Mechanisms of fibroblast cell therapy for dystrophic epidermolysis bullosa: high stability of collagen VII favors long-term skin integrity

Abstract

Here, we report on the first systematic long-term study of fibroblast therapy in a mouse model for recessive dystrophic epidermolysis bullosa (RDEB), a severe skin-blistering disorder caused by loss-of-function of collagen VII. Intradermal injection of wild-type (WT) fibroblasts in >50 mice increased the collagen VII content at the dermal-epidermal junction 3.5- to 4.7-fold. Although the active biosynthesis lasted <28 days, collagen VII remained stable and dramatically improved skin integrity and resistance to mechanical forces for at least 100 days, as measured with a digital 3D-skin sensor for shear forces. Experiments using species-specific antibodies, collagen VII-deficient fibroblasts, gene expression analyses, and cytokine arrays demonstrated that the injected fibroblasts are the major source of newly deposited collagen VII. Apart from transitory mild inflammation, no adverse effects were observed. The cells remained within an area <or=10 mm of the injection site, and did not proliferate, form tumors, or cause fibrosis. Instead, they became gradually apoptotic within 28 days. These data on partial restoration of collagen VII in the skin demonstrate the excellent ratio of clinical effects to biological parameters, support suitability of fibroblast-based therapy approaches for RDEB, and, as a preclinical test, pave way to human clinical trials.

Figure 1

Schematic representation of the cell therapy regimen applied in this study. (a) Fibroblasts were isolated from the skin of newborn WT pups of the C57Bl/6-TgH(Col7a1flNeo)288LBT inbred strain or from the C57BL/6-Tg(ACTB-EGFP)1Osb/J strain (EGFP⁺ fibroblasts). (b) Fibroblasts in passages 2–3 were trypsinized, extensively washed and adjusted to a concentration of 40 × 10⁶ cells/ml in sterile saline. (c) A volume of 0.5 ml of the cell suspension (i.e., 20 × 10⁶ cells) were injected intradermally into a 1.5 × 2.5 cm skin area on caudal back of the collagen VII hypomorph. After 7 days, the procedure was repeated. (d) One hour before dissection, skin stability was assessed. Skin samples were obtained from symmetrically marked injected (green), neighboring (yellow), and untreated (blue) areas for (immuno)histopathological, ultrastructural, and molecular analysis. (e) The animals were killed 1–120 days after the second injection. EGFP, enhanced green fluorescent protein; WT, wild type.

Figure 2

EGFP⁺ fibroblasts are present in the dermis for at least 21 days after intradermal injection, and actively synthesize protein. (a) At 24 hours after the second intradermal injection of 20 × 10⁶ EGFP⁺ fibroblasts, the cells were found in all layers of the dermis within the injected area. (b) Some fibroblasts migrated into the adjacent skin, ≤10 mm from the marked area. (c) No cells were present in the skin >10 mm from the injection site. (d,e) The number of EGFP⁺ fibroblasts decreases with time: (d) 7 days after treatment, (e) 21 days after treatment, and (f) 28 days after treatment; at this point only very few EGFP⁺ fibroblasts were seen. EGFP⁺ fibroblasts appear in green, nuclei in blue. The interrupted white line depicts the DEJZ. Bar = 50 µm. (g) RT-PCR demonstrating EGFP mRNA expression by the cells within the time span of 1–28 days. The expression increased between 1 and 7 days (−, untreated; +, treated), but gradually decreased thereafter, until at day 28 no expression was seen. GAPDH expression was used as internal standard. EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

Fibroblasts gradually undergo apoptosis in the dermis. TUNEL assays of the injected areas showed that both EGFP⁺ and WT fibroblasts slowly become apoptotic in a similar manner. (a) In untreated control occasional apoptotic cells were seen in the epidermis. In treated skin, apoptotic cells were found in the dermis (b) 7 days and (c) 21 days after injection of EGFP⁺ fibroblasts. The situation was similar after injection of WT fibroblasts from the same inbred strain: (d) untreated control, (e) 7 days, and (f) 21 days after injection. Apoptotic cells appear in red, nuclei in blue. Bar = 50 µm. EGFP, enhanced green fluorescent protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild type.

Figure 4

Injected fibroblasts actively express Col7a1 mRNA in the skin. Whole skin specimens obtained from WT, untreated (−), and treated (+) areas of the collagen VII hypomorph were used for total RNA extraction and RT-PCR amplification of collagen VII mRNA. In untreated skin, the expression of Col7a1 mRNA corresponds to ~8% of WT levels. In the areas treated with fibroblast injections, the expression of collagen VII increased between (a) 1 and (b) 7 days after injection and decreased gradually thereafter (21 days, c), until at (d) day 28, (e) day 70, and (f) day 100 the expression levels were similar to those in untreated areas. (g) Col7a1 mRNA expression in skin injected with fibroblasts isolated from a hypomorphic mouse. Note that in contrast to (b) WT cells, the injection of hypomorphic fibroblasts did not lead to increased Col7a1 mRNA expression at day 7 (g). Such an increase would have been expected, if the therapeutic cells exerted paracrine effects and stimulated keratinocytes to express collagen VII. (h) Semiquantification of Col7a1 mRNA levels by densitometry. Individual bands in panels a–g were normalized to GAPDH, and the WT band was set as 100%. The relative amount of Col7a1 mRNA in treated and untreated areas in % of WT are shown. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hypo, collagen VII hypomorph fibroblasts; RT, reverse transcription; WT, wild type.

Figure 5

Collagen VII deposition is restored at the DEJZ. (a) In untreated hypomorphic skin, collagen VII is strongly reduced. (b) Increased collagen VII levels at the DEJZ 70 days after injection of murine WT fibroblasts. (c) The increased levels persisted for at least 100 days. (d) WT control. (e) Injection of normal human fibroblasts increased deposition of human collagen VII at the DEJZ 7 days after injection, as shown with the NC1-F3 antibody, specific to human collagen VII.⁴⁴ This clearly demonstrates that fibroblast-derived collagen VII is incorporated into the DEJZ. Collagen VII appears in green, nuclei in red. Bar = 25 µm. (f) Semiquantitative confocal microscopy (n = 5 per group) using identical image settings demonstrated that the relative mean intensity of collagen VII fluorescence signals at the DEJZ was significantly increased after treatment with WT fibroblasts, as compared to untreated skin of the hypomorphic mice. At 70 days after injection, collagen VII signals were 4.7-fold higher and 100 days after injection 3.5-fold higher than in untreated skin (Mean plus standard deviation, the asterisks denote statistical significance, ***P < 0.001; *P < 0.05 in Student's t-test). After 100 days, the levels continued to decline, but were still about twofold higher at 120 days than in untreated skin (P < 0.05). (g) Ultrastructural analysis showed normal hemidesmosomes, but lack of anchoring fibrils in untreated skin of the collagen VII hypomorph. Magnification ×35,000. (h) Some filamentous aggregates with frayed ends (inset, arrows) could be discerned 70 days after fibroblast treatment in the skin of the collagen VII hypomorph. Magnification ×39,000. DEJZ, dermal–epidermal junction zone; WT, wild type.

Figure 6

3D force sensor shows enhanced resistance against mechanical forces after fibroblast treatment. (a) The silicon-based 3D force sensor with the tactile element, i.e., a silicone cylinder with hemispherical tip (red). The silicon chip is mounted and wire-bonded to a printed circuit board. The silicone cylinder serves as tactile element to apply frictional stress to the skin and to mechanically stabilize the fragile silicon structure. (b) Shear forces applied to the skin as a function of time. The forces were detected using the 3D force sensor shown in a. In this specific case, shear forces of 380.6 ± 134.6 mN were extracted. Fibroblast-treated skin resisted average forces of 341 mN, applied 20 times within 20 seconds, without blistering (c,e), whereas in untreated skin (d,f) dermal–epidermal separation and blister formation occurred (arrows), as demonstrated with hematoxylin/eosin staining of skin sections. (c,d) 70 days and (e,f) 100 days after fibroblast treatment. Bar = 50 µm. (g) Morphometric quantification of skin integrity after application of frictional stress, 70 and 100 days after fibroblast treatment. The graph depicts the percentage of blistered DEJZ. White columns: untreated skin. Black columns: treated skin (mean plus standard deviation). DEJZ, dermal–epidermal junction zone.

Figure 7

Fibroblast injections cause a transient inflammatory reaction but no immune response to collagen VII. (a) Positive immunofluorescence staining of CD11b, a marker for monocytes, dendritic cells, and macrophages, revealed a mild inflammatory infiltrate in the dermis 24 hours after fibroblast injection. (b) The number of CD11b-positive inflammatory cells was reduced after 7 days. (c) No inflammatory cells were observed in the skin 21 days after injection. (d) To analyze the inflammatory response to multiple fibroblast injections, which could be necessary in the treatment of DEB patients, collagen VII hypomorphs were injected four times with 1-week intervals. Seven days after the fourth injection a mild inflammatory infiltrate was observed, similar to that seen in c. CD11b-positive cells appear in red, nuclei in blue. The interrupted white line depicts the DEJZ. (e–h) Potential therapy-induced immune reactions to collagen VII were tested with indirect immunofluorescence staining. (e) Positive control with collagen VII antibody shows a linear signal at the DEJZ. Circulating antibodies to the DEJZ were screened with indirect immunofluorescence staining of normal mouse skin with 25 sera of fibroblast-treated mice. (f) A serum obtained 120 days after fibroblast injections showed no reactivity. (g) Only one of 25 sera of fibroblast-treated mice (obtained 56 days after injection) displayed a very faint positive reaction with the DEJZ. (h) However, when this serum was tested with immunoblotting, it failed to recognize collagen VII. 1: positive control, 2: the serum used in h. Arrow: collagen VII. Bar = 50 µm. DEJZ, dermal–epidermal junction zone.

Figure 8

Long-term responses of RDEB skin to fibroblast injections. This graphic illustration summarizes the molecular, cellular, and functional responses of RDEB skin to fibroblast injections as correlation of time. The values were obtained as shown in the Results and Figures 2–6. The X-axis represents days after the second fibroblast injection (not drawn to scale), the Y-axis depicts relative units for each parameter. Green line: EGFP⁺ fibroblasts in the skin. The amount was determined on the basis of EGFP mRNA expression in the skin. The maximal amount of EGFP mRNA found at 7 days was set as the value of 1.0. With time, the number of the EGFP⁺ fibroblasts gradually decreased, and after 28 days, no cells/EGFP mRNA synthesis were observed any more. Grey line: Col7a1 mRNA expression in the skin. The maximal amount of Col7a1 mRNA found at 7 days was set as the value of 1.0. Similar to EGFP expression, collagen VII mRNA decreased gradually and reached pretreatment levels after 28 days. Blue line: Collagen VII protein at the DEJZ. The injected cells generated a maximally 4.7-fold increase of collagen VII content at the DEJZ. This was set as the value of 1.0, present still 70 days after the second injection. The collagen VII content then slowly decreased to 0.75 relative units at 100 days, still a remarkably high value. Red line: Skin blistering as an indicator of adhesive collagen VII functions at the DEJZ. The pretreatment level of mechanically induced blistering was set as the value of 1.0. Within 7 days, the fibroblast injections resulted in a significant increase in adhesive collagen VII functions and in drastic reduction of skin blistering. These positive effects persisted for at least 70 days. Thereafter, blistering tendency slowly increased, but as compared to pretreatment levels was still low at 100 days. Taken together, it is notable that even though protein biosynthesis by the injected fibroblasts lasted maximally 28 days, the long half-life of collagen VII protein at the DEJZ contributed to significantly increased skin integrity during extended periods of time. DEJZ, dermal–epidermal junction zone; EGFP, enhanced green fluorescent protein; RDEB, recessive dystrophic epidermolysis bullosa.