Development of xeno-free epithelial differentiation media for adherent, non-expanded adipose stromal vascular cell cultures.

INTRODUCTION: Reconstruction of respiratory epithelium is critical for the fabrication of bioengineered airway implants. Epithelial differentiation is typically achieved using bovine pituitary extract (BPE). Due to the xenogenic nature and undefined composition of BPE, an alternative for human clinical applications, devoid of BPE, must be developed. The goal of this study was to develop two different BPE-free media, with and without select pituitary hormone (PH), which could initiate epithelial differentiation for use in human implantation.

METHODS: The ability of the two BPE-free media to initiate epithelial differentiation of adherent, non-expanded stromal-vascular cells grown on porcine small intestinal submucosa was compared to traditional BPE-containing media (M1). Nanostring® was used to measure differences in gene expression of stemness (MSC), basal cell (basal), and ciliated markers (muco-cil), and staining was performed support the gene data.

RESULTS: Compared to baseline, both BPE-free media upregulated epithelial and stemness genes, however this was to a lower degree than BPE-containing media. In general, the expression of basal cell markers (COL17A1, DSG3, ITGA6, KRT6A, LOXL2) and secreted mucous proteins (PLUNC, MUC5B, SCGB2A1) was upregulated. The gene expression of ciliated markers C9orf24, TUBA3 and DNCL2B but not of the key transcription factor for cilagenesis FOXJ1 were upregulated, indicating that mucus-secreting cell differentiation occurs more rapidly than ciliogenesis. The ability of the adherent stromal vascular cells to upregulate gene expression of both epithelial and stemness markers suggests maintenance of the self-renewal capacity of undifferentiated and/or basal cell-like cells contributing to proliferation and ensuring a persisting source of cells for regenerative medicine applications.

CONCLUSION: This study provides the initial step to defining a BPE-free epithelial differentiation medium for clinical translation. Thus, either of the proposed BPE-free medium are viable alternatives to BPE-containing medium for partial epithelial differentiation for human translational applications.