Zhiyong Sheng

Enhanced wound‐healing quality with bone marrow mesenchymal stem cells autografting after skin injury

Adult stem cells exist in various tissues and organs and have the potential to differentiate into different cell lineages, including bone, cartilage, fat, tendon, muscle, and epithelial cells of the gastrointestinal tract. Here, we report that the in vitro expanded and purified bone marrow mesenchymal stem cells (MSCs) might take on phenotypes with characteristics of vascular endothelial cells (7% on day 3 and 15% on day 1) or epidermal cells (3% on day 3 and 13% on day 1) after being cultured under different lineage‐specific culture conditions. Also, in vivo grafting experiments showed that 5‐bromodeoxyuridine‐labeled MSCs could convert into the phenotypes of vascular endothelial cells (3.43, 3.46, and 2.94% on days 7, 14, and 28, respectively) in granulation tissues, sebaceous duct cells, and epidermal cells (0–1.49%) in regenerated skin, implying that these grafted MSCs might have transdifferentiated into the above three cell types. Animal autografting experiments with MSCs further confirmed that indices pertaining to wound healing quality, such as the speed of reepithelialization, the number of epidermal ridges and thickness of the regenerated epidermis, the morphology and the number and arrangement of microvasculature, fibroblasts and collagen, were much enhanced. Our results indicate that locally delivered bone marrow MSCs can enhance wound healing quality, and may generate de novo intact skin, resulting in perfect skin regeneration after full‐thickness injury.

Engineered growth factors and cutaneous wound healing: Success and possible questions in the past 10 years

In the past 10 years, many engineered growth factors, including recombinant human epidermal growth factor, basic fibroblast growth factor, and platelet‐derived growth factor, have been produced and used in the clinic. After screening the results from different centers, some results are found to be encouraging, while others are discouraging. Although the interpretation of these results may depend on your perspective, it may also depend on different criteria, different wounds, and even different aims. In this article, successful experiences and failures concerning the use of growth factors and cutaneous wound healing are summarized. Based on this information and our clinical experience, we address peoples concerns such as whether growth factors have altered clinical practice thus far and whether growth factor treatments have solved all problems involved in wound healing. Is there a need for exogenous application of growth factors in acute or chronic wounds, and if so, is it safe to use growth factors to promote wound healing? Last, can we achieve perfect wound healing in those wounds treated with growth factors?

Migration of bone marrow-derived mesenchymal stem cells induced by tumor necrosis factor-α and its possible role in wound healing

We aimed to investigate the effect of tumor necrosis factor‐α (TNF‐α) on the expression of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) in the migration ability of mesenchymal stem cells (MSCs) in the context of wound healing. We also explored the role of p38 mitogen‐activated protein kinase and extracellular signal‐regulated kinase (ERK) signaling pathways in the migration of MSCs. MSCs were isolated from the bone marrow and cultured. Immunocytochemistry, Western blotting, and reverse transcription‐polymerase chain reaction were used to observe the effect of TNF‐α on the expression of ICAM‐1 and VCAM‐1 in MSCs. The chemotaxis effect of TNF‐α on MSCs was investigated by the trans‐well system and the inhibition effect of TNF‐α using its antibody. Western blotting analysis was used to observe the activation of JAK‐STAT and mitogen‐activated protein kinase signaling pathways, and ERK was inhibited with PD98059 and p38 with SB203580 to observe the effect of TNF‐α on MSC migration and ICAM‐1 expression. The expression of ICAM‐1 could be up‐regulated by 50u2003μg/L TNF‐α (p<0.05), whereas that of VCAM‐1 remained unchanged (p>0.05). Also, TNF‐α showed a chemotaxis effect by enhancing the migration ability of MSCs (p<0.05). TNF‐α at 50u2003μg/L increased the expression of phospho‐ERK and phospho‐p38, and SB203580, but not PD98059, could suppress the chemotaxis effect and up‐regulation of ICAM‐1 induced by TNF‐α in MSCs (p<0.05). Thus, TNF‐α could up‐regulate the expression of ICAM‐1 in MSCs and enhance the cells migration ability, and the p38 signaling pathway might be involved in the TNF‐α–induced migration ability for a role in wound repair and regeneration.

Regeneration of functional sweat gland-like structures by transplanted differentiated bone marrow mesenchymal stem cells.

Regeneration of sweat glands after deep burns has been an unsolved problem. Owing to lack of perspiration, survivors of an extensive deep burn injury are leading a miserable life in sultry months. It was our contemplation to solve this problem by inducing bone marrow mesenchymal stem cells (MSCs) to acquire the phenotype of sweat gland cells in vitro. Then these cells were transplanted into fresh skin wounds resulting from excision of anhydrotic scars after healing of deep burn injury in five patients. Two to 12 months after the procedure, it was proved that there was recovery of perspiration function in all the MSCs transplanted areas, as evidenced by positive iodine–starch perspiration test. Histological and biochemical observation confirmed the involvement of MSCs transformed sweat gland cells in the recovery of functional sweat glands, and the components of sweat collected from these areas were similar to that collected from normal skin. This is the first report of successful transplantation of MSCs in regenerating functional sweat glands, which may help solve the problem of depletion of sweat glands in patients surviving extensive deep burns in the future.

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.

Adipose tissue extract enhances skin wound healing.

The main function of adipose tissue has been considered as storage of triglycerides. Adipose tissue was considered harmful for healing extensive and deep burns because of poor circulation and easy liquefaction in wound beds, which offer an excellent culture medium for bacteria. However, these traditional concepts have been challenged with the discovery of the endocrine function of adipose tissue. To investigate the effects of adipose tissue extract on wound healing, we created four 3.0 × 2.5u2003cm full‐thickness wounds on each side of the back of male Wu Zhi Shan minipigs (n=6), for eight wounds in each animal. The wounds were randomly divided to receive normal saline (0.5u2003mL; controls), adipose tissue extract (1.5u2003g), basic fibroblast growth factor (50u2003U/cm2), and epidermal growth factor (50u2003U/cm2). Reduction in wound area and wound volume was accelerated with adipose tissue treatment as compared with growth factor or control treatment. The thickness of the regenerated epidermis and the number of new vascular nets were markedly increased in adipose tissue‐treated wounds. Biopsy of adipose tissue‐treated wounds showed enhanced expression of proliferation cell nuclear antigen (PCNA) and Factor VIII‐related antigen, which indicated active cell differentiation and proliferation. In vitro study in rat tissue showed adipose tissue extracts stimulating skin growth. Bacteriology results showed no significant differences in amount or type of bacteria, whatever the treatment. These results may challenge the traditional concept that adipose tissue plays a negative role in wound healing and may offer direct evidence for encouraging the retention of adipose tissue in autologous skin grafting for skin wounds.

Dedifferentiation: A New Approach in Stem Cell Research

ABSTRACT Dedifferentiation is an important biological phenomenon whereby cells regress from a specialized function to a simpler state reminiscent of stem cells. Stem cells are self-renewing cells capable of giving rise to differentiated cells when supplied with the appropriate factors. Stem cells that are derived by dedifferentiation of ones own cells could be a new resource for regenerative medicine, one that poses no risk of genetic incompatibility or immune rejection and provokes fewer ethical debates than the use of stem cells derived from embryonic tissue. Until now, it has not been quite clear why some differentiated cell types can dedifferentiate and proliferate, whereas others cannot. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine and even tumor biology. If so, dedifferentiation will offer an ethically acceptable alternative route to obtain an abundant source of stem cells. Dedifferentiation is likely to become a new focus of stem cell research. Here we compile recent advances in this emerging but significant research, highlighting its central concepts, research findings, possible signaling pathways, and potential applications.

Promising new potential for mesenchymal stem cells derived from human umbilical cord Wharton's jelly: sweat gland cell‐like differentiative capacity

Mesenchymal stem cells derived from Whartons jelly of the human umbilical cord (hUC‐MSCs) possess various advantageous properties, similar to bone marrow‐derived mesenchymal stem cells (BM‐MSCs), including self‐renewal, extended proliferation potential and multilineage differentiation potential. In this study, we hoped to determine whether hUC‐MSCs could be induced to differentiate into sweat gland cell‐like cells, that would be potential in sweat glands restoration after injury. In this study, the results of flow cytometry analysis revealed that hUC‐MSCs showed the typical antigen profile of MSCs and were positive for CD29, CD44, CD90, CD105 and Oct‐4; they were negative for the antigens of CD34, CEA and CK14. Remarkably, hUC‐MSCs maintained proper proliferation and differentiation ability. After culture in sweat gland cell‐conditioned medium (induction group 1) for 3u2009weeks, hUC‐MSCs possessed sweat gland cell‐like morphology and expressed markers of sweat gland cells (CEA, CK14 and CK19) more efficiently than those of induction group 2. In reverse‐transcription PCR and western blotting analysis, it was further confirmed that induced hUC‐MSCs (group 1) also expressed a higher level of sweat gland developmental genes (EDA and EDAR) than group 2. These results together provided evidence that hUC‐MSCs could possess a new emerging potential to differentiate into sweat gland cell‐like cells with a higher efficacy under our new induction system. Thus, hUC‐MSCs could be considered a new strategy for sweat glands restoration after skin injury as well as improvement of cutaneous regeneration. Copyright

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.