Human Umbilical Cord Lining-Derived Epithelial Cells: A Potential Source of Non-Native Epithelial Cells That Accelerate Healing in a Porcine Cutaneous Wound Model.

Human umbilical cord lining epithelial cells [CLECs) are naïve in nature and can be ethically recovered from cords that are routinely discarded. The success of using oral mucosal epithelial cells for cornea defects hints at the feasibility of treating cutaneous wounds using non-native CLECs. Herein, we characterized CLECs using flow cytometry (FC) and skin organotypic cultures in direct comparison with skin keratinocytes (KCs). This was followed by wound healing study to compare the effects of CLEC application and the traditional use of human skin allografts (HSGs) in a porcine wound model. While CLECs were found to express all the epidermal cell markers probed, the major difference between CLECs and KCs lies in the level of expression (in FC analysis) as well as in the location of expression (of the epithelium in organotypic cultures) of some of the basal cell markers probed. On the pig wounds, CLEC application promoted accelerated healing with no adverse reaction compared to HSG use. Though CLECs, like HSGs, elicited high levels of local and systemic immune responses in the animals during the first week, these effects were tapered off more quickly in the CLEC-treated group. Overall, the in vivo porcine data point to the potential of CLECs as a non-native and safe source of cells to treat cutaneous wounds.

Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices.

The basal keratinocyte progenitor cells in cultured epithelial autografts (CEAs) regenerate human epidermis after transplantation, a curative therapy for severe burns and, recently, diseases with epidermal loss, such as junctional epidermolysis bullosa (EB). Although a culturing technique for skin keratinocytes was developed four decades ago, the xenogeneic nature of that conventional CEA culture system restricts its use to the treatment of critical and life-threatening cases, such as severe burns on >30% of total body surface area and EB. In the present protocol, we describe how to implement a defined, xeno-free culture system that supports long-term ex vivo expansion of functional human epidermal keratinocytes. Skin-specific basement membrane proteins called laminins play important roles in the maintenance of phenotypic integrity and in supporting the survival of keratinocytes that are adhered to them. This fully human keratinocyte culture system is 'regulatory friendly' and increases the potential of epithelial cellular therapy, which can be expanded to treat less severe burns and other skin defects, such as chronic diabetic wounds. It takes between 7 and 14 d to obtain an initial culture. Conservatively, a secondary culture from the primary culture can be expanded up to 20-fold within 4-5 d once cells reach confluency.

Biologically relevant laminin as chemically defined and fully human platform for human epidermal keratinocyte culture.

The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system. Through our characterization efforts of the human skin basement membrane and murine feeder layer 3T3-J2, we identified two biologically relevant recombinant laminins-LN-511 and LN-421- as potential candidates to replace the murine feeder. Herein, we report a completely xeno-free and defined culture system utilizing these laminins which enables robust expansion of adult human skin keratinocytes. We demonstrate that our laminin system is comparable to the 3T3-J2 co-culture system in terms of basal markers' profile, colony-forming efficiency and the ability to form normal stratified epidermal structure in both in vitro and in vivo models. These results show that the proposed system may not only provide safer keratinocyte use in the clinics, but also facilitate the broader use of other cultured human epithelial cells in regenerative medicine.

Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.