Yolk sac-derived fetal macrophages suppress skin fibrosis and contribute to scarless wound healing

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Furthermore, after E17, neither the texture nor the dermis regenerate, resulting in dermal fibrosis. Currently, there are no detailed reports on the changes in inflammatory cells during this series of changes in the embryonic period. Thus, in this study, we identified the membrane surface markers of fetal macrophages and investigated their potential for use in inhibiting fibrosis. We found that fetal macrophages, which are derived from the yolk sac, accumulate at the wound site until E13 when the skin wound is completely regenerated. Using microarrays, we successfully identified specific markers of fetal macrophages. Furthermore, we demonstrated that transplantation of fetal macrophages into wounds at E14 or in adult animals suppressed fibrosis. These effects were confirmed using microarray results. Recent reports have suggested that yolk sac-derived macrophages are involved in fibrosis in various organs. The utilization of fetal macrophages suggests potential clinical applications in the treatment of fibrotic diseases such as myocardial infarction, pulmonary fibrosis, cirrhosis, and scleroderma, and may lead to innovative therapeutic approaches. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Medical research Biological sciences/Stem cells yolk sac-derived macrophages fetal macrophages scarless wound healing skin regeneration fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In mammals, skin wounds heal completely without scarring during early to mid-gestation. We previously reported that in mice, skin wounds created on embryonic day 13 (E13) regenerate completely, including restoration of the skin texture. In contrast, wounds created between E14 and E16 regenerate the dermal structure but not the skin texture, and wounds created after E17 do not regenerate either the skin texture or the dermis, resulting in dermal fibrosis [ 1 ] . There are no detailed reports on the changes in inflammatory cells during this series of changes in the embryonic period. On the other hand, approximately 80% of macrophages present in the skin up to E13 are derived from the yolk sac [ 2 – 4 ] . After E14, in addition to yolk sac-derived macrophages, the number of fetal-liver-derived monocyte-derived macrophages, which migrate throughout the body via monocytes from the fetal liver, gradually increases, replacing yolk sac-derived macrophages. Since the transition from complete skin regeneration to no regeneration correlates with changes in macrophage types, we termed the macrophages in E13 skin, which may contribute to complete skin regeneration, as “fetal macrophages.” For simplicity, this study refers to monocyte-derived macrophages that pass through the fetal liver as “adult macrophages.” Therefore, in this study, we identified the membrane surface markers of fetal macrophages and investigated their potential for use in inhibiting fibrosis. Results Accumulation of inflammatory cells in wounds created at each stage of mouse development When the embryonic wound site was stained using whole-mount immunostaining and observed under a confocal microscope, F4/80-positive macrophages accumulated at the wound site created at E13 (Fig. 1a). To quantify these findings, sections of each wound site were prepared, and immunofluorescence staining was performed using F4/80 as a macrophage marker. The results showed that macrophages accumulated at all wound sites at E13, E15, and E17 (Fig. 1b). The number of macrophages accumulating in the wound gradually increased from E13 and remained stable from E17 onwards (Supplementary Fig. S1). In contrast, Gr-1-positive granulocytes were detected in the liver at E15, but their accumulation in the wound was first observed from E17 onwards (Fig. 1b). The number of granulocytes increased further at P1. Search for fetal macrophage cell markers First, we enzymatically treated the entire skin to isolate macrophages and measured their numbers. At E13, approximately 8–10% of all skin cells were F4/80-positive, whereas at E18, only about 2% of all skin cells were F4/80-positive (Fig. 2a). Microarray analysis was performed using total RNA recovered from macrophages, and the cell membrane surface markers of each macrophage were analyzed. Gene Ontology (GO) analysis by microarray showed that pathways related to tissue regeneration, such as wound healing (GO:0042060) and cell adhesion (GO:0007155), were significantly enriched in E13 macrophages. In contrast, in E18 macrophages, pathways related to inflammatory responses, such as inflammatory response (GO:0006954), response to viruses (GO:0009615), and antigen processing and presentation (GO:0019882), were elevated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also showed that inflammation-related signaling pathways, such as the Jak-STAT signaling pathway (mmu04630) and the chemokine signaling pathway (mmu04062), were upregulated in E18 compared to E13. This suggests that while E13 macrophages contribute to regeneration without inducing inflammation, E18 macrophages enhance the inflammatory response [5, 6] . A complete list of GO terms and KEGG pathways with significant differences is presented in Supplementary Table S1. We isolated and identified the cell surface antigens expressed on E13 fetal macrophages. Cell surface antigens, including CD276, CD24, CD69, CD180, CD9, CD59, and CD38, were more than twice as abundant in fetal macrophages. We performed FACS using these candidates. CD276 and CD69 were not detected by FACS in fetal macrophages. Among these, CD9, CD24, CD38, and CD180, which could be clearly detected using existing fluorescent antibodies, were compared. CD9 and CD24 showed similar trends, while CD38 and CD180 also showed similar trends. Therefore, fetal macrophages could be separated using CD9 and CD180, which enabled cell identification, as shown in Fig. 2b. In fetal macrophages, CD180 continued to be expressed at each stage of development, whereas CD9 expression gradually decreased as the fetus developed (Fig. 2b). Adult macrophages showed low expression levels of CD180 and CD9. Next, we confirmed the expression of CD180 and CD9 in fetal macrophages in the skin of E13 by immunostaining and observed colocalization of F4/80-positive cells with CD180- and CD9-positive cells (Fig. 2c). Distribution of fetal macrophages in adult tissues We examined whether cells expressing surface antigens similar to those of fetal macrophages were present in adult mice. As a result, we confirmed that a small number of F4/80 + CD180 + CD9 + macrophages exist in the spleen of adult mice (Supplementary Fig. S2a). Conversely, they were found in high proportions in E18 placentas during the fetal period (Supplementary Fig. S2b). Transplantation of fetal macrophages into E14 skin wounds To investigate whether skin regeneration was promoted by increasing the population of fetal macrophages in the skin at E14, green fluorescent protein (GFP)-fetal macrophages (1×10 5 cells) were transplanted into the dermis at E14 using a capillary tube, and a wound was immediately created using a 1-mm diameter derma punch. The wound site was observed 72 h later. In the control E14 wound, the skin texture did not regenerate, and wound contraction, surrounding fibrosis, and edema-like changes were observed. However, in the fetal macrophage local injection group, these changes were reduced, and scarring was less noticeable (Fig. 3a). The adult macrophage local injection group showed strong white changes suggestive of fibrosis. Hematoxylin and eosin (HE) staining revealed that the wound diameter at 72 h was significantly smaller in the fetal macrophage injection group than in the controls (two-sided Student’s t-test; n = 3 per group; P = 0.0043). The local phosphate-buffered saline (PBS)-injected group showed a wound contraction to an average of 0.67 mm. The adult macrophage local injection group showed smaller wound contractions with an average of 0.90 mm. On the other hand, the fetal macrophage local injection group showed a wound contraction to an average of 0.44 mm (Fig. 3b, c). The number of blood vessels per unit area was significantly higher in the fetal macrophage injection group than in the controls (two-sided Student’s t-test, n = 9 per group; fetal macrophages vs. adult macrophages, P = 0.0016 ; PBS vs. adult macrophages, P = 0.0494 ). Masson’s trichrome (MT) staining showed that the PBS local injection group had an average of 6.16 vessels/0.01 mm², while the fetal macrophage local injection group had an average of 4.05 vessels/0.01 mm². Thus, no significant difference was observed in comparison with normal skin. The adult macrophage local injection group showed a significant increase in the number of blood vessels, with an average of 10.11 per 0.01 mm² (Fig. 3b, d). In addition, the number of alpha smooth muscle actin (αSMA)-positive cells was significantly lower in the fetal macrophage local injection group compared with the adult macrophage local injection group or controls (two-sided Student’s t-test; n = 6 per group; fetal macrophages vs. adult macrophages, P = 1.8×10⁻¹⁰ ; PBS vs. adult macrophages, P = 8.2×10⁻¹¹ ; adult macrophages vs. normal, P = 1.7×10⁻⁷ ). αSMA staining showed no significant difference between the PBS local injection group and the fetal macrophage local injection group compared to normal skin. The number of αSMA-positive cells increased in the adult macrophage local injection group (Fig. 3b, e). Three groups of wounds were analyzed using Primos🄬, and the unevenness of the wounds was observed. In the fetal macrophage local injection group, the wounds were flattened, while in the PBS local injection group, the unevenness was distinct. In the adult macrophage local injection group, the unevenness was not distinct due to overall edema-like changes, but the wounds were deep (Fig. 3f). To confirm whether the administered GFP-fetal macrophages were undergoing transformation into other cells, immunostaining for vimentin, pan-cytokeratin, αSMA, Ly6G, and CD31 was performed using frozen sections 72 h after administration. GFP-positive and strongly vimentin-positive cells were confirmed. Since other macrophages were weakly positive for vimentin, the possibility of their transformation into fibroblasts was suggested. Administration of fetal macrophages to adult wounds Next, we examined the effects of fetal macrophages on adult skin wounds. We created 8-mm bilateral full-thickness skin defects on the dorsal skin of B6 mice and observed them over time (Fig. 4a). There was no significant difference in wound size between the fetal macrophage-treated and control groups (Fig. 4b). Vascular regeneration was observed macroscopically when tissue sections were collected on day 4 (Fig. 4c). αSMA staining on day 4 revealed relatively thick vessels within the wound and muscle tissue (Fig. 4d). When the tissue sections on day 4 were compared using MT staining, no significant changes were observed in the central region. However, promotion of epithelialization was confirmed (Fig. 4e). When MT staining was performed on tissue sections collected on day 11, differences in the collagen fibers stained with aniline blue were observed in the scarred area (Fig. 4f). In the fetal macrophage-treated group, aniline blue was stained at the same intensity as in normal skin, indicating randomly arranged mature collagen fibers. In contrast, in the PBS-treated group, aniline blue stained weakly, the collagen fibers were immature, and the fibers exhibited a unidirectional regular arrangement typical of the wound healing process. These findings suggest that fetal macrophage administration may normalize collagen fibers within scars. The results were evaluated using a Modified Mouse Masson Trichrome Scar Scale (MMTSS). The MMTSS score was significantly reduced in the fetal macrophage local injection groups compared to that in the controls (two-sided Student’s t-test without correction for multiple comparisons; n = 3 per group; P = 0.0325 ). (Supplementary Fig. S3). A comparison using αSMA was also performed, but no significant differences were observed. In summary, the administration of fetal macrophages to adult skin wounds resulted in angiogenesis and epithelialization by day 4; however, no significant effect on wound healing was observed, although dermal collagen fibers appeared normalized. Fetal macrophages were collected from GFP mice and transplanted into the wound sites of B6 mice. On day 4, tissue sections were prepared, and F4/80-positive and GFP-positive cells were observed at the wound site (Fig. 5). Some of the GFP-fetal macrophages remained F4/80-positive, while others became negative. This suggests that fetal macrophages undergo transformation and control fibrosis. Immunohistochemical staining with vimentin, pan-cytokeratin, αSMA, Ly6G, and CD31 was performed to determine the cell types into which the administered fetal macrophages had differentiated into, but no obvious transformation was confirmed. In summary, these results suggest that fetal macrophages transform into fibroblasts at the wound site during the fetal period. However, as this was not confirmed in adult animals, it is possible that regeneration does not occur under mature inflammatory conditions. It has been suggested that fetal macrophages in adult animals control inflammation and suppress scarring and fibrosis. Discussion In mammals, skin injuries inflicted before mid-gestation can completely regenerate. Longaker et al. reported that when wounds were inflicted on the lips of macaques, which are similar to humans, during gestation, the presence or absence of scars changed depending on the gestational age, and they referred to this as “transition wound” [ 7 – 9 ] . Similarly, in mice, complete regeneration occurs up to E13, whereas incomplete regeneration occurs at E14 and E15, with the disappearance of skin appendages and preservation of the dermal structure. From E16 onwards, the dermis becomes scarred [ 1 ] . Macrophages actively accumulated at the E13 wound site, where the skin was completely regenerated. Based on a report by Hoeffel et al., these macrophages were considered to correspond to the yolk sac-derived macrophages [ 2 , 3 ] . Macrophages derived from the yolk sac are intermediate cells between erythro-myeloid progenitor cells and tissue-resident macrophages, and are considered immature tissue-resident macrophages. Tissue-resident macrophages maintain homeostasis through repeated self-replication. By focusing on the role of macrophages in this switch in regenerative capacity, we have demonstrated that yolk sac-derived fetal macrophages suppress fibrosis and scar formation, which is a novel finding. Complete skin regeneration in fetuses is caused by a combination of multiple factors, rather than a single factor. These factors include the suppression of inflammation and fibrosis, balance of specific growth factors, fetus-specific extracellular matrix, weak mechanical contraction environment, and fetal cell activity [ 10 , 11 ] . Inflammatory responses typically promote fibrosis and hinder regeneration. Macrophages are classified into M1 and M2 subtypes; however, in adult skin wound healing, M2 macrophages mainly promote fibrosis at the wound site [ 12 ] . Microarray results showed that fetal macrophages did not exhibit increased expression of markers specific to M1 (Nos2, IL12b, Ciita, IL6) or M2 (Arg1, Retnla (Fizz1), Ch3l3 (Ym1), and Mrc1 (CD206)) compared to adult macrophages, indicating that they possess properties distinct from those of M1 and M2 macrophages. Furthermore, as shown in the results, fetal macrophages exhibited an immature inflammatory response, with underdeveloped Jak-STAT and chemokine signaling pathways, and no signs of exacerbated inflammation were observed. In particular, fetal macrophages accumulating at the wound site on E13 exhibited low inflammatory induction and abundant expression of wound healing-related genes. In contrast, inflammatory responses and immune-related gene expression were predominant in the adult macrophages. These results indicate that fetal macrophages have non-inflammatory characteristics that differ from those of general macrophages and do not contribute to scar formation. Furthermore, the establishment of isolation methods using surface markers, such as CD180 and CD9, will serve as a bridge to future functional analyses and clinical applications. Although the results of this microarray study cannot directly demonstrate the molecular mechanisms based on previously reported wound healing and inflammatory response pathways, our results support these pathways through their association with known signaling pathways. This study complements and expands upon existing reports by clarifying the role of macrophages in embryonic skin wound regeneration from both gene expression and transplantation perspectives. The transplantation of fetal macrophages into E14 skin and adult skin wounds suppressed fibrosis and improved the properties of collagen fibers. This is consistent with the anti-fibrotic effects of yolk sac-derived macrophages on the heart and other organs. In the heart, approximately 80% of the resident macrophages are yolk sac-derived, as reported in a study using tamoxifen-treated Cx3cr1CreER-YFP:R26Td mice [ 13 ] . Tissue-resident macrophages derived from the yolk sac of the heart are not involved in inflammation during the acute phase of myocardial infarction, but play an important role in remodeling after myocardial infarction. Furthermore, tissue-resident macrophages in the heart suppress fibrosis and limit harmful cardiac remodeling [ 14 ] . Microglia are derived almost entirely from yolk-sac macrophages in the brain and spinal cord. The role of microglia in spinal cord injury is to promote the repair of the central nervous system, including phagocytosis of growth-inhibiting debris and stimulation of nerve fiber extension. However, these processes also depend on interactions with other cells, and the exact mechanisms remain unclear. Nevertheless, it has been reported that microglia-dominant inflammation leads to preservation and increased axons at the injury site, as well as functional recovery [ 15 ] . Macrophages in fetal testes are almost entirely derived from the yolk sac [ 16 ] . Macrophages derived from the yolk sac have been suggested to regulate fibrosis during cirrhosis [ 17 ] . Yolk sac-derived macrophages are involved in suppressing fibrosis in various organs. Their effects—such as direct action on fibroblasts, regulation of profibrotic pathways, and protection of surrounding cells—have been proposed; however, the mechanism remains unclear. It is possible that inflammation is controlled and fibrosis is suppressed through interactions with fibroblasts. Although the molecular mechanism is unclear, the microarray analysis results of this study, such as Jak-STAT signaling and chemokine pathway immaturity, support the anti-inflammatory effects of fetal macrophages. Clinically, the findings obtained in this study suggest the possibility of new cell therapies not only for inhibiting skin fibrosis and scarring but also for treating multi-organ fibrotic diseases such as myocardial infarction, pulmonary fibrosis, and cirrhosis. The direct application of fetal macrophages is ethically constrained. However, by mimicking their characteristics and developing soluble factors or cell induction methods, their applications in regenerative medicine and fibrosis treatment are anticipated. A limitation of this study is that complete regeneration was not observed in adult mice or at E14 wound sites. In particular, owing to significant differences in inflammatory responses and tissue environments between adult animals and fetuses, transplantation of fetal macrophages alone may have suppressed fibrosis but failed to promote adequate wound healing. This issue remains a challenge for future research, and it is necessary to consider treatment methods that combine other factors with the cells. Materials and Methods Collection of macrophages All the experiments were conducted in accordance with the Keio University guidelines for animal and genetically modified animal experiments (approval numbers A2022-279 and D2013-021). Additional ARRIVE-compliant reporting: Study design: Fetal macrophage transplantation groups were compared to sham-operated controls. A single mouse was used as the experimental unit. Sample size: Three mice were assigned to each treatment group. Sample sizes were based on previous studies, and no a priori power calculations were performed. Inclusion/exclusion criteria: All mice were included and no exclusion criteria were applied. Randomization: Animals were randomly assigned to treatment groups using a random number generator. Blinding: Investigators assessing histological outcomes were blinded to group allocation. Outcome measures The primary outcome measure was collagen deposition (MT staining). Secondary outcomes included angiogenesis and fibrosis scores. Experimental animals: C57BL/6J mice (E13–14 embryos) were obtained from Sankyo Labo Service Corporation, Inc. Experimental procedures: Skin wounds were generated on E13. Fetal macrophages (1×10 5 ) were transplanted into the wound edges. The procedures were performed under isoflurane anesthesia. Animal housing and husbandry: Mice were housed in specific pathogen-free conditions under a 12-h light/dark cycle, with standard chow and water ad libitum. Animal care and monitoring: The mice were monitored daily. No unexpected adverse events were observed. No humane endpoints were included. Female Slc:ICR mice, C57BL/6JmsSlc mice, and C57BL/6-Tg (CAG-EGFP) mice at 13–18 days of gestation were purchased from Sankyo Labo Service Corporation, Inc. On the day of delivery, the mice were euthanized by cervical dislocation after inhalation of anesthesia in the morning. The abdomen was incised, fetuses were removed, washed with PBS to remove maternal blood, and the skin was collected using scissors. The collected skin samples were minced with scissors. After homogenization, collagenase type 1 treatment was performed, followed by stirring at 37°C in an incubator. The mixture was then filtered through a 100 µm cell strainer to recover the cells. The E13 and E18 placenta and E18 umbilical cord were processed using the same method described above. Adult spleens were obtained from 8-week-old male ICR mice. The spleens were removed under general anesthesia, homogenized, treated with collagenase type 1, stirred in a 37°C incubator, filtered through a 100 µm cell strainer, and the cells were recovered. The collected cells were isolated by magnetic-activated cell sorting (MACS). Anti-F4/80 Micro Beads UltraPure (Miltenyi Biotec) was used as the antibody, and an MS column was used for collection. The cells were washed with MACS (FACS) buffer and centrifuged at 300 g for 5 min. The supernatant was discarded, and the cells were incubated at 4°C with a 1/100 dilution of Anti-F4/80 Micro Beads UltraPure while rotating for 15 min. The antibody-labeled cells were washed with MACS buffer and centrifuged at 300 g for 5 min. The supernatant was discarded, the cells were diluted with MACS buffer, passed through an MS column set up in a magnetic field, and F4/80-positive cells were recovered. As described above in the animal experiment, antibodies used included anti-CD45, anti-CD11b, anti-F4/80, anti-CD180, and anti-CD9 (BioLegend), which were incubated at 4°C for 30 min with stirring. Fetal and adult macrophages were obtained using flow cytometry (Beckman MoFlo XDP). Microarray and analysis Microarray analysis was performed using the Clariom_S_mouse (Thermo Fisher Scientific). The analysis was normalized using the RMA method. The R package “limma” was used to test for gene expression variations. Creation of mouse fetal skin wounds Mice at 13–18 days of gestation were anesthetized with 5% isoflurane, and anesthesia was maintained with 2–3% isoflurane. The abdomen was incised to confirm the presence of the uterus. The uterus was incised under a stereomicroscope to confirm fetal development. The amniotic membrane was incised, and a wound was created on the dorsal skin using microsurgical microscissors. At E13–E14, the amniotic membrane was sutured with 9-0 nylon. At E15 and beyond, the uterine wall was sutured using 9-0 nylon. Prior to closing the abdomen, ritodrine hydrochloride was administered to the abdominal cavity, and the abdomen was sutured with 5-0 nylon to complete the fetal surgery. The animals were subsequently kept warm and observed. At 24 h and 72 h postoperatively, the animals were euthanized via cervical dislocation after inhalation anesthesia with 5% isoflurane, and the abdomen was opened to retrieve the fetus. Administration of fetal macrophages to E14 fetal wound sites Using the above method, fetal surgery was performed on mice at day 14 of gestation. The amniotic membrane was incised, and capillary tubes were used to administer 1×10 5 GFP-fetal macrophages (N = 4), PBS (N = 4) to the dermis where the wound was to be created, or 1×10 5 E18 GFP-adult macrophages (N = 4). Immediately afterward, a circular wound of uniform size was created on the dorsal skin using a 1-mm dermal punch. The amniotic membrane was sutured using 9-0 nylon. Ritodrine hydrochloride was administered to the abdominal cavity, and the abdomen was sutured with 5-0 nylon to complete the fetal surgery. Subsequently, the fetus was kept warm and observed. After 72 h, 5% isoflurane inhalation anesthesia was administered, cervical dislocation was performed, the abdomen was incised to retrieve the fetus, and the wound site was examined. Surface irregularities of the skin were measured using Primos® (Integral Co., Ltd.). Local administration of fetal macrophages to adult wound sites Male mice aged 8–10 weeks were shaved after inhalation anesthesia and depilated using a depilatory agent. The skin was stretched, and identical wounds were created on the dorsal skin using an 8-mm derma punch on both sides. The skin collected adjacent to the wound was used as normal skin for tissue sections. On the left wound site, GFP-fetal macrophages recovered using MACS were diluted in 100 μL of PBS at a concentration of 1 × 10 5 cells and administered locally. The right side received an equal volume of PBS. The wounds were protected with Permirole® and Tegaderm®, and kept warm with frequent observations. Wound contraction rates were analyzed using the ImageJ. Histological stain and immunohistochemistry The collected tissue was fixed in 4% paraformaldehyde (PFA) at 4°C for 24 h and then paraffin-embedded. After paraffin embedding, tissue sections were prepared at a thickness of 5 µm. The tissue sections were deparaffinized using slides. MT staining was performed using Ponceau xylidine, acid fuchsin, and aniline blue. HE staining was performed. αSMA was stained using DAB and a Leica automatic staining device. Frozen sections were fixed in 4% PFA at 4°C for 24 h, replaced with 20% sucrose, replaced with 30% sucrose, replaced with 40% sucrose, and then frozen and embedded in an OCT compound. After frozen embedding, immunostaining was performed on tissue sections sliced to a thickness of 10 μm. For whole-mount staining, after fixation with 4% PFA at 4°C for 24 h, the samples were washed with PBS, incubated with antibody for 24 h at 4°C, washed again with PBS, and then immunostained. The following antibodies were used: Anti-F4/80Micro Beads Ultra Pure (Miltenyi Biotec) Alexa Fluor 594 anti-mouse/human CD11b (BioLegend) APC-Cy7 anti-mouse/human CD11b (BioLegend） APC anti-mouse F4/80 (BioLegend) FITC anti-mouse F4/80 (BioLegend) PE anti-mouse CD180 (BioLegend) APC anti-mouse CD9 (BioLegend) PE-Cy7 anti-mouse CD45 (BioLegend) Anti α-SMA (SIGMA) Anti-IBA1 (Abcam) Vimentin (Santa Cruz) Anti-mouse Ly6G (Gr-1) (Abcam) Anti-CD31 (Millipore) Observations were performed using Keyence BZ-X700 and Canon FV3000 confocal fluorescence microscopes. Organizational assessment The Manchester Scar Scale is an index for the pathological evaluation of human scars; therefore, it was adapted for mice, and the number of evaluation items was reduced [ 18 ] . Subsequently, three individuals conducted a blinded evaluation. Analyses All the statistical analyses were performed using Microsoft Excel. Data are expressed as mean ± standard error of the mean (SEM). For comparisons between the two groups, a two-sided unpaired Student’s t-test was used without correction for multiple comparisons. The normality of data distribution was assessed using the Shapiro–Wilk test, and variance between groups was assumed to be equal. A significance level of α = 0.05 was adopted. The exact P values, sample sizes (n), and details of the statistical tests are reported in the figures. In each analysis, n refers to biologically independent samples; for fetal experiments, the number of individual fetuses analyzed per group (typically n = 3–4); for adult wound healing experiments, the number of mice analyzed (typically n = 3–9, depending on the experiment); and for histological quantification, the number of tissue sections or fields examined per animal. The error bars in the figures represent the SEM. All the tests were two-sided. Declarations Acknowledgments This study was conducted with the cooperation of Toshiko Toda, Hiromi Fujita, and university student Kazuhiro Takada, who assisted in experiment preparation and animal care. We express our gratitude toward them. Funding This work was supported by JSPS KAKENHI, Grant Numbers JP21K16926 and JP25K12938. Author contributions Shigeki Sakai conducted the experiments, collected data, and wrote the manuscript. Keisuke Okabe conducted wound healing experiments in the dorsal region of ICR mice from E13 to P1. Kento Takaya and Yukari Nakajima took the photographs. Kazuo Kishi designed the study, supervised the experiments, and revised the manuscript. Data availability The microarray data generated in this study were deposited in the NCBI Gene Expression Omnibus (GEO) GSE306564. During peer review, reviewers can access data using the following token: [sxaxmceuhxgnpix]. Other datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request. Additional Information The authors declare the following competing interests: S. (Shigeki Sakai) and K. (Kazuo Kishi) are listed as inventors of patent applications related to the methods described in this study. The authors declare no conflicts of interest. This study was supported by a Grant-in-Aid for Scientific Research (C). The funders had no role in the study design, data collection, or data interpretation. References Takaya, K. et al. Actin cable formation and epidermis-dermis positional relationship during complete skin regeneration. Sci. Rep. 12 , 15913 (2022). Hoeffel, G. et al. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. J. Exp. Med. 209 , 1167–1181 (2012). Hoeffel, G. et al. 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Emerging concepts in myeloid cell biology after spinal cord injury. Neurotherapeutics 8 , 252–261 (2011). DeFalco, T., Bhattacharya, I., Williams, A. V., Sams, D. M. & Capel, B. Yolk-sac–derived macrophages regulate fetal testis vascularization and morphogenesis. Proc. Natl Acad. Sci. U. S. A. 111 , E2384–E2393 (2014). Ramachandran, P. et al . Resolving the fibrotic niche of human liver cirrhosis at single cell level. Nature 575 , 512–518 (2019). Sakai, S., Aramaki-Hattori, N. & Kishi, K. Fetal fibroblast transplantation via ablative fractional laser irradiation reduces scarring. Biomedicines 11 , 347 (2023). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigureS1.tif SupplementarytableS1a.tif SupplementarytableS1b.tif SupplementarytableS1c.tif SupplementaryFigureS2a.tif SupplementaryFigureS2b.tif SupplementaryFigureS3.tif SupplementaryTableS1GOtermsfull.xlsx Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":741863,"visible":true,"origin":"","legend":"\u003cp\u003eE13 back skin wound, 24 h later. Center: E14 back skin wound, 24 h later. Right: E15 back skin wound, 24 h later. Scale bar: 200 μm. Green: F4/80; Red: CD31.\u003c/p\u003e\n\u003cp\u003eFig. 1b\u003c/p\u003e\n\u003cp\u003eMacrophages and granulocytes 24 h after dorsal skin wounding at E13, E15, E17, and P1. Green: F4/80; blue: DAPI; red: Gr-1. Scale bar: 200 μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/bcca0083a46589353d045560.png"},{"id":92400996,"identity":"17d24094-ce38-47ce-b242-f3dc0c043e1d","added_by":"auto","created_at":"2025-09-29 10:13:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":475309,"visible":true,"origin":"","legend":"\u003cp\u003ea\u003c/p\u003e\n\u003cp\u003eBack skin cells on day 18.5 of gestation, sorted using CD11b and F4/80.\u003c/p\u003e\n\u003cp\u003eFig. 2b\u003c/p\u003e\n\u003cp\u003eRelationship between F4/80-positive cells and CD180/CD9 in the skin, compared at 13.5, 15.5, and 17.5 days of gestation.\u003c/p\u003e\n\u003cp\u003eFig. 2c\u003c/p\u003e\n\u003cp\u003eF4/80-positive cells with CD180 and CD9 expression in normal E13 skin. Blue: DAPI; Green: F4/80; Purple: CD9; Gray: CD180. Scale bar: 40 μm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/5580fd64675fa9642b1031d1.png"},{"id":92401195,"identity":"2ebc7270-bc3a-4f4f-86c2-0b380933ec44","added_by":"auto","created_at":"2025-09-29 10:21:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1132473,"visible":true,"origin":"","legend":"\u003cp\u003ea\u003c/p\u003e\n\u003cp\u003eBack skin wound at 14.5 days post-conception. Macroscopic findings 72 h after wound creation. Top: fetal macrophage injection group; Middle: PBS injection group; Bottom: adult macrophage injection group. Scale bar: 1000 μm.\u003c/p\u003e\n\u003cp\u003eFig. 3b\u003c/p\u003e\n\u003cp\u003eHE, MT, and αSMA staining. Top row: fetal macrophage injection group; Middle row: PBS injection group; Bottom row: adult macrophage injection group. Green dotted line: wound site; Red arrows: blood vessels; Yellow arrow: αSMA-positive cells. Scale bar: 100 μm.\u003c/p\u003e\n\u003cp\u003eFig. 3c\u003c/p\u003e\n\u003cp\u003eDiameter of the wound site 72 h after creation of an E14 dorsal skin wound. Data are presented as mean ± SEM (n = 3 per group). Statistical significance was determined using a two-sided Student’s \u003cem\u003et\u003c/em\u003e-test; \u003cem\u003eP\u003c/em\u003e = 0.0043.\u003c/p\u003e\n\u003cp\u003eFig. 3d\u003c/p\u003e\n\u003cp\u003eNumber of blood vessels per unit area. Data are presented as mean ± SEM (n = 9 per group). Two-sided Student’s \u003cem\u003et\u003c/em\u003e-test; fetal macrophages vs. adult macrophages, \u003cem\u003eP\u003c/em\u003e = 0.0016; PBS vs. adult macrophages, \u003cem\u003eP\u003c/em\u003e = 0.0494.\u003c/p\u003e\n\u003cp\u003eFig. 3e\u003c/p\u003e\n\u003cp\u003eNumber of αSMA-positive cells per unit area. Data are presented as mean ± SEM (n = 6 per group). Two-sided Student’s \u003cem\u003et\u003c/em\u003e-test; fetal macrophages vs. adult macrophages, \u003cem\u003eP\u003c/em\u003e= 1.8 × 10⁻¹⁰; PBS vs. adult macrophages, \u003cem\u003eP\u003c/em\u003e = 8.2× 10⁻¹¹; adult macrophages vs. normal, \u003cem\u003eP\u003c/em\u003e = 1.7× 10⁻⁷.\u003c/p\u003e\n\u003cp\u003eFig. 3f\u003c/p\u003e\n\u003cp\u003eThree-dimensional images of wound unevenness obtained using Primos® (laser depth gauge). Top: fetal macrophage local injection group; middle: PBS local injection group; bottom: adult macrophage local injection group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/2bc3d8d10b40ac4d421b6d51.png"},{"id":92400999,"identity":"58e88f41-23e7-4921-8bf3-cf2704f7f786","added_by":"auto","created_at":"2025-09-29 10:13:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1460264,"visible":true,"origin":"","legend":"\u003cp\u003ea\u003c/p\u003e\n\u003cp\u003ePaired injury model in the same mouse. Left: fetal macrophage local injection side; right: PBS local injection side.\u003c/p\u003e\n\u003cp\u003eFig. 4b\u003c/p\u003e\n\u003cp\u003eTemporal contraction rate of wounds in adult mice. Orange: PBS local injection group; blue: fetal macrophage local injection group.\u003c/p\u003e\n\u003cp\u003eFig. 4c\u003c/p\u003e\n\u003cp\u003eMacroscopic appearance of wounds on day 4 after creation, during tissue collection. Left: fetal macrophage local injection side; right: PBS local injection side.\u003c/p\u003e\n\u003cp\u003eFig. 4d\u003c/p\u003e\n\u003cp\u003eαSMA staining of the fetal macrophage local administration group on day 4 after wound creation. Red arrow: blood vessel. Scale bar = 500 μm.\u003c/p\u003e\n\u003cp\u003eFig. 4e\u003c/p\u003e\n\u003cp\u003eMasson’s trichrome (MT) staining on day 4 after wound creation. Left: fetal macrophage local injection side; right: PBS local injection side. Blue arrow: epithelialization tip. Scale bar = 500 μm.\u003c/p\u003e\n\u003cp\u003eFig. 4f\u003c/p\u003e\n\u003cp\u003eMT staining of tissue sections on day 11 after wound creation. Left: fetal macrophage local injection side; right: PBS local injection side. Scale bar = 500 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/dcbc1b3074aaa3fcbb24c407.png"},{"id":92399903,"identity":"e8a0a81f-fa2c-41e4-b4b9-fcb05c9ea8bd","added_by":"auto","created_at":"2025-09-29 10:05:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":762518,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining on day 4 in the group that received local injections of fetal macrophages into an 8-mm dorsal skin wound in B6 mice. Red: F4/80; green: GFP; blue: DAPI. Scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/61e8c3f76ea84a9c06e0d179.png"},{"id":97139356,"identity":"b6a327c9-f810-45fd-b461-8b77f4c39322","added_by":"auto","created_at":"2025-12-01 10:00:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4804782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/ff300290-f7bf-4aac-b3f9-4c86fbea1ed4.pdf"},{"id":92399899,"identity":"e9691641-c9e6-4c22-a536-8c344aa6084b","added_by":"auto","created_at":"2025-09-29 10:05:36","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2765105,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/ab2e658e96e188e76e812621.tif"},{"id":92399900,"identity":"60b7dbfc-d832-440a-8a86-63c8ba1dc2db","added_by":"auto","created_at":"2025-09-29 10:05:36","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2765101,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS1a.tif","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/d0c57929bee2f0cac4beb3fb.tif"},{"id":92399915,"identity":"99b593f3-2c7f-40b3-bc81-cfdefff1ca8d","added_by":"auto","created_at":"2025-09-29 10:05:37","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2765101,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS1b.tif","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/47dae63c2efbcc05ad70fa98.tif"},{"id":92399916,"identity":"54ebebff-b658-437c-b61e-d48df090f63a","added_by":"auto","created_at":"2025-09-29 10:05:37","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2765101,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS1c.tif","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/8efb517290b3a7babee449ae.tif"},{"id":92399907,"identity":"6a77d187-306c-4249-bcf1-c2c436c89ce3","added_by":"auto","created_at":"2025-09-29 10:05:37","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2765100,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2a.tif","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/78d7792011969ff21dd147b8.tif"},{"id":92399919,"identity":"0936c10f-2005-48db-aa43-2aae2a108f5a","added_by":"auto","created_at":"2025-09-29 10:05:37","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2765100,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2b.tif","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/55293f62605ab4ebbae34a5e.tif"},{"id":92399908,"identity":"fee54ed8-2b5c-4102-90a9-35ed09990f2a","added_by":"auto","created_at":"2025-09-29 10:05:37","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2765101,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/59a2d0b244a23b58769b2ee4.tif"},{"id":92402006,"identity":"f7985d5f-7727-4374-a84d-06527df9ee71","added_by":"auto","created_at":"2025-09-29 10:29:37","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":11312,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1GOtermsfull.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/902cadecba914e8c7e173460.xlsx"},{"id":92399911,"identity":"8fdd7d83-de01-4314-a667-1e2277badecf","added_by":"auto","created_at":"2025-09-29 10:05:37","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":13959,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7478492/v1/8c356bd4c12619cdd1f7c9c1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Yolk sac-derived fetal macrophages suppress skin fibrosis and contribute to scarless wound healing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn mammals, skin wounds heal completely without scarring during early to mid-gestation. We previously reported that in mice, skin wounds created on embryonic day 13 (E13) regenerate completely, including restoration of the skin texture. In contrast, wounds created between E14 and E16 regenerate the dermal structure but not the skin texture, and wounds created after E17 do not regenerate either the skin texture or the dermis, resulting in dermal fibrosis\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThere are no detailed reports on the changes in inflammatory cells during this series of changes in the embryonic period. On the other hand, approximately 80% of macrophages present in the skin up to E13 are derived from the yolk sac\u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. After E14, in addition to yolk sac-derived macrophages, the number of fetal-liver-derived monocyte-derived macrophages, which migrate throughout the body via monocytes from the fetal liver, gradually increases, replacing yolk sac-derived macrophages.\u003c/p\u003e\u003cp\u003eSince the transition from complete skin regeneration to no regeneration correlates with changes in macrophage types, we termed the macrophages in E13 skin, which may contribute to complete skin regeneration, as \u0026ldquo;fetal macrophages.\u0026rdquo; For simplicity, this study refers to monocyte-derived macrophages that pass through the fetal liver as \u0026ldquo;adult macrophages.\u0026rdquo; Therefore, in this study, we identified the membrane surface markers of fetal macrophages and investigated their potential for use in inhibiting fibrosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eAccumulation of inflammatory cells in wounds created at each stage of mouse development\u003c/h2\u003e\n \u003cp\u003eWhen the embryonic wound site was stained using whole-mount immunostaining and observed under a confocal microscope, F4/80-positive macrophages accumulated at the wound site created at E13 (Fig.\u0026nbsp;1a). To quantify these findings, sections of each wound site were prepared, and immunofluorescence staining was performed using F4/80 as a macrophage marker. The results showed that macrophages accumulated at all wound sites at E13, E15, and E17 (Fig.\u0026nbsp;1b). The number of macrophages accumulating in the wound gradually increased from E13 and remained stable from E17 onwards (Supplementary Fig. S1). In contrast, Gr-1-positive granulocytes were detected in the liver at E15, but their accumulation in the wound was first observed from E17 onwards (Fig.\u0026nbsp;1b). The number of granulocytes increased further at P1.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSearch for fetal macrophage cell markers\u003c/h3\u003e\n\u003cp\u003eFirst, we enzymatically treated the entire skin to isolate macrophages and measured their numbers. At E13, approximately 8–10% of all skin cells were F4/80-positive, whereas at E18, only about 2% of all skin cells were F4/80-positive (Fig.\u0026nbsp;2a). Microarray analysis was performed using total RNA recovered from macrophages, and the cell membrane surface markers of each macrophage were analyzed.\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) analysis by microarray showed that pathways related to tissue regeneration, such as wound healing (GO:0042060) and cell adhesion (GO:0007155), were significantly enriched in E13 macrophages. In contrast, in E18 macrophages, pathways related to inflammatory responses, such as inflammatory response (GO:0006954), response to viruses (GO:0009615), and antigen processing and presentation (GO:0019882), were elevated.\u003c/p\u003e\n\u003cp\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also showed that inflammation-related signaling pathways, such as the Jak-STAT signaling pathway (mmu04630) and the chemokine signaling pathway (mmu04062), were upregulated in E18 compared to E13. This suggests that while E13 macrophages contribute to regeneration without inducing inflammation, E18 macrophages enhance the inflammatory response\u003csup\u003e[5, 6]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA complete list of GO terms and KEGG pathways with significant differences is presented in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003eWe isolated and identified the cell surface antigens expressed on E13 fetal macrophages. Cell surface antigens, including CD276, CD24, CD69, CD180, CD9, CD59, and CD38, were more than twice as abundant in fetal macrophages. We performed FACS using these candidates. CD276 and CD69 were not detected by FACS in fetal macrophages. Among these, CD9, CD24, CD38, and CD180, which could be clearly detected using existing fluorescent antibodies, were compared. CD9 and CD24 showed similar trends, while CD38 and CD180 also showed similar trends. Therefore, fetal macrophages could be separated using CD9 and CD180, which enabled cell identification, as shown in Fig.\u0026nbsp;2b.\u003c/p\u003e\n\u003cp\u003eIn fetal macrophages, CD180 continued to be expressed at each stage of development, whereas CD9 expression gradually decreased as the fetus developed (Fig.\u0026nbsp;2b). Adult macrophages showed low expression levels of CD180 and CD9. Next, we confirmed the expression of CD180 and CD9 in fetal macrophages in the skin of E13 by immunostaining and observed colocalization of F4/80-positive cells with CD180- and CD9-positive cells (Fig.\u0026nbsp;2c).\u003c/p\u003e\n\u003ch3\u003eDistribution of fetal macrophages in adult tissues\u003c/h3\u003e\n\u003cp\u003eWe examined whether cells expressing surface antigens similar to those of fetal macrophages were present in adult mice. As a result, we confirmed that a small number of F4/80 + CD180 + CD9 + macrophages exist in the spleen of adult mice (Supplementary Fig. S2a). Conversely, they were found in high proportions in E18 placentas during the fetal period (Supplementary Fig. S2b).\u003c/p\u003e\n\u003ch3\u003eTransplantation of fetal macrophages into E14 skin wounds\u003c/h3\u003e\n\u003cp\u003eTo investigate whether skin regeneration was promoted by increasing the population of fetal macrophages in the skin at E14, green fluorescent protein (GFP)-fetal macrophages (1×10\u003csup\u003e5\u003c/sup\u003e cells) were transplanted into the dermis at E14 using a capillary tube, and a wound was immediately created using a 1-mm diameter derma punch. The wound site was observed 72 h later. In the control E14 wound, the skin texture did not regenerate, and wound contraction, surrounding fibrosis, and edema-like changes were observed. However, in the fetal macrophage local injection group, these changes were reduced, and scarring was less noticeable (Fig.\u0026nbsp;3a).\u003c/p\u003e\n\u003cp\u003eThe adult macrophage local injection group showed strong white changes suggestive of fibrosis.\u003c/p\u003e\n\u003cp\u003eHematoxylin and eosin (HE) staining revealed that the wound diameter at 72 h was significantly smaller in the fetal macrophage injection group than in the controls (two-sided Student’s t-test; n = 3 per group; P = 0.0043). The local phosphate-buffered saline (PBS)-injected group showed a wound contraction to an average of 0.67 mm. The adult macrophage local injection group showed smaller wound contractions with an average of 0.90 mm. On the other hand, the fetal macrophage local injection group showed a wound contraction to an average of 0.44 mm (Fig.\u0026nbsp;3b, c). The number of blood vessels per unit area was significantly higher in the fetal macrophage injection group than in the controls (two-sided Student’s t-test, n = 9 per group; fetal macrophages vs. adult macrophages, \u003cem\u003eP = 0.0016\u003c/em\u003e; PBS vs. adult macrophages, \u003cem\u003eP = 0.0494\u003c/em\u003e). Masson’s trichrome (MT) staining showed that the PBS local injection group had an average of 6.16 vessels/0.01 mm², while the fetal macrophage local injection group had an average of 4.05 vessels/0.01 mm². Thus, no significant difference was observed in comparison with normal skin. The adult macrophage local injection group showed a significant increase in the number of blood vessels, with an average of 10.11 per 0.01 mm² (Fig.\u0026nbsp;3b, d).\u003c/p\u003e\n\u003cp\u003eIn addition, the number of alpha smooth muscle actin (αSMA)-positive cells was significantly lower in the fetal macrophage local injection group compared with the adult macrophage local injection group or controls (two-sided Student’s t-test; n = 6 per group; fetal macrophages vs. adult macrophages, \u003cem\u003eP = 1.8×10⁻¹⁰\u003c/em\u003e; PBS vs. adult macrophages, \u003cem\u003eP = 8.2×10⁻¹¹\u003c/em\u003e; adult macrophages vs. normal, \u003cem\u003eP = 1.7×10⁻⁷\u003c/em\u003e). αSMA staining showed no significant difference between the PBS local injection group and the fetal macrophage local injection group compared to normal skin. The number of αSMA-positive cells increased in the adult macrophage local injection group (Fig.\u0026nbsp;3b, e).\u003c/p\u003e\n\u003cp\u003eThree groups of wounds were analyzed using Primos🄬, and the unevenness of the wounds was observed. In the fetal macrophage local injection group, the wounds were flattened, while in the PBS local injection group, the unevenness was distinct. In the adult macrophage local injection group, the unevenness was not distinct due to overall edema-like changes, but the wounds were deep (Fig.\u0026nbsp;3f).\u003c/p\u003e\n\u003cp\u003eTo confirm whether the administered GFP-fetal macrophages were undergoing transformation into other cells, immunostaining for vimentin, pan-cytokeratin, αSMA, Ly6G, and CD31 was performed using frozen sections 72 h after administration. GFP-positive and strongly vimentin-positive cells were confirmed. Since other macrophages were weakly positive for vimentin, the possibility of their transformation into fibroblasts was suggested.\u003c/p\u003e\n\u003ch3\u003eAdministration of fetal macrophages to adult wounds\u003c/h3\u003e\n\u003cp\u003eNext, we examined the effects of fetal macrophages on adult skin wounds. We created 8-mm bilateral full-thickness skin defects on the dorsal skin of B6 mice and observed them over time (Fig.\u0026nbsp;4a). There was no significant difference in wound size between the fetal macrophage-treated and control groups (Fig.\u0026nbsp;4b). Vascular regeneration was observed macroscopically when tissue sections were collected on day 4 (Fig.\u0026nbsp;4c). αSMA staining on day 4 revealed relatively thick vessels within the wound and muscle tissue (Fig.\u0026nbsp;4d). When the tissue sections on day 4 were compared using MT staining, no significant changes were observed in the central region. However, promotion of epithelialization was confirmed (Fig.\u0026nbsp;4e). When MT staining was performed on tissue sections collected on day 11, differences in the collagen fibers stained with aniline blue were observed in the scarred area (Fig.\u0026nbsp;4f). In the fetal macrophage-treated group, aniline blue was stained at the same intensity as in normal skin, indicating randomly arranged mature collagen fibers. In contrast, in the PBS-treated group, aniline blue stained weakly, the collagen fibers were immature, and the fibers exhibited a unidirectional regular arrangement typical of the wound healing process. These findings suggest that fetal macrophage administration may normalize collagen fibers within scars.\u003c/p\u003e\n\u003cp\u003eThe results were evaluated using a Modified Mouse Masson Trichrome Scar Scale (MMTSS). The MMTSS score was significantly reduced in the fetal macrophage local injection groups compared to that in the controls (two-sided Student’s t-test without correction for multiple comparisons; n = 3 per group; \u003cem\u003eP = 0.0325\u003c/em\u003e). (Supplementary Fig. S3).\u003c/p\u003e\n\u003cp\u003eA comparison using αSMA was also performed, but no significant differences were observed. In summary, the administration of fetal macrophages to adult skin wounds resulted in angiogenesis and epithelialization by day 4; however, no significant effect on wound healing was observed, although dermal collagen fibers appeared normalized. Fetal macrophages were collected from GFP mice and transplanted into the wound sites of B6 mice. On day 4, tissue sections were prepared, and F4/80-positive and GFP-positive cells were observed at the wound site (Fig.\u0026nbsp;5).\u003c/p\u003e\n\u003cp\u003eSome of the GFP-fetal macrophages remained F4/80-positive, while others became negative. This suggests that fetal macrophages undergo transformation and control fibrosis. Immunohistochemical staining with vimentin, pan-cytokeratin, αSMA, Ly6G, and CD31 was performed to determine the cell types into which the administered fetal macrophages had differentiated into, but no obvious transformation was confirmed. In summary, these results suggest that fetal macrophages transform into fibroblasts at the wound site during the fetal period. However, as this was not confirmed in adult animals, it is possible that regeneration does not occur under mature inflammatory conditions. It has been suggested that fetal macrophages in adult animals control inflammation and suppress scarring and fibrosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn mammals, skin injuries inflicted before mid-gestation can completely regenerate. Longaker et al. reported that when wounds were inflicted on the lips of macaques, which are similar to humans, during gestation, the presence or absence of scars changed depending on the gestational age, and they referred to this as \u0026ldquo;transition wound\u0026rdquo;\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Similarly, in mice, complete regeneration occurs up to E13, whereas incomplete regeneration occurs at E14 and E15, with the disappearance of skin appendages and preservation of the dermal structure. From E16 onwards, the dermis becomes scarred\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Macrophages actively accumulated at the E13 wound site, where the skin was completely regenerated. Based on a report by Hoeffel et al., these macrophages were considered to correspond to the yolk sac-derived macrophages\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Macrophages derived from the yolk sac are intermediate cells between erythro-myeloid progenitor cells and tissue-resident macrophages, and are considered immature tissue-resident macrophages. Tissue-resident macrophages maintain homeostasis through repeated self-replication. By focusing on the role of macrophages in this switch in regenerative capacity, we have demonstrated that yolk sac-derived fetal macrophages suppress fibrosis and scar formation, which is a novel finding.\u003c/p\u003e\u003cp\u003eComplete skin regeneration in fetuses is caused by a combination of multiple factors, rather than a single factor. These factors include the suppression of inflammation and fibrosis, balance of specific growth factors, fetus-specific extracellular matrix, weak mechanical contraction environment, and fetal cell activity\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Inflammatory responses typically promote fibrosis and hinder regeneration. Macrophages are classified into M1 and M2 subtypes; however, in adult skin wound healing, M2 macrophages mainly promote fibrosis at the wound site\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Microarray results showed that fetal macrophages did not exhibit increased expression of markers specific to M1 (Nos2, IL12b, Ciita, IL6) or M2 (Arg1, Retnla (Fizz1), Ch3l3 (Ym1), and Mrc1 (CD206)) compared to adult macrophages, indicating that they possess properties distinct from those of M1 and M2 macrophages. Furthermore, as shown in the results, fetal macrophages exhibited an immature inflammatory response, with underdeveloped Jak-STAT and chemokine signaling pathways, and no signs of exacerbated inflammation were observed. In particular, fetal macrophages accumulating at the wound site on E13 exhibited low inflammatory induction and abundant expression of wound healing-related genes. In contrast, inflammatory responses and immune-related gene expression were predominant in the adult macrophages. These results indicate that fetal macrophages have non-inflammatory characteristics that differ from those of general macrophages and do not contribute to scar formation. Furthermore, the establishment of isolation methods using surface markers, such as CD180 and CD9, will serve as a bridge to future functional analyses and clinical applications. Although the results of this microarray study cannot directly demonstrate the molecular mechanisms based on previously reported wound healing and inflammatory response pathways, our results support these pathways through their association with known signaling pathways. This study complements and expands upon existing reports by clarifying the role of macrophages in embryonic skin wound regeneration from both gene expression and transplantation perspectives.\u003c/p\u003e\u003cp\u003eThe transplantation of fetal macrophages into E14 skin and adult skin wounds suppressed fibrosis and improved the properties of collagen fibers. This is consistent with the anti-fibrotic effects of yolk sac-derived macrophages on the heart and other organs. In the heart, approximately 80% of the resident macrophages are yolk sac-derived, as reported in a study using tamoxifen-treated Cx3cr1CreER-YFP:R26Td mice\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Tissue-resident macrophages derived from the yolk sac of the heart are not involved in inflammation during the acute phase of myocardial infarction, but play an important role in remodeling after myocardial infarction. Furthermore, tissue-resident macrophages in the heart suppress fibrosis and limit harmful cardiac remodeling\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Microglia are derived almost entirely from yolk-sac macrophages in the brain and spinal cord. The role of microglia in spinal cord injury is to promote the repair of the central nervous system, including phagocytosis of growth-inhibiting debris and stimulation of nerve fiber extension. However, these processes also depend on interactions with other cells, and the exact mechanisms remain unclear. Nevertheless, it has been reported that microglia-dominant inflammation leads to preservation and increased axons at the injury site, as well as functional recovery\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Macrophages in fetal testes are almost entirely derived from the yolk sac\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Macrophages derived from the yolk sac have been suggested to regulate fibrosis during cirrhosis\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Yolk sac-derived macrophages are involved in suppressing fibrosis in various organs. Their effects\u0026mdash;such as direct action on fibroblasts, regulation of profibrotic pathways, and protection of surrounding cells\u0026mdash;have been proposed; however, the mechanism remains unclear. It is possible that inflammation is controlled and fibrosis is suppressed through interactions with fibroblasts. Although the molecular mechanism is unclear, the microarray analysis results of this study, such as Jak-STAT signaling and chemokine pathway immaturity, support the anti-inflammatory effects of fetal macrophages. Clinically, the findings obtained in this study suggest the possibility of new cell therapies not only for inhibiting skin fibrosis and scarring but also for treating multi-organ fibrotic diseases such as myocardial infarction, pulmonary fibrosis, and cirrhosis. The direct application of fetal macrophages is ethically constrained. However, by mimicking their characteristics and developing soluble factors or cell induction methods, their applications in regenerative medicine and fibrosis treatment are anticipated.\u003c/p\u003e\u003cp\u003eA limitation of this study is that complete regeneration was not observed in adult mice or at E14 wound sites. In particular, owing to significant differences in inflammatory responses and tissue environments between adult animals and fetuses, transplantation of fetal macrophages alone may have suppressed fibrosis but failed to promote adequate wound healing. This issue remains a challenge for future research, and it is necessary to consider treatment methods that combine other factors with the cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCollection of macrophages\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experiments were conducted in accordance with the Keio University guidelines for animal and genetically modified animal experiments (approval numbers A2022-279 and D2013-021).\u0026nbsp;Additional ARRIVE-compliant reporting:\u003c/p\u003e\n\u003cp\u003eStudy design: Fetal macrophage transplantation groups were compared to sham-operated controls. A single mouse was used as the experimental unit.\u003c/p\u003e\n\u003cp\u003eSample size: Three mice were assigned to each treatment group. Sample sizes were based on previous studies, and no a priori power calculations were performed.\u003c/p\u003e\n\u003cp\u003eInclusion/exclusion criteria: All mice were included and no exclusion criteria were applied.\u003c/p\u003e\n\u003cp\u003eRandomization: Animals were randomly assigned to treatment groups using a random number generator.\u003c/p\u003e\n\u003cp\u003eBlinding: Investigators assessing histological outcomes were blinded to group allocation.\u003c/p\u003e\n\u003cp\u003eOutcome measures The primary outcome measure was collagen deposition (MT staining). Secondary outcomes included angiogenesis and fibrosis scores.\u003c/p\u003e\n\u003cp\u003eExperimental animals: C57BL/6J mice (E13\u0026ndash;14 embryos) were obtained from Sankyo Labo Service Corporation, Inc.\u003c/p\u003e\n\u003cp\u003eExperimental procedures: Skin wounds were generated on E13. Fetal macrophages (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were transplanted into the wound edges. The procedures were performed under isoflurane anesthesia.\u003c/p\u003e\n\u003cp\u003eAnimal housing and husbandry: Mice were housed in specific pathogen-free conditions under a 12-h light/dark cycle, with standard chow and water ad libitum.\u003c/p\u003e\n\u003cp\u003eAnimal care and monitoring: The mice were monitored daily. No unexpected adverse events were observed. No humane endpoints were included.\u003c/p\u003e\n\u003cp\u003eFemale Slc:ICR mice, C57BL/6JmsSlc mice, and C57BL/6-Tg (CAG-EGFP) mice at 13\u0026ndash;18 days of gestation were purchased from Sankyo Labo Service Corporation, Inc. On the day of delivery, the mice were euthanized by cervical dislocation after inhalation of anesthesia in the morning. The abdomen was incised, fetuses were removed, washed with PBS to remove maternal blood, and the skin was collected using scissors. The collected skin samples were minced with scissors. After homogenization, collagenase type 1 treatment was performed, followed by stirring at 37\u0026deg;C in an incubator. The mixture was then filtered through a 100 \u0026micro;m cell strainer to recover the cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe E13 and E18 placenta and E18 umbilical cord were processed using the same method described above. Adult spleens were obtained from 8-week-old male ICR mice. The spleens were removed under general anesthesia, homogenized, treated with collagenase type 1, stirred in a 37\u0026deg;C incubator, filtered through a 100 \u0026micro;m cell strainer, and the cells were recovered. The collected cells were isolated by magnetic-activated cell sorting (MACS). Anti-F4/80 Micro Beads UltraPure (Miltenyi Biotec) was used as the antibody, and an MS column was used for collection. The cells were washed with MACS (FACS) buffer and centrifuged at 300 g for 5 min. The supernatant was discarded, and the cells were incubated at 4\u0026deg;C with a 1/100 dilution of Anti-F4/80 Micro Beads UltraPure while rotating for 15 min. The antibody-labeled cells were washed with MACS buffer and centrifuged at 300 g for 5 min. The supernatant was discarded, the cells were diluted with MACS buffer, passed through an MS column set up in a magnetic field, and F4/80-positive cells were recovered.\u003c/p\u003e\n\u003cp\u003eAs described above in the animal experiment, antibodies used included anti-CD45, anti-CD11b, anti-F4/80, anti-CD180, and anti-CD9 (BioLegend), which were incubated at 4\u0026deg;C for 30 min with stirring. Fetal and adult macrophages were obtained using flow cytometry (Beckman MoFlo XDP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMicroarray and analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroarray analysis was performed using the Clariom_S_mouse (Thermo Fisher Scientific). The analysis was normalized using the RMA method. The R package \u0026ldquo;limma\u0026rdquo; was used to test for gene expression variations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCreation of mouse fetal skin wounds\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice at 13\u0026ndash;18 days of gestation were anesthetized with 5% isoflurane, and anesthesia was maintained with 2\u0026ndash;3% isoflurane. The abdomen was incised to confirm the presence of the uterus. The uterus was incised under a stereomicroscope to confirm fetal development. The amniotic membrane was incised, and a wound was created on the dorsal skin using microsurgical microscissors. At E13\u0026ndash;E14, the amniotic membrane was sutured with 9-0 nylon. At E15 and beyond, the uterine wall was sutured using 9-0 nylon. Prior to closing the abdomen, ritodrine hydrochloride was administered to the abdominal cavity, and the abdomen was sutured with 5-0 nylon to complete the fetal surgery. The animals were subsequently kept warm and observed. At 24 h and 72 h postoperatively, the animals were euthanized via cervical dislocation after inhalation anesthesia with 5% isoflurane, and the abdomen was opened to retrieve the fetus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdministration of fetal macrophages to E14 fetal wound sites\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the above method, fetal surgery was performed on mice at day 14 of gestation. The amniotic membrane was incised, and capillary tubes were used to administer 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e GFP-fetal macrophages (N = 4), PBS (N = 4) to the dermis where the wound was to be created, or 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e E18 GFP-adult macrophages (N = 4). Immediately afterward, a circular wound of uniform size was created on the dorsal skin using a 1-mm dermal punch. The amniotic membrane was sutured using 9-0 nylon. Ritodrine hydrochloride was administered to the abdominal cavity, and the abdomen was sutured with 5-0 nylon to complete the fetal surgery. Subsequently, the fetus was kept warm and observed. After 72 h, 5% isoflurane inhalation anesthesia was administered, cervical dislocation was performed, the abdomen was incised to retrieve the fetus, and the wound site was examined. Surface irregularities of the skin were measured using Primos\u0026reg; (Integral Co., Ltd.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLocal administration of fetal macrophages to adult wound sites\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale mice aged 8\u0026ndash;10 weeks were shaved after inhalation anesthesia and depilated using a depilatory agent. The skin was stretched, and identical wounds were created on the dorsal skin using an 8-mm derma punch on both sides. The skin collected adjacent to the wound was used as normal skin for tissue sections. On the left wound site, GFP-fetal macrophages recovered using MACS were diluted in 100 \u0026mu;L of PBS at a concentration of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells and administered locally. The right side received an equal volume of PBS. The wounds were protected with Permirole\u0026reg; and Tegaderm\u0026reg;, and kept warm with frequent observations. Wound contraction rates were analyzed using the ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistological stain and immunohistochemistry\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected tissue was fixed in 4% paraformaldehyde (PFA) at 4\u0026deg;C for 24 h and then paraffin-embedded. After paraffin embedding, tissue sections were prepared at a thickness of 5 \u0026micro;m. The tissue sections were deparaffinized using slides. MT staining was performed using Ponceau xylidine, acid fuchsin, and aniline blue. HE staining was performed. \u0026alpha;SMA was stained using DAB and a Leica automatic staining device. Frozen sections were fixed in 4% PFA at 4\u0026deg;C for 24 h, replaced with 20% sucrose, replaced with 30% sucrose, replaced with 40% sucrose, and then frozen and embedded in an OCT compound. After frozen embedding, immunostaining was performed on tissue sections sliced to a thickness of 10 \u0026mu;m. For whole-mount staining, after fixation with 4% PFA at 4\u0026deg;C for 24 h, the samples were washed with PBS, incubated with antibody for 24 h at 4\u0026deg;C, washed again with PBS, and then immunostained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following antibodies were used:\u003c/p\u003e\n\u003cp\u003eAnti-F4/80Micro Beads Ultra Pure (Miltenyi Biotec)\u003c/p\u003e\n\u003cp\u003eAlexa Fluor 594 anti-mouse/human CD11b (BioLegend)\u003c/p\u003e\n\u003cp\u003eAPC-Cy7 anti-mouse/human CD11b (BioLegend）\u003c/p\u003e\n\u003cp\u003eAPC anti-mouse F4/80 (BioLegend)\u003c/p\u003e\n\u003cp\u003eFITC anti-mouse F4/80 (BioLegend)\u003c/p\u003e\n\u003cp\u003ePE anti-mouse CD180 (BioLegend)\u003c/p\u003e\n\u003cp\u003eAPC anti-mouse CD9 (BioLegend)\u003c/p\u003e\n\u003cp\u003ePE-Cy7 anti-mouse CD45 (BioLegend)\u003c/p\u003e\n\u003cp\u003eAnti \u0026alpha;-SMA (SIGMA)\u003c/p\u003e\n\u003cp\u003eAnti-IBA1 (Abcam)\u003c/p\u003e\n\u003cp\u003eVimentin (Santa Cruz)\u003c/p\u003e\n\u003cp\u003eAnti-mouse Ly6G (Gr-1) (Abcam)\u003c/p\u003e\n\u003cp\u003eAnti-CD31 (Millipore)\u003c/p\u003e\n\u003cp\u003eObservations were performed using Keyence BZ-X700 and Canon FV3000 confocal fluorescence microscopes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOrganizational assessment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Manchester Scar Scale is an index for the pathological evaluation of human scars; therefore, it was adapted for mice, and the number of evaluation items was reduced\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e18\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. Subsequently, three individuals conducted a blinded evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the statistical analyses were performed using Microsoft Excel. Data are expressed as mean \u0026plusmn; standard error of the mean (SEM). For comparisons between the two groups, a two-sided unpaired Student\u0026rsquo;s t-test was used without correction for multiple comparisons. The normality of data distribution was assessed using the Shapiro\u0026ndash;Wilk test, and variance between groups was assumed to be equal. A significance level of \u0026alpha; = 0.05 was adopted. The exact P values, sample sizes (n), and details of the statistical tests are reported in the figures. In each analysis, n refers to biologically independent samples; for fetal experiments, the number of individual fetuses analyzed per group (typically n = 3\u0026ndash;4); for adult wound healing experiments, the number of mice analyzed (typically n = 3\u0026ndash;9, depending on the experiment); and for histological quantification, the number of tissue sections or fields examined per animal. The error bars in the figures represent the SEM. All the tests were two-sided.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted with the cooperation of Toshiko Toda, Hiromi Fujita, and university student Kazuhiro Takada, who assisted in experiment preparation and animal care. We express our gratitude toward them.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI, Grant Numbers JP21K16926 and JP25K12938.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShigeki Sakai conducted the experiments, collected data, and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003eKeisuke Okabe conducted wound healing experiments in the dorsal region of ICR mice from E13 to P1.\u003c/p\u003e\n\u003cp\u003eKento Takaya and Yukari Nakajima took the photographs.\u003c/p\u003e\n\u003cp\u003eKazuo Kishi designed the study, supervised the experiments, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microarray data generated in this study were deposited in the NCBI Gene Expression Omnibus (GEO) GSE306564. During peer review, reviewers can access data using the following token: [sxaxmceuhxgnpix]. Other datasets generated and/or analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare the following competing interests:\u0026nbsp;S. (Shigeki Sakai) and K. (Kazuo Kishi) are listed as inventors of patent applications related to the methods described in this study. The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eThis study was supported by a Grant-in-Aid for Scientific Research (C). The funders had no role in the study design, data collection, or data interpretation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTakaya, K. \u003cem\u003eet al.\u003c/em\u003e Actin cable formation and epidermis-dermis positional relationship during complete skin regeneration. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 15913 (2022).\u003c/li\u003e\n\u003cli\u003eHoeffel, G. \u003cem\u003eet al.\u003c/em\u003e Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. \u003cem\u003eJ. Exp. 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KEGG: Kyoto Encyclopedia of Genes and Genomes. \u003cem\u003eNucleic Acids Res\u003c/em\u003e. \u003cstrong\u003e28\u003c/strong\u003e, 27\u0026ndash;30 (2000)\u003c/li\u003e\n\u003cli\u003eKanehisa, M., Furumichi, M., Sato, Y., Kawashima, M. \u0026amp; Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. \u003cem\u003eNucleic Acids Res\u003c/em\u003e. \u003cstrong\u003e51\u003c/strong\u003e, D587\u0026ndash;D592 (2023)..\u003c/li\u003e\n\u003cli\u003eWhitby, D. J., Longaker, M. T., Harrison, M. R., Adzick, N. S. \u0026amp; Ferguson, M. W. Rapid epithelialisation of fetal wounds is associated with the early deposition of tenascin. \u003cem\u003eJ. Cell Sci.\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 583\u0026ndash;586 (1991).\u003c/li\u003e\n\u003cli\u003eWhitby, D. J. \u0026amp; Ferguson, M. W. The extracellular matrix of lip wounds in fetal, neonatal and adult mice. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, 651\u0026ndash;668 (1991).\u003c/li\u003e\n\u003cli\u003eWhitby, D. J. \u0026amp; Ferguson, M. W. Immunohistochemical localization of growth factors in fetal wound healing. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, 207\u0026ndash;215 (1991).\u003c/li\u003e\n\u003cli\u003ePierce, G. F. \u003cem\u003eet al\u003c/em\u003e. Platelet-derived growth factor (BB homodimer), transforming growth factor-beta 1, and basic fibroblast growth factor in dermal wound healing. Neovessel and matrix formation and cessation of repair. \u003cem\u003eAm. J. Pathol.\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 1375\u0026ndash;1388 (1992). \u003c/li\u003e\n\u003cli\u003eLawrence, T. \u0026amp; Natoli, G. 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Emerging concepts in myeloid cell biology after spinal cord injury. \u003cem\u003eNeurotherapeutics\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 252\u0026ndash;261 (2011).\u003c/li\u003e\n\u003cli\u003eDeFalco, T., Bhattacharya, I., Williams, A. V., Sams, D. M. \u0026amp; Capel, B. Yolk-sac\u0026ndash;derived macrophages regulate fetal testis vascularization and morphogenesis. \u003cem\u003eProc. Natl Acad. Sci. \u003c/em\u003e\u003cem\u003eU. S. A.\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, E2384\u0026ndash;E2393 (2014).\u003c/li\u003e\n\u003cli\u003eRamachandran, P. \u003cem\u003eet al\u003c/em\u003e. Resolving the fibrotic niche of human liver cirrhosis at single cell level. \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e575\u003c/strong\u003e, 512\u0026ndash;518 (2019).\u003c/li\u003e\n\u003cli\u003eSakai, S., Aramaki-Hattori, N. \u0026amp; Kishi, K. Fetal fibroblast transplantation via ablative fractional laser irradiation reduces scarring. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 347 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"yolk sac-derived macrophages, fetal macrophages, scarless wound healing, skin regeneration, fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-7478492/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7478492/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn mice, skin wounds created on embryonic day 13 (E13) regenerate completely, including skin texture restoration; however, after E14, the skin texture does not regenerate, while the dermis regenerates. Furthermore, after E17, neither the texture nor the dermis regenerate, resulting in dermal fibrosis. Currently, there are no detailed reports on the changes in inflammatory cells during this series of changes in the embryonic period. Thus, in this study, we identified the membrane surface markers of fetal macrophages and investigated their potential for use in inhibiting fibrosis. We found that fetal macrophages, which are derived from the yolk sac, accumulate at the wound site until E13 when the skin wound is completely regenerated. Using microarrays, we successfully identified specific markers of fetal macrophages. Furthermore, we demonstrated that transplantation of fetal macrophages into wounds at E14 or in adult animals suppressed fibrosis. These effects were confirmed using microarray results. Recent reports have suggested that yolk sac-derived macrophages are involved in fibrosis in various organs. The utilization of fetal macrophages suggests potential clinical applications in the treatment of fibrotic diseases such as myocardial infarction, pulmonary fibrosis, cirrhosis, and scleroderma, and may lead to innovative therapeutic approaches.\u003c/p\u003e","manuscriptTitle":"Yolk sac-derived fetal macrophages suppress skin fibrosis and contribute to scarless wound healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 10:05:31","doi":"10.21203/rs.3.rs-7478492/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2a81268-c68f-4ba5-b344-c16982e95558","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55453650,"name":"Biological sciences/Cell biology"},{"id":55453651,"name":"Health sciences/Diseases"},{"id":55453652,"name":"Health sciences/Medical research"},{"id":55453653,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-11-28T13:38:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 10:05:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7478492","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7478492","identity":"rs-7478492","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}