Tongzhu Sun

Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skin appendages

Adult bone-marrow-derived mesenchymal stem cells (MSCs) are well-established as having the capacity to differentiate into cells with mesodermal, ectodermal, and endodermal characteristics and can leave their niche to home toward and engraft within foreign tissues. To investigate whether adult MSCs contribute to the repair of skin appendages after injury, BrdU-labeled MSCs were co-cultured with heat-shocked confluent sweat gland cells (SGCs) in vitro and later intravenously injected into full-thickness skin wounds in rats. When adult MSCs were co-cultured with heat-shocked SGCs, a subset of adult MSCs differentiated into SGCs, the percentage of differentiation being enhanced by epidermal growth factor and the injured microenviroment, but weakened by PD98059. The ERK (extracellular signal-regulated kinase) pathway, especially pERK, was involved in the phenotype conversion of human MSCs into human SGC. Labeled MSCs were noted in hair follicles, sebaceous glands, blood vessels, and dermis in full-thickness wounds, and the incorporated cells in hair follicles and sebaceous glands were also positive for pan-cytokeratin. After wound healing, some labeled MSCs returned to the bone marrow, whereas other were retained in the dermis. We conclude that adult MSCs have the capacity to dock at specific sites, to contribute to wound healing of skin appendages, and to home toward marrow, and that engraftment of bone-marrow-derived cells is a functional event.

Analysis of differentially expressed genes in keloids and normal skin with cDNA microarray

BACKGROUND Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of diseases. Keloid is an intricate lesion that is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skin with the aid of a cDNA microarray to explore the molecular mechanism underlying keloid formation. MATERIALS AND METHODS The polymerase chain reaction products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs were isolated from freshly excised human keloids and normal skins and then were purified to mRNAs by Oligotex. Both the mRNAs from keloids and normal skins were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. RESULTS Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250 genes, including TGF-beta1 and NGF, were upregulated (2.98%) and 152 downregulated (1.81%). Analyses of collagen, fibronectin, proteoglycan, growth factors, and apoptosis-related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of keloids. Higher expression of TGF-beta(1) and NGF in keloids versus normal skins was also testified with reverse transcription polymerase chain reaction method. CONCLUSIONS DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help to understand the molecular mechanism of keloid formation.

Ontogeny of expression of transforming growth factor‐β and its receptors and their possible relationship with scarless healing in human fetal skin

Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor‐β (TGF‐β) isoforms and their receptors in the wound healing process, their roles in scarless wound repair observed in early gestation and their functions in human fetal skin development, and structural and functional maintenance are still not well understood. In this study, we explore the expression and distribution characteristics of three TGF‐β isoforms and their receptors, TGF‐βRI (TBRI) and TGF‐βRII (TBRII), in fetal and postnatal skins to understand the relevance of these five proteins to skin development and elucidate the mechanism(s) underlying the phenotypic transition from scarless to scar‐forming healing observed during fetal gestation. Fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages from 13 to 32 weeks and postnatal skin specimens were collected from patients undergoing plastic surgery. Gene expression and positive immunohistochemical signals of TGF‐β1, TGF‐β2, TGF‐β3, TBRI, and TBRII could all be detected in fetal and postnatal skins. In early gestation, gene expression of TGF‐β1, TBRI, and TBRII was weaker and protein contents were less compared with postnatal skins (p < 0.05). In contrast, more TGF‐β2 mRNA transcript was found in early gestation than in late gestation and in postnatal skins, whereas protein content of this growth factor increased during gestation. Lastly, mRNA transcript and protein contents of TGF‐β3 were apparently higher in early gestation compared to postnatal skin (p < 0.05). In postnatal skin, granules containing the three TGF‐β isoforms were mainly distributed in the cytoplasm and extracellular matrix of epidermal cells, interfollicular keratinocytes, and some fibroblasts. TBRI and TBRII were chiefly located in the cellular membrane of epidermal keratinocytes and some fibroblasts. The endogenous three TGF‐β isoforms and their receptors may be involved in the development of embryonic skin and in the maintenance of cutaneous structure and function, and also in postnatal wound healing. The differential levels of TGF‐β isoforms may provide either a predominantly antiscarring or profibrotic signal upon wounding depending on the gestational period. Lower expression of their receptors in early gestational skins may be a reason for the reduced ability to perceive ligands, ultimately leading to scar‐free healing.

In vivo dedifferentiation of human epidermal cells

Consistent with our previous study, we herein offer further evidence to demonstrate the dedifferentiation of differentiating epidermal cells into stem cells or stem cells ‐like in vivo. The epidermal sheets eliminated of basal cells were labeled with 6‐diamidino‐2‐phenylindole (DAPI), and then were transplanted onto the full‐thickness skin wounds nude mice. Immunohistochemical examination of the survival sheets showed that some cells were positive for both DAPI and either cytokeratins (CK19, CK14) or β1 integrin in spinous and granular layers at day 7 after transplantation. Furthermore, there was a significant increase in the percentages of both α6briCDdim and α6briCD71bri populations in survival epidermal sheet grafts 7 d after transplantation compared with those before xenotransplantation (P < 0.05), as determined by flow cytometry. The results collectively indicated that some of the differentiated cells in engrafted epidermal sheets dedifferentiated into stem cells or stem cells‐like in vivo, which offer us new evidence and insights into the dedifferentiation.

Profiling of genes differentially expressed in a rat of early and later gestational ages with high-density oligonucleotide DNA array.

The early gestational fetus heals dermal wounds rapidly and scarlessly. This phenomenon appears to be intrinsic to fetal skin and is probably modulated by interplay of many genes. We ventured to study differences in gene expression between earlier gestational skin (EGS) and later gestational skin (LGS) with the aid of high‐density oligonucleotide DNA array to explore the molecular mechanism underlying scarless healing. Total RNA was isolated from fetal Wistar rat skin of the scarless (E15) and scar‐forming (E18) periods of gestation (term=21.5 days), and purified to mRNAs. Both the mRNAs from EGS and LGS were reversely transcribed to cDNAs, and were labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes through single primer amplification reaction and Klenow labeling methods. The mixed probes were then hybridized to the oligonucleotide DNA arrays that contained 5,705 DNA fragments representing 5,705 rat genes. After highly stringent washing, the microarray was scanned for fluorescent signals to display the differentially expressed genes between two groups of tissues. Among 5,705 rat genes, there were 53 genes (0.93%) with differentially expressed levels between EGS and LGS; 27 genes, including fibroblast growth factor 8 and follistatin, were up‐regulated (0.47%); and 26 genes, containing lymphoid enhancer binding factor‐1 and β‐catenin, were down‐regulated (0.46%) in fetal skin of scarless period vs. scar‐forming period. Analyses of genes related to ion channels, growth factors, extracellular matrix and cellular skeleton, and movement confirmed that our molecular data obtained by oligonucleotide DNA array were consistent with the published biochemical and clinical findings of fetal scarless healing. Stronger expression of fibroblast growth factor 8, follistatin, and weaker expression of lymphoid enhancer binding factor‐1 and β‐catenin in EGS vs. LGS were also testified with reverse transcription‐polymerase chain reaction and Western blotting methods. Oligonucleotide DNA array was a powerful tool for investigating different gene expression between scarless and scar‐forming periods of gestation in the rat fetal skin. Many genes were involved in the phenotypic transition from scarless to scar‐forming wound repair during gestation. Further analysis of the obtained genes will help to understand the molecular mechanism of fetal scarless healing.

Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell‐like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation‐derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c‐nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β1‐integrin emerged at the wound‐neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β1‐integrin+ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10+ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation‐derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β1‐integrin, Oct4 and Nanog in dedifferentiation‐derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation‐derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9‐fold and 6.7‐fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 105 cells versus 1 × 1012 cells) were all significantly increased and (4) dedifferentiation‐derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation‐derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.

Wnt/β‐catenin signaling is critical for dedifferentiation of aged epidermal cells in vivo and in vitro

Aged epidermal cells have the capacity to dedifferentiate into stem cell‐like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/β‐catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell‐like cells that were positive for CK19 and β1 integrin but negative for CK10. In addition, Wnt/β‐catenin pathway was activated during this process. There was increased expression of Wnt‐1, Wnt‐4, Wnt‐7a, β‐catenin, cyclin D1, and c‐myc. Secreted frizzled‐related protein 1, a Wnt/β‐catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)‐3β inhibitor, of Wnt/β‐catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, β1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony‐forming efficiency, long‐term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/β‐catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell‐like cells.

Basic fibroblast growth factor reduces the gut and liver morphologic and functional injuries after ischemia and reperfusion.

OBJECTIVE To explore the possible effects of basic fibroblast growth factor (bFGF) on ischemic gut and liver injuries after trauma. METHODS Animal models of superior mesenteric artery occlusion (45 minutes) and reperfusion (3 days) were used in this study. Seventy-two Wistar rats were divided into three groups of 24 rats each. The animals in bFGF-treated group were injected with 4 microg bFGF/rat in 0.15 mL normal saline solution containing heparin 0.1% (w/v) through the jugular vein at the onset of reperfusion. In the normal saline control group, all rats received the same vehicle, but without bFGF. Group 3 (sham-operated) underwent the same laparotomy procedure, but without superior mesenteric artery occlusion. Liver function parameters, the levels of serum tumor necrosis factor alpha, nitric oxide, superoxide dismutase, malondialdehyde (MDA), tissue bacterial examination, and pathologic study were used to evaluate the results. RESULTS In bFGF-treated rats, the amounts of serum alanine transaminase and aspartate aminotransferase and serum tumor necrosis factor-alpha were reduced significantly at 6, 24, and 48 hours when compared with normal saline-treated rats. However, the changes in nitric oxide, superoxide dismutase, and MDA varied from each other as a function of time after injury. The amounts of nitric oxide were increased significantly at 6 hours in intestine in normal saline-treated rats and in liver in bFGF-treated rats (p < 0.05). At 6 hours after reperfusion, the activity of superoxide dismutase in normal saline-treated rats were much lower in liver than those in bFGF-treated and sham-operated rats (p < 0.05), but the levels of MDA were increased in intestine in bFGF-treated rats and in liver in normal saline-treated rats when compared with sham-operated rats (p < 0.05). At 24 hours, the levels of MDA in normal saline-treated rats were much higher than those in both bFGF and sham-operated rats (p < 0.05). Bacterial examination revealed that the ratio and the amounts of bacterial translocation from gut to liver, spleen, and mesenteric lymph nodes in bFGF-treated rats were much lower than those in normal saline-treated rats. The results of pathologic study support the assumption that bFGF provided protective effects against reperfusion injury. CONCLUSIONS Intravenous administration of bFGF may benefit in reducing gut and liver injuries after ischemia and reperfusion. The mechanisms of those effects may involve mitogenic and nonmitogenic effects of bFGF.

Non-mitogenic acidic fibroblast growth factor reduces intestinal dysfunction induced by ischemia and reperfusion injury in rats.

Background:  Acidic fibroblast growth factor (aFGF) has potentially therapeutic uses in some diseases, but the mitogenic activity of aFGF has been found to contribute to several human pathologies, so the extensive applications of wild‐type aFGF have been limited. The purpose of the present study was to explore the effects and mechanisms of wild‐type (aFGF) and non‐mitogenic aFGF on gut ischemia–reperfusion injury in rats.