Research ArticleBlood-derived small Dot cells reduce scar in wound healing
Introduction
Scarring can be caused by traumatic injury, surgery and fibrotic disease. Patients with large scars have life long psychological and physical burdens since no proven therapy for scarring exists. However, observations that fetuses heal skin wounds without scar have been reported in human, monkey, sheep and rodents [1], [2], [3]. Until now, the mechanism of fetal scarless healing is not clear. In mice, the scarless wound healing time period is before E17.5, when hair follicles are not fully developed and have no bulge region ready either [4].
In recent years, studies of skin stem cells suggest a potential treatment for scarring. However most studies focus on postnatal epidermal stem cells, as these can regenerate hair or epidermis. The niche for postnatal epidermal stem cells is suggested to be in the bulge region of hair follicles [5], [6], [7]. However, the niche in fetal skin is unknown, as the bulge region within hair follicles is not developed. Some researchers report that epidermal stem cells are in the follicular epithelium in fetal skin, and relocate to the bulge area when the skin has fully developed [8]. Cell surface markers that identify epidermal stem cells include integrin β1, CD34, integrin α6, P63 and keratin19 [5], [7], [9], [10]. Other skin-derived precursor cells have been isolated from neonatal and adult skin. These cells express nestin, fibronectin and βIII tubulin and can differentiate into both neural and mesodermal cell types [11]. One group also reported that epithelial stem cells, isolated from the fetal dermis express E-cadherin, cytokeratin-8, -18, -19, p63 and integrin β1 [12]. In addition, participation of bone marrow (BM)- or blood-derived hematopoietic stem cells (HSCs) have been demonstrated in tissue development and regeneration [13], [14]. Unfractionated bone marrow cells can regenerate myocytes, neurons, hepatocytes, smooth muscle cells, and other tissues specific cells [15], [16], [17], [18], indicating the presence of stem cells in BM. Most researchers believe that BM contains two types of stem cells: HSCs and stromal stem cells. HSCs express c-kit(+), lin(−) and sca1(+) and only differentiate to hematopoietic tissues [19], [20], [21]. Stromal stem cells undergo differentiation towards osteogenic, adipogenic, myogenic and chondrogenic lineages. Stromal stem cells include multipotent adult progenitor cells (MAPCs) which have been characterized as CD34(−), CD44(−), CD45(−) and c-kit(−) [22], marrow-isolated adult multi-lineage inducible (MIAMI) cells characterized as CD29(+), CD63(+), Cd81(+), CD122(+), CD164(+),Cd34(−), Cd36(−), Cd45(−) and c-kit(−) [23], unrestricted somatic stem cells (USSC) that express CD34(low), CD45(−) and c-kit(low) [24], and amniotic fluid-derived stem (AFS) cells that express similar surface markers as USSCs [25]. Although attempts to isolate HSCs with multi-lineage potential have been made for years, the results remain unclear or controversial [25], [26]. The tissue repair effects of HSCs have been found through either lineage differentiation or cellular fusion[18], [27].
E-cadherin is a transmembrane protein that is mainly expressed on epithelial cells [28]. However, it is also expressed in mast cells, brain endothelial cells, and skin Langerhans cells [29], [30], [31]. In addition, E-cadherin is expressed on embryos at the two-cell stage. E-cadherin null embryos die due to failed implantation [32], suggesting a critical function of E-cadherin during development. In addition, over expression of E-cadherin also represses TGF-β induced epithelia-mesenchymal transition [33]. Here we provide evidence that a group of blood-derived E-cadherin positive cells, Dot cells, are found in fetal dermal blood with their highest numbers on E16.5, when scarless wound healing occurs. We also identified Dot cells from the blood of human and mice, however, with much lower ratio of total blood cells compared to that in fetal mice. Dot cells migrate to wounds and repair the damaged tissues through cellular differentiation. Transplantation of isolated Dot cells to wounded adult mice induces scarless healing, suggesting that Dot cells are the fetal cells responsible for scarless repair. Our data are the first to describe the Dot cells and their function during tissue repair.
Section snippets
Animals and materials
Time dated 16 days (E16.5) pregnant Balb/C mice and GFP (FVB.Cg-Tg(ACTB-EGFP)B5Nagy/J, Jackson Lab) mice were bred and maintained in the Stanford Animal Care Laboratory. Mice received food and water ad libitum. All procedures with animals were conducted in accordance with university-approved protocols according to NIH guidelines. E-cadherin, integrin β1, PECAM-1, and c-kit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). CD34 antibody was from Abcam. CD45, Sca-1 and c-kit
Identification of Dot cells in fetal mouse skin
We found a group of small cells that heavily express E-cadherin and are located in the dermis of fetal mice. Fig. 1 shows small cells that express E-cadherin in the dorsal skin of embryonic days E14.5 to E18.5 mice. On E14.5 and E15.5, only a few scattered small cells stained densely for E-cadherin in the dermal area (arrows). On E16.5, these small cells grouped together in clustered patterns (arrows) without clear cell–cell boundaries with dense E-cadherin staining in the dermis. Meanwhile,
Discussion
In the present study, we introduced a new group of small cells, Dot cells that were detected within both fetal and adult mouse bloods. We believe that Dot cells are a previously unidentified group of cells since no other reports have described a similar cell morphology or marker profile. Dot cells strongly express to E-cadherin, integrin β1, CD184. They have weaker to CD34, CD13 and low to Sca1 expression. Because E-cadherin is expressed mainly by epithelial cells, and integrin β1 is a
Acknowledgments
This work was supported by Oak Foundation and NIH grant RO1-GM041343. The authors thank John Perrino for the electron microscope image processing. The authors thank Dr. Edward P. Buchanan for editing the manuscript and thank Bryan W. Lin for histology data analysis.
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