotective effects on NR degeneration. NR explants were obtained from porcine eyes and cultured alone or co-cultured with commercially available MSCs from Valladolid (MSCV; Citospin S.L.; Valladolid, Spain), currently used for several approved treatments. Cultures were maintained for 72 h. MSC surface markers were evaluated before and after co-culture with NRs. Culture supernatants were collected and the concentration of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial-derived neurotrophic factor (GDNF) were determined by enzyme-linked immunosorbent assays. NR sections were stained by haematoxylin/eosin or immunostained for terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), glial fibrillary acidic protein, β-tubulin III, and neuronal nuclei marker. NR morphology, morphometry, nuclei count, apoptosis rate, retinal ganglion cells, and glial cell activation were evaluated. Treatment effects were statistically analysed by parametric or non-parametric tests. The MSCs retained stem cell surface markers after co-culture with NR. BDNF and CNTF concentrations in NR-MSCV co-cultures were higher than other experimental conditions at 72 h (p < 0.05), but no GDNF was detected. NR general morphology, total thickness, and cell counts were broadly preserved in co-cultures, and the apoptosis rate determined by TUNEL assay was lower than for NR monocultures (all p < 0.05). Co-cultures with MSCV also protected retinal ganglion cells from degenerative changes and reduced reactive gliosis (both p < 0.05). In this in vitro model of spontaneous NR degeneration, the presence of co-cultured MSCs retarded neuroglial degeneration. This effect was associated with elevated concentrations of the neurotrophic factors BDNF and CNTF. Our data suggest that the paracrine secretion of these, and possibly other molecules, are a potential resource for the treatment of several neuroretinal diseases.
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Periodontal Ligament Stem Cells: Current Knowledge and Future Perspectives
Article
  • Apr 2019
Teeth represent a fascinating area of study in regenerative medicine, because of their unique and complex developmental origin. Several types of mesenchymal stem cells (MSCs) have been characterized in the oral cavity, and those derived from the periodontal ligament (PDL) first isolated by our group in 2005, can be expanded in a xeno-free medium preserving morphological features and markers associated with pluripotency. These postnatal MSCs can be easily recovered by noninvasive procedures and cultured. This could facilitate the use of adult stem cells in human clinical regeneration therapy. In this review we summarize the results of our studies describing morphofunctional features, surface markers, and multilineage differentiation capacity in vitro of PDL MSCs obtained in our laboratories. In vivo characterization of PDL stem cell (PDLSC) location and heterogeneity are still lacking. However, we describe studies exploring the potential use of PDLSC to treat both periodontal diseases and regeneration of other tissues. These MSCs may have an advantage in possessing also angiogenetic, immunoregulatory, and anti-inflammatory properties. The secretome of such cells contains several interesting molecules mimicking the effects of the producer cells. We describe some recent studies from our group on the use of conditioned medium from PDL MSCs, and purified extracellular vesicles therein contained, in animal models of experimental autoimmune encephalomyelitis and their potential application to human disease.
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Endothelial committed oral stem cells as modelling in the relationship between periodontal and cardiovascular disease
Article
  • Mar 2018
In the present study we have mimicked, in vitro, an inflammatory process using Lipopolysaccharide derived from Porphyromonas Gingivalis (LPS‐G) and human Periodontal Ligament Stem Cells induced to endothelial differentiation (e‐hPDLSCs). The research project has been organized into the three following steps: i) induction of hPDLSCs toward endothelial differentiation; ii) evaluation of the molecular signaling pathway involved in the response to the LPS‐G, and iii) functional response evaluation of the living construct constituted by porcine decellularized valve/e‐hPDLSCs treated with LPS‐G. Obtained results showed that 5 μg/ml LPS‐G stimulus provokes: a slowdown of cell growth starting from 24 hr and the release of IL6, IL8, and MCP1 molecules. Signaling network analyzed showed the activation of TLR4/ NFkB/ERK1/2/p‐ERK1/2 signaling mediated by MyD88 in LPS‐G stimulated e‐hPDLSCs, moreover a time course put in evidence a nuclear traslocation of ERK1/2 and p‐ERK1/2 in differentiated samples. Following, the ability of e‐hPDLSCs to expand and colonize the decellularized porcine heart valves was appraised at ultrastructural level. Considering that, the Reactive Oxygen Species (ROS) play an important role in the progression and development of cardiovascular disease (CVD), in LPS‐G living construct model e‐hPDLSCs/decellularized porcine heart valves (dPHV), ROS production was assessed. Time lapse experiments evidenced that LPS‐G provokes in e‐hPDLSCs a rapid and sustained increase in ROS generation, negligible on undifferentiated cells. From obtained data, by multiparametric analyses, a reasonable conclusion may be that the inflammation process activated by LPS‐G can affect endothelial cells and could represent in vivo a possible pathological and predictor state of CVD.
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Bone marrow or adipose tissue mesenchymal stem cells: Comparison of the therapeutic potentials in mice model of acute liver failure
Article
  • Mar 2018
Acute liver failure (ALF) is a lethal disease with limited life‐saving therapy. Because lack of whole organ donors for liver transplantation, a substitute treatment strategy is needed for these patients. Preclinical and clinical findings have proved that treatment with mesenchymal stem cells (MSCs) is beneficial for recovery from ALF. In this approach, however, the appropriate sources of these cells are unclear. In the present study, we investigated and compared the therapeutic potentials of bone marrow‐mesenchymal stem cells (BM‐MSC) with those of adipose tissue (AT‐MSC) in carbon tetrachloride (CCL4)‐induced acute liver failure in mice. Murine BM‐ and AT‐MSCs obtained from normal mice were cultured and labelled. The cells were transplanted to CCL4‐induced ALF mice models intravenously. After cell transplantation, blood samples and liver tissues were collected daily for 72 h to analyze liver enzymes and liver histopathology, respectively. We found that survival rate of AT‐MSC transplanted (AT‐TR) mice was significantly higher than that of control (ALF) group. Liver histopathology was superior in the AT‐TR mice, but not significantly, compared to that in BM‐MSC transplanted (BM‐TR) ones. Furthermore, in the AT‐TR mice the level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), in some time points were significantly less than those of BM‐TR. Taken together, these data suggest that in comparison to BM‐MSC, AT‐MSCs is an appropriate choice for cell therapy in the case of acute liver failure.
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