Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Oct 26, 2020; 12(10): 1152-1170
Published online Oct 26, 2020. doi: 10.4252/wjsc.v12.i10.1152
Pericyte-like differentiation of human adipose-derived mesenchymal stem cells: An in vitro study
Giuliana Mannino, Florinda Gennuso, Giovanni Giurdanella, Federica Conti, Filippo Drago, Salvatore Salomone, Debora Lo Furno, Claudio Bucolo, Rosario Giuffrida
Giuliana Mannino, Debora Lo Furno, Rosario Giuffrida, Physiology Section, Department of Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, Italy
Florinda Gennuso, Federica Conti, Filippo Drago, Salvatore Salomone, Claudio Bucolo, Pharmacology Section, Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
Giovanni Giurdanella, Biochemistry Section, Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
Author contributions: Mannino G and Gennuso F made substantial contributions to conception and design, participated in the acquisition, analysis and interpretation of data; Giurdanella G and Conti F participated in data acquisition; Salomone S and Drago F were involved in data analysis and interpretation, and revising the manuscript critically for important intellectual content; Lo Furno D, Bucolo C and Giuffrida R conceived the project and developed the experimental design, analyzed data and wrote the manuscript. All authors reviewed the manuscript and approved the version to be published.
Supported by “Piano Triennale per la Ricerca 2016-2018 – Linea Intervento 2”, University of Catania, Italy, No. 20722142118.
Institutional review board statement: This study was reviewed and approved by the local ethics committee (Comitato etico Catania1; Authorization n. 155/2018/PO).
Conflict-of-interest statement: The authors declare no conflict of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Debora Lo Furno, PhD, Assistant Professor, Physiology Section, Department of Biomedical and Biotechnological Sciences, University of Catania, No. 95125 Catania, Catania 95123, Italy. lofurno@unict.it
Received: May 15, 2020
Peer-review started: May 15, 2020
First decision: June 7, 2020
Revised: June 18, 2020
Accepted: August 25, 2020
Article in press: August 25, 2020
Published online: October 26, 2020
Processing time: 163 Days and 18.5 Hours
Abstract
BACKGROUND

Adipose-derived mesenchymal stem cells (ASCs) are characterized by long-term self-renewal and a high proliferation rate. Under adequate conditions, they may differentiate into cells belonging to mesodermal, endodermal or ectodermal lineages. Pericytes support endothelial cells and play an important role in stabilizing the vessel wall at the microcirculation level. The loss of pericytes, as occurs in diabetic retinopathy, results in a breakdown of the blood-retina barrier (BRB) and infiltration of inflammatory cells. In this context, the use of pericyte-like differentiated ASCs may represent a valuable therapeutic strategy for restoring BRB damage.

AIM

To test in vitro strategies to obtain pericyte-like differentiation of human ASCs (hASCs).

METHODS

Different culture conditions were tested: hASCs cultured in a basal medium supplemented with transforming growth factor β1; and hASCs cultured in a specific pericyte medium (PM-hASCs). In a further sample, pericyte growth supplement was omitted from the PM. In addition, cultures of human retinal pericytes (hRPCs) were used for comparison. Pericyte-like differentiation of hASCs was tested by immunocytochemical staining and western blotting to evaluate the expression of α-smooth muscle actin (α-SMA) and neural/glial antigen 2 (NG2). Interactions between human retinal endothelial cells (hRECs) and different groups of hASCs were investigated in co-culture experiments. In these cases, the expression of typical junctional proteins such as vascular endothelial-Cadherin, zonula occludens-1 and Occludin were assessed in hRECs. In an in vitro model of the BRB, values of trans-endothelial electrical resistance were measured when hRECs were co-cultured with various groups of pretreated hASCs. The values observed were compared with co-cultures of hRECs and hRPCs as well as with cultures of hRECs alone. Three-dimensional co-cultures of hRECs and hRPCs or pericyte-like hASCs in Matrigel were designed to assess their reciprocal localization.

RESULTS

After 3-6 d of culture, α-SMA and NG2 immunocytochemistry showed that the closest pericyte-like phenotype was observed when hASCs were cultured in Pericyte Medium (PM-hASCs). In particular, α-SMA immunoreactivity, already visible at the basal level in pericytes and ASCs, was strongly increased only when transforming growth factor was added to the culture medium. NG2 expression, almost undetectable in most conditions, was substantially increased only in PM-hASCs. Immunocytochemical results were confirmed by western blot analysis. The presence of pericyte growth supplement seems to increase NG2 expression rather than α-SMA, in agreement with its role in maintaining pericytes in the proliferative state. In co-culture experiments, immunoreactivity of vascular endothelial-Cadherin, zonula occludens-1 and Occludin was considerably increased in hRECs when hRPCs or PM-hASCs were also present. Supporting results were found by trans-endothelial electrical resistance measurements, gathered at 3 and 6 d of co-culture. The highest resistance values were obtained when hRECs were co-cultured with hRPCs or PM-hASCs. The pericyte-like phenotype of PM-hASCs was also confirmed in three-dimensional co-cultures in Matrigel, where PM-hASCs and hRPCs similarly localized around the tubular formations made by hRECs.

CONCLUSION

PM-hASCs seem able to strengthen the intercellular junctions between hRECs, likely reinforcing the BRB; thus, hASC-based therapeutic approaches may be developed to restore the integrity of retinal microcirculation.

Keywords: Adipose-derived mesenchymal stem cells; Pericyte-like differentiation; Retinal endothelial cells; Retinal pericytes; Blood-retina barrier; Junction proteins

Core Tip: Pericyte-like differentiation was achieved in human adipose-derived mesenchymal stem cells (hASCs) by a culture medium specific for optimal pericyte growth. When co-cultured with retinal endothelial cells, pre-differentiated hASCs increased endothelial junction protein expression and trans-endothelial electrical resistance. Similar to pericytes, differentiated hASCs localized in the typical perivascular position.