Human Amniotic Fluid Stem Cell-Derived Exosomes as a Novel Cell-Free Therapy for Cutaneous Regeneration

Abstract

Adult wound healing often results in fibrotic scarring that is caused by myofibroblast aggregation. Human amniotic fluid stem cells (hAFSCs) exhibit significantly anti-fibrotic scarring properties during wound healing. However, it is little known whether hAFSCs directly or indirectly (paracrine) contribute to this process. Using the full-thickness skin-wounded rats, we investigated the therapeutic potential of hAFSC-derived exosomes (hAFSC-exo). Our results showed that hAFSC-exo accelerated the wound healing rate and improved the regeneration of hair follicles, nerves, and vessels, as well as increased proliferation of cutaneous cells and the natural distribution of collagen during wound healing. Additionally, hAFSC-exo suppressed the excessive aggregation of myofibroblasts and the extracellular matrix. We identified several miRNAs, including let-7-5p, miR-22-3p, miR-27a-3p, miR-21-5p, and miR-23a-3p, that were presented in hAFSC-exo. The functional analysis demonstrated that these hAFSC-exo-miRNAs contribute to the inhibition of the transforming growth factor-β (TGF-β) signaling pathway by targeting the TGF-β receptor type I (TGF-βR1) and TGF-β receptor type II (TGF-βR2). The reduction of TGF-βR1 and TGF-βR2 expression induced by hAFSC-exo was also confirmed in the healing tissue. Finally, using mimics of miRNAs, we found that hAFSC-exo-miRNAs were essential for myofibroblast suppression during the TGF-β1-induced human dermal fibroblast-to-myofibroblast transition in vitro. In summary, this study is the first to show that exosomal miRNAs used in hAFSC-based therapy inhibit myofibroblast differentiation. Our study suggests that hAFSC-exo may represent a strategic tool for suppressing fibrotic scarring during wound healing.

Small Extracellular Vesicles from Human Amniotic Fluid Samples as Promising Theranostics

“…This can be easily obtained by either incubating cells in xeno- and serum-free medium (SF) formulations or by employing commercially available or home-made FBS-EV-free supplements. Most studies focusing on hAFSC-EVs apply SF culture conditions [ 19 , 46 , 47 , 54 , 57 , 58 , 60 , 67 , 84 , 87 , 89 ], while fewer groups have reported using EV-free culture medium to collect the cell-CM [ 55 , 56 ]. Likewise, other variables have been shown to prime stem cell and MSC secretory potential, such as oxygen tension or pro-inflammatory stimuli (i.e., IFNγ) [ 84 ].…”

“…Indeed, several groups reported that in vitro hypoxic preconditioning may lead to an increased secretome yield, including EV fractions [ 47 , 54 , 89 ]. The duration of preconditioning is another sensible aspect, since hAF-MSC/hAFSC-CM has been reported to be collected after 24 h [ 46 , 47 , 67 , 89 ], 48 h [ 19 , 60 ], 72 h [ 56 ] or 96 h [ 57 , 58 ]. Notably, hAF-MSC and hAFSC have been suggested to mature different miRNA profiles which can be further enriched in their EV cargo, depending on culture conditions [ 54 ].…”

“…Topic administration with 20 µg hAFSC-EVs once a week improved also wound healing in a preclinical rat model of cutaneous injury [ 60 ]. After 28 days, EVs treatment resulted in increased angiogenesis by a high level of CD31 expression, with inhibition of fibrotic scarring.…”

“…Several independent studies have reported that the hAF-MSC and hAFSC secretome exert pro-survival, anti-apoptotic effects [ 47 ], while quenching inflammation [ 48 ], stimulating local angiogenesis [ 49 , 50 ], and supporting cardiac protection following myocardial infarction or cardiotoxicity [ 49 , 51 , 52 , 53 ]. In this scenario, hAFSC-EVs have been widely profiled to assess whether they can recapitulate such pro-regenerative potential with promising results in preclinical models of skeletal and cardiac muscle injury [ 19 , 47 , 49 , 54 ], lung [ 55 ] and neurodegenerative disease [ 56 , 57 ], osteoarthritis [ 58 ], osteoporosis [ 59 ] and cutaneous injury [ 60 ].…”

Mesenchymal Stem-Cell Derived Exosome Therapy as a Potential Future Approach for Treatment of Male Infertility Caused by Chlamydia Infection

“…Exosomes secrete from T cells ( Nolte-‘t Hoen et al, 2009 ), B cells ( Clayton et al, 2005 ), macrophages ( Bhatnagar et al, 2007 ), epithelial cells ( Skogberg et al, 2015 ), endothelial cells ( Song et al, 2014 , 2015 ), as well as MSCs ( Yeo et al, 2013 ). The vesicles with exosomal characteristics have been also founded in the various body fluids such as semen ( Fabiani et al, 1994 ; Arienti et al, 1999 ; Park et al, 2011 ; Aalberts et al, 2012 ), blood ( Blanc et al, 2005 ; Caby et al, 2005 ; Yunusova et al, 2016 ), breast milk ( Admyre et al, 2007 ; Qin et al, 2016 ; Miyake et al, 2020 ), ascites fluid ( Andre et al, 2002 ; Navabi et al, 2005 ; Runz et al, 2007 ), saliva ( Ogawa et al, 2008 ; Michael et al, 2010 ), amniotic fluid ( Asea et al, 2008 ; Zhang et al, 2021 ), urine ( Gonzales et al, 2010 ; Street et al, 2017 ), and bile ( Masyuk et al, 2010 ; Sagredo et al, 2017 ). Because exosomes are in nano sized range, they spread through body fluids and easily penetrate through tissues and affect targeted cells ( Phinney and Pittenger, 2017 ), even if those cells are far away ( François et al, 2006 ).…”