Human Adipose-Derived Stem Cell Conditioned Media and Exosomes Containing MALAT1 Promote Human Dermal Fibroblast Migration and Ischemic Wound Healing

Abstract

Objective: Chronically ill patients heal recalcitrant ulcerative wounds more slowly. Human adipose-derived stem cells (hADSCs) play an important role in tissue regeneration and exosomes secreted by hADSC contribute to their paracrine signaling. In addition to cytokines, lipids and growth factors, hADSC secrete mRNA, miRNA, and long noncoding (lnc) RNA into exosomes. In this study we examined the role of lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), an abundant lncRNA in exosomes from conditioned media (CM), on cell migration and ischemic wound healing.Approach: CM and isolated exosomes from hADSC were applied to human dermal fibroblast (HDF) in scratch assays and electric cell-substrate impedance sensing (ECIS) assays. CM was also applied to a rat model of ischemic wound healing and wound closure was followed.Results: CM stimulated cell migration of HDFs in vitro by 48%. CM stimulated the closure of ischemic wounds in a rat model 50% faster than unconditioned media. The depletion of MALAT1 in adipose-derived stem cell (ADSC) CM significantly reduced cell migration. Since MALAT1 is secreted into exosomes, a purified population of exosomes was applied to HDF where they enhanced cell migration in a similar manner to FGF-2 or basic fibroblast growth factor (bFGF) in ECIS wound healing assays. The uptake of exosomes by HDF was shown using dynasore, an inhibitor that blocks clathrin- and caveolin-dependent endocytosis. Depletion of MALAT1 in hADSC with antisense oligonucleotides resulted in exosomes without MALAT1. These exosomes had an effect similar to the unconditioned, control media in ECIS assays.Innovation: Exosomes contain lncRNA MALAT1 and other factors that have the potential to stimulate HDF cell migration and angiogenesis involved in wound healing without applying stem cells to wounds.Conclusion: Our results show the potential of using topically applied ADSC-derived exosomes containing MALAT1 for treating ischemic wounds. This allows for harnessing the power of stem cell paracrine signaling capabilities without applying the cells.

Extracellular Vesicles Derived From Mesenchymal Stem Cells (MSC) in Regenerative Medicine: Applications in Skin Wound Healing

“…Also, it has been shown that conditioned media from human adipose-derived stem cell (ADSC) cultures stimulated both the human dermal-fibroblast migration and closure of ischemic wounds in a rat model. That is due to the presence of long ncRNA (lncRNA) of metastasisassociated lung-adenocarcinoma transcript 1 (MALAT-1) in EV (Cooper et al, 2018).…”

Secretome of Adipose Tissue-Derived Stem Cells (ASCs) as a Novel Trend in Chronic Non-Healing Wounds: An Overview of Experimental In Vitro and In Vivo Studies and Methodological Variables

“…In this study, ASC-CM was prepared by incubating ASCs with serum-free DMEM for 24 h. No details on the concentration of the CM used were provided by the authors. In another study [61], ASC-CM, previously collected from ASCs cultured for 48 h in serum-free MSC basal medium, was shown to stimulate HDF migration in vitro by 43% when compared to untreated cells as analyzed through a scratch and electric cell-substrate impedance assays. To investigate the underlying molecular mechanisms driving the therapeutic impact of the ASC-secretome, Park et al [46] used CM samples collected from cultured ASCs previously isolated from adipose tissues of breast cancer patients.…”

“…The evaluation of lesions at day 0, 2, 5, and 10 indicated that ASC-secretome treatment was able to stimulate skin thickening, angiogenesis, and recruitment of immune cells, thus accelerating the wound healing process. Using a rat model of an ischemic wound (Fisher 344 rats, full-thickness excisional wounds created with a 6 mm punch biopsy), Cooper et al [61] reported that ASC-CM collected from ASCs cultured for 48 h in serum-free MSC basal medium and applied at 20 µL to each wound daily significantly accelerated ischemic wound closure compared to unconditioned medium. Irons et al [68] recently showed that after topical application of CM collected from ASCs isolated from the gluteal region of Yorkshire pigs, the diabetic wound healing in the same animals was significantly enhanced, with concomitant increased angiogenesis and immunomodulation.…”

“…Moreover, in the same article, the stimulating effects of ASC-Exos at 0, 25, 50, and 100 µg/mL on HDF migration through scratch closure test and transwell assay as well as cell proliferation and collagen synthesis in vitro were also reported. Cooper et al [61], in addition to investigating the effects of ASC-CM described in Section 2.1, using the in vitro HDF model also evaluated the effectiveness of ASC-Exos in accelerating the wound healing process. Exos, quantitated using a NanoSight system, showed an average size of 90 to 110 nm at a concentration of 1.1 × 10 8 /mL of CM derived from 10 6 ASCs.…”

“…Increase of cell migration rate. [61] Adult fat samples. HUVEC Stimulation of angiogenesis, upregulation and downregulation of angiogenesis-stimulating or -inhibitory genes, respectively.…”