Chimeric composite skin substitutes for delivery of autologous keratinocytes to promote tissue regeneration

Abstract

Objective: We hypothesize that the pathogen-free NIKS human keratinocyte progenitor cell line cultured in a chimeric fashion with patient's primary keratinocytes would produce a fully stratified engineered skin substitute tissue and serve to deliver autologous keratinocytes to a cutaneous wound.

Summary of background data: Chimeric autologous/allogeneic bioengineered skin substitutes offer an innovative regenerative medicine approach for providing wound coverage and restoring cutaneous barrier function while delivering autologous keratinocytes to the wound site. NIKS keratinocytes are an attractive allogeneic cell source for this application.

Methods: Mixed populations of green fluorescent protein (GFP)-labeled NIKS and unlabeled primary keratinocytes were used to model the allogeneic and autologous components in chimeric monolayer and organotypic cultures.

Results: In monolayer coculture, GFP-labeled NIKS had no effect on the growth rate of primary keratinocytes and cell-cell junction formation between labeled and unlabeled keratinocytes was observed. In organotypic culture employing dermal and epidermal compartments, chimeric composite skin substitutes generated using up to 90% GFP-labeled NIKS exhibited normal tissue architecture and possessed substantial regions attributable to the primary keratinocytes. Tissues expressed proteins essential for the structure and function of a contiguous, fully-stratified squamous epithelia and exhibited barrier function similar to that of native skin. Furthermore, chimeric human skin substitutes stably engrafted in an in vivo mouse model, with long-term retention of primary keratinocytes but loss of the GFP-labeled NIKS population by 28 days after surgical application.

Conclusions: This study provides proof of concept for the use of NIKS keratinocytes as an allogeneic cell source for the formation of bioengineered chimeric skin substitute tissues, providing immediate formal wound coverage while simultaneously supplying autologous cells for tissue regeneration.

Figure 1

(A) Phase contrast, GFP localization (green), and E-cadherin (red) detection in a confluent co-culture of NIKS^GFP and primary keratinocytes plated in equal proportions. Arrows indicate junction between individual NIKS^GFP and primary keratinocytes. Scale bar: 50 μm. (B) Growth rate of NIKS^GFP and primary keratinocytes plated separately and at specific ratios of 50%:50%, 10%:90%, or 1%:99% (NIKS^GFP : primary keratinocytes). Results are shown as percent of expected value correcting for differences in plating efficiency between the two strains of keratinocytes.

Figure 2

Assessment of stratified squamous epithelia generated from 100% NIKS^GFP cells (A,E), 90%:10% (NIKS^GFP : primary keratinocytes) (B,F), 50%:50% (C,G) and 100% primary keratinocytes (D,H). H&E staining confirmed formation of distinct basal, spinous, granular, and squamous layers (A-D). Fluorescence microscopy was employed to discriminate tissue regions generated by NIKS^GFP cells (green) from tissue produced by unlabeled primary keratinocytes (EH). Sections were counterstained with Hoechst 33258 (blue) to visualize nuclei. A white dashed line in E-H denotes the dermal / epidermal boundary. Scale bar represents 100 μm.

Figure 3

Barrier function properties of composite skin substitute tissues as measured by skin surface electrical impedance (DPM units). The values obtained for native skin, and skin where the barrier had been disrupted by tape-stripping, are also presented. Data represents the mean from triplicate samples. Error bars represent the standard error of the mean.

Figure 4

90%:10% (NIKS^GFP : primary keratinocytes) chimeric skin substitute evaluated for expression of E-cadherin (A, E) keratin 1 (B, F) transglutaminase-1 (C, G) and filaggrin (D, H). GFP fluorescence (green) permitted visualization of tissue chimerism, whereas Hoechst 33258 (blue) identified nuclei localization. A white dashed line denotes the dermal / epidermal boundary. Scale bar represents 100 μm.

Figure 5

Detection of engrafted NIKS^GFP tissue. A representative section spanning the human / mouse interface of the wound edge from NIKS^GFP tissues 28 days after surgery was stained with H&E (A). Fluorescence microscopy was employed to visualize tissue generated by NIKS^GFP cells (green) in the adjacent section (B). Expression of human MHC Class I (red) in the epidermal region co-localized with NIKS^GFP tissue (C). Hoechst 33258 (blue) permitted visualization of nuclei. Scale bar represents 200 μm.

Figure 6

Detection of GFP fluorescence in engrafted skin substitute tissues generated from either 100% NIKS^GFP cells (A-C), 90%:10% (NIKS^GFP : primary keratinocytes) (D-F) or 50%:50% (G-I). At post-operative days 7 (A, D, G) and 14 (B, E, H), GFP-labeled cells were detected in all tissues in a dose-dependent manner. Although GFP fluorescence was detected in 100% NIKS^GFP tissues, no labeled cells were observed in chimeric tissues 28 days (C, F, I) after surgical application. Hoechst 33258 (blue) permitted visualization of nuclei. A white dashed line in D-I denotes the dermal / epidermal boundary. Scale bar represents 100 μm.

Figure 7

Detection of NIKS^GFP cells by genomic DNA analysis in chimeric tissues prior to and 28 days after surgical application. Whereas GFP-containing cells were detected in the positive control sample and chimeric tissues, no amplification of DNA encoding GFP was detected by PCR analysis in engrafted chimeric tissue 28 days after surgical application. Detection of Pv92 confirmed the presence of human DNA in tissues both prior to and after engraftment.