One of the main stages during wound healing is inflammation. There are indications that extracellular vesicles (EV) from mesenchymal stem cells (MSC) can ameliorate inflammation during this process. MSC have immunomodulatory properties and can induce regeneration. MSC from various sources have been used in wound healing studies. Moreover, several studies have demonstrated that conditioned media from MSC cultures have similar, or even higher, regenerative capacity than the MSC themselves when applied to wounds. Thus, the regenerative capacity of MSC could be due to their paracrine activity, including EV. EV contain molecules including nucleic acids and proteins and the composition depends on the cell type and its physiological conditions. MSC derived from term Amniotic Fluid (TAF) are neonatal and can be propagated to extremely high amounts for use in cell therapy. Furthermore, sorting strategies using tissue-specific markers can prepare a sorted population of cells that can be used for each tissue-type. EV derived from skin-specific TAF-MSC are highly interesting for wound healing applications.
RNASeq data from TAF-MSC clones was used to identify skin-specific markers present on these MSC. Several prospective skin markers were tested by flow cytometry on cultured TAF-MSC. One of these markers was used for cell-sorting using Tyto MACSQuant cell sorter and the positive cell population was expanded to final product stage (CutiStem). The secretome of CutiStem cells was collected after starvation. Extracellular vesicles were purified by ultra-centrifugation. Nanoparticle tracking (NTA) measurements were used to assess size and concentration by Zetaview. MACSPlex was used for multiplexed flow cytometry marker analysis. An immunomodulation assay was used to test action on the NFkB pathway. THP-1 dual cell monocytic cell line was constructed by stable integration of two inducible reporter constructs. This THP-1 cell assay was assessed by monitoring the activity of SEAP, the reporter protein. Samples were treated with LPS (positive control), dexamethasone (negative control), or a combination thereof.
Several prospective markers were tested of which one was used for skin-MSC sorting. Sorting at passage 2 increased positivity of this marker from 17% to 88%. The positive fraction was further propagated until passage 7 (CutiStem). CutiStem cells were cultured to 70% confluence whereafter growth medium was exchanged to starvation medium. Secretome was collected after 72 h of starvation. EV were purified by ultracentrifugation and analyzed. NTA revealed high yield of pure EV with mean particle size of 210 ± 5 nm. MACSPlex and flow cytometry showed high presence of exosome markers CD9, CD24, CD29, CD63 and CD81 and MSC markers CD105 and CD146. NFkB pathway activity was assessed by THP-1 assay, indicating that EV derived from CutiStem reduce inflammation. The unpurified secretome did not reduce inflammation, indicating that the EV fraction is responsible for the immunomodulatory activities and not the free proteins of the secretome.
One marker was found to be a good skin-specific marker for sorting of TAF-MSC using the Tyto MACSQuant cell sorter instrument. Sorted cells were further propagated and the secretome was collected for further analysis. EV derived from skin-sorted TAF-MSC (CutiStem) reduce inflammation.