Peng Chen

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.

Investigation of laser-diode end-pumped Er:YSGG/YSGG composite crystal lasers at 2.79 μm

The advantages of laser-diode end-pumped Er:YSGG/YSGG composite crystals in reducing thermal effects and improving output power are investigated theoretically and experimentally. Compared with Er:YSGG, the temperature rise and total additional optical path difference of Er:YSGG/YSGG are evidently reduced because of the thermal conduction effects of the undoped YSGG crystal. The maximum continuous-wave output power of 504 mW with slope efficiency of 11.2%, and 900 mW with slope efficiency of 12.1%, at 2.79 μm is obtained in a 970 nm laser-diode end-pumped Er:YSGG crystal and Er:YSGG/YSGG crystal, respectively. To our knowledge, the output power of the Er:YSGG/YSGG crystal is the highest value for a laser-diode end-pumped Er:YSGG crystal. The thermal focal length of Er:YSGG and Er:YSGG/YSGG is respectively 36 and 51.8 mm when the pump power is 5.94 W. Investigations demonstrate that the Er:YSGG/YSGG composite crystal has great advantages in reducing the influence of thermal effects and improving output power.

A diode-end-pumped Nd:GYSGG continuous wave laser at 1104 nm

The continuous wave (CW) laser performance of Nd:GYSGG at 1104 nm is investigated for the first time, to our knowledge. A CW laser output power of 4.7 W is obtained when the pump power of the 808 nm fiber coupled laser diode is 19.1 W, corresponding to a conversion efficiency of 24.6% and slope efficiency of 37%.

Effect of low level laser irradiation on the proliferation of myoblasts—the skeletal muscle precursor cells: an experimental in vitro study

The aim of this paper is to study the effect of low-level laser irradiation (LLLI) on proliferation of myoblasts in culture. Myoblasts derived from rat skeletal muscle were irradiated by He-Ne laser with different doses. Compared with nonirradiated control group, the number of myoblasts increased when the cells in normal culture conditions were exposed to the laser of specific energy density. The amount of cells with proliferating cell nuclear antigen (PCNA) positive expression and the 5-bromo-2′-deoxyuridine (BrdU) incorporation rate after laser irradiation were also higher than that of the control group, suggesting that LLLL at certain doses can effectively enhance myoblasts growth activity in vitro. This study firstly demonstrated that stimulating myoblasts to enter into proliferative stage from initial resting state was an important mechanism of regeneration and repair of injured skeletal muscle promoted by LLLI in clinical treatment.

Inducing myoblast re-entry into the cell cycle: a potential mechanism for laser-enhanced skeletal muscle regeneration

This study investigated the effect of low-level laser irradiation (LLLI) on the cell cycle and proliferative activity of cultured myoblasts, and sought to elucidate the possible cellular mechanism by which LLLI promotes the regeneration of skeletal muscle in vivo. Primary myoblasts isolated from rat hindlegs were irradiated with helium-neon laser light at different energy densities. Distributions of cell-cycle subpopulations and the expression of cell-cycle regulatory proteins in myoblasts were assessed using flow cytometric analysis and western blot assay. It was found that laser irradiation stimulated cell-cycle entry; induced the expression of cyclin A and cyclin D; and increased cell proliferation index and bromodeoxyuridine incorporation as compared to the unirradiated control cells, indicating LLLI augmented the number of proliferative myoblasts in the S phase and G2/M phase of the cell cycle. These results suggest that LLLI at certain fluxes and wavelengths could activate quiescent myoblasts, leading to cell division and facilitating new myofiber formation. This could contribute to the improvement of skeletal muscle regeneration following trauma and myopathic diseases.

Pilot study about dose-effect relationship of ocular injury in argon laser photocoagulation

The aim of this article was to study the injury effect of either convergent or parallel argon laser beam on rabbit retina, get the dose-effect relationship for the two types of laser beams, and calculate the damage threshold of argon laser for human retinas. An argon laser therapeutic instrument for ophthalmology was used in this study. A total of 80 rabbit eyes were irradiated for 600 lesions, half of which were treated by convergent laser and the other half were done with parallel laser beam. After irradiation, slit lamp microscope and fundus photography were used to observe the lesions, change and the incidence of injury was processed statistically to get the damage threshold of rabbit retina. Based on results from the experiments on animals and the data from clinical cases of laser treatment, the photocoagulation damage thresholds of human retinas for convergent and parallel argon laser were calculated to be 0.464 and 0.285 mJ respectively. These data provided biological reference for safely operation when employing laser photocoagulation in clinical practice and other fields.

Stimulative Effects of Low Intensity He-Ne Laser Irradiation on the Proliferative Potential and Cell-Cycle Progression of Myoblasts in Culture

Low intensity laser irradiation (LILI) was found to promote the regeneration of skeletal muscle in vivo but the cellular mechanisms are not fully understood. Myoblasts, normally quiescent and inactivated in adult skeletal muscle, are a type of myogenic progenitor cells and considered as the major candidates responsible for muscle regeneration. The aim of the present study was to study the effect of LILI on the growth potential and cell-cycle progression of the cultured myoblasts. Primary myoblasts isolated from rat hind legs were cultured in nutrient-deficient medium for 36 hours and then irradiated by helium-neon laser at a certain energy density. Immunohistochemical and flow cytometric analysis revealed that laser irradiation could increase the expression of cellular proliferation marker and the amount of cell subpopulations in the proliferative phase as compared with the nonirradiated control group. Meanwhile, the expressions of cell-cycle regulatory proteins in the laser-treated myoblasts were markedly upregulated as compared to the unirradiated cells, indicating that LILI could promote the reentry of quiescent myoblasts into the cell division cycle. These results suggest that LILI at certain fluences could promote their proliferation, thus contributing to the skeletal muscle regeneration following trauma and myopathic diseases.

Effect of Low Power Laser Irradiation on the Ability of Cell Growth and Myogenic Differentiation of Myoblasts Cultured In Vitro

As a therapeutic modality, low power laser irradiation (LPLI) has been used clinically in the treatment of skeletal muscle injuries and other myopathic conditions, but the cellular and molecular mechanisms attributed to this therapy were still unclear. Myoblasts are a type of myogenic stem cells quiescence in mature skeletal muscle fibers and are considered as the source cells during the regenerating process. The purpose of this paper was to investigate the effects of LPLI on the proliferation and myogenic differentiation of the cultured myoblasts and to find out the major candidates responsible for LPLI-induced muscle regeneration in vivo. In this study, primary rat myoblasts were exposed to helium-neon (He-Ne) laser. Cell proliferation, differentiation, and the cellular responses to LPLI were monitored by using morphological observation and molecular biological methods. It was found that LPLI at a certain fluence could increase the cell growth potential for myoblasts and further induce more cells entering into S phase of the mitotic cycle as indicated by high levels of bromodeoxyuridine (BrdU) incorporation, while at the same time inhibiting their in vitro differentiation and decreasing the expression of myogenic regulatory genes to a certain extent. Taken together, these results provide experimental evidence for the clinical applications of LPLI in regenerating skeletal muscle.

Effects of low-intensity laser irradiation on the apoptosis of rabbit vascular smooth muscle cells in culture

Restenosis is a major complication after coronary intervention therapy. Excessive proliferation of vascular smooth muscle cells (VSMCs) and a decline in their apoptosis, which eventually leads to excessive neointimal thickening in coronary arteries, are the main causes of restenosis. Induction of the apoptosis of VSMCs and inhibition of excessive proliferation of VSMCs are therefore crucial for the prevention of restenosis, and low-intensity laser irradiation of coronary arteries may play a promising role in keeping this in balance. In this study, we used in vitro cultured rabbit VSMCs to investigate the effects of low-intensity laser irradiation at a wavelength of 532 nm on the apoptosis of VSMCs via morphological observation and molecular biology. The results showed that apoptotic bodies and obvious intranuclear apoptosis-positive particles formed within VSMCs 24 h after laser irradiation, suggesting that low-intensity laser irradiation at certain doses can inhibit the proliferation of VSMCs by promoting their apoptosis. This experiment provides evidences for further animal experiments and clinical trials on prevention and treatment of restenosis by intracoronary low-intensity laser irradiation at a wavelength of 532 nm.