Journal: Development (Cambridge, England)

Article Title: Natural killer cells act as an extrinsic barrier for in vivo reprogramming

doi: 10.1242/dev.200361

Figure Lengend Snippet: Mφ and Gr1 + cell depletion during in vivo reprogramming. (A) Representative flow cytometry plots and quantification of F4/80 and CD11b double-positive populations in blood 16 h after liposome (LP) were administered intraperitoneally on day 3 of reprogramming. Mice were randomly selected ( n =4). Cells were gated from CD45 + cells. (B) Representative H&E and F4/80 immunohistochemistry of the spleen of mice treated with doxycycline and empty or clodronate LP for 7 days. (C) Dysplasia quantification in the pancreas of mice reprogrammed with empty or clodronate LP for 7 days ( n =6 for WT, n =10 for empty LP and n =11 for clodronate LP groups). (D) Representative flow cytometry plots and quantification of Ly6C and CD11b, and Ly6G and CD11b populations in blood on day 5 from reprogramming from mice treated with anti- Gr1 or isotype control antibodies. Cells were gated from CD45 + cells. (E) Representative H&E, neutrophil elastase (NE) and Gr1 immunohistochemistry in the spleen of partially reprogrammed mice treated with anti- Gr1 or isotype control antibodies for 7 days. (F) Dysplasia quantification in the pancreas of mice reprogrammed for 7 days with either anti- Gr1 or isotype control ( n =8 for WT and n =11 for i4F and anti- Gr1 groups). All data are mean±s.d.; * P <0.05, *** P <0.001 evaluated using the unpaired two-tailed Student's t -test. ns, not significant.

Article Snippet: For the depletion of Mφ, mice were treated with 200 μl of either clodronate or empty liposomes (Liposoma BV, CP-010-010) retro-orbitally on days 1, 3 and 6 of reprogramming.

Techniques: In Vivo, Flow Cytometry, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: Mechanical stretch induces hair regeneration through the alternative activation of macrophages

doi: 10.1038/s41467-019-09402-8

Figure Lengend Snippet: Macrophages are major mediators in stretch-induced hair regeneration. a Immunostaining of F4/80 indicated extensive macrophage infiltration in response to stretch (day 1 and day 7) that was sustained after stretch was released (day 9). b Representative FACS scatterplots display the percentages of CD45 + F4/80 + cells activated in response to stretch (day 1 and day 7) and release of stretch (day 9). c Immunostaining of F4/80 revealed decreased macrophage infiltration after skin was stretched for 3 days and 7 days in the clodronate injection (Clo) group compared with the control liposome injection (Ctrl) group. d Schematic of subcutaneous injection of clodronate liposomes to target macrophages. e Quantification of F4/80 + cells in the Clo and Ctrl groups; n = 3 for each group. f Stretch-induced hair regeneration is halted by clodronate injection; n = 6 for each group. g Real-time PCR for Ccl2 , Ccl3 , Ccl6 , Ccl7 , Ccl12 , and Ccl22 in response to stretch (day 1 and day 7) and release of stretch (day 9); n = 6 for each group. Statistical significance was determined using Student’s two-tailed t -test ( e ) or ANOVA followed by a Bonferroni post hoc test ( g ). Data are presented as means ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. Scale bars = 100 μm. Source data are provided as a

Article Snippet: For macrophage ablation, Clodrosome® (Liposomal Clodronate, Encapsula NanoSciences) or control liposomes were injected subcutaneously (0.05 mg/g) every other day from 4 days before to 4 days after stretching device was fixed.

Techniques: Immunostaining, Injection, Control, Liposomes, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Nature Communications

Article Title: Mechanical stretch induces hair regeneration through the alternative activation of macrophages

doi: 10.1038/s41467-019-09402-8

Figure Lengend Snippet: M2 polarization induced by strain alteration is crucial for regeneration. a , b Representative FACS histograms ( a ) and quantification ( b ) of the percentages of CD45 + F4/80 + CD206 + CD86 - M2 macrophages activated in response to stretch (day 1 and day 7) and release of stretch (day 9 and day 14); n = 6 for each group. c – e Real-time PCR for Arginase-1 (Arg1) ( c ), Ym1 ( d ), and Il4 ( e ) in response to stretch (day 1 and day 7) and release of stretch (day 9 and day 14); n = 6 for each group. f Quantification using flow cytometry of M2 macrophages in response to 20 or 33% strain; n = 3 for each group. g Dual immunostaining of F4/80 and Arginase indicated that double positive M2 macrophages were more conspicuous at day 14. h Schematic of subcutaneous injection with mannosylated clodronate liposomes (M-Clodronate) to deplete M2 macrophages. i Stretch-induced hair regeneration was perturbed by mannosylated clodronate liposomes (M-Clo) injection; n = 6 for each group. j Transplantation of unpolarized and polarized M1 or M2 macrophages into the back skin of mice during the telogen phase. Note only M2 macrophages caused hair regeneration, and the intensity of hair growth was proportional to the number of transplanted cells. The red circle at day 0 indicates the injection area; n = 6 for each group. Statistical significance was determined by conducting ANOVA followed by a Bonferroni post hoc test ( b – e ) or Student’s two-tailed t -test ( f ). Data are presented as means ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. Scale bar = 50 μm. Source data are provided as a

Article Snippet: For macrophage ablation, Clodrosome® (Liposomal Clodronate, Encapsula NanoSciences) or control liposomes were injected subcutaneously (0.05 mg/g) every other day from 4 days before to 4 days after stretching device was fixed.

Techniques: Real-time Polymerase Chain Reaction, Flow Cytometry, Immunostaining, Injection, Liposomes, Transplantation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Mechanical stretch induces hair regeneration through the alternative activation of macrophages

doi: 10.1038/s41467-019-09402-8

Figure Lengend Snippet: M2 macrophages produce growth factors to facilitate hair regeneration. a Real-time PCR of the sorted macrophages revealed the kinetics of gene expression of various growth factors in response to stretch (day 7) and release of stretch (day 9); n = 6 for each group. b Expression levels of growth factors within M2 (CD45 + F4/80 + CD206 + ) and non-M2 (CD45 + F4/80 + CD206 - ) macrophages according to real-time PCR; n = 6 for each group. c Dual immunostaining of K15 and Ki67 demonstrated that the injection of HGF-coated or IGF-1- coated beads during the telogen phase precociously activates K15 + hair stem cells. *Autofluorescence of hair shafts. Statistical significance was determined by conducting ANOVA followed by a Bonferroni post hoc test ( a ) or Student’s two-tailed t -test ( b ). Data are presented as means ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. Scale bar = 50 μm. Source data are provided as a

Article Snippet: For macrophage ablation, Clodrosome® (Liposomal Clodronate, Encapsula NanoSciences) or control liposomes were injected subcutaneously (0.05 mg/g) every other day from 4 days before to 4 days after stretching device was fixed.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Immunostaining, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Mechanical stretch induces hair regeneration through the alternative activation of macrophages

doi: 10.1038/s41467-019-09402-8

Figure Lengend Snippet: Schematic of the molecular basis of stretch-induced hair regeneration. a Mechanical stretch promotes BMP-2 production by activating fibroblasts and adipocytes, which inhibit hair regeneration; however, mechanical stretch also stimulates chemokine production to recruit macrophages into the stretched skin. The recruited macrophages undergo M2 polarization in response to a stretched macroenvironment. b Macrophages continue to undergo M2 polarization after stretch release and produce growth factors to facilitate anagen initiation

Article Snippet: For macrophage ablation, Clodrosome® (Liposomal Clodronate, Encapsula NanoSciences) or control liposomes were injected subcutaneously (0.05 mg/g) every other day from 4 days before to 4 days after stretching device was fixed.

Techniques: