Fig 2: Expression of CD200 on freshly isolated as well as in vitro cultured juvenile/adult and fetal HDMEC. (a) Flow cytometry analysis of freshly isolated HDMEC derived from j/a human skin. CD31 and podoplanin were used to discriminate between blood endothelial cells (BEC, CD31+Podo−) and lymphatic endothelial cells (LEC, CD31+Podo+). HDMEC obtained from freshly isolated j/a contained 60.13 ± 17.35% of j/aBEC. Note the presence of two distinct subpopulations of LEC expressing different levels of podoplanin, namely LEC PodoHigh, which comprise 26.8 ± 6.75%, as well as LEC PodoLow representing 11.5 ± 2.95% of all freshly isolated j/aHDMEC. (d,e) Flow cytometric analysis of CD200 expression in freshly isolated j/aBEC and PodoHigh and PodoLow LEC. Whereas only 12.91 ± 5.86% of freshly harvested j/aBEC stained positive for CD200, 58.73 ± 16.97% of all j/aLEC expressed this marker. Please note that LEC PodoHigh are almost entirely positive for CD200 (98.1 ± 15.6%) (b,e), while LEC PodoLow exhibit only a moderate expression of this marker (36.9 ± 5.1%) (p = 0.0041) (c,e). (f–i) Flow cytometric analysis of cultured HDMEC at passage 1 (P1) derived from j/a skin. Cultured j/aHDMEC contains approximately 67.5 ± 18.21% of BEC as well as 31.8 ± 10.35% of LEC (f). Whereas 11.3 ± 2.0% of cultured j/aBEC exhibit CD200 expression (g,i), a significantly higher number of cultured j/aLEC was CD200-positive (48.7 ± 17.2, p < 0.0001) (h,i). (j–m) Flow cytometric analysis of cultured fHDMEC at (P1) consisting of 58.4 ± 17.4% of fBEC and 41.6 ± 11.5 of fLEC. Further, 9.06 ± 2.0% of fBEC show the expression of CD200 (k,m), while 51.9 ± 15.7% of fLEC are positive for CD200 (l,m) (p < 0.0001). Two-way unpaired student t-test with ns = not significant (p > 0.05); ** for p-value 0.001 to 0.01 (very significant); and *** for p < 0.001 (extremely significant) was used for statistical analysis, n = 5 independent j/a and fetal skin donors each.

Fig 3: Gene expression analysis in distinct LEC and BEC fractions. HDMEC were harvested from j/a skin, cultured, separated at P0 into CD200−/CD200+ LEC and CD200− BEC fractions and used directly for qRT-PCR. The relative gene expression levels were normalized to GAPDH housekeeping gene and displayed relative to CD200− BEC samples. (a–c) Analysis of adhesion molecules confirmed 11-fold upregulation of CD200 mRNA in j/aCD200+ LEC (31.70 ± 0.67) as compared to j/aCD200− LEC (2.88 ± 0.6; p < 0.0001) and j/aCD200− BEC (1 ± 0.41; p < 0.0001) (a). Another adhesion molecule, CD157 was also enriched in j/aCD200+ LEC (4.44 ± 0.48) as compared to j/aCD200− LEC (2.68 ± 0.54; p = 0.0107) and j/aCD200− BEC (1 ± 0.5; p = 0.0003) (b). The expression of CD62P selectin mRNA was reduced in j/aCD200+ LEC (0.22 ± 0.09) in comparison to j/aCD200− LEC (0.43 ± 0.35; p = 0.5773, ns) and j/aCD200− BEC (1.0 ± 0.25; p = 0.0186) (c). (d,e) Assessment of mRNA expression of lymphatic markers. J/aCD200+ LEC showed upregulated expression of PROX1 (66.34 ± 0.74) and podoplanin (76.93 ± 0.77) as compared to j/aCD200− LEC (PROX1: 43.55 ± 0.78, podoplanin: 55.13 ± 0.76; both p < 0.0001) and j/aCD200− BEC (PROX1: 1 ± 0.41, podoplanin: 1 ± 0.09; both p < 0.0001). (f,g) Chemokines CCL27 and CCL21 showed various mRNA expressions, whereas CCL27 mRNA was downregulated in j/aCD200+ LEC (2.32 ± 0.11), CCL27 mRNA was upregulated in j/aCD200− LEC (9.96 ± 0.30; p < 0.0001) as compared to j/aCD200− BEC expression (1 ± 0.33; p = 0.0018). In contrast, CCL21 was significantly upregulated in j/aCD200+ LEC (19.86 ± 0.41), whereas CCL27 showed reduced mRNA levels in j/aCD200− LEC (11.17 ± 0.66; p < 0.0001). Each bar represents mean ± SD of n = 3 biological samples. Experimental groups were compared using one-way ANOVA with Turkey’s multiple comparisons test. Asterisks denote significance as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Fig 4: Quantification of CD200 expression in transplanted vascDESS. (a,b) Immunofluorescence co-staining of j/a human skin against huCD200 (green) and huPodoplanin (a) or collagen IV (b). Podoplanin depicts human lymphatic endothelium, while collagen IV is expressed exclusively around human blood capillaries. White arrows in magnification insets indicate double-positive microvascular structures. (c,d) Immunoflorescence co-staining of vascDESS against huCD200 (green) as well as huPodoplanin (red), visualizing the human lymphatic capillaries (c) and huCollagen IV (red) depicting the human blood endothelium (d). (e) Quantification revealed that vascDESS contains a significantly higher number of blood (66.59 ± 20.95) than lymphatic capillaries (32 ± 8.34; ** p = 0.009). Presented values are the mean (±SD) of total numbers of human CD31+ or podoplanin+ vessels counted per 10× high-power field, n = 6 biological samples in each group. White arrows in magnification insets indicate double-positive microvascular structures. (f) Quantification of CD200 expression on blood and lymphatic vessels in vascDESS. Please note that CD200 is expressed mainly on the lymphatic vessels (41.67 ± 5.89), while blood vessels exhibit only minor expression of that marker (8.63 ± 2.95; **** p < 0.0001). The results are expressed as mean ± SD from n = 3 biological donors from 3 independent animal transplantation (6 animals in total). Cell nuclei are stained with Hoechst (blue). Scale bars 50 μm.

Fig 5: Expression of CD200R in human j/a skin as well as in non-vascDESS and vascDESS. (a,a’) Immunofluorescence pictures of j/a skin stained against CD31 (red) and CD200R (green). Note that CD200R+ immune cells (9.65 ± 3.56) are closely associated with CD31+ capillaries. (b,c) The expression of CD200R in non-vascDESS at one week (4 ± 1.55, p = 0.1551 vs. normal skin) (b) and 3 weeks (2.67 ± 1.55, p = 0.0565 vs. normal skin) (c) after transplantation. Once again, CD200R-expressing cells were located in close proximity to the CD31+ capillaries. (d,e) The expression of CD200R in vascDESS one week (26.5 ± 7.71, p < 0.0001 vs. non-vascDESS 1w, **** p < 0.0001 vs. human skin) (d) and three weeks (21.31 ± 5.4, p < 0.0001 vs. non-vascDESS 1w, p < 0.0001 vs. human skin) (e) post-transplantation. Numerous CD200R+ cells were visible throughout the entire dermal layer and adhered mainly to the CD200+ endothelium. (f) Quantification of CD200R-expressing cells in the dermal compartments of j/a human skin as well as in non-vasc and vascDESS after one and three weeks in vivo. Please note the significantly higher number of CD200R+ cells in the dermis of vascDESS, as compared to the native skin or non-vascDESS. In contrast, human vascDESS containing human blood and lymphatic capillaries demonstrated enhanced density of rat CD200R-positive cells at one (26.5 ± 7.71, p < 0.0001 vs. non-vascDESS one week, **** p < 0.0001 vs. human skin) and three (21.31 ± 5.4, **** p < 0.0001 vs. non-vascDESS 1w, p < 0.0001 vs. human skin) weeks in vivo (d,e). White arrows in magnification insets indicate CD200–CD200R interactions. (g) In vivo, significantly more CD200+ endothelial cells were associated with rat CD200R+ immune cells 1 w post-transplantation compared to CD200R+ cells that were non-associated with CD200+ endothelial cells (**** p < 0.0001). This difference disappears after 3 w in vivo (ns, p > 0.05). The results are expressed as mean ± SD from n = 3 biological donors from 3 independent animal transplantation (6 animals in total). Cell nuclei are stained with Hoechst (blue). Scale bars 100 μm and 50 μm (magnification insets).