1
|
Wolint P, Hofmann S, von Atzigen J, Böni R, Miescher I, Giovanoli P, Calcagni M, Emmert MY, Buschmann J. Standardization to Characterize the Complexity of Vessel Network Using the Aortic Ring Model. Int J Mol Sci 2024; 26:291. [PMID: 39796147 PMCID: PMC11719671 DOI: 10.3390/ijms26010291] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2024] [Revised: 12/05/2024] [Accepted: 12/30/2024] [Indexed: 01/13/2025] Open
Abstract
Regeneration after ischemia requires to be promoted by (re)perfusion of the affected tissue, and, to date, there is no therapy that covers all needs. In treatment with mesenchymal stem cells (MSC), the secretome acts via paracrine mechanisms and has a positive influence on vascular regeneration via proangiogenic factors. A lack of standardization and the high complexity of vascular structures make it difficult to compare angiogenic readouts from different studies. This emphasizes the need for improved approaches and the introduction of an index in the preclinical setting. A characterization of human MSC secretomes obtained from one of the three formats-single cells, small, and large spheroids-was performed using the chicken aortic ring assay in combination with a modified angiogenic activity index (AAI) and an angiogenic profile. While the secretome of the small spheroid group showed an inhibitory effect on angiogenesis, the large spheroid group impressed with a fully pro-angiogenic response, and a higher AAI compared to the single cell group, underlying the suitability of these three-stem cell-derived secretomes with their distinct angiogenic properties to validate the AAI and the novel angiogenic profile established here.
Collapse
Affiliation(s)
- Petra Wolint
- Division of Surgical Research, University Hospital of Zurich, 8091 Zurich, Switzerland
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland; (S.H.); (J.v.A.); (I.M.); (P.G.); (M.C.)
| | - Silvan Hofmann
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland; (S.H.); (J.v.A.); (I.M.); (P.G.); (M.C.)
| | - Julia von Atzigen
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland; (S.H.); (J.v.A.); (I.M.); (P.G.); (M.C.)
| | - Roland Böni
- White House Center for Liposuction, 8044 Zurich, Switzerland;
| | - Iris Miescher
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland; (S.H.); (J.v.A.); (I.M.); (P.G.); (M.C.)
| | - Pietro Giovanoli
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland; (S.H.); (J.v.A.); (I.M.); (P.G.); (M.C.)
| | - Maurizio Calcagni
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland; (S.H.); (J.v.A.); (I.M.); (P.G.); (M.C.)
| | - Maximilian Y. Emmert
- Institute for Regenerative Medicine (IREM), University of Zurich, 8952 Zurich, Switzerland;
- Deutsches Herzzentrum der Charité (DHZC), Department of Cardiothoracic and Vascular Surgery, 13353 Berlin, Germany
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, 13353 Berlin, Germany
- BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Johanna Buschmann
- Division of Surgical Research, University Hospital of Zurich, 8091 Zurich, Switzerland
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland; (S.H.); (J.v.A.); (I.M.); (P.G.); (M.C.)
| |
Collapse
|
2
|
Trotzier C, Bellanger C, Abdessadeq H, Delannoy P, Mojallal A, Auxenfans C. Deciphering influence of donor age on adipose-derived stem cells: in vitro paracrine function and angiogenic potential. Sci Rep 2024; 14:27589. [PMID: 39528480 PMCID: PMC11555058 DOI: 10.1038/s41598-024-73875-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2024] [Accepted: 09/23/2024] [Indexed: 11/16/2024] Open
Abstract
BACKGROUND As fat grafting is commonly used as a filler, Adipose-derived stem/stromal cells (ASC) have been reported to be key player in retention rate. Paracrine and differentiation potential of those cells confer them strong pro-angiogenic capacities. However, a full characterization of the influence of aging on ASC has not been reported yet. Here we've investigated the effect of age on paracrine function, stemness and angiogenic potential of ASC. METHODS ASC were extracted from young and old adult donors. We assessed stromal vascular fraction cell populations repartition, ASC stemness potential, capability to differentiate into mesenchymal lineages as well as their secretome. Angiogenic potential was assessed using a sprouting assay, an indirect co-culture of ASC and dermal microvascular endothelial cells (EC). Total vascular sprout length was measured, and co-culture soluble factors were quantified. Pro-angiogenic factors alone or in combination as well as ASC-conditioned medium (CM) were added to EC to assess sprouting induction. RESULTS Decrease of endothelial cells yield and percentage is observed in cells extracted from adipose tissue of older patients, whereas ASC percentage increased with age. Clonogenic potential of ASC is stable with age. ASC can differentiate into adipocytes, chondrocytes and osteoblasts, and aging does not alter this potential. Among the 25 analytes quantified, high levels of pro-angiogenic factors were found, but none is significantly modulated with age. ASC induce a significantly longer vascular sprouts compared to fibroblasts, and no difference was found between young and old ASC donors on that parameter. Higher concentrations of FGF-2, G-CSF, HGF and IL-8, and lower concentrations of VEGF-C were quantified in EC/ASC co-cultures compared to EC/fibroblasts co-cultures. EC/ASC from young donors secrete higher levels of VEGF-A compared to old ones. Neither soluble factor nor CM without cells are able to induce organized sprouts, highlighting the requirement of cell communication for sprouting. CM produced by ASC supporting development of long vascular sprouts promote sprouting in co-cultures that establish shorter sprouts. CONCLUSION Our results show cells from young and old donors exhibit no difference in all assessed parameters, suggesting all patients could be included in clinical applications. We emphasized the leading role of ASC in angiogenesis, without impairment with age, where secretome is a key but not sufficient actor.
Collapse
Affiliation(s)
- Chloe Trotzier
- Advanced Research, L'Oréal Research and Innovation, 1, Av. Eugene Schueller, 93600, Aulnay sous Bois, France.
| | - Clement Bellanger
- Advanced Research, L'Oréal Research and Innovation, 1, Av. Eugene Schueller, 93600, Aulnay sous Bois, France
| | - Hakima Abdessadeq
- Advanced Research, L'Oréal Research and Innovation, 1, Av. Eugene Schueller, 93600, Aulnay sous Bois, France
| | - Philippe Delannoy
- Advanced Research, L'Oréal Research and Innovation, 1, Av. Eugene Schueller, 93600, Aulnay sous Bois, France
| | - Ali Mojallal
- Department of Plastic, Reconstructive and Aesthetic Surgery, La Croix Rousse Hospital, Bernard Lyon 1 University, Lyon, France
| | - Celine Auxenfans
- Banque de Tissus et de Cellules des Hospices Civils de Lyon, Edouard Herriot Hospital, Lyon, France
| |
Collapse
|
3
|
Foti R, Storti G, Palmesano M, Scioli MG, Fiorelli E, Terriaca S, Cervelli G, Kim BS, Orlandi A, Cervelli V. Senescence in Adipose-Derived Stem Cells: Biological Mechanisms and Therapeutic Challenges. Int J Mol Sci 2024; 25:8390. [PMID: 39125960 PMCID: PMC11312747 DOI: 10.3390/ijms25158390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2024] [Revised: 07/24/2024] [Accepted: 07/25/2024] [Indexed: 08/12/2024] Open
Abstract
Adipose tissue-derived stem cells (ADSCs) represent a subset of the mesenchymal stem cells in every adipose compartment throughout the body. ADSCs can differentiate into various cell types, including chondrocytes, osteocytes, myocytes, and adipocytes. Moreover, they exhibit a notable potential to differentiate in vitro into cells from other germinal lineages, including endothelial cells and neurons. ADSCs have a wide range of clinical applications, from breast surgery to chronic wounds. Furthermore, they are a promising cell population for future tissue-engineering uses. Accumulating evidence indicates a decreased proliferation and differentiation potential of ADSCs with an increasing age, increasing body mass index, diabetes mellitus, metabolic syndrome, or exposure to radiotherapy. Therefore, the recent literature thoroughly investigates this cell population's senescence mechanisms and how they can hinder its possible therapeutic applications. This review will discuss the biological mechanisms and the physio-pathological causes behind ADSC senescence and how they can impact cellular functionality. Moreover, we will examine the possible strategies to invert these processes, re-establishing the full regenerative potential of this progenitor population.
Collapse
Affiliation(s)
- Riccardo Foti
- Plastic Surgery, Department of Surgical Sciences, University of Rome “Tor Vergata”, 00133 Rome, Italy; (R.F.); (M.P.); (V.C.)
| | - Gabriele Storti
- Plastic Surgery, Department of Surgical Sciences, University of Rome “Tor Vergata”, 00133 Rome, Italy; (R.F.); (M.P.); (V.C.)
| | - Marco Palmesano
- Plastic Surgery, Department of Surgical Sciences, University of Rome “Tor Vergata”, 00133 Rome, Italy; (R.F.); (M.P.); (V.C.)
| | - Maria Giovanna Scioli
- Anatomy Pathology Institute, Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, 00133 Rome, Italy; (M.G.S.); (E.F.); (S.T.); (A.O.)
| | - Elena Fiorelli
- Anatomy Pathology Institute, Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, 00133 Rome, Italy; (M.G.S.); (E.F.); (S.T.); (A.O.)
| | - Sonia Terriaca
- Anatomy Pathology Institute, Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, 00133 Rome, Italy; (M.G.S.); (E.F.); (S.T.); (A.O.)
| | - Giulio Cervelli
- Department of Experimental Medicine, University of Rome “Tor Vergata”, 00133 Rome, Italy;
| | - Bong Sung Kim
- Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8006 Zurich, Switzerland;
| | - Augusto Orlandi
- Anatomy Pathology Institute, Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, 00133 Rome, Italy; (M.G.S.); (E.F.); (S.T.); (A.O.)
| | - Valerio Cervelli
- Plastic Surgery, Department of Surgical Sciences, University of Rome “Tor Vergata”, 00133 Rome, Italy; (R.F.); (M.P.); (V.C.)
| |
Collapse
|
4
|
Kang Y, Na J, Karima G, Amirthalingam S, Hwang NS, Kim HD. Mesenchymal Stem Cell Spheroids: A Promising Tool for Vascularized Tissue Regeneration. Tissue Eng Regen Med 2024; 21:673-693. [PMID: 38578424 PMCID: PMC11187036 DOI: 10.1007/s13770-024-00636-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 02/29/2024] [Accepted: 03/05/2024] [Indexed: 04/06/2024] Open
Abstract
BACKGROUND Mesenchymal stem cells (MSCs) are undifferentiated cells that can differentiate into specific cell lineages when exposed to the right conditions. The ability of MSCs to differentiate into particular cells is considered very important in biological research and clinical applications. MSC spheroids are clusters of MSCs cultured in three dimensions, which play an important role in enhancing the proliferation and differentiation of MSCs. MSCs can also participate in vascular formation by differentiating into endothelial cells and secreting paracrine factors. Vascularization ability is essential in impaired tissue repair and function recovery. Therefore, the vascularization ability of MSCs, which enhances angiogenesis and accelerates tissue healing has made MSCs a promising tool for tissue regeneration. However, MSC spheroids are a relatively new research field, and more research is needed to understand their full potential. METHODS In this review, we highlight the importance of MSC spheroids' vascularization ability in tissue engineering and regenerative medicine while providing the current status of studies on the MSC spheroids' vascularization and suggesting potential future research directions for MSC spheroids. RESULTS Studies both in vivo and in vitro have demonstrated MSC spheroids' capacity to develop into endothelial cells and stimulate vasculogenesis. CONCLUSION MSC spheroids show potential to enhance vascularization ability in tissue regeneration. Yet, further research is required to comprehensively understand the relationship between MSC spheroids and vascularization mechanisms.
Collapse
Affiliation(s)
- Yoonjoo Kang
- Department of IT Convergence (Brain Korea Plus 21), Korea National University of Transportation, Chungju, 27469, Republic of Korea
| | - Jinwoo Na
- Department of Polymer Science and Engineering, Korea National University of Transportation, 50 Daehak-ro, Chungju, 27469, Republic of Korea
| | - Gul Karima
- Department of Polymer Science and Engineering, Korea National University of Transportation, 50 Daehak-ro, Chungju, 27469, Republic of Korea
| | - Sivashanmugam Amirthalingam
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea
- Institute of Engineering Research, Seoul National University, Seoul, 08826, Republic of Korea
| | - Nathaniel S Hwang
- School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 08826, Republic of Korea
- Institute of Engineering Research, Seoul National University, Seoul, 08826, Republic of Korea
| | - Hwan D Kim
- Department of IT Convergence (Brain Korea Plus 21), Korea National University of Transportation, Chungju, 27469, Republic of Korea.
- Department of Polymer Science and Engineering, Korea National University of Transportation, 50 Daehak-ro, Chungju, 27469, Republic of Korea.
- Department of Biomedical Engineering, Korea National University of Transportation, Chungju, 27469, Republic of Korea.
| |
Collapse
|
5
|
Montemurro T, Lavazza C, Montelatici E, Budelli S, La Rosa S, Barilani M, Mei C, Manzini P, Ratti I, Cimoni S, Brasca M, Prati D, Saporiti G, Astori G, Elice F, Giordano R, Lazzari L. Off-the-Shelf Cord-Blood Mesenchymal Stromal Cells: Production, Quality Control, and Clinical Use. Cells 2024; 13:1066. [PMID: 38920694 PMCID: PMC11202005 DOI: 10.3390/cells13121066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Revised: 06/04/2024] [Accepted: 06/13/2024] [Indexed: 06/27/2024] Open
Abstract
Background Recently, mesenchymal stromal cells (MSCs) have gained recognition for their clinical utility in transplantation to induce tolerance and to improve/replace pharmacological immunosuppression. Cord blood (CB)-derived MSCs are particularly attractive for their immunological naivety and peculiar anti-inflammatory and anti-apoptotic properties. OBJECTIVES The objective of this study was to obtain an inventory of CB MSCs able to support large-scale advanced therapy medicinal product (ATMP)-based clinical trials. STUDY DESIGN We isolated MSCs by plastic adherence in a GMP-compliant culture system. We established a well-characterized master cell bank and expanded a working cell bank to generate batches of finished MSC(CB) products certified for clinical use. The MSC(CB) produced by our facility was used in approved clinical trials or for therapeutic use, following single-patient authorization as an immune-suppressant agent. RESULTS We show the feasibility of a well-defined MSC manufacturing process and describe the main indications for which the MSCs were employed. We delve into a regulatory framework governing advanced therapy medicinal products (ATMPs), emphasizing the need of stringent quality control and safety assessments. From March 2012 to June 2023, 263 of our Good Manufacturing Practice (GMP)-certified MSC(CB) preparations were administered as ATMPs in 40 subjects affected by Graft-vs.-Host Disease, nephrotic syndrome, or bronco-pulmonary dysplasia of the newborn. There was no infusion-related adverse event. No patient experienced any grade toxicity. Encouraging preliminary outcome results were reported. Clinical response was registered in the majority of patients treated under therapeutic use authorization. CONCLUSIONS Our 10 years of experience with MSC(CB) described here provides valuable insights into the use of this innovative cell product in immune-mediated diseases.
Collapse
Affiliation(s)
- Tiziana Montemurro
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Cristiana Lavazza
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Elisa Montelatici
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Silvia Budelli
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Salvatore La Rosa
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Mario Barilani
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Cecilia Mei
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Paolo Manzini
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Ilaria Ratti
- Milano Cord Blood Bank and Center of Transfusion Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (I.R.); (S.C.); (M.B.); (D.P.)
| | - Silvia Cimoni
- Milano Cord Blood Bank and Center of Transfusion Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (I.R.); (S.C.); (M.B.); (D.P.)
| | - Manuela Brasca
- Milano Cord Blood Bank and Center of Transfusion Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (I.R.); (S.C.); (M.B.); (D.P.)
| | - Daniele Prati
- Milano Cord Blood Bank and Center of Transfusion Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (I.R.); (S.C.); (M.B.); (D.P.)
| | - Giorgia Saporiti
- Bone Marrow Transplantation and Cellular Therapy Center, Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy;
| | - Giuseppe Astori
- Laboratory of Advanced Cellular Therapies and Haematology Unit, San Bortolo Hospital, AULSS8 “Berica”, 36100 Vicenza, Italy; (G.A.); (F.E.)
| | - Francesca Elice
- Laboratory of Advanced Cellular Therapies and Haematology Unit, San Bortolo Hospital, AULSS8 “Berica”, 36100 Vicenza, Italy; (G.A.); (F.E.)
| | - Rosaria Giordano
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| | - Lorenza Lazzari
- Unit of Cellular and Gene Therapy, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milano, Italy; (T.M.); (C.L.); (E.M.); (S.B.); (S.L.R.); (M.B.); (C.M.); (P.M.); (L.L.)
| |
Collapse
|
6
|
Phinney DG. Alexander Friedenstein, Mesenchymal Stem Cells, Shifting Paradigms and Euphemisms. Bioengineering (Basel) 2024; 11:534. [PMID: 38927770 PMCID: PMC11201071 DOI: 10.3390/bioengineering11060534] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2024] [Revised: 05/20/2024] [Accepted: 05/21/2024] [Indexed: 06/28/2024] Open
Abstract
Six decades ago, Friedenstein and coworkers published a series of seminal papers identifying a cell population in bone marrow with osteogenic potential, now referred to as mesenchymal stem cells (MSCs). This work was also instrumental in establishing the identity of hematopoietic stem cell and the identification of skeletal stem/progenitor cell (SSPC) populations in various skeletal compartments. In recognition of the centenary year of Friedenstein's birth, I review key aspects of his work and discuss the evolving concept of the MSC and its various euphemisms indorsed by changing paradigms in the field. I also discuss the recent emphasis on MSC stromal quality attributes and how emerging data demonstrating a mechanistic link between stromal and stem/progenitor functions bring renewed relevance to Friedenstein's contributions and much needed unity to the field.
Collapse
Affiliation(s)
- Donald G Phinney
- Department of Molecular Medicine, Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL 33458, USA
| |
Collapse
|
7
|
Matveeva D, Kashirina D, Ezdakova M, Larina I, Buravkova L, Ratushnyy A. Senescence-Associated Alterations in Matrisome of Mesenchymal Stem Cells. Int J Mol Sci 2024; 25:5332. [PMID: 38791371 PMCID: PMC11120844 DOI: 10.3390/ijms25105332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2024] [Revised: 04/26/2024] [Accepted: 04/30/2024] [Indexed: 05/26/2024] Open
Abstract
The process of aging is intimately linked to alterations at the tissue and cellular levels. Currently, the role of senescent cells in the tissue microenvironment is still being investigated. Despite common characteristics, different cell populations undergo distinctive morphofunctional changes during senescence. Mesenchymal stem cells (MSCs) play a pivotal role in maintaining tissue homeostasis. A multitude of studies have examined alterations in the cytokine profile that determine their regulatory function. The extracellular matrix (ECM) of MSCs is a less studied aspect of their biology. It has been shown to modulate the activity of neighboring cells. Therefore, investigating age-related changes in the MSC matrisome is crucial for understanding the mechanisms of tissue niche ageing. This study conducted a broad proteomic analysis of the matrisome of separated fractions of senescent MSCs, including the ECM, conditioned medium (CM), and cell lysate. This is the first time such an analysis has been conducted. It has been established that there is a shift in production towards regulatory molecules and a significant downregulation of the main structural and adhesion proteins of the ECM, particularly collagens, fibulins, and fibrilins. Additionally, a decrease in the levels of cathepsins, galectins, S100 proteins, and other proteins with cytoprotective, anti-inflammatory, and antifibrotic properties has been observed. However, the level of inflammatory proteins and regulators of profibrotic pathways increases. Additionally, there is an upregulation of proteins that can directly cause prosenescent effects on microenvironmental cells (SERPINE1, THBS1, and GDF15). These changes confirm that senescent MSCs can have a negative impact on other cells in the tissue niche, not only through cytokine signals but also through the remodeled ECM.
Collapse
Affiliation(s)
| | | | | | | | | | - Andrey Ratushnyy
- Institute of Biomedical Problems, Russian Academy of Sciences, Khoroshevskoye Shosse, 76a, 123007 Moscow, Russia; (D.M.); (D.K.); (M.E.); (I.L.); (L.B.)
| |
Collapse
|
8
|
Zhang Z, Bao Y, Wei P, Yan X, Qiu Q, Qiu L. Melatonin attenuates dental pulp stem cells senescence due to vitro expansion via inhibiting MMP3. Oral Dis 2024; 30:2410-2424. [PMID: 37448325 DOI: 10.1111/odi.14649] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2022] [Revised: 05/07/2023] [Accepted: 06/05/2023] [Indexed: 07/15/2023]
Abstract
OBJECTIVE We aimed to identify the crucial genes involved in dental pulp stem cell (DPSC) senescence and evaluate the impact of melatonin on DPSC senescence. METHODS Western blotting, SA-β-Gal staining and ALP staining were used to evaluate the senescence and differentiation potential of DPSCs. The optimal concentration of melatonin was determined using the CCK-8 assay. Differentially expressed genes (DEGs) involved in DPSC senescence were obtained via bioinformatics analysis, followed by RT-qPCR. Gain- and loss-of-function studies were conducted to explore the role of MMP3 in DPSC in vitro expansion and in response to melatonin. GSEA was employed to analyse MMP3-related pathways in cellular senescence. RESULTS Treatment with 0.1 μM melatonin attenuated cellular senescence and differentiation potential suppression in DPSCs due to long-term in vitro expansion. MMP3 was a crucial gene in senescence, as confirmed by bioinformatics analysis, RT-qPCR and Western blotting. Furthermore, gain- and loss-of-function studies revealed that MMP3 played a regulatory role in cellular senescence. Rescue assays showed that overexpression of MMP3 reversed the effect of melatonin on senescence. GSEA revealed that the MMP3-dependent anti-senescence effect of melatonin was associated with the IL6-JAK-STAT3, TNF-α-Signalling-VIA-NF-κB, COMPLEMENT, NOTCH Signalling and PI3K-AKT-mTOR pathways. CONCLUSION Melatonin attenuated DPSC senescence caused by long-term expansion by inhibiting MMP3.
Collapse
Affiliation(s)
- Zeying Zhang
- Department of Endodontics, Liaoning Provincial Key Laboratory of Oral Diseases, School and Hospital of Stomatology, China Medical University, Shenyang, China
| | - Yandong Bao
- Department of Cardiology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China
| | - Penggong Wei
- Department of Endodontics, Liaoning Provincial Key Laboratory of Oral Diseases, School and Hospital of Stomatology, China Medical University, Shenyang, China
| | - Xiaoyuan Yan
- Department of Endodontics, Liaoning Provincial Key Laboratory of Oral Diseases, School and Hospital of Stomatology, China Medical University, Shenyang, China
| | - Qiujing Qiu
- Department of Endodontics, Liaoning Provincial Key Laboratory of Oral Diseases, School and Hospital of Stomatology, China Medical University, Shenyang, China
| | - Lihong Qiu
- Department of Endodontics, Liaoning Provincial Key Laboratory of Oral Diseases, School and Hospital of Stomatology, China Medical University, Shenyang, China
| |
Collapse
|
9
|
Ushakov R, Ratushnyy A, Buravkova L, Tolkunova E, Burova E. The Decellularized Cell-Derived Extracellular Matrix Enhances the Paracrine Function of Human Mesenchymal Stromal/Stem Cells. Int J Mol Sci 2024; 25:2419. [PMID: 38397096 PMCID: PMC10889327 DOI: 10.3390/ijms25042419] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Revised: 02/12/2024] [Accepted: 02/17/2024] [Indexed: 02/25/2024] Open
Abstract
The mesenchymal stromal/stem cells (MSCs) are known to secrete pleiotropic paracrine factors, contributing to tissue regeneration. This unique ability makes MSCs promising therapeutic tools for many diseases, including even those that were previously untreatable. Thus, the development of preconditioning approaches aimed at enhancing the paracrine function of MSCs attracts great interest. In the present work, we studied how the extracellular matrix, the essential part of the native tissue microenvironment, affects the secretory capacity of MSCs of various origins. The MSC-derived decellularized extracellular matrix (dECM), used as the cell culture substrate, triggered strong upregulation of FGF-2, MMP-1, HGF, GRO-α, GRO-β, CXCL-5, CXCL-6, IL-6, IL-8, G-CSF and MCP-1. Functional in vitro tests revealed that conditioned media derived from MSCs cultured on dECM significantly improved 3T3 fibroblast and HaCaT keratinocyte scratch wound healing, stimulated THP-1 monocyte migration and promoted capillary-like HUVEC-based tube formation compared to conditioned media from MSCs grown on plastic. In addition, we found that FAK inhibition promoted dECM-induced upregulation of paracrine factors, suggesting that this kinase participates in the MSCs' paracrine response to dECM. Together, these findings demonstrate that dECM provides cues that considerably enhance the secretory function of MSCs. Thus, dECM usage as a cell culture substrate alone or in combination with a FAK inhibitor may be viewed as a novel MSC preconditioning technique.
Collapse
Affiliation(s)
- Roman Ushakov
- Institute of Cytology, Russian Academy of Sciences, 194064 Saint Petersburg, Russia; (R.U.); (E.T.)
| | - Andrey Ratushnyy
- Institute of Biomedical Problems, Russian Academy of Sciences, 123007 Moscow, Russia; (A.R.); (L.B.)
| | - Ludmila Buravkova
- Institute of Biomedical Problems, Russian Academy of Sciences, 123007 Moscow, Russia; (A.R.); (L.B.)
| | - Elena Tolkunova
- Institute of Cytology, Russian Academy of Sciences, 194064 Saint Petersburg, Russia; (R.U.); (E.T.)
| | - Elena Burova
- Institute of Cytology, Russian Academy of Sciences, 194064 Saint Petersburg, Russia; (R.U.); (E.T.)
| |
Collapse
|
10
|
Xue Z, Liao Y, Li Y. Effects of microenvironment and biological behavior on the paracrine function of stem cells. Genes Dis 2024; 11:135-147. [PMID: 37588208 PMCID: PMC10425798 DOI: 10.1016/j.gendis.2023.03.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2023] [Revised: 02/14/2023] [Accepted: 03/05/2023] [Indexed: 08/18/2023] Open
Abstract
Mesenchymal stem cells (MSCs), the most well-studied cell type in the field of stem cell therapy, have multi-lineage differentiation and self-renewal potential. MSC-based therapies have been used to treat diverse diseases because of their ability to potently repair tissue and locally restore function. An increasing body of evidence demonstrates that paracrine function is central to the effects of MSC-based therapy. Growth factors, cytokines, chemokines, extracellular matrix components, and extracellular vehicles all contribute to the beneficial effects of MSCs on tissue regeneration and repair. The paracrine substances secreted by MSCs change depending on the tissue microenvironment and biological behavior. In this review, we discuss the bioactive substances secreted by MSCs depending on the microenvironment and biological behavior and their regulatory mechanisms, which explain their potential to treat human diseases, to provide new ideas for further research and clinical cell-free therapy.
Collapse
Affiliation(s)
- Zhixin Xue
- The Department of Plastic and Cosmetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Yunjun Liao
- The Department of Plastic and Cosmetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Ye Li
- The Department of Plastic and Cosmetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
| |
Collapse
|
11
|
Turlo AJ, Hammond DE, Ramsbottom KA, Soul J, Gillen A, McDonald K, Peffers MJ. Mesenchymal Stromal Cell Secretome Is Affected by Tissue Source and Donor Age. Stem Cells 2023; 41:1047-1059. [PMID: 37591507 PMCID: PMC10631804 DOI: 10.1093/stmcls/sxad060] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Accepted: 07/21/2023] [Indexed: 08/19/2023]
Abstract
Variation in mesenchymal stromal cell (MSC) function depending on their origin is problematic, as it may confound clinical outcomes of MSC therapy. Current evidence suggests that the therapeutic benefits of MSCs are attributed to secretion of biologically active factors (secretome). However, the effect of donor characteristics on the MSC secretome remains largely unknown. Here, we examined the influence of donor age, sex, and tissue source, on the protein profile of the equine MSC secretome. We used dynamic metabolic labeling with stable isotopes combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify secreted proteins in MSC conditioned media (CM). Seventy proteins were classified as classically secreted based on the rate of label incorporation into newly synthesized proteins released into the extracellular space. Next, we analyzed CM of bone marrow- (n = 14) and adipose-derived MSCs (n = 16) with label-free LC-MS/MS. Clustering analysis of 314 proteins detected across all samples identified tissue source as the main factor driving variability in MSC CM proteomes. Linear modelling applied to the subset of 70 secreted proteins identified tissue-related difference in the abundance of 23 proteins. There was an age-related decrease in the abundance of CTHRC1 and LOX, further validated with orthogonal techniques. Due to the lack of flow cytometry characterization of MSC surface markers, the analysis could not account for the potential effect of cell population heterogeneity. This study provides evidence that tissue source and donor age contribute to differences in the protein composition of MSC secretomes which may influence the effects of MSC therapy.
Collapse
Affiliation(s)
- Agnieszka J Turlo
- Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK
| | - Dean E Hammond
- epartment of Cellular and Molecular Physiology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK
| | - Kerry A Ramsbottom
- Computational Biology Facility, Liverpool Shared Research Facilities, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, UK
| | - Jamie Soul
- Computational Biology Facility, Liverpool Shared Research Facilities, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, UK
| | - Alexandra Gillen
- Department of Veterinary Science, Philip Leverhulme Equine Hospital, University of Liverpool, UK
| | | | - Mandy J Peffers
- Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK
| |
Collapse
|
12
|
Barone L, Gallazzi M, Rossi F, Papait R, Raspanti M, Zecca PA, Buonarrivo L, Bassani B, Bernardini G, Bruno A, Gornati R. Human Dental Pulp Mesenchymal Stem Cell-Derived Soluble Factors Combined with a Nanostructured Scaffold Support the Generation of a Vascular Network In Vivo. NANOMATERIALS (BASEL, SWITZERLAND) 2023; 13:2479. [PMID: 37686988 PMCID: PMC10489993 DOI: 10.3390/nano13172479] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/11/2023] [Revised: 08/28/2023] [Accepted: 08/31/2023] [Indexed: 09/10/2023]
Abstract
Among all strategies directed at developing new tools to support re-vascularization of damaged tissues, the use of pro-angiogenic soluble factors, derived from mesenchymal stem cells (MSCs), appears a promising approach for regenerative medicine. Here, we compared the feasibility of two devices, generated by coupling soluble factors of human dental pulp mesenchymal stem cells (DPSCs), with a nanostructured scaffold, to support angiogenesis once transplanted in mice. DPSCs were obtained from impacted wisdom tooth removal, usually considered surgical waste material. After 28 days, we verified the presence of active blood vessels inside the scaffold through optical and scansion electron microscopy. The mRNA expression of surface antigens related to macrophage polarization (CD68, CD80, CD86, CD163, CD206), as well as pro-angiogenic markers (CD31, CD34, CD105, Angpt1, Angpt2, CDH5) was evaluated by real-time PCR. Our results demonstrate the capability of DPSC-scaffold and DPSC soluble factors-scaffold to support angiogenesis, similarly to adipose stem cells, whereas the absence of blood vessels was found in the scaffold grafted alone. Our results provide evidence that DPSC-conditioned medium can be proposed as a cell-free preparation able to support angiogenesis, thus, providing a relevant tool to overcome the issues and restrictions associated with the use of cells.
Collapse
Affiliation(s)
- Ludovica Barone
- Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy; (L.B.); (F.R.); (R.P.); (L.B.); (G.B.)
| | - Matteo Gallazzi
- Laboratory of Innate Immunity, Unit of Molecular Pathology, Biochemistry, and Immunology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) MultiMedica, 20138 Milan, Italy; (M.G.); (B.B.)
| | - Federica Rossi
- Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy; (L.B.); (F.R.); (R.P.); (L.B.); (G.B.)
| | - Roberto Papait
- Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy; (L.B.); (F.R.); (R.P.); (L.B.); (G.B.)
| | - Mario Raspanti
- Department of Medicine and Innovative Technology, University of Insubria, 21100 Varese, Italy; (M.R.); (P.A.Z.)
| | - Piero Antonio Zecca
- Department of Medicine and Innovative Technology, University of Insubria, 21100 Varese, Italy; (M.R.); (P.A.Z.)
| | - Luca Buonarrivo
- Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy; (L.B.); (F.R.); (R.P.); (L.B.); (G.B.)
| | - Barbara Bassani
- Laboratory of Innate Immunity, Unit of Molecular Pathology, Biochemistry, and Immunology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) MultiMedica, 20138 Milan, Italy; (M.G.); (B.B.)
| | - Giovanni Bernardini
- Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy; (L.B.); (F.R.); (R.P.); (L.B.); (G.B.)
| | - Antonino Bruno
- Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy; (L.B.); (F.R.); (R.P.); (L.B.); (G.B.)
- Laboratory of Innate Immunity, Unit of Molecular Pathology, Biochemistry, and Immunology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) MultiMedica, 20138 Milan, Italy; (M.G.); (B.B.)
| | - Rosalba Gornati
- Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy; (L.B.); (F.R.); (R.P.); (L.B.); (G.B.)
| |
Collapse
|
13
|
Matveeva DK, Ezdakova MI, Ratushnyy AY. Modification of the Properties of Extracellular Matrix of Senescent Mesenchymal Stem Cells. Bull Exp Biol Med 2023; 175:569-575. [PMID: 37770790 DOI: 10.1007/s10517-023-05905-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Indexed: 09/30/2023]
Abstract
Cell senescence leads to changes in the secretory activity of mesenchymal stem cells (MSC), including proteins of extracellular matrix (ECM). Here we studied the regulatory properties of ECM of senescent MSC in a model with endothelial cells (EC). EC were seeded onto a decellularized extracellular matrix of senescent MSC. Changes in cell morphology and a decrease in cell growth were observed. In addition, increased production of inflammatory chemokines MCP-1 and GROα and reduced synthesis of proangiogenic growth factor FGF-2 were revealed. Analysis of ECM showed quantitative and qualitative changes, including fibronectin layer morphology, total protein content, and concentration of deposited growth factors such as VEGF. Thus, our work demonstrates that senescence of MSC can lead to modification of the effects of their ECM on EC activity.
Collapse
Affiliation(s)
- D K Matveeva
- Institute for Biomedical Problems, Russian Academy of Sciences, Moscow, Russia
| | - M I Ezdakova
- Institute for Biomedical Problems, Russian Academy of Sciences, Moscow, Russia
| | - A Yu Ratushnyy
- Institute for Biomedical Problems, Russian Academy of Sciences, Moscow, Russia.
| |
Collapse
|
14
|
Suzdaltseva Y, Kiselev SL. Mesodermal Derivatives of Pluripotent Stem Cells Route to Scarless Healing. Int J Mol Sci 2023; 24:11945. [PMID: 37569321 PMCID: PMC10418846 DOI: 10.3390/ijms241511945] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Revised: 07/07/2023] [Accepted: 07/24/2023] [Indexed: 08/13/2023] Open
Abstract
Scar formation during normal tissue regeneration in adults may result in noticeable cosmetic and functional defects and have a significant impact on the quality of life. In contrast, fetal tissues in the mid-gestation period are known to be capable of complete regeneration with the restitution of the initial architecture, organization, and functional activity. Successful treatments that are targeted to minimize scarring can be realized by understanding the cellular and molecular mechanisms of fetal wound regeneration. However, such experiments are limited by the inaccessibility of fetal material for comparable studies. For this reason, the molecular mechanisms of fetal regeneration remain unknown. Mesenchymal stromal cells (MSCs) are central to tissue repair because the molecules they secrete are involved in the regulation of inflammation, angiogenesis, and remodeling of the extracellular matrix. The mesodermal differentiation of human pluripotent stem cells (hPSCs) recapitulates the sequential steps of embryogenesis in vitro and provides the opportunity to generate the isogenic cell models of MSCs corresponding to different stages of human development. Further investigation of the functional activity of cells from stromal differon in a pro-inflammatory microenvironment will procure the molecular tools to better understand the fundamental mechanisms of fetal tissue regeneration. Herein, we review recent advances in the generation of clonal precursors of primitive mesoderm cells and MSCs from hPSCs and discuss critical factors that determine the functional activity of MSCs-like cells in a pro-inflammatory microenvironment in order to identify therapeutic targets for minimizing scarring.
Collapse
Affiliation(s)
- Yulia Suzdaltseva
- Department of Epigenetics, Vavilov Institute of General Genetics of the Russian Academy of Sciences, 119333 Moscow, Russia;
| | | |
Collapse
|
15
|
Leone A, Nicolò A, Prevenzano I, Zatterale F, Longo M, Desiderio A, Spinelli R, Campitelli M, Conza D, Raciti GA, Beguinot F, Nigro C, Miele C. Methylglyoxal Impairs the Pro-Angiogenic Ability of Mouse Adipose-Derived Stem Cells (mADSCs) via a Senescence-Associated Mechanism. Cells 2023; 12:1741. [PMID: 37443775 PMCID: PMC10340470 DOI: 10.3390/cells12131741] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2023] [Revised: 06/23/2023] [Accepted: 06/26/2023] [Indexed: 07/15/2023] Open
Abstract
Adipose-derived stem cells (ADSCs) play a crucial role in angiogenesis and repair of damaged tissues. However, in pathological conditions including diabetes, ADSC function is compromised. This work aims at evaluating the effect of Methylglyoxal (MGO), a product of chronic hyperglycemia, on mouse ADSCs' (mADSCs) pro-angiogenic function and the molecular mediators involved. The mADSCs were isolated from C57bl6 mice. MGO-adducts and p-p38 MAPK protein levels were evaluated by Western Blot. Human retinal endothelial cell (hREC) migration was analyzed by transwell assays. Gene expression was measured by qRT-PCR, and SA-βGal activity by cytofluorimetry. Soluble factor release was evaluated by multiplex assay. MGO treatment does not impair mADSC viability and induces MGO-adduct accumulation. hREC migration is reduced in response to both MGO-treated mADSCs and conditioned media from MGO-treated mADSCs, compared to untreated cells. This is associated with an increase of SA-βGal activity, SASP factor release and p53 and p21 expression, together with a VEGF- and PDGF-reduced release from MGO-treated mADSCs and a reduced p38-MAPK activation in hRECs. The MGO-induced impairment of mADSC function is reverted by senolytics. In conclusion, MGO impairs mADSCs' pro-angiogenic function through the induction of a senescent phenotype, associated with the reduced secretion of growth factors crucial for hREC migration.
Collapse
Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | - Cecilia Nigro
- URT Genomics of Diabetes, Institute of Experimental Endocrinology and Oncology, National Research Council & Department of Translational Medical Sciences, Federico II University of Naples, 80131 Naples, Italy; (A.L.); (A.N.); (I.P.)
| | | |
Collapse
|
16
|
Hodge JG, Robinson JL, Mellott AJ. Tailoring the secretome composition of mesenchymal stem cells to augment specific functions of epidermal regeneration: an in vitro diabetic model. FRONTIERS IN MEDICAL TECHNOLOGY 2023; 5:1194314. [PMID: 37378005 PMCID: PMC10291509 DOI: 10.3389/fmedt.2023.1194314] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Accepted: 05/26/2023] [Indexed: 06/29/2023] Open
Abstract
Introduction Wound healing consists of a dynamic series of events that are highly dependent on paracrine factors for proper progression through the phases of wound healing. Inappropriate progression through the phases is associated with insufficient epidermal regeneration (i.e., re-epithelialization) of wounds and subsequent propagation of chronic wounds, such as diabetic ulcers, which are associated with increased patient morbidity. Recently, investigation into the dynamic secretome of Adipose-derived Mesenchymal Stem Cells (ASCs), have shown promise in augmenting the wound healing response of chronic diabetic wounds. However, currently utilized 2D culture techniques are known to drastically alter the regenerative phenotype of ASCs. In this study a novel tissue-mimetic 3D system was utilized as a means to culture ASCs. Methods The capacity for the ASC secretome to augment epidermal regeneration activity was then evaluated after exposure of ASCs to "wound priming stimuli" in 2D and 3D. The priming stimuli consisted of coating the 2D and 3D systems with the wound matrix proteins, collagen type I, fibronectin, and fibrin. To understand the potential benefit of the ASC secretome in the context of diabetic wounds, keratinocytes (KCs) were exposed to super-physiological glucose levels to induce a diabetic-like phenotype (idKCs). Results Relative to KCs, idKC exhibited a 52% and 23% decline in proliferation and migration, respectively. Subsequently, analyses of the ASC secretome were performed. ASC conditioned media (ASC-CM) from tissue-mimetic culture demonstrated a > 50% increase secretion of proteins and a 2-fold increase in secreted EVs, relative to 2D culture. Interestingly, the different priming stimuli did not alter the total amount of protein or EVs secreted within the tissue-mimetic system. However, evaluation of specific soluble proteins via ELISA revealed significant differences in key epidermal regeneration factors, such as EGF, IGF-1, FGF-2, MMP-1, TIMP-1, and TGFβ-1. Additionally, the relative effect of ASC-EVs from the 2D and 3D system on idKCs epidermal regeneration functionality varied significantly, with EVs from 3D-Collagen culture providing the most significant benefit on idKC activity. Discussion Together, these data support the utilization of tissue-mimetic culture system to enhance the adaptability and secretory activity of MSC-like populations in order to generate tailored biologics, via priming stimuli, for specific wound healing applications.
Collapse
Affiliation(s)
- Jacob G. Hodge
- Bioengineering Graduate Program, University of Kansas, Lawrence, KS, United States
- Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, KS, United States
| | - Jennifer L. Robinson
- Bioengineering Graduate Program, University of Kansas, Lawrence, KS, United States
- Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS, United States
- Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, United States
- Department of Mechanical Engineering, University of Washington, Seattle, WA, United States
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, United States
| | - Adam J. Mellott
- Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, KS, United States
- Ronawk Inc., Olathe, KS, United States
| |
Collapse
|
17
|
Phinney DG, Hwa Lee R, Boregowda SV. Revisiting the Mesenchymal "Stem vs. Stromal" Cell Dichotomy and Its Implications for Development of Improved Potency Metrics. Stem Cells 2023; 41:444-452. [PMID: 36891977 PMCID: PMC10183967 DOI: 10.1093/stmcls/sxad019] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Accepted: 02/21/2023] [Indexed: 03/10/2023]
Abstract
Mesenchymal stem/stromal cell (MSC)-based therapies have been evaluated in over 1500 human clinical trials for a diverse array of disease indication, but outcomes remain unpredictable due to knowledge gaps in the quality attributes that confer therapeutic potency onto cells and their mode of action in vivo. Based on accumulated evidence from pre-clinical models, MSCs exert therapeutic effects by repressing inflammatory and immune-mediated response via paracrine action following reprogramming by the host injury microenvironment, and by polarization of tissue resident macrophages following phagocytosis to an alternatively activated (M2) state. An important tenet of this existing paradigm is that well-established stem/progenitor functions of MSCs are independent of paracrine function and dispensable for their anti-inflammatory and immune suppressive functions. Herein, we review evidence that stem/progenitor and paracrine functions of MSCs are mechanistically linked and organized hierarchically and describe how this link may be exploited to develop metrics that predict MSC potency across a spectrum of activities and regenerative medicine applications.
Collapse
Affiliation(s)
- Donald G Phinney
- Department of Molecular Medicine, Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, USA
| | - Ryang Hwa Lee
- Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, TX, USA
| | - Siddaraju V Boregowda
- Department of Molecular Medicine, Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, USA
| |
Collapse
|
18
|
Siraj Y, Galderisi U, Alessio N. Senescence induces fundamental changes in the secretome of mesenchymal stromal cells (MSCs): implications for the therapeutic use of MSCs and their derivates. Front Bioeng Biotechnol 2023; 11:1148761. [PMID: 37229499 PMCID: PMC10203235 DOI: 10.3389/fbioe.2023.1148761] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Accepted: 04/24/2023] [Indexed: 05/27/2023] Open
Abstract
Mesenchymal stromal cells (MSCs) are a heterogeneous population containing multipotent adult stem cells with a multi-lineage differentiation capacity, which differentiated into mesodermal derivatives. MSCs are employed for therapeutic purposes and several investigations have demonstrated that the positive effects of MSC transplants are due to the capacity of MSCs to modulate tissue homeostasis and repair via the activity of their secretome. Indeed, the MSC-derived secretomes are now an alternative strategy to cell transplantation due to their anti-inflammatory, anti-apoptotic, and regenerative effects. The cellular senescence is a dynamic process that leads to permanent cell cycle arrest, loss of healthy cells' physiological functions and acquiring new activities, which are mainly accrued through the release of many factors, indicated as senescence-associated secretory phenotype (SASP). The senescence occurring in stem cells, such as those present in MSCs, may have detrimental effects on health since it can undermine tissue homeostasis and repair. The analysis of MSC secretome is important either for the MSC transplants and for the therapeutic use of secretome. Indeed, the secretome of MSCs, which is the main mechanism of their therapeutic activity, loses its beneficial functions and acquire negative pro-inflammatory and pro-aging activities when MSCs become senescent. When MSCs or their derivatives are planned to be used for therapeutic purposes, great attention must be paid to these changes. In this review, we analyzed changes occurring in MSC secretome following the switch from healthy to senescence status.
Collapse
Affiliation(s)
- Yesuf Siraj
- Department of Experimental Medicine, University of Campania, Naples, Italy
- Department of Medical Laboratory Sciences, School of Health Sciences, College of Medicine and Health Sciences, Bahir Dar University, Bahir Dar, Ethiopia
| | - Umberto Galderisi
- Department of Experimental Medicine, University of Campania, Naples, Italy
- Department of Biology, Faculty of Science, Erciyes University, Kayseri, Türkiye
- Center for Biotechnology, Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, United States
| | - Nicola Alessio
- Department of Experimental Medicine, University of Campania, Naples, Italy
| |
Collapse
|
19
|
Hodge JG, Decker HE, Robinson JL, Mellott AJ. Tissue-mimetic culture enhances mesenchymal stem cell secretome capacity to improve regenerative activity of keratinocytes and fibroblasts in vitro. Wound Repair Regen 2023; 31:367-383. [PMID: 36866522 DOI: 10.1111/wrr.13076] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2022] [Revised: 01/18/2023] [Accepted: 02/21/2023] [Indexed: 03/04/2023]
Abstract
Mesenchymal stem/stromal cells (MSCs) are a heterogenous population of multipotent and highly secretory cells currently being investigated in the field of wound healing for their ability to augment tissue responses. The adaptive response of MSC populations to the rigid substrate of current 2D culture systems has been considered to result in a deterioration of regenerative 'stem-like' properties. In this study, we characterise how the improved culture of adipose-derived mesenchymal stem cells (ASCs) within a tissue-mimetic 3D hydrogel system, that is mechanically similar to native adipose tissue, enhances their regenerative capabilities. Notably, the hydrogel system contains a porous microarchitecture that permits mass transport, enabling efficient collection of secreted cellular compounds. By utilising this 3D system, ASCs retained a significantly higher expression of ASC 'stem-like' markers while demonstrating a significant reduction in senescent populations, relative to 2D. Additionally, culture of ASCs within the 3D system resulted in enhanced secretory activity with significant increases in the secretion of proteinaceous factors, antioxidants and extracellular vesicles (EVs) within the conditioned media (CM) fraction. Lastly, treatment of wound healing cells, keratinocytes (KCs) and fibroblasts (FBs), with ASC-CM from the 2D and 3D systems resulted in augmented functional regenerative activity, with ASC-CM from the 3D system significantly increasing KC and FB metabolic, proliferative and migratory activity. This study demonstrates the potential beneficial role of MSC culture within a tissue-mimetic 3D hydrogel system that more closely mimics native tissue mechanics, and subsequently how the improved phenotype augments secretory activity and potential wound healing capabilities of the MSC secretome.
Collapse
Affiliation(s)
- Jacob G Hodge
- Bioengineering Graduate Program, University of Kansas, Lawrence, Kansas, USA
- Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, Kansas, USA
| | | | - Jennifer L Robinson
- Bioengineering Graduate Program, University of Kansas, Lawrence, Kansas, USA
- Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, Kansas, USA
| | - Adam J Mellott
- Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, Kansas, USA
- Ronawk, LLC, Olathe, Kansas, USA
| |
Collapse
|
20
|
Chiu FY, Kvadas RM, Mheidly Z, Shahbandi A, Jackson JG. Could senescence phenotypes strike the balance to promote tumor dormancy? Cancer Metastasis Rev 2023; 42:143-160. [PMID: 36735097 DOI: 10.1007/s10555-023-10089-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Accepted: 01/23/2023] [Indexed: 02/04/2023]
Abstract
After treatment and surgery, patient tumors can initially respond followed by a rapid relapse, or respond well and seemingly be cured, but then recur years or decades later. The state of surviving cancer cells during the long, undetected period is termed dormancy. By definition, the dormant tumor cells do not proliferate to create a mass that is detectable or symptomatic, but also never die. An intrinsic state and microenvironment that are inhospitable to the tumor would bias toward cell death and complete eradication, while conditions that favor the tumor would enable growth and relapse. In neither case would clinical dormancy be observed. Normal cells and tumor cells can enter a state of cellular senescence after stress such as that caused by cancer therapy. Senescence is characterized by a stable cell cycle arrest mediated by chromatin modifications that cause gene expression changes and a secretory phenotype involving many cytokines and chemokines. Senescent cell phenotypes have been shown to be both tumor promoting and tumor suppressive. The balance of these opposing forces presents an attractive model to explain tumor dormancy: phenotypes of stable arrest and immune suppression could promote survival, while reversible epigenetic programs combined with cytokines and growth factors that promote angiogenesis, survival, and proliferation could initiate the emergence from dormancy. In this review, we examine the phenotypes that have been characterized in different normal and cancer cells made senescent by various stresses and how these might explain the characteristics of tumor dormancy.
Collapse
Affiliation(s)
- Fang-Yen Chiu
- Department of Biochemistry and Molecular Biology, Tulane School of Medicine, 1430 Tulane Avenue, New Orleans, LA, 70112, USA
| | - Raegan M Kvadas
- Department of Biochemistry and Molecular Biology, Tulane School of Medicine, 1430 Tulane Avenue, New Orleans, LA, 70112, USA
| | - Zeinab Mheidly
- Department of Biochemistry and Molecular Biology, Tulane School of Medicine, 1430 Tulane Avenue, New Orleans, LA, 70112, USA
| | - Ashkan Shahbandi
- Department of Biochemistry and Molecular Biology, Tulane School of Medicine, 1430 Tulane Avenue, New Orleans, LA, 70112, USA
| | - James G Jackson
- Department of Biochemistry and Molecular Biology, Tulane School of Medicine, 1430 Tulane Avenue, New Orleans, LA, 70112, USA.
| |
Collapse
|
21
|
Oliinyk D, Eigenberger A, Felthaus O, Haerteis S, Prantl L. Chorioallantoic Membrane Assay at the Cross-Roads of Adipose-Tissue-Derived Stem Cell Research. Cells 2023; 12:cells12040592. [PMID: 36831259 PMCID: PMC9953848 DOI: 10.3390/cells12040592] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2023] [Revised: 02/09/2023] [Accepted: 02/10/2023] [Indexed: 02/15/2023] Open
Abstract
With a history of more than 100 years of different applications in various scientific fields, the chicken chorioallantoic membrane (CAM) assay has proven itself to be an exceptional scientific model that meets the requirements of the replacement, reduction, and refinement principle (3R principle). As one of three extraembryonic avian membranes, the CAM is responsible for fetal respiration, metabolism, and protection. The model provides a unique constellation of immunological, vascular, and extracellular properties while being affordable and reliable at the same time. It can be utilized for research purposes in cancer biology, angiogenesis, virology, and toxicology and has recently been used for biochemistry, pharmaceutical research, and stem cell biology. Stem cells and, in particular, mesenchymal stem cells derived from adipose tissue (ADSCs) are emerging subjects for novel therapeutic strategies in the fields of tissue regeneration and personalized medicine. Because of their easy accessibility, differentiation profile, immunomodulatory properties, and cytokine repertoire, ADSCs have already been established for different preclinical applications in the files mentioned above. In this review, we aim to highlight and identify some of the cross-sections for the potential utilization of the CAM model for ADSC studies with a focus on wound healing and tissue engineering, as well as oncological research, e.g., sarcomas. Hereby, the focus lies on the combination of existing evidence and experience of such intersections with a potential utilization of the CAM model for further research on ADSCs.
Collapse
Affiliation(s)
- Dmytro Oliinyk
- Department of Plastic, Hand and Reconstructive Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
- Correspondence:
| | - Andreas Eigenberger
- Department of Plastic, Hand and Reconstructive Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
| | - Oliver Felthaus
- Department of Plastic, Hand and Reconstructive Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
| | - Silke Haerteis
- Institute for Molecular and Cellular Anatomy, Faculty for Biology and Preclinical Medicine, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany
| | - Lukas Prantl
- Department of Plastic, Hand and Reconstructive Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
| |
Collapse
|
22
|
Poplawski P, Alseekh S, Jankowska U, Skupien-Rabian B, Iwanicka-Nowicka R, Kossowska H, Fogtman A, Rybicka B, Bogusławska J, Adamiok-Ostrowska A, Hanusek K, Hanusek J, Koblowska M, Fernie AR, Piekiełko-Witkowska A. Coordinated reprogramming of renal cancer transcriptome, metabolome and secretome associates with immune tumor infiltration. Cancer Cell Int 2023; 23:2. [PMID: 36604669 PMCID: PMC9814214 DOI: 10.1186/s12935-022-02845-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Accepted: 12/30/2022] [Indexed: 01/06/2023] Open
Abstract
BACKGROUND Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer. The molecules (proteins, metabolites) secreted by tumors affect their extracellular milieu to support cancer progression. If secreted in amounts detectable in plasma, these molecules can also serve as useful, minimal invasive biomarkers. The knowledge of ccRCC tumor microenvironment is fragmentary. In particular, the links between ccRCC transcriptome and the composition of extracellular milieu are weakly understood. In this study, we hypothesized that ccRCC transcriptome is reprogrammed to support alterations in tumor microenvironment. Therefore, we comprehensively analyzed ccRCC extracellular proteomes and metabolomes as well as transcriptomes of ccRCC cells to find molecules contributing to renal tumor microenvironment. METHODS Proteomic and metabolomics analysis of conditioned media isolated from normal kidney cells as well as five ccRCC cell lines was performed using mass spectrometry, with the following ELISA validation. Transcriptomic analysis was done using microarray analysis and validated using real-time PCR. Independent transcriptomic and proteomic datasets of ccRCC tumors were used for the analysis of gene and protein expression as well as the level of the immune infiltration. RESULTS Renal cancer secretome contained 85 proteins detectable in human plasma, consistently altered in all five tested ccRCC cell lines. The top upregulated extracellular proteins included SPARC, STC2, SERPINE1, TGFBI, while downregulated included transferrin and DPP7. The most affected extracellular metabolites were increased 4-hydroxy-proline, succinic acid, cysteine, lactic acid and downregulated glutamine. These changes were associated with altered expression of genes encoding the secreted proteins (SPARC, SERPINE1, STC2, DPP7), membrane transporters (SLC16A4, SLC6A20, ABCA12), and genes involved in protein trafficking and secretion (KIF20A, ANXA3, MIA2, PCSK5, SLC9A3R1, SYTL3, and WNTA7). Analogous expression changes were found in ccRCC tumors. The expression of SPARC predicted the infiltration of ccRCC tumors with endothelial cells. Analysis of the expression of the 85 secretome genes in > 12,000 tumors revealed that SPARC is a PanCancer indicator of cancer-associated fibroblasts' infiltration. CONCLUSIONS Transcriptomic reprogramming of ccRCC supports the changes in an extracellular milieu which are associated with immune infiltration. The proteins identified in our study represent valuable cancer biomarkers detectable in plasma.
Collapse
Affiliation(s)
- Piotr Poplawski
- grid.414852.e0000 0001 2205 7719Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, ul. Marymoncka 99/103, 01-813 Warsaw, Poland
| | - Saleh Alseekh
- grid.418390.70000 0004 0491 976XMax-Planck Institute of Molecular Plant Physiology, Golm, 14476 Potsdam, Germany ,grid.510916.a0000 0004 9334 5103Center for Plant Systems Biology and Biotechnology, 4000 Plovdiv, Bulgaria
| | - Urszula Jankowska
- grid.5522.00000 0001 2162 9631Proteomics and Mass Spectrometry Core Facility, Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Bozena Skupien-Rabian
- grid.5522.00000 0001 2162 9631Proteomics and Mass Spectrometry Core Facility, Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Roksana Iwanicka-Nowicka
- grid.12847.380000 0004 1937 1290Laboratory of Systems Biology, Faculty of Biology, University of Warsaw, 02-106 Warsaw, Poland ,grid.413454.30000 0001 1958 0162Laboratory for Microarray Analysis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
| | - Helena Kossowska
- grid.12847.380000 0004 1937 1290Laboratory of Systems Biology, Faculty of Biology, University of Warsaw, 02-106 Warsaw, Poland
| | - Anna Fogtman
- grid.413454.30000 0001 1958 0162Laboratory for Microarray Analysis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
| | - Beata Rybicka
- grid.414852.e0000 0001 2205 7719Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, ul. Marymoncka 99/103, 01-813 Warsaw, Poland
| | - Joanna Bogusławska
- grid.414852.e0000 0001 2205 7719Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, ul. Marymoncka 99/103, 01-813 Warsaw, Poland
| | - Anna Adamiok-Ostrowska
- grid.414852.e0000 0001 2205 7719Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, ul. Marymoncka 99/103, 01-813 Warsaw, Poland
| | - Karolina Hanusek
- grid.414852.e0000 0001 2205 7719Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, ul. Marymoncka 99/103, 01-813 Warsaw, Poland
| | - Jan Hanusek
- grid.414852.e0000 0001 2205 7719Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, ul. Marymoncka 99/103, 01-813 Warsaw, Poland
| | - Marta Koblowska
- grid.12847.380000 0004 1937 1290Laboratory of Systems Biology, Faculty of Biology, University of Warsaw, 02-106 Warsaw, Poland ,grid.413454.30000 0001 1958 0162Laboratory for Microarray Analysis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
| | - Alisdair R. Fernie
- grid.418390.70000 0004 0491 976XMax-Planck Institute of Molecular Plant Physiology, Golm, 14476 Potsdam, Germany ,grid.510916.a0000 0004 9334 5103Center for Plant Systems Biology and Biotechnology, 4000 Plovdiv, Bulgaria
| | - Agnieszka Piekiełko-Witkowska
- grid.414852.e0000 0001 2205 7719Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, ul. Marymoncka 99/103, 01-813 Warsaw, Poland
| |
Collapse
|
23
|
Hodge JG, Robinson JL, Mellott AJ. Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations. Regen Med 2023; 18:23-36. [PMID: 36222003 PMCID: PMC9732917 DOI: 10.2217/rme-2022-0140] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2022] [Accepted: 09/22/2022] [Indexed: 12/14/2022] Open
Abstract
Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6 weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, 'stem-like' MSC markers, and ability for their secretome to augment a secondary cell population. Results: The 3D hydrogel system resulted in an enhanced retention of more regenerative, nonsenescent ASC populations that exhibited increased expression of 'stem-like' MSC surface markers. Conclusion: This study introduces a proof-of-concept design for a novel modular 3D system that can improve in vitro expansion of stem-like cell populations for future regenerative therapies.
Collapse
Affiliation(s)
- Jacob G Hodge
- Bioengineering Graduate Program, University of Kansas, Lawrence, KS 66045, USA
- Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Jennifer L Robinson
- Bioengineering Graduate Program, University of Kansas, Lawrence, KS 66045, USA
- Department of Chemical & Petroleum Engineering, University of Kansas, Lawrence, KS 66045, USA
| | - Adam J Mellott
- Department of Plastic Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA
- Ronawk, LLC, Olathe, KS 66062, USA
| |
Collapse
|
24
|
Marote A, Santos D, Mendes-Pinheiro B, Serre-Miranda C, Anjo SI, Vieira J, Ferreira-Antunes F, Correia JS, Borges-Pereira C, Pinho AG, Campos J, Manadas B, Teixeira MR, Correia-Neves M, Pinto L, Costa PM, Roybon L, Salgado AJ. Cellular Aging Secretes: a Comparison of Bone-Marrow-Derived and Induced Mesenchymal Stem Cells and Their Secretome Over Long-Term Culture. Stem Cell Rev Rep 2023; 19:248-263. [PMID: 36152233 DOI: 10.1007/s12015-022-10453-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/09/2022] [Indexed: 01/29/2023]
Abstract
Mesenchymal stem cells (MSCs) hold promising therapeutic potential in several clinical applications, mainly due to their paracrine activity. The implementation of future secretome-based therapeutic strategies requires the use of easily accessible MSCs sources that provide high numbers of cells with homogenous characteristics. MSCs obtained from induced pluripotent stem cells (iMSCs) have been put forward as an advantageous alternative to the gold-standard tissue sources, such as bone marrow (BM-MSCs). In this study, we aimed at comparing the secretome of BM-MSCs and iMSCs over long-term culture. For that, we performed a broad characterization of both sources regarding their identity, proteomic secretome analysis, as well as replicative senescence and associated phenotypes, including its effects on MSCs secretome composition and immunomodulatory action. Our results evidence a rejuvenated phenotype of iMSCs, which is translated into a superior proliferative capacity before the induction of replicative senescence. Despite this significant difference between iMSCs and BM-MSCs proliferation, both untargeted and targeted proteomic analysis revealed a similar secretome composition for both sources in pre-senescent and senescent states. These results suggest that shifting from the use of BM-MSCs to a more advantageous source, like iMSCs, may yield similar therapeutic effects as identified over the past years for this gold-standard MSC source.
Collapse
Affiliation(s)
- Ana Marote
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal. .,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal.
| | - Diogo Santos
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Bárbara Mendes-Pinheiro
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Cláudia Serre-Miranda
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Sandra I Anjo
- CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.,Multidisciplinary Institute of Ageing (MIA), University of Coimbra, Coimbra, Portugal
| | - Joana Vieira
- Department of Laboratory Genetics, Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center, Porto, Portugal.,Cancer Genetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (HealthResearch Network), Portuguese Oncology Institute of Porto (IPO Porto) / PortoComprehensive Cancer Center, Porto, Portugal
| | - Filipa Ferreira-Antunes
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Joana Sofia Correia
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Caroline Borges-Pereira
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Andreia G Pinho
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Jonas Campos
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Bruno Manadas
- CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
| | - Manuel R Teixeira
- Department of Laboratory Genetics, Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center, Porto, Portugal.,Cancer Genetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (HealthResearch Network), Portuguese Oncology Institute of Porto (IPO Porto) / PortoComprehensive Cancer Center, Porto, Portugal.,School of Medicine and Biomedical Sciences (ICBAS), University of Porto, Porto, Portugal
| | - Margarida Correia-Neves
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Luísa Pinto
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal.,Behavioral and Molecular Lab (Bn'ML), University of Minho, Braga, Portugal
| | - Pedro M Costa
- Stemmatters, Biotecnologia e Medicina Regenerativa S.A., Guimarães, Portugal
| | - Laurent Roybon
- Stem Cell Laboratory for CNS Disease Modeling, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, BMC A10, Lund, Sweden.,Strategic Research Area MultiPark, Lund University, Lund, Sweden.,Lund Stem Cell Center, Lund University, Lund, Sweden
| | - António J Salgado
- Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057, Braga, Portugal.,ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal
| |
Collapse
|
25
|
Urrata V, Trapani M, Franza M, Moschella F, Di Stefano AB, Toia F. Analysis of MSCs' secretome and EVs cargo: Evaluation of functions and applications. Life Sci 2022; 308:120990. [PMID: 36155182 DOI: 10.1016/j.lfs.2022.120990] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Revised: 09/12/2022] [Accepted: 09/20/2022] [Indexed: 11/25/2022]
Affiliation(s)
- Valentina Urrata
- BIOPLAST-Laboratory of BIOlogy and Regenerative Medicine-PLASTic Surgery, Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, 90127 Palermo, Italy
| | - Marco Trapani
- BIOPLAST-Laboratory of BIOlogy and Regenerative Medicine-PLASTic Surgery, Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, 90127 Palermo, Italy; Plastic and Reconstructive Surgery, Department of Oncology, Azienda Ospedaliera Universitaria Policlinico "Paolo Giaccone", 90127 Palermo, Italy
| | - Mara Franza
- Plastic and Reconstructive Surgery, Department of Oncology, Azienda Ospedaliera Universitaria Policlinico "Paolo Giaccone", 90127 Palermo, Italy; Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, 90127 Palermo, Italy
| | - Francesco Moschella
- BIOPLAST-Laboratory of BIOlogy and Regenerative Medicine-PLASTic Surgery, Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, 90127 Palermo, Italy
| | - Anna Barbara Di Stefano
- BIOPLAST-Laboratory of BIOlogy and Regenerative Medicine-PLASTic Surgery, Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, 90127 Palermo, Italy.
| | - Francesca Toia
- BIOPLAST-Laboratory of BIOlogy and Regenerative Medicine-PLASTic Surgery, Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, 90127 Palermo, Italy; Plastic and Reconstructive Surgery, Department of Oncology, Azienda Ospedaliera Universitaria Policlinico "Paolo Giaccone", 90127 Palermo, Italy; Plastic and Reconstructive Surgery, Department of Surgical, Oncological and Oral Sciences, University of Palermo, 90127 Palermo, Italy
| |
Collapse
|
26
|
Gresham RC, Kumar D, Copp J, Lee MA, Leach JK. Characterization of Induction and Targeting of Senescent Mesenchymal Stromal Cells. Tissue Eng Part C Methods 2022; 28:239-249. [PMID: 35438548 PMCID: PMC9247679 DOI: 10.1089/ten.tec.2022.0048] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Mesenchymal stromal cells (MSCs) from older donors have limited potential for bone tissue formation compared with cells from younger donors, and cellular senescence has been postulated as an underlying cause. There is a critical need for methods to induce premature senescence to study this phenomenon efficiently and reproducibly. However, the field lacks consensus on the appropriate method to induce and characterize senescence. Moreover, we have a limited understanding of the effects of commonly used induction methods on senescent phenotype. To address this significant challenge, we assessed the effect of replicative, hydrogen peroxide, etoposide, and irradiation-induced senescence on human MSCs using a battery of senescent cell characteristics. All methods arrested proliferation and resulted in increased cell spreading compared with low passage controls. Etoposide and irradiation increased expression of senescence-related genes in MSCs at early time points, proinflammatory cytokine secretion, DNA damage, and production of senescence-associated β-galactosidase. We then evaluated the effect of fisetin, a flavonoid and candidate senolytic agent, to clear senescent cells and promote osteogenic differentiation of MSCs entrapped in gelatin methacryloyl (GelMA) hydrogels in vitro. When studying a mixture of nonsenescent and senescent MSCs, we did not observe decreases in senescent markers or increases in osteogenesis with fisetin treatment. However, the application of the same treatment toward a heterogeneous population of human bone marrow-derived cells entrapped in GelMA decreased senescent markers and increased osteogenesis after 14 days in culture. These results identify best practices for inducing prematurely senescent MSCs and motivate the need for further study of fisetin as a senolytic agent. Impact Statement The accumulation of senescent cells within the body has detrimental effects on tissue homeostasis. To study the role of senescent cells on tissue repair and regeneration, there is a need for effective means to induce premature cell senescence. Herein, we characterized the influence of common stressors to induce premature senescence in human mesenchymal stromal cells (MSCs). Irradiation of MSCs resulted in a phenotype most similar to quiescent, high-passage cells. These studies establish key biomarkers for evaluation when studying senescent cells in vitro.
Collapse
Affiliation(s)
- Robert C.H. Gresham
- Department of Orthopedic Surgery, School of Medicine, University of California Davis Health, Sacramento, California, USA
| | - Devanshi Kumar
- Department of Biomedical Engineering, University of California, Davis, Davis, California, USA
| | - Jonathan Copp
- Department of Orthopedic Surgery, School of Medicine, University of California Davis Health, Sacramento, California, USA.,Department of Orthopedic Trauma Surgery, Forrest General Hospital, Hattiesburg, Mississippi, USA
| | - Mark A. Lee
- Department of Orthopedic Surgery, School of Medicine, University of California Davis Health, Sacramento, California, USA
| | - J. Kent Leach
- Department of Orthopedic Surgery, School of Medicine, University of California Davis Health, Sacramento, California, USA.,Department of Biomedical Engineering, University of California, Davis, Davis, California, USA.,Address correspondence to: J. Kent Leach, PhD, Department of Orthopaedic Surgery, School of Medicine, University of California, Davis Health, 4800 Y Street, Suite 3600, Sacramento, CA 95817, USA
| |
Collapse
|
27
|
Kawagishi-Hotta M, Hasegawa S, Hasebe Y, Inoue Y, Okuno R, Arima M, Iwata Y, Sugiura K, Akamatsu H. Increase in Inhibin beta A/Activin-A expression in the human epidermis and the suppression of epidermal stem/progenitor cell proliferation with aging. J Dermatol Sci 2022; 106:150-158. [DOI: 10.1016/j.jdermsci.2022.05.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Revised: 03/21/2022] [Accepted: 05/05/2022] [Indexed: 11/25/2022]
|
28
|
Human Adipose-Derived Stem Cell-Conditioned Medium Promotes Vascularization of Nanostructured Scaffold Transplanted into Nude Mice. NANOMATERIALS 2022; 12:nano12091521. [PMID: 35564230 PMCID: PMC9100239 DOI: 10.3390/nano12091521] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 04/26/2022] [Accepted: 04/27/2022] [Indexed: 12/10/2022]
Abstract
Several studies have been conducted on the interaction between three-dimensional scaffolds and mesenchymal stem cells for the regeneration of damaged tissues. Considering that stem cells do not survive for sufficient time to directly sustain tissue regeneration, it is essential to develop cell-free systems to be applied in regenerative medicine. In this work, by in vivo experiments, we established that a collagen-nanostructured scaffold, loaded with a culture medium conditioned with mesenchymal stem cells derived from adipose tissue (hASC-CM), exerts a synergic positive effect on angiogenesis, fundamental in tissue regeneration. To this aim, we engrafted athymic BALB-C nude mice with four different combinations: scaffold alone; scaffold with hASCs; scaffold with hASC crude protein extract; scaffold with hASC-CM. After their removal, we verified the presence of blood vessels by optical microscopy and confirmed the vascularization evaluating, by real-time PCR, several vascular growth factors: CD31, CD34, CD105, ANGPT1, ANGPT2, and CDH5. Our results showed that blood vessels were absent in the scaffold grafted alone, while all the other systems appeared vascularized, a finding supported by the over-expression of CD31 and CDH5 mRNA. In conclusion, our data sustain the capability of hASC-CM to be used as a therapeutic cell-free approach for damaged tissue regeneration.
Collapse
|
29
|
Sanmartin MC, Borzone FR, Giorello MB, Yannarelli G, Chasseing NA. Mesenchymal Stromal Cell-Derived Extracellular Vesicles as Biological Carriers for Drug Delivery in Cancer Therapy. Front Bioeng Biotechnol 2022; 10:882545. [PMID: 35497332 PMCID: PMC9046597 DOI: 10.3389/fbioe.2022.882545] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Accepted: 03/25/2022] [Indexed: 12/11/2022] Open
Abstract
Cancer is the second leading cause of death worldwide, with 10.0 million cancer deaths in 2020. Despite advances in targeted therapies, some pharmacological drawbacks associated with anticancer chemo and immunotherapeutic agents include high toxicities, low bioavailability, and drug resistance. In recent years, extracellular vesicles emerged as a new promising platform for drug delivery, with the advantage of their inherent biocompatibility and specific targeting compared to artificial nanocarriers, such as liposomes. Particularly, mesenchymal stem/stromal cells were proposed as a source of extracellular vesicles for cancer therapy because of their intrinsic properties: high in vitro self-renewal and proliferation, regenerative and immunomodulatory capacities, and secretion of extracellular vesicles that mediate most of their paracrine functions. Moreover, extracellular vesicles are static and safer in comparison with mesenchymal stem/stromal cells, which can undergo genetic/epigenetic or phenotypic changes after their administration to patients. In this review, we summarize currently reported information regarding mesenchymal stem/stromal cell-derived extracellular vesicles, their proper isolation and purification techniques - from either naive or engineered mesenchymal stem/stromal cells - for their application in cancer therapy, as well as available downstream modification methods to improve their therapeutic properties. Additionally, we discuss the challenges associated with extracellular vesicles for cancer therapy, and we review some preclinical and clinical data available in the literature.
Collapse
Affiliation(s)
- María Cecilia Sanmartin
- Laboratorio de Inmunohematología, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMeTTyB), Universidad Favaloro - CONICET, Buenos Aires, Argentina
| | - Francisco Raúl Borzone
- Laboratorio de Inmunohematología, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - María Belén Giorello
- Laboratorio de Inmunohematología, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Gustavo Yannarelli
- Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMeTTyB), Universidad Favaloro - CONICET, Buenos Aires, Argentina
| | - Norma Alejandra Chasseing
- Laboratorio de Inmunohematología, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| |
Collapse
|
30
|
Suzdaltseva Y, Goryunov K, Silina E, Manturova N, Stupin V, Kiselev SL. Equilibrium among Inflammatory Factors Determines Human MSC-Mediated Immunosuppressive Effect. Cells 2022; 11:1210. [PMID: 35406773 PMCID: PMC8997511 DOI: 10.3390/cells11071210] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Revised: 03/29/2022] [Accepted: 04/02/2022] [Indexed: 02/07/2023] Open
Abstract
Mesenchymal stem cells (MSCs) are thought to be a promising therapeutic agent due to their multiple paracrine and immunomodulatory properties, providing protection from chronic inflammation and promoting tissue repair. MSCs can regulate the balance of pro-inflammatory and anti-inflammatory factors in inflamed tissues, creating a microenvironment necessary for successful healing; however, their interactions with immune cells are still poorly studied. We examined the temporal and spatial changes in gene regulation and the paracrine milieu accompanying the MSC-mediated immunosuppression effect in mixed cultures with activated peripheral blood mononuclear cells (PBMCs). Our data reveal that the peak of suppression of PBMC proliferation was achieved within 48 h following co-culture with MSCs and subsequently did not undergo a significant change. This effect was accompanied by an increase in COX-2 expression and an induction of IDO synthesis in MSCs. At this point, the expression of IL-1, IL-6, IL-8, IFN-γ, MCP-1, and G-CSF was upregulated in co-cultured cells. On the contrary, we observed a decrease in the concentrations of IL-10, IL-13, IL-5, and MIP-1b in co-culture supernatants compared to intact cultures of activated PBMCs. The regulation of IDO, IL-1, IL-6, and G-CSF production was accomplished with the involvement of direct cell-cell contact between MSCs and PBMCs. These findings provide new insights into the use of potential precondition inducers or their combinations to obtain functionally qualified MSCs for more effective treatment of inflammatory diseases.
Collapse
Affiliation(s)
- Yulia Suzdaltseva
- Department of Epigenetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, 119333 Moscow, Russia;
| | - Kirill Goryunov
- Department of Cell Technologies, National Medical Research Center for Obstetrics, Gynecology and Perinatology, 117997 Moscow, Russia;
| | - Ekaterina Silina
- Institute of Biodesign and Modeling of Complex Systems, I.M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia;
| | - Natalia Manturova
- Department of Plastic and Reconstructive surgery, Cosmetology and Cell Technologies, Pirogov Russian National Research Medical University, 117997 Moscow, Russia;
| | - Victor Stupin
- Department of Hospital Surgery, Pirogov Russian National Research Medical University, 117997 Moscow, Russia;
| | - Sergey L. Kiselev
- Department of Epigenetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, 119333 Moscow, Russia;
| |
Collapse
|
31
|
Tan KX, Chang T, Lin XL. Secretomes as an emerging class of bioactive ingredients for enhanced cosmeceutical applications. Exp Dermatol 2022; 31:674-688. [PMID: 35338666 DOI: 10.1111/exd.14570] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Revised: 02/23/2022] [Accepted: 03/22/2022] [Indexed: 11/30/2022]
Abstract
Skin aging is predominantly caused by either intrinsic or extrinsic factors, leading to undesirable skin features. Advancements in both molecular and cellular fields have created possibilities in developing novel stem cell-derived active ingredients for cosmeceutical applications and the beauty industry. Mesenchymal stromal cell (MSC)-derived secretomes or conditioned media hold great promise for advancing skin repair and regeneration due to the presence of varying cytokines. These cytokines signal our cells and trigger biological mechanisms associated with anti-inflammatory, antioxidant, anti-aging, proliferative, and immunomodulatory effects. In this review, we discuss the potential of MSC secretomes as novel biomaterials for skincare and rejuvenation by illustrating their mechanism of action related to wound healing, anti-aging, and whitening properties. The advantages and disadvantages of secretomes are compared to both plant-based and animal-derived extracts. In addition, this paper reviews the current safety standards, regulations, market products and research work related to the cosmeceutical applications of secretomes along with strategies to maintain and improve the therapeutic efficacy and production of secretomes. The future outlook of beauty industry is also presented. Lastly, we highlight significant challenges to be addressed for the clinical realization of MSC secretomes-based skin therapies as well as providing perspectives for the future direction of secretomes.
Collapse
Affiliation(s)
- Kei-Xian Tan
- Esco Aster, Block 67, Ayer Rajah Crescent, 139950, Singapore
| | - Trixie Chang
- Esco Aster, Block 67, Ayer Rajah Crescent, 139950, Singapore
| | - Xiang-Liang Lin
- Esco Aster, Block 67, Ayer Rajah Crescent, 139950, Singapore
| |
Collapse
|
32
|
Spinelli R, Florese P, Parrillo L, Zatterale F, Longo M, D’Esposito V, Desiderio A, Nerstedt A, Gustafson B, Formisano P, Miele C, Raciti GA, Napoli R, Smith U, Beguinot F. ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first-degree relatives of type 2 diabetics. Aging Cell 2022; 21:e13557. [PMID: 35146866 PMCID: PMC8920444 DOI: 10.1111/acel.13557] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 12/16/2021] [Accepted: 01/09/2022] [Indexed: 12/18/2022] Open
Abstract
Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age-related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First-degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top-ranked senescence-related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3-overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.
Collapse
Affiliation(s)
- Rosa Spinelli
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Pasqualina Florese
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Luca Parrillo
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Federica Zatterale
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Michele Longo
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Vittoria D’Esposito
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Antonella Desiderio
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Annika Nerstedt
- Lundberg Laboratory for Diabetes ResearchDepartment of Molecular and Clinical MedicineSahlgrenska AcademyUniversity of GothenburgGothenburgSweden
| | - Birgit Gustafson
- Lundberg Laboratory for Diabetes ResearchDepartment of Molecular and Clinical MedicineSahlgrenska AcademyUniversity of GothenburgGothenburgSweden
| | - Pietro Formisano
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Claudia Miele
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Gregory Alexander Raciti
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| | - Raffaele Napoli
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
| | - Ulf Smith
- Lundberg Laboratory for Diabetes ResearchDepartment of Molecular and Clinical MedicineSahlgrenska AcademyUniversity of GothenburgGothenburgSweden
| | - Francesco Beguinot
- Department of Translational Medical SciencesFederico II University of NaplesNaplesItaly
- URT Genomics of DiabetesInstitute of Experimental Endocrinology and OncologyNational Research CouncilNaplesItaly
| |
Collapse
|
33
|
Krawczenko A, Klimczak A. Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells and Their Contribution to Angiogenic Processes in Tissue Regeneration. Int J Mol Sci 2022; 23:ijms23052425. [PMID: 35269568 PMCID: PMC8910401 DOI: 10.3390/ijms23052425] [Citation(s) in RCA: 45] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 02/17/2022] [Accepted: 02/21/2022] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem/stromal cells (MSCs) are widely described in the context of their regenerative and immunomodulatory activity. MSCs are isolated from various tissues and organs. The most frequently described sources are bone marrow and adipose tissue. As stem cells, MSCs are able to differentiate into other cell lineages, but they are usually reported with respect to their paracrine potential. In this review, we focus on MSCs derived from adipose tissue (AT-MSCs) and their secretome in regeneration processes. Special attention is given to the contribution of AT-MSCs and their derivatives to angiogenic processes described mainly in the context of angiogenic dysfunction. Finally, we present clinical trials registered to date that concern the application of AT-MSCs and their secretome in various medical conditions.
Collapse
|
34
|
Yang K, Xie D, Lin W, Xiang P, Peng C. Adipose mesenchymal stem cells and gingival mesenchymal stem cells have a comparable effect in endothelium repair. Exp Ther Med 2021; 22:1415. [PMID: 34676008 PMCID: PMC8524764 DOI: 10.3892/etm.2021.10851] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2020] [Accepted: 07/14/2021] [Indexed: 12/14/2022] Open
Abstract
Restenosis is the major factor influencing the long-term success rate of angioplasty and stent implantation and effective strategies to prevent restenosis remain limited. Mesenchymal stem cells (MSCs) are pluripotent stem cells capable of self-renewal and multidirectional differentiation, which may be able to promote endothelium repair, thereby reducing restenosis. The present study aimed to evaluate the effects of adipose MSCs (AMSCs) and gingival MSCs (GMSCs) on endothelium repair. MSCs were isolated from two human tissue types, namely adipose tissue and gingival tissue, and the effects of AMSCs and GMSCs in ex vivo endothelium repair and on vascular smooth muscle cell (SMC) growth were examined. To compare the feasibility of using AMSCs and GMSCs for the repair of endothelium damage in endothelial cell (EC) damage and vasoproliferative disorders, an ex vivo model of endothelium repair in a co-culture system was developed. It was indicated that AMSCs and GMSCs expressed characteristic MSC markers (CD105 and CD166). 3H-thymidine incorporation in the co-culture group of AMSCs and SMCs in the presence of ECs was lower compared with that in the GMSC and SMC co-culture group. The protein expression level of proliferating cell nuclear antigen in the co-culture group of AMSCs and SMCs in the presence of ECs were lower compared with that in the GMSC and SMC co-culture group. After co-culture with ECs for 5 days, 25.71±3.08% of AMSCs began to express CD31 protein and 20.06±2.09% of GMSCs began to express CD31 protein. Furthermore, anti-VEGF antibody was able to inhibit MSC differentiation. Collectively, the present results suggested that seeding of AMSCs had a stronger effect to inhibit the proliferation and migration of SMCs compared with GMSCs.
Collapse
Affiliation(s)
- Ke Yang
- Department of Cardiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, P.R. China
| | - Dongmei Xie
- Department of Cardiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, P.R. China
| | - Wanwen Lin
- Department of Cardiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, P.R. China
| | - Peng Xiang
- Key Laboratory for Stem Cells and Tissue Engineering, Center for Stem Cell Biology and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong 510600, P.R. China
| | - Chaoquan Peng
- Department of Cardiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, P.R. China
| |
Collapse
|
35
|
TNF-α and IFN-γ Participate in Improving the Immunoregulatory Capacity of Mesenchymal Stem/Stromal Cells: Importance of Cell-Cell Contact and Extracellular Vesicles. Int J Mol Sci 2021; 22:ijms22179531. [PMID: 34502453 PMCID: PMC8431422 DOI: 10.3390/ijms22179531] [Citation(s) in RCA: 73] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Revised: 08/10/2021] [Accepted: 08/26/2021] [Indexed: 12/14/2022] Open
Abstract
Mesenchymal stem/stromal cells (MSCs) have an immunoregulatory capacity and have been used in different clinical protocols requiring control of the immune response. However, variable results have been obtained, mainly due to the effect of the microenvironment on the induction, increase, and maintenance of MSC immunoregulatory mechanisms. In addition, the importance of cell–cell contact for MSCs to efficiently modulate the immune response has recently been highlighted. Because these interactions would be difficult to achieve in the physiological context, the release of extracellular vesicles (EVs) and their participation as intermediaries of communication between MSCs and immune cells becomes relevant. Therefore, this article focuses on analyzing immunoregulatory mechanisms mediated by cell contact, highlighting the importance of intercellular adhesion molecule-1 (ICAM-1) and the participation of EVs. Moreover, the effects of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), the main cytokines involved in MSC activation, are examined. These cytokines, when used at the appropriate concentrations and times, would promote increases in the expression of immunoregulatory molecules in the cell and allow the acquisition of EVs enriched with these molecules. The establishment of certain in vitro activation guidelines will facilitate the design of conditioning protocols to obtain functional MSCs or EVs in different pathophysiological conditions.
Collapse
|
36
|
Protective Effect of Adipose-Derived Mesenchymal Stem Cell Secretome against Hepatocyte Apoptosis Induced by Liver Ischemia-Reperfusion with Partial Hepatectomy Injury. Stem Cells Int 2021; 2021:9969372. [PMID: 34457008 PMCID: PMC8390152 DOI: 10.1155/2021/9969372] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2021] [Revised: 07/07/2021] [Accepted: 07/29/2021] [Indexed: 12/12/2022] Open
Abstract
Ischemia-reperfusion injury (IRI) is an inevitable complication of liver surgery and liver transplantation. Hepatocyte apoptosis plays a significant role in the pathological process of hepatic IRI. Adipose-derived stem cells (ADSCs) are known to repair and regenerate damaged tissues by producing bioactive factors, including cytokines, exosomes, and extracellular matrix components, which collectively form the secretome of these cells. The aim of this study was to assess the protective effects of the ADSCs secretome after liver ischemia-reperfusion combined with partial hepatectomy in miniature pigs. We successfully established laparoscopic liver ischemia-reperfusion with partial hepatectomy in miniature pigs and injected saline, DMEM, ADSC-secretome, and ADSCs directly into the liver parenchyma immediately afterwards. Both ADSCs and the ADSC-secretome improved the IR-induced ultrastructural changes in hepatocytes and significantly decreased the proportion of TUNEL-positive apoptotic cells along with caspase activity. Consistent with this, P53, Bax, Fas, and Fasl mRNA and protein levels were markedly decreased, while Bcl-2 was significantly increased in the animals treated with ADSCs and ADSC-secretome. Our findings indicate that ADSCs exert therapeutic effects in a paracrine manner through their secretome, which can be a viable alternative to cell-based regenerative therapies.
Collapse
|
37
|
Local and Remote Effects of Mesenchymal Stem Cell Administration on Skin Wound Regeneration. PATHOPHYSIOLOGY 2021; 28:355-372. [PMID: 35366280 PMCID: PMC8830469 DOI: 10.3390/pathophysiology28030024] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2021] [Revised: 08/10/2021] [Accepted: 08/10/2021] [Indexed: 12/14/2022] Open
Abstract
Wound healing is an important medical problem. We evaluated the efficacy of locally administered mesenchymal stem cells (MSCs) isolated from human umbilical cords on the dynamics of skin wound healing. The study was conducted on the backs of Wistar rats, where two square wounds were created by removing all layers of the skin. Four groups were studied in two series of experiments: (1) a Control_NaCl group (the wounds were injected with 0.9% NaCl solution) and a Control_0 group (intact wounds on the opposite side of the same rat's back); (2) an MSC group (injected MSCs, local effect) and a Control_sc group (intact wounds on the opposite side of the back, remote MSC effect). The area and temperature of the wounds and the microcirculation of the wound edges were measured. Histological and morphometric studies were performed on days 3 and 7 after the wounds were created. The results showed that the injection trauma (Control_NaCl) slowed the regeneration process. In both MSC groups (unlike in either control group), we observed no increase in the area of the wounds; in addition, we observed inhibition of the inflammatory process and improved wound regeneration on days 1-3 in the remote group and days 1-5 in the local (injected) group. The MSC and Control_sc groups demonstrated improved microcirculation and suppression of leukocyte infiltration on day 3. On day 7, all the studied parameters of the wounds of the Control_0 group were the same as those of the wounds that received cell therapy, although in contrast to the results of the Control_ NaCl group, fibroblast proliferation was greater in the MSC and Control_sc groups. The dynamics of the size of the wounds were comparable for both local and remote application of MSCs. Thus, even a one-time application of MSCs was effective during the first 3-5 days after injury due to anti-inflammatory processes, which improved the regeneration process. Remote application of MSC, as opposed to direct injection, is advisable, especially in the case of multiple wounds, since the results were indistinguishable between the groups and injection trauma was shown to slow healing.
Collapse
|
38
|
Human Adipose-Derived Stem/Stromal Cells Promote Proliferation and Migration in Head and Neck Cancer Cells. Cancers (Basel) 2021; 13:cancers13112751. [PMID: 34206064 PMCID: PMC8199568 DOI: 10.3390/cancers13112751] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2021] [Revised: 05/25/2021] [Accepted: 05/27/2021] [Indexed: 11/17/2022] Open
Abstract
Simple Summary Fat grafts obtained from a minimal invasive liposuction device contain multipotent stem cells termed adipose-derived stem/stromal cells (ASCs). ASCs can be used for their proposed wound healing relevant characteristics, including for tissue defects in cancer patients. For head and neck cancers, little is known about the effects of ASCs on tumor cells. Using supernatants of ASCs from five patients in different functional experiments, this study aimed to investigate how ASCs influence tumor growth, invasive properties, and neoangiogenesis. The data show that all mentioned characteristics are promoted by fat graft stem cells in vitro in head and neck cancer cell lines. Although clinical relevance of these in vitro findings is unclear, due to the lack of in vivo and clinical data, fat grafts should be used cautiously and complete removal of tumor should be ensured before augmentation in head and neck cancer patients is performed. Abstract Human adipose-derived stem/stromal cells (ASCs) are increasingly used as auto-transplants in regenerative medicine to restore tissue defects or induce wound healing, especially in cancer patients. The impact of ASCs on squamous cell carcinoma of the upper aerodigestive tract (UAT) including head and neck and esophageal squamous cell carcinoma (HNSCC and ESCC) is not yet fully understood. ASCs were cultured from subcutaneous, abdominal lipoaspirates of five patients, who received auto-transplants to the head and neck. Supernatants were tested for paracrine effects in functional in vitro assays of proliferation of HNSCC tumor cell line FaDu and ESCC cell line Kyse30, and their cell migration/invasion capacities in Boyden chambers, in addition to endothelial tube formation assay using human umbilical vein endothelial cells (HUVECs). All ASC-derived supernatants enhanced proliferation of FaDu cells, invasive migration, and tube formation by HUVECs, compared to controls. Of five patients’ lipoaspirates, ASC-derived supernatants of four patients increased proliferation and invasive migration in Kyse30 cells. The data suggests that ASCs can promote tumor cell proliferation, invasiveness, and neo-angiogenesis in these tumor cell lines of the UAT and HUVEC in a paracrine manner. Although clinical studies on the subject of oncological safety are still needed, these findings emphasize the importance of complete tumor removal before ASCs are used in the head and neck.
Collapse
|
39
|
Ghodrat S, Hoseini SJ, Asadpour S, Nazarnezhad S, Alizadeh Eghtedar F, Kargozar S. Stem cell-based therapies for cardiac diseases: The critical role of angiogenic exosomes. Biofactors 2021; 47:270-291. [PMID: 33606893 DOI: 10.1002/biof.1717] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2020] [Accepted: 01/25/2021] [Indexed: 12/26/2022]
Abstract
Finding effective treatments for cardiac diseases is among the hottest subjects in medicine; cell-based therapies have brought great promises for managing a broad range of life-threatening heart complications such as myocardial infarction. After clarifying the critical role of angiogenesis in tissue repair and regeneration, various stem/progenitor cell were utilized to accelerate the healing of injured cardiac tissue. Embryonic, fetal, adult, and induced pluripotent stem cells have shown the appropriate proangiogenic potential for tissue repair strategies. The capability of stem cells for differentiating into endothelial lineages was initially introduced as the primary mechanism involved in improving angiogenesis and accelerated heart tissue repair. However, recent studies have demonstrated the leading role of paracrine factors secreted by stem cells in advancing neo-vessel formation. Genetically modified stem cells are also being applied for promoting angiogenesis regarding their ability to considerably overexpress and secrete angiogenic bioactive molecules. Yet, conducting further research seems necessary to precisely identify molecular mechanisms behind the proangiogenic potential of stem cells, including the signaling pathways and regulatory molecules such as microRNAs. In conclusion, stem cells' pivotal roles in promoting angiogenesis and consequent improved cardiac healing and remodeling processes should not be ignored, especially in the case of stem cell-derived extracellular vesicles.
Collapse
Affiliation(s)
- Sara Ghodrat
- Department of Nutrition, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Seyed Javad Hoseini
- Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Shiva Asadpour
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies, Shahrekord University of Medical Sciences, Shahrekord, Iran
| | - Simin Nazarnezhad
- Tissue Engineering Research Group (TERG), Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Fariba Alizadeh Eghtedar
- Tissue Engineering Research Group (TERG), Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Saeid Kargozar
- Tissue Engineering Research Group (TERG), Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| |
Collapse
|
40
|
Kundeková B, Máčajová M, Meta M, Čavarga I, Bilčík B. Chorioallantoic Membrane Models of Various Avian Species: Differences and Applications. BIOLOGY 2021; 10:biology10040301. [PMID: 33917385 PMCID: PMC8067367 DOI: 10.3390/biology10040301] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Revised: 03/31/2021] [Accepted: 04/01/2021] [Indexed: 12/11/2022]
Abstract
The chorioallantoic membrane model (CAM) of an avian embryo is used as an experimental model in various fields of research, including angiogenesis research and drug testing, xenografting and cancer research, and other scientific and commercial disciplines in microbiology, biochemistry, cosmetics, etc. It is a low-cost, low-maintenance, and well-available in vivo animal model that is non-sentient and can be used as an alternative for other mammal experimental models. It respects the principles of the "3R" rule (Replacement, Reduction, and Refinement)-conditions set out for scientific community providing an essential framework for conducting a more human animal research, which is also in line with constantly raising public awareness of welfare and the ethics related to the use of animal experimental models. In this review, we describe the chorioallantoic membrane of an avian embryo, focusing on its properties and development, its advantages and disadvantages as an experimental model, and the possibilities of its application in various fields of biological research. Since the most common chicken CAM model is already well known and described in many publications, we are particularly focusing on the advantages and application of less known avian species that are used for the CAM model-quail, turkey, and duck.
Collapse
Affiliation(s)
- Barbora Kundeková
- Institute of Animal Biochemistry and Genetics, CBs SAS, 840 05 Bratislava, Slovakia; (M.M.); (M.M.); (I.Č.); (B.B.)
- Correspondence:
| | - Mariana Máčajová
- Institute of Animal Biochemistry and Genetics, CBs SAS, 840 05 Bratislava, Slovakia; (M.M.); (M.M.); (I.Č.); (B.B.)
| | - Majlinda Meta
- Institute of Animal Biochemistry and Genetics, CBs SAS, 840 05 Bratislava, Slovakia; (M.M.); (M.M.); (I.Č.); (B.B.)
| | - Ivan Čavarga
- Institute of Animal Biochemistry and Genetics, CBs SAS, 840 05 Bratislava, Slovakia; (M.M.); (M.M.); (I.Č.); (B.B.)
- St. Elizabeth Cancer Institute, 812 50 Bratislava, Slovakia
| | - Boris Bilčík
- Institute of Animal Biochemistry and Genetics, CBs SAS, 840 05 Bratislava, Slovakia; (M.M.); (M.M.); (I.Č.); (B.B.)
| |
Collapse
|
41
|
Liu K, Gao R, Wu H, Wang Z, Han G. Single-cell analysis reveals metastatic cell heterogeneity in clear cell renal cell carcinoma. J Cell Mol Med 2021; 25:4260-4274. [PMID: 33759378 PMCID: PMC8093989 DOI: 10.1111/jcmm.16479] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2020] [Revised: 02/20/2021] [Accepted: 02/25/2021] [Indexed: 12/12/2022] Open
Abstract
Renal cell carcinoma (RCC) is one of the leading causes of cancer-related death worldwide. Tumour metastasis and heterogeneity lead to poor survival outcomes and drug resistance in patients with metastatic RCC (mRCC). In this study, we aimed to assess intratumoural heterogeneity (ITH) in mRCC cells by performing a combined analysis of bulk data and single-cell RNA-sequencing data, and develop novel biomarkers for prognosis prediction on the basis of the potential molecular mechanisms underlying tumorigenesis. Eligible single-cell cohorts related to mRCC were acquired using the Gene Expression Omnibus (GEO) dataset to identify potential mRCC subpopulations. We then performed gene set variation analysis to understand the differential function in primary RCC and mRCC samples. Subsequently, we applied weighted correlation network analysis to identify coexpressing gene modules that were related to the external trait of metastasis. Protein-protein interactions were used to screen hub subpopulation-difference (sub-dif) markers (ACTG1, IL6, CASP3, ACTB and RAP1B) that might be involved in the regulation of RCC metastasis and progression. Cox regression analysis revealed that ACTG1 was a protective factor (HR < 1), whereas the other four genes (IL6, CASP3, ACTB and RAP1B) were risk factors (HR > 1). Kaplan-Meier survival analysis suggested the potential prognostic value of these sub-dif markers. The expression of sub-dif markers in mRCC was further evaluated in clinical samples by immunohistochemistry (IHC). Additionally, the genetic features of sub-dif marker expression patterns, such as genetic variation profiles, correlations with tumour-infiltrating lymphocytes (TILs), and targeted signalling pathway activities, were assessed in bulk RNA-seq datasets. In conclusion, we established novel subpopulation markers as key prognostic factors affecting EMT-related signalling pathway activation in mRCC, which could facilitate the implementation of a treatment for mRCC patients.
Collapse
Affiliation(s)
- Kun Liu
- Department of Anesthesiology, Shengjing Hospital of China Medical University, Shenyang, China
| | - Rui Gao
- Department of Anesthesiology, Shengjing Hospital of China Medical University, Shenyang, China
| | - Hao Wu
- Department of Anesthesiology, Shengjing Hospital of China Medical University, Shenyang, China
| | - Zhe Wang
- Department of Gastrointestinal Oncology, Cancer Hospital of China Medical University Liaoning Cancer Hospital & Institute, Shenyang, China
| | - Guang Han
- Department of Anesthesiology, Shengjing Hospital of China Medical University, Shenyang, China
| |
Collapse
|
42
|
Khazaei S, Keshavarz G, Bozorgi A, Nazari H, Khazaei M. Adipose tissue-derived stem cells: a comparative review on isolation, culture, and differentiation methods. Cell Tissue Bank 2021; 23:1-16. [PMID: 33616792 DOI: 10.1007/s10561-021-09905-z] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 02/01/2021] [Indexed: 01/14/2023]
Abstract
Adipose tissue-derived stem cells (ADSCs) are an available source of mesenchymal stem cells with the appropriate capacity to in vitro survive, propagate, and differentiate into cells from three lineages of ectoderm, mesoderm, and endoderm. The biological features of ADSCs depend on the donor physiology and health status, isolation procedure, culture conditions, and differentiation protocols used. Adipose tissue samples are provided by surgery and lipoaspiration-based methods and subjected to various mechanical and chemical digestion techniques to finally generate a heterogeneous mixture named stromal vascular fraction (SVF). ADSCs are purified through varied cell populations that exist within SVF and cultured under standard conditions to give rise to a highly rich resource of stem cells directly applied in the clinic or differentiated into a wide range of cells. The development and optimization of conventional isolation, expansion, and differentiation methods seem noteworthy to preserve the desirable biological functions of ADSCs in pre-clinical and clinical investigations.
Collapse
Affiliation(s)
- Saber Khazaei
- Department of Endodontics, School of Dentistry, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Ghazal Keshavarz
- Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Azam Bozorgi
- Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.,Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Hamed Nazari
- Department of Orofacial Surgery, School of Dentistry, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Mozafar Khazaei
- Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
| |
Collapse
|
43
|
Zha K, Yang Y, Tian G, Sun Z, Yang Z, Li X, Sui X, Liu S, Zhao J, Guo Q. Nerve growth factor (NGF) and NGF receptors in mesenchymal stem/stromal cells: Impact on potential therapies. Stem Cells Transl Med 2021; 10:1008-1020. [PMID: 33586908 PMCID: PMC8235142 DOI: 10.1002/sctm.20-0290] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2020] [Revised: 12/27/2020] [Accepted: 01/12/2021] [Indexed: 12/13/2022] Open
Abstract
Mesenchymal stem/stromal cells (MSCs) are promising for the treatment of degenerative diseases and traumatic injuries. However, MSC engraftment is not always successful and requires a strong comprehension of the cytokines and their receptors that mediate the biological behaviors of MSCs. The effects of nerve growth factor (NGF) and its two receptors, TrkA and p75NTR, on neural cells are well studied. Increasing evidence shows that NGF, TrkA, and p75NTR are also involved in various aspects of MSC function, including their survival, growth, differentiation, and angiogenesis. The regulatory effect of NGF on MSCs is thought to be achieved mainly through its binding to TrkA. p75NTR, another receptor of NGF, is regarded as a novel surface marker of MSCs. This review provides an overview of advances in understanding the roles of NGF and its receptors in MSCs as well as the effects of MSC‐derived NGF on other cell types, which will provide new insight for the optimization of MSC‐based therapy.
Collapse
Affiliation(s)
- Kangkang Zha
- Medical School of Chinese PLA, Beijing, People's Republic of China.,Institute of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma and War Injuries, PLA, Beijing, People's Republic of China.,School of Medicine, Nankai University, Tianjin, People's Republic of China
| | - Yu Yang
- Department of Othopaedics, Trauma and Hand Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, People's Republic of China
| | - Guangzhao Tian
- Medical School of Chinese PLA, Beijing, People's Republic of China.,Institute of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma and War Injuries, PLA, Beijing, People's Republic of China.,School of Medicine, Nankai University, Tianjin, People's Republic of China
| | - Zhiqiang Sun
- Medical School of Chinese PLA, Beijing, People's Republic of China.,Institute of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma and War Injuries, PLA, Beijing, People's Republic of China.,School of Medicine, Nankai University, Tianjin, People's Republic of China
| | - Zhen Yang
- Medical School of Chinese PLA, Beijing, People's Republic of China.,Institute of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma and War Injuries, PLA, Beijing, People's Republic of China.,School of Medicine, Nankai University, Tianjin, People's Republic of China
| | - Xu Li
- Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, Innovative Orthopaedic Biomaterial and Drug Translational Research Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, People's Republic of China
| | - Xiang Sui
- Institute of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma and War Injuries, PLA, Beijing, People's Republic of China
| | - Shuyun Liu
- Institute of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma and War Injuries, PLA, Beijing, People's Republic of China
| | - Jinmin Zhao
- Department of Othopaedics, Trauma and Hand Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, People's Republic of China
| | - Quanyi Guo
- Institute of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma and War Injuries, PLA, Beijing, People's Republic of China
| |
Collapse
|
44
|
Aging of Bone Marrow Mesenchymal Stromal Cells: Hematopoiesis Disturbances and Potential Role in the Development of Hematologic Cancers. Cancers (Basel) 2020; 13:cancers13010068. [PMID: 33383723 PMCID: PMC7794884 DOI: 10.3390/cancers13010068] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 12/16/2020] [Accepted: 12/24/2020] [Indexed: 12/13/2022] Open
Abstract
Simple Summary As for many other cancers, the risk of developing hematologic malignancies increases considerably as people age. In recent years, a growing number of studies have highlighted the influence of the aging microenvironment on hematopoiesis and tumor progression. Mesenchymal stromal cells are a major player in intercellular communication inside the bone marrow microenvironment involved in hematopoiesis support. With aging, their functions may be altered, leading to hematopoiesis disturbances which can lead to hematologic cancers. A good understanding of the mechanisms involved in mesenchymal stem cell aging and the consequences on hematopoiesis and tumor progression is therefore necessary for a better comprehension of hematologic malignancies and for the development of therapeutic approaches. Abstract Aging of bone marrow is a complex process that is involved in the development of many diseases, including hematologic cancers. The results obtained in this field of research, year after year, underline the important role of cross-talk between hematopoietic stem cells and their close environment. In bone marrow, mesenchymal stromal cells (MSCs) are a major player in cell-to-cell communication, presenting a wide range of functionalities, sometimes opposite, depending on the environmental conditions. Although these cells are actively studied for their therapeutic properties, their role in tumor progression remains unclear. One of the reasons for this is that the aging of MSCs has a direct impact on their behavior and on hematopoiesis. In addition, tumor progression is accompanied by dynamic remodeling of the bone marrow niche that may interfere with MSC functions. The present review presents the main features of MSC senescence in bone marrow and their implications in hematologic cancer progression.
Collapse
|
45
|
González-González A, García-Sánchez D, Dotta M, Rodríguez-Rey JC, Pérez-Campo FM. Mesenchymal stem cells secretome: The cornerstone of cell-free regenerative medicine. World J Stem Cells 2020; 12:1529-1552. [PMID: 33505599 PMCID: PMC7789121 DOI: 10.4252/wjsc.v12.i12.1529] [Citation(s) in RCA: 74] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 10/07/2020] [Accepted: 11/11/2020] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem cells (MSCs) are the most frequently used stem cells in clinical trials due to their easy isolation from various adult tissues, their ability of homing to injury sites and their potential to differentiate into multiple cell types. However, the realization that the beneficial effect of MSCs relies mainly on their paracrine action, rather than on their engraftment in the recipient tissue and subsequent differentiation, has opened the way to cell-free therapeutic strategies in regenerative medicine. All the soluble factors and vesicles secreted by MSCs are commonly known as secretome. MSCs secretome has a key role in cell-to-cell communication and has been proven to be an active mediator of immune-modulation and regeneration both in vitro and in vivo. Moreover, the use of secretome has key advantages over cell-based therapies, such as a lower immunogenicity and easy production, handling and storage. Importantly, MSCs can be modulated to alter their secretome composition to better suit specific therapeutic goals, thus, opening a large number of possibilities. Altogether these advantages now place MSCs secretome at the center of an important number of investigations in different clinical contexts, enabling rapid scientific progress in this field.
Collapse
Affiliation(s)
- Alberto González-González
- Department of Molecular Biology_IDIVAL, Faculty of Medicine, University of Cantabria, Santander 39011, Cantabria, Spain
| | - Daniel García-Sánchez
- Department of Molecular Biology_IDIVAL, Faculty of Medicine, University of Cantabria, Santander 39011, Cantabria, Spain
| | - Monica Dotta
- Department of Molecular Biology_IDIVAL, Faculty of Medicine, University of Cantabria, Santander 39011, Cantabria, Spain
| | - José C Rodríguez-Rey
- Department of Molecular Biology_IDIVAL, Faculty of Medicine, University of Cantabria, Santander 39011, Cantabria, Spain
| | - Flor M Pérez-Campo
- Department of Molecular Biology_IDIVAL, Faculty of Medicine, University of Cantabria, Santander 39011, Cantabria, Spain.
| |
Collapse
|
46
|
Ratushnyy AY, Rudimova YV, Buravkova LB. Replicative Senescence and Expression of Autophagy Genes in Mesenchymal Stromal Cells. BIOCHEMISTRY (MOSCOW) 2020; 85:1169-1177. [PMID: 33202202 DOI: 10.1134/s0006297920100053] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Cell senescence leads to a number of changes in the properties of mesenchymal stromal cells (MSCs). In particular, the number of damaged structures is increased producing negative effect on intracellular processes. Elimination of the damaged molecules and organelles occurs via autophagy that can be important in the context of aging. Cultivation under low oxygen level can be used as an approach for enhancement of MSC therapeutic properties and "slowing down" cell senescence. The goal of this work was to study some morphological and functional characteristics and expression of autophagy-associated genes during replicative senescence of MSCs under different oxygen concentration. The study revealed changes in the regulation of autophagy at the transcriptional level. Upregulation of the expression of autophagosome membrane growth genes ATG9A and ULK1, of the autophagosome maturation genes CTSD, CLN3, GAA, and GABARAPL1, of the autophagy regulation genes TP53, TGFB1, BCL2L1, FADD, and HTT was shown. These changes were accompanied by downregulation of IGF1 and TGM2 expression. Increase of the lysosomal compartment volume was observed in the senescent MSCs that also indicated increase of their degradation activity. The number of lysosomes was decreased following prolonged cultivation under low oxygen concentration (5%). The replicative senescence of MSCs under conditions of different oxygen levels led to the similar modifications in the expression of the autophagy-associated genes.
Collapse
Affiliation(s)
- A Y Ratushnyy
- Institute of Biomedical Problems (IBMP), Russian Academy of Sciences, Moscow, 123007, Russia.
| | - Y V Rudimova
- Institute of Biomedical Problems (IBMP), Russian Academy of Sciences, Moscow, 123007, Russia
| | - L B Buravkova
- Institute of Biomedical Problems (IBMP), Russian Academy of Sciences, Moscow, 123007, Russia
| |
Collapse
|
47
|
Battram AM, Bachiller M, Martín-Antonio B. Senescence in the Development and Response to Cancer with Immunotherapy: A Double-Edged Sword. Int J Mol Sci 2020; 21:ijms21124346. [PMID: 32570952 PMCID: PMC7352478 DOI: 10.3390/ijms21124346] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2020] [Revised: 06/11/2020] [Accepted: 06/13/2020] [Indexed: 12/12/2022] Open
Abstract
Cellular senescence was first described as a physiological tumor cell suppressor mechanism that leads to cell growth arrest with production of the senescence-associated secretory phenotype known as SASP. The main role of SASP in physiological conditions is to attract immune cells to clear senescent cells avoiding tumor development. However, senescence can be damage-associated and, depending on the nature of these stimuli, additional types of senescence have been described. In the context of cancer, damage-associated senescence has been described as a consequence of chemotherapy treatments that were initially thought of as a tumor suppressor mechanism. However, in certain contexts, senescence after chemotherapy can promote cancer progression, especially when immune cells become senescent and cannot clear senescent tumor cells. Moreover, aging itself leads to continuous inflammaging and immunosenescence which are responsible for rewiring immune cells to become defective in their functionality. Here, we define different types of senescence, pathways that activate them, and functions of SASP in these events. Additionally, we describe the role of senescence in cancer and its treatments, including how aging and chemotherapy contribute to senescence in tumor cells, before focusing on immune cell senescence and its role in cancer. Finally, we discuss potential therapeutic interventions to reverse cell senescence.
Collapse
Affiliation(s)
- Anthony M. Battram
- Department of Hematology, Hospital Clinic, IDIBAPS, 08036 Barcelona, Spain; (A.M.B.); (M.B.)
| | - Mireia Bachiller
- Department of Hematology, Hospital Clinic, IDIBAPS, 08036 Barcelona, Spain; (A.M.B.); (M.B.)
| | - Beatriz Martín-Antonio
- Department of Hematology, Hospital Clinic, IDIBAPS, 08036 Barcelona, Spain; (A.M.B.); (M.B.)
- Department of Hematology, Hospital Clinic, IDIBAPS/Josep Carreras Leukaemia Research Institute, Carrer Rosselló 149-153, 08036 Barcelona, Spain
- Correspondence: ; Tel.: +34-93-227-45-28; Fax: +34-93-312-94-07
| |
Collapse
|
48
|
Domnina A, Ivanova J, Alekseenko L, Kozhukharova I, Borodkina A, Pugovkina N, Smirnova I, Lyublinskaya O, Fridlyanskaya I, Nikolsky N. Three-Dimensional Compaction Switches Stress Response Programs and Enhances Therapeutic Efficacy of Endometrial Mesenchymal Stem/Stromal Cells. Front Cell Dev Biol 2020; 8:473. [PMID: 32612993 PMCID: PMC7308716 DOI: 10.3389/fcell.2020.00473] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Accepted: 05/20/2020] [Indexed: 12/16/2022] Open
Abstract
Mesenchymal stem cells are currently tested as a promising tool for the treatment of a wide range of human diseases. Enhanced therapeutic potential of spheroids formed from these cells has been proved in numerous studies, however, the fundamental basics of this effect are still being discussed. In this work, we showed that endometrial mesenchymal stem/stromal cells (eMSCs) assembled in spheroids possess a higher therapeutic efficacy compared to cells grown in monolayer in the treatment of the defects that are non-specific for eMSC tissue origin – skin wounds. With the purpose to elucidate the possible causes of superior spheroid potency, we compared the tolerance of eMSC cultivated in spheres and monolayer to the stress insults. Using genetically encoded hydrogen peroxide biosensor HyPer, we showed that three-dimensional configuration (3D) helped to shield the inner cell layers of spheroid from the external H2O2-induced oxidative stress. However, the viability of oxidatively damaged eMSCs in spheroids appeared to be much lower than that of monolayer cells. An extensive analysis, which included administration of heat shock and irradiation stress, revealed that cells in spheroids damaged by stress factors activate the apoptosis program, while in monolayer cells stress-induced premature senescence is developed. We found that basal down-regulation of anti-apoptotic and autophagy-related genes provides the possible molecular basis of the high commitment of eMSCs cultured in 3D to apoptosis. We conclude that predisposition to apoptosis provides the programmed elimination of damaged cells and contributes to the transplant safety of spheroids. In addition, to investigate the role of paracrine secretion in the wound healing potency of spheroids, we exploited the in vitro wound model (scratch assay) and found that culture medium conditioned by eMSC spheroids accelerates the migration of adherent cells. We showed that 3D eMSCs upregulate transcriptional activator, hypoxia-inducible factor (HIF)-1, and secret ten-fold more HIF-1-inducible pro-angiogenic factor VEGF (vascular endothelial growth factor) than monolayer cells. Taken together, these findings indicate that enhanced secretory activity can promote wound healing potential of eMSC spheroids and that cultivation in the 3D cell environment alters eMSC vital programs and therapeutic efficacy.
Collapse
Affiliation(s)
- Alisa Domnina
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Julia Ivanova
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Larisa Alekseenko
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Irina Kozhukharova
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Aleksandra Borodkina
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Natalia Pugovkina
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Irina Smirnova
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Olga Lyublinskaya
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Irina Fridlyanskaya
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| | - Nikolay Nikolsky
- Department of Intracellular Signaling and Transport, Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
| |
Collapse
|