OBJECTIVETo study the effects of pulsed ultrasound (PUS) and pulsed electromagnetic fields (PEMF) on the secretion of extracellular matrix from a culture complex during in vitro chondrogenesis.

METHODSAll the rat bone marrow mesen- chymal stem cell pellets were cultured in achondrogenic medium. Different intensities of PUS (100, 150, and 200 mW · cm⁻²) and PEMF (1, 2, and 5 mT) were applied to the cell pellets for 2 weeks. Group N was cultured without PUS and PEMF stimu- lation as control. The culture medium was collected after 2 weeks of culture. Enzyme-linked immunosorbent assay (ELISA) was used to detect the type of collagen and glycosaminoglycan (GAG) in the culture medium.

RESULTSPUS increased the secreting-type collagen and GAG from cell pellets compared with group N (P < 0.05), whereas there was no difference in different intensities (P > 0.05). PEMF had no significant effect on the secretion of the type of collagen (P > 0.05). A PEMF of 1 mT had no significant effect on the secretion of GAG (P > 0.05). A PEMF 2 and 5 mT decreased the secretion of GAG (P < 0.05).

CONCLUSIONTo prevent the secretary of extracellular matrix may play a role in chondrogenic effect of PEMF.

Animals ; Bone Marrow Cells ; radiation effects ; Cells, Cultured ; Chondrogenesis ; radiation effects ; Electromagnetic Fields ; Extracellular Matrix ; Glycosaminoglycans ; Hematopoietic Stem Cells ; Mesenchymal Stromal Cells ; radiation effects ; Rats ; Ultrasonic Waves

BACKGROUNDThe stem-cell compartment is the primary target for the accumulation of oncogenic mutations. Overexposure to solar ultraviolet radiation is responsible for the development and progression of > 90% of skin cancers. Ultraviolet B (UVB) light-induced keratinocyte apoptosis is a strong preventive mechanism against carcinogenesis. The aim of this study was to isolate keratinocytes enriched with putative human epidermal stem cells and to investigate their apoptotic induction by UVB.

METHODSKeratinocytes enriched with putative human epidermal stem cells were isolated by adherence to collagen IV and the expressions of β1-integrin and p63 were investigated. Keratinocytes enriched with putative human epidermal stem cells and normal keratinocytes were irradiated with UVB at 0 - 80 mJ/cm(2). The apoptotic response was investigated with phase-contrast microscopy, Hoechst 33342 staining, flow cytometry of annexin V/PI, and procaspase-3 Western blotting.

RESULTSKeratinocyte enriched with stem cells expressed high levels of p63 protein and β1-integrin and low level of pan-keratin (C11). In comparison to non-irradiated cells, significant apoptosis of keratinocyte enriched with stem cells was found with 40 and 80 mJ/cm(2) UVB. However, significant apoptosis of normal keratinocytes was only found for 80 mJ/cm(2) UVB.

CONCLUSIONSHuman epidermal stem cells can undergo apoptosis in response to UVB radiation and are more susceptible than other keratinocytes. The method could be used in vitro studies of human epidermal stem cells.

Apoptosis ; radiation effects ; Blotting, Western ; Cells, Cultured ; Flow Cytometry ; Humans ; Keratinocytes ; cytology ; radiation effects ; Stem Cells ; cytology ; radiation effects ; Ultraviolet Rays

OBJECTIVE: To explore the effects of low level laser irradiation (LLLI) on the osteogenic capacity of three-dimensional (3D) structure by 3D bio-printing construct used human adipose-derived stem cells (hASCs) as seed cells. METHODS: Using hASCs as seed cells, we prepared sodium alginate/gelatin/hASCs 3D bio-printing construct, and divided them into four groups: PM (proliferative medium), PM+LLLI, OM (osteogenic medium) and OM+LLLI, and the total doses of LLLI was 4 J/cm². Immunofluorescence microscopy was used to observe the viability of the cells, and analyze the expression of the osteogenesis-related protein Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). RESULTS: The 3D constructs obtained by printing were examined by microscope. The sizes of these 3D constructs were 10 mm×10 mm×1.5 mm. The wall thickness of the printed gelatin mold was approximately 1 mm, and the pores were round and had a diameter of about 700 μm. The cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct was high, and the difference among the four groups was not significant. On day 7, the expression of OCN from high to low was group OM+LLLI, PM+LLLI, OM and PM. There were significant differences among these groups (P<0.01), but there was no significant difference between group PM+LLLI and OM. On day 14, the expression of OCN in each group was higher than that on day 7, and there was no significant difference between group OM+LLLI and OM. The expression of Runx2 in group OM+LLLI was more than 90%, significantly higher than that in group OM (P<0.01). But the expression of Runx2 in group PM+LLLI and OM+LLLI were significantly lower than that in the non-irradiated groups. The expression of osteogenesis-related protein Runx2 and OCN were higher in OM groups than in PM groups. Furthermore, the irradiated groups were significantly higher than the non-irradiated groups. CONCLUSION LLLI does not affect the cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct, and may promote the osteogenic differentiation of hASCs.
Adipocytes/radiation effects* ; Alginates ; Cell Differentiation ; Cell Proliferation ; Gelatin ; Humans ; Lasers ; Osteogenesis ; Printing, Three-Dimensional ; Stem Cells/radiation effects*

Low-intensity pulsed ultrasound (LIPUS) is a promising therapy that has been increasingly explored in basic research and clinical applications. LIPUS is an appealing therapeutic option as it is a noninvasive treatment that has many advantages, including no risk of infection or tissue damage and no known adverse reactions. LIPUS has been shown to have many benefits including promotion of tissue healing, angiogenesis, and tissue regeneration; inhibition of inflammation and pain relief; and stimulation of cell proliferation and differentiation. The biophysical mechanisms of LIPUS remain unclear and the studies are ongoing. In recent years, more and more research has focused on the relationship between LIPUS and stem/progenitor cells. A comprehensive search of the PubMed and Embase databases to July 2020 was performed. LIPUS has many effects on stem cells. Studies show that LIPUS can stimulate stem cells in vitro; promote stem cell proliferation, differentiation, and migration; maintain stem cell activity; alleviate the problems of insufficient seed cell source, differentiation, and maturation; and circumvent the low efficiency of stem cell transplantation. The mechanisms involved in the effects of LIPUS are not fully understood, but the effects demonstrated in studies thus far have been favorable. Much additional research is needed before LIPUS can progress from basic science research to large-scale clinical dissemination and application.
Cell Proliferation ; Humans ; Signal Transduction ; Stem Cells/radiation effects* ; Ultrasonic Therapy/methods* ; Ultrasonic Waves

This study was aimed to investigate the radioprotective effects of recombinant human interleukin-12 (rhIL-12) on monkey hematopoietic system, and to provide experimental evidence for future clinical prophylaxis and treatment for patients who suffered from acute radiation syndrome. In in vitro study, the effect of rhIL-12 in different concentrations (0, 1, 5, 25, 125 and 625 ng/ml) on colony forming capacity of human or monkey bone marrow-derived mononuclear cells was examined in methylcellulose H4434 medium. In in vivo study, the acute radiation syndrome model was established in 11 Rhesus monkeys which received lethal total body irradiation by 6 Gy (60)Co γ in single time irradiation. The irradiated monkeys were randomly divided into 3 subgroups: control group (n = 4) which received subcutaneous PBS injection, rhIL-12 single-dose group (n = 3) which received subcutaneous single injection of rhIL-12 (4 µg/kg) at 2 h after irradiation, and multiple-dose group (n = 4) which received subcutaneous injection of rhIL-12 (1 µg/kg per injection) at 2 h, day 3, 6 and 9 after irradiation respectively. Peripheral blood cells were counted before and after irradiation every other day. The survival status of animals were observed daily. In vitro test results showed that different concentrations of rhIL-12 obviously promoted human and healthy monkeys' bone marrow mononuclear cells to form various hematopoietic progenitor cell colonies, especial CFU-E and CFU-GM. All animals in control group died within 22 d after lethal total body irradiation, average survival time was (20.3 ± 1.2) d. Only one monkey in multiple-dose group died due to anemia on day 17. All monkeys in single-dose group survived. Compared with control group, rhIL-12-administrated monkeys' white blood cell count, hemoglobin level, platelet and reticulocyte counts showed faster recovery from high dose radiation. It is concluded that the rhIL-12 treatment can promote the bone marrow hematopoietic stem/progenitor cell colony formation in vitro and protect lethally-irradiated monkeys. There is an obvious therapeutic effect of rhIL-12 on monkeys suffered from bone marrow failure caused by severe acute radiation exposure.
Animals ; Bone Marrow Cells ; cytology ; drug effects ; radiation effects ; Cells, Cultured ; Hematopoietic Stem Cells ; drug effects ; radiation effects ; Humans ; Interleukin-12 ; pharmacology ; Macaca mulatta ; Radiation-Protective Agents ; pharmacology ; Recombinant Fusion Proteins ; pharmacology

The present paper is aimed to investigate the effects of pulse electrical stimulation on mutual adhesion of vascular endothelial cell and endothelial progenitor cell (EPC). EPC was induced from periphery blood, labeled with fluorescence dye and then co-cultured with vascular endothelial cell. With a fixed electric voltage and frequency of 5V and 5Hz, respectively, the co-culture system was continually stimulated for 24h under different pulse width, 1, 3, 6 and 9ms. After pulse stimulation, fluorescence intensity of adherent labeled EPC was measured and converted to fluorescence ratio. Compared to that in the control group, fluorescence ratio of 3 ms and 6 ms group were significantly larger, while that in the 9 ms group was lower. The peak fluorescence ratio value was appeared at 6 ms group. It is indicated that suitable pulse electrical stimulation could benefit the adhesion of endothelial cell and EPC. All these results provide a new theoretical basis about why electrical stimulation could contribute to neovascularization.
Adult ; Cell Adhesion ; radiation effects ; Cells, Cultured ; Coculture Techniques ; Electric Stimulation ; Female ; Hematopoietic Stem Cells ; cytology ; radiation effects ; Human Umbilical Vein Endothelial Cells ; cytology ; radiation effects ; Humans ; Male ; Young Adult

The aim of this study was to investigate the effect of WR2721(amifostine) against bone marrow hematopoietic damage of mice exposed to 6.5 Gy of (60)Co-γ ray. A total of 60 C57/BL6J mice was divided into 3 groups:normal group (mice were injected with physiological salt solution), irradiation group (mice were injected with physiologic salt solution before irradiation) and WR2721 group (mice were injected with WR2721 before irradiation). The WBC, neutrophil (Neut), Plt and RBC levels in peripheral blood of 3 group mice were counted within 60 days after irradiation; the bone marrow nuclear cells (BMNC) were counted at 2 and 24 hours after irradiation; the hematopoietic stem/progenitor cell (LK/LSK) level and colony formation capability were detected by flow cytometry at 2 and 24 hours after irradiation. The results indicated that the counts of WBC and neut at 4 and 18 days, Plt at 7-18 days and RBC at 10-30 day after irradiation in WR2721 group were higher than those in irradiation group (P < 0.05); the BMNC, LSK and LK levels obviously increased at 24 hours after irradiation (P < 0.05), the CFU-GEMM, CFU-GM, CFU-MK BFU-E and CFU-E all significantly increased at 2 and 24 hours after irradiation (P < 0.01), as compared with irradiation group. It is concluded that WR2721 can effectively alleviate early hematopoietic damage and promote the fast recovery of peripheral blood cells in mice exposed to γ-ray, suggesting that the WR2721 has significant radioprotective effect on hematopoietic system.
Amifostine ; pharmacology ; Animals ; Blood Cell Count ; Bone Marrow Cells ; cytology ; drug effects ; radiation effects ; Gamma Rays ; Hematopoietic Stem Cells ; cytology ; drug effects ; radiation effects ; Male ; Mice ; Mice, Inbred C57BL ; Radiation-Protective Agents ; pharmacology ; Whole-Body Irradiation

OBJECTIVETo investigate radiation-induced cell cycle changes of human breast cancer stem cells enriched by suspension culture.

METHODSThe tumorigenicity of human breast cancer stem cell line MCF-7 cultured in serum-free media was confirmed in NOD/SCID mice, and the radiosensitivity of the cells was tested by clone formation assay following radiation exposure. Flow cytometry was performed to evaluate radiation-induced cell cycle changes, and the protein expression of pCDC25C (ser216) was measured by Western blotting.

RESULTSAfter the exposure to 2 Gy radiation, the survived fraction of the cells in suspension culture and those in adherent culture was 0.856 ∓ 0.061 and 0.783 ∓ 0.097, respectively, and the cells in suspension culture showed an obviously greater capacity of tumorigenicity in NOD/SCID mice. The radiation exposure resulted in an obvious increase in the proportion of G2 phase cells from (22.03 ∓ 2.12)% to (45.83 ∓ 2.25)% and significantly increased the expression of pCDC25C (ser216).

CONCLUSIONRadiation- induced G2 phase arrest may contribute to the resistance of the breast cancer stem cells to radiotherapy.

Animals ; Breast Neoplasms ; pathology ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; radiation effects ; Female ; G2 Phase Cell Cycle Checkpoints ; radiation effects ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplastic Stem Cells ; pathology ; radiation effects ; Radiation Tolerance

OBJECTIVEAdipose tissue distributes widely in human body. The irradiation response of the adipose cells in vivo remains to be investigated. In this study we investigated irradiation response of adipose-derived stem cells (ASCs) under three-dimensional culture condition.

METHODSASCs were isolated and cultured in low attachment dishes to form three-dimensional (3D) spheres in vitro. The neuronal differentiation potential and stem-liked characteristics was monitored by using immunofluoresence staining and flow cytometry in monolayer and 3D culture. To investigate the irradiation sensitivity of 3D sphere culture, the fraction of colony survival and micronucleus were detected in monolayer and 3D culture. Soft agar assays were performed for measuring malignant transformation for the irradiated monolayer and 3D culture.

RESULTSThe 3D cultured ASCs had higher differentiation potential and an higher stem-like cell percentage. The 3D cultures were more radioresistant after either high linear energy transfer (LET) carbon ion beam or low LET X-ray irradiation compared with the monolayer cell. The ASCs' potential of cellular transformation was lower after irradiation by soft agar assay.

CONCLUSIONThese findings suggest that adipose tissue cell are relatively genomic stable and resistant to genotoxic stress.

Adipose Tissue ; cytology ; radiation effects ; Cell Culture Techniques ; Cell Differentiation ; Flow Cytometry ; Humans ; Neurons ; cytology ; Stem Cells ; cytology ; radiation effects ; X-Rays

The aim of study was to investigate the injury of bone marrow microenvironment after γ ray irradiation conditioning in mouse allogeneic hematopoietic stem cell transplantation (allo-HSCT). The mononuclear cells collected from mice bone marrow for culture in vitro, were identified by flow cytometry with double staining when cultured for 5 - 7 days. Mice were separated randomly into 4 groups, namely, the control group, irradiation group, endothelial progenitor cell (EPC) transplantation group and irradiation combined EPC transplantation group. Peripheral blood was collected to assay the circulating white blood cells. The histological, electron microscopic and immunofluorescence analyses of bone marrow were performed in the same time, furthermore the distribution of labeled EPC was determined. The results showed that EPC were identified as CD45(low/-)CD133(+)CD31(+), double positive of Dil-Ac-LDL and FITC-UEA-1. The bone marrow microenvironment injury of recipient mice was shown in the irradiation group in which the number of WBC began to decrease after conditioning, and the mice were all died at 8 days (p < 0.05). The intramedullary hemorrhage could be detected by light microscopy at 3 days after irradiation, when the destruction of connection between endothelial cell and the basement membrane was observed by TEM. There were CFSE labeled cells in bone marrow in irradiation combined EPC transplantation group at 18 hours after transplanted cultured EPC in vitro, the number of CFSE(+) cells was 58-folds of EPC transplantation group (p < 0.05). It is concluded that the irradiation can cause the severe endothelium injury that drives extrinsic EPC homing to the injured bone marrow microenvironment.
Animals ; Bone Marrow ; pathology ; radiation effects ; Bone Marrow Cells ; cytology ; radiation effects ; Bone Marrow Transplantation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; radiation effects ; Gamma Rays ; adverse effects ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Stem Cells ; cytology ; radiation effects ; Transplantation Conditioning ; adverse effects