Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Nov 26, 2021; 13(11): 1783-1796
Published online Nov 26, 2021. doi: 10.4252/wjsc.v13.i11.1783
ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells
Giuliana Mannino, Martina Cristaldi, Giovanni Giurdanella, Rosario Emanuele Perrotta, Debora Lo Furno, Rosario Giuffrida, Dario Rusciano
Giuliana Mannino, Giovanni Giurdanella, Debora Lo Furno, Rosario Giuffrida, Department of Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, CT, Italy
Martina Cristaldi, Dario Rusciano, Research Center, SOOFT-Italia S.p.A., Catania 95123, CT, Italy
Rosario Emanuele Perrotta, Department of General Surgery and Medical-Surgery Specialties, University of Catania, Catania 95100, CT, Italy
Author contributions: Mannino G, Lo Furno D and Rusciano D conceptualized and designed the study, and drafted the article; Cristaldi M and Giurdanella G contributed to the acquisition, analysis and interpretation of data; Giuffrida R and Perrotta RE contributed to critical revisions related to important intellectual content of the manuscript; All authors contributed to the collection of literature, reviewed the manuscript and approved the version to be published.
Supported by University of Catania, Italy, “Piano Triennale per la Ricerca 2020-2022–Grant PIACERI, project “NanoRet”.
Institutional review board statement: This study was reviewed and approved by the local ethics committee (Comitato etico Catania1; Authorization n. 155/2018/PO).
Institutional animal care and use committee statement: No animals were used in the present study.
Conflict-of-interest statement: The authors declare no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines. No animals were used in the present study.
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, Department of Biomedical and Biotechnological Sciences, University of Catania, Via Santa Sofia 97, Catania 95123, CT, Italy. lofurno@unict.it
Received: May 5, 2021
Peer-review started: May 5, 2021
First decision: June 23, 2021
Revised: June 25, 2021
Accepted: October 15, 2021
Article in press: October 15, 2021
Published online: November 26, 2021
Processing time: 203 Days and 16.5 Hours
Abstract
BACKGROUND

Adipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.

AIM

To test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).

METHODS

ASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).

RESULTS

Depending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5.

CONCLUSION

The presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.

Keywords: Adipose-derived mesenchymal stem cells, Retinal pigment epithelium, Neural markers, Neural differentiation, Retina damage, Cell-based medicine

Core Tip: Neural-like differentiation of adipose-derived stem cells (ASCs) was tested using a conditioned medium from ARPE-19 cells, a cell line derived from human retinal pigment epithelium. Following this treatment, the expression of typical glial and neuronal markers increased in a time-dependent manner. Neural-like differentiated ASCs may represent a valuable tool for cell-based therapeutic approaches in the field of regenerative medicine for the treatment of eye diseases.