Abstract: The concept of using "non-native" sources of cultured epithelial cells as a form of regenerative medicine is not new. Corneal reconstruction with cultured autologous oral mucosal epithelium remains in practice today. However, the question of whether cultured human umbilical cord lining-derived epithelial cells (hCLECs) can engraft as a non-native source for skin epidermal reconstruction of severe wound defects is still not fully answered.
We first characterised and compared human umbilical cord and skin tissues through histological analysis and identified common proteins in both tissues. These include keratin combinations 5/14 and 1/10, epithelial stem cell markers Tp63 and ΔNp63, integrins α6 and β1, as well as the differentiation marker involucrin.
Single-cell RNA sequencing of hCLECs isolated directly from the lining of umbilical cords revealed that epithelial cells constitute the majority of the four main cell populations detected, and which share many common genes associated with human epidermal keratinocytes (hEKs). The transcriptomic signature of one epithelial cell population cluster showed a 44.9% overlap with the holoclone of hEKs, and GO analysis suggested that this cluster possesses stem-cell-like characteristics.
We evaluated the culture of hCLECs in a classical mouse feeder system using 3T3-J2, based on Green and Rheinwald's method. Colony forming efficiencies (CFE) of hCLECs were comparable to hEKs, averaging 20-40% for the first three passages. Subsequently, the majority of hCLEC colonies became aborted, with a significant reduction in size. Similarly, cumulative population doublings of hCLECs were considerably lower at approximately 20, reaching a plateau at passages 4-5, compared to hEKs with 60 cumulative doublings. Addition of ROCK inhibitor (Y-27632) into the culture system rescued the low growth potential and CFE of hCLECs.
Functional assays on early-passage hCLECs demonstrated that these cells could attach and stratify on 3D skin organotypic cultures in vitro, and similarly engraft and stratify on a nude mouse flap in vivo. These findings suggest that hCLECs expanded ex vivo at early generations could serve as an alternative to hEKs, providing a non-native and autologous cell source for treating major burns, extensive congenital aplasia cutis, and epidermolysis bullosa.
Funding Source: National Medical Research Council, Ministry of Health, Singapore (OFIRG20nov-0024)
