Differentiation of Porcine Mesenchymal Stem Cells to Lung Epithelial Cells Using Media Conditioned by Human Primary Small Airway Epithelial Cells
Authors: Kelly Gillian C. , Murata Lee‐Ann M. , Lee Benjamin C. , Uyehara Catherine F.T. , Ho Dao H. 
Published: June 2021

Mesenchymal stem cells (MSCs) under defined conditions, can differentiate into many different cell types, and may be the key to treating a multitude of ailments including chronic lung injury. Although MSCs can reduce inflammation and alter immune response when injected into sites of injury, differentiating MSCs in vitro prior to injection may enhance their therapeutic actions. The aim of this study was to develop a simple and reliable method to differentiate porcine adipose‐derived (pAMSCs) and bone marrow‐derived mesenchymal stem cells (pBMSCs) to small airway epithelium (SAE) for future testing in a pig model of lung injury. Traditional induction of MSCs to SAE involves treatment with an artificial combination of factors that mimic the local milieu in vivo. A simpler and more physiologically‐relevant approach is to use naturally conditioned media that may yield more biologically active cells. We hypothesized that media conditioned with human primary small airway epithelial cells (hSAECs) can induce differentiation of pAMSCs and pBMSCs to SAE. hSAECs (ATCC) were seeded in serum‐free M199 media to create conditioned media which was collected 4 days after seeding for immediate use. pAMSCs and pBMSCs were isolated in‐house from juvenile Yorkshire pigs, and expanded to passage 3 prior to start of experiments. Fibronectin‐coated, 8‐well slides (for immunohistochemistry) or T75 flasks (to examine morphology) were seeded with either pAMSCs or pBMSCs (10,000 cells/cm2). Every 4 days for 16 days, a subset of cells were labeled with stem cell markers (anti‐FOXA1, anti‐CD44, anti‐CD105), small airway cell markers (anti‐SCGB3A2, anti‐RAGE), and neurofilament markers (anti‐neurofilament heavy polypeptide, anti‐68kDa neurofilament)), and media was exchanged with either conditioned media or unconditioned media. After 5 days, pBMSCs exposed to conditioned media transitioned to cuboidal and epithelial‐like cells. pBMSCs exposed to unconditioned media remained elongated and stem cell‐like. pAMSCs proliferated faster than pBMSCs, but yielded spindly overgrown cells in high density that did not differentiate in either conditioned or unconditioned media. Expression of proteins did not correlate with the morphological changes in pBMSCs. Although RAGE expression increased initially in response to conditioned media, levels were reduced after day 12. SCGB3A2 expression also increased over time, but was not different between the conditioned and unconditioned groups. The distinct change in morphology observed in pBMSCs exposed to conditioned media indicates a promising phenotypic change. Verification of the epithelial cell phenotype induced by the conditioned media with semi‐quantitative gene and protein expression of cell markers for small airway cells and their functional ability to produce surfactant remains to be determined. Our study indicates that conditioned media is able to induce differentiation of MSCs into epithelial cells. Further, our results revealed that the source of MSCs (adipose versus bone marrow), and the density of MSCs may be important factors in successful differentiation of MSCs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.