Fig 2: Evaluation of CD200 expression in normal human juvenile/adult and fetal skin. (a–a’’’) Immunofluorescence triple staining of juvenile/adult (j/a) human skin with antibodies against CD31 (a) for human blood capillaries (red), podoplanin (a’) depicting human lymphatic endothelium (white), and CD200 (a’’) (green). (a’’’) represents a merged confocal Z stack. Whereas blood capillaries stain only CD31+ (empty arrows), lymphatic capillaries are CD31+Podo+ (white arrows). Note the presence of blood capillaries (CD31+Podo−) which are mainly negative for CD200 (empty arrows), as well as the presence of lymphatic capillaries (CD31+Podo+) showing the expression of CD200 marker (white filled arrows). (b–b’’’) Immunofluorescence triple staining of human fetal skin (f skin) against CD31 (b) delineating human blood capillaries (red), podoplanin (b’) depicting human lymphatic capillaries (white), and CD200 (b’’) (green). Note the presence of blood and lymphatic capillaries expressing CD200 (empty and filled white arrows, respectively). (b’’’) represents a merged overlay. (c) Quantification of blood and lymphatic capillaries in j/a and f skin. In the dermis of j/a, blood capillaries constitute approximately 68.8 ± 13%, whereas lymphatic endothelium represents 31.0 ± 10.6% of all vessels present in this skin type (*** p < 0.0001). In the f skin, blood endothelium comprises 73.0 ± 19% while lymphatic vessels constitute 27.0 ± 13% of all capillaries (** p < 0.001). (d) Quantification of CD200 expression on blood and lymphatic capillaries of j/a or f skin. Note the significantly higher expression of CD200 on j/a lymphatic capillaries as compared to the j/a blood capillaries (67.0 ± 16% vs. 14.0 ± 2.0%, respectively, *** p < 0.0001), whereas the expression of CD200 was similar in blood and lymphatic capillaries of human f skin (44.0 ± 8.0% vs. 64.0 ± 5.0%, respectively, p = ns), n = 5 independent j/a and fetal skin donors each. Cell nuclei are stained with Hoechst (blue). Scale bars 100 μm.

Fig 3: 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 4: Expression of CD200 on in vitro cultured blood and lymphatic capillaries in 3D collagen. (a) Collagen type I-based hydrogel, and stained for endothelial-specific marker CD31 (red) and lymphatic lineage marker PROX1 (green) (n = 5). Whereas double-positive CD31+PROX1+ capillaries represent lymphatics, single positive CD31+PROX1− demonstrate blood capillaries. The lower inset shows a higher magnification of the single nuclear PROX1 staining in lymphatic capillaries (arrows). (b) The merged confocal immunofluorescence showing CD31+ blood capillaries (red) and lymphatic capillaries positive for CD31+ (red) and LYVE1 (green), which is a lymphatic-specific marker. Insets show a magnification of single-stained LYVE1 lymphatic capillaries. (c) Triple stained image of human capillary network co-stained for PROX1+ (green) CD200+ (white) CD31+ (red). Accordingly, PROX1+CD200+CD31+.triple positive capillaries represent lymphatics expressing CD200 marker (filled arrows), whereas double-positive PROX1−CD200+CD31+ staining demonstrates blood capillaries positive for CD200 (empty arrows). (d) Triple co-staining for LYVE1+ (green) CD200+ (white) CD31+ (red). Whereas the majority of lymphatic capillaries (CD31+LYVE1+) were positive for CD200 (filled arrow), only a few blood capillaries (CD31+LYVE1−) expressed CD200 (empty arrows). The results are representative from n = 3 biological samples of EC populations. Cell nuclei are stained with Hoechst (blue). Scale bars: (a–d): 50 μm.

Fig 5: 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.