Objective To use direct adherent method to induce human embryonic stem cells （hESCs） to become cardiomyocytes in vitro and examine their differentiation rate. Methods Undifferentiated hESCs were seeded onto Matrigel-coated plates at a density of 1×105 cells/cm2 and cultured in MEF-conditioned medium （MEF-CM） with 8 ng/ml basic fibroblast growth factor （bFGF） for 6 days. Then MEF-CM was replaced with RPMI 1640/B27 medium supplemented with 100 ng/ml human recombinant activin A for 24 hours in hESCs culture，followed by supplementation of 10 ng/ml human recombinant bone morphogenetic protein 4 （BMP4） for 4 days in hESCs culture. The medium was then replaced with RPMI 1640/B27 medium without supplementary cytokines，and hESCs were refed every 2-3 days for 2-3 additional weeks. Self-beating cardiomyocytes and the beating frequency were observed under the microscope，and the percentage of colonies showing beating cardiomyocytes was calculated. Cardiac troponin T （cTnT），a specific marker of cardiomyocytes，was examined by immunofluorescence. Spontaneous action potentials of cardiomyocytes were measured with patch clamp technique.Apoptotic rate of cardiomyocytes was detected with apoptosis-hoechst staining kit after beating cardiomyocytes were culturedunder hypoxia for 24 hours. Results A large number of spontaneous beating cardiomyocytes were observed 13 days after induction. The average time to show beating cardiomyocytes was 13.0±1.1 days after induction，the percentage of colonies showing beating cardiomyocytes was 66.7%，and the beating frequency of cardiomyocytes was 63.0±7.0 times/minutes. Beating cardiomyocytes were cTnT-positive. Spontaneous action potentials were detected in beating cardiomyocytes.Apoptotic rate of cardiomyocytes was 8.0%±0.5% after beating cardiomyocytes were cultured under hypoxia for 24 hours. Conclusion It’s the first time to use direct adherent method to induce hESCs to become cardiomyocytes in vitro in China with the differentiation rate of 66.7% and differentiation time of 13 days.
Abstract: Objective To study the integration of transplanted cells and host cells by detection of the cellcell junction after transplantation of the myocardiumlike cell derived from the canine umbilical cord blood. Methods The mesenchymal stem cell(MSCs) was transfected by Laz-Z after harvest, culture, induced by 5-azacytidine(5-aza). Thirty-six adult hybrid dogs were randomly divided into cell transplantation group and control group. The canine of myocardium infarction was established. 107 MSCs were transplanted into dogs with acute myocardium infarction by coronary artery infusion and local injection in cell transplantation group and physiologic saline was used in the control group. The specimens were harvested and detected by immunofluorescence for 2, 4 and 8 weeks respectively. Results The umbilical cord blood MSCs were fusiform or spindleshaped. They presented clonal and knittinglike growth.The MSCs could differentiate into myocardium-like cell by the induction of 5-aza and express α-actin, desmin, connexin43.The transplanted cells could survive more than 8 weeks after transplantation. Cadherin and connexin 43 were found in the position of cellcell junction of transplanted cells group and between transplanted cells and host cells. Cadherin and connexin 43 were found in the hose cells of the control group. Conclusion The umbilical cord blood MSCs is able to differentiate into myocardiumlike cell in vitro and form cellcell junction in vivo to communicate with surrounding cells.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To study the effects of the human keratinocyte growth factor 2 (hKGF-2) on the survival and differentiation of human neural stem cells (hNSCs). Methods The hNSCs at 17 passages preserved in liquid nitrogen were resuscitated and cultured for 7 days with normal methods to form neural spheres. The specific Nestin antigen and differentiated cells antigen were identified using immunohistochemistry technology. Some concentrated hNSCs were incubated in 12-well culture plate with 1 mL basic medium [(DMEM/F12 + N2 (1 ∶ 100) + epidermal growth factor (EGF) (20 ng/mL)] and divided into 7 groups, 6 wells each group. hKGF-2 (0, 10, 30, 60, 90, and 120 ng/mL) and bFGF (10 ng/mL) were added in groups A (control), B, C, D, E, F, and G, respectively. The neurospheres and the cell number were recorded for analyzing growth and multiplication of neural spheres. Some concentrated hNSCs were incubated in 6-well culture plate (cover glass coated with polylysine) with 3 mL DMEM/F12 medium and divided into 4 groups, 6 wells each group. N2 (1 ∶ 100), N2 (1 ∶ 100) + hKGF-2 (90 ng/mL), FBS (1 ∶ 20), and FBS (1 ∶ 20) + hKGF-2 (90 ng/mL) were added in groups A1, B1, C1, and D1, respectively. Then, the growth and multiplication of neural spheres were observed during culture; the separated neural spheres was identified and analyzed with indirect immunofluorescence and flow cytometry. Results Reanimated hNSCs could form neural spheres containing a lot of Nestin antigen; differentiated cells by induction expressed the specific antigens of neurofilament 200 (NF- 200) and glial fibrillary acidic protein (GFAP). At 7 days after culture, enlarged neural spheres were observed in each group. The neurospheres and the cell number of hNSCs increased with increased concentration of hKGF-2, showing a gradually increasing tendency; they were significantly higher in groups E, F, and G than that in groups A, B, C, and D (P lt; 0.05); significant differences were found among groups B, C, and D (P lt; 0.05), but no significant difference between groups A and B, and among groups E, F, and G (P gt; 0.05). After induction in vitro, the cell growth showed a progressive increase, significant difference was found among groups (P lt; 0.05); the percentage of NF-200 positive cells in group B1 was significantly higher than that in the other 3 groups (P lt; 0.05); the percentage of GFAP positive cells in group B1 was significantly lower than that in the other 3 groups (P lt; 0.05), but no significant difference among groups A1, C1, and D1 (P gt; 0.05). At 14 days after culture, cell growth reached the peak, which were mainly astero-cells. Conclusion The hNSCs are pure after incubated to 17 passages in vitro. hKGF-2 can promote the clone and the growth of differentiated cells, and increase the proportion of neuron.
Objective To observe whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into the smooth muscle cells (SMCs) induced by bladder SMCs (BSMCs) conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system. Methods UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1 ∶ 1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium (induced group, group A) and complete medium (control group, group B), respectively; simple BSMCs served as positive control group (group C). The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of α-smooth muscle actin (α-SMA), Calponin, and smooth muscle myosin heavy chain (SM-MHC) of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days. Results The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of α-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of α-SMA could be observed in groups A and B; at 14 days, the positive expression of α-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of α-SMA, Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining. Conclusion UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.
Objective To review the research progress of mesenchymal stem cells (MSCs) in burn wound repair. Methods The recent literature about MSCs involved in burn wound repair and mechanism was extensively reviewed and analyzed. Results MSCs have the capacity of self-renew, rapid proliferation, differentiation and paracrine, and promote burn wound repair through differentiating into a series of skin wound cells and regulating wound microenvironment. Conclusion MSCs have great potentials in the burn field. However, the cell survival and outcome are also facing challenges from poor microenvironment of the burn wound.
Objective To observe the chondrogenic differentiation of adipose-derived stem cells (ADSCs) by co-culturing chondrocytes and ADSCs. Methods ADSCs and chondrocytes were isolated and cultured from 8 healthy 4-month-old New Zealand rabbits (male or female, weighing 2.2-2.7 kg). ADSCs and chondrocytes at passage 2 were used. The 1 mL chondrocytes at concentration 2 × 104/mL and 1 mL ADSCs at concentration 2 × 104/mL were seeded on the upper layer and lower layer of Transwell 6-well plates separately in the experimental group, while ADSCs were cultured alone in the control group. The morphology changes of the induced ADSCs were observed by inverted phase contrast microscope. The glycosaminoglycan and collagen type II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA expressions of collagen type II, aggrecan, and SOX9 were detected with real-time fluorescent quantitative PCR. Results ADSCs in the experimental group gradually became chondrocytes-like in morphology and manifested as round; while ADSCs in the control group manifested as long spindle in morphology with whirlool growth pattern. At 14 days after co-culturing, the results of toluidine blue staining and immunohistochemistry staining were positive in the experimental group, while the results were negative in the control group. The results of real-time fluorescent quantitative PCR indicated that the expression levels of collagen type II, aggrecan, and SOX9 mRNA in the experimental group (1.43 ± 0.07, 2.13 ± 0.08, and 1.08 ± 0.08) were significantly higher than those in the control group (0.04 ± 0.03, 0.13 ± 0.04, and 0.10 ± 0.02) (P lt; 0.05). Conclusion ADSCs can differentiate into chondrocytes-like after co-culturing with chondrocytes.
Objective To explore the effects of mechanical stretch with variant frequencies on the alignment and differentiation of the multilayer myotubes cultured in vitro, and to select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro. Methods C2C12 myoblasts cultured in vitro in the groove casts of Sylgard 184 were induced into the multilayer myotubes. Meanwhile the myoblasts were treated with various mechanical stretch withcells tensile instrument, at the amplitude of 10% and the frequency of 0 (group A), 0.25 (group B), 0.50 (group C), and 1.00 Hz (group D) for 1 hour, 3 times a day. The myotubes morphology was observed by inverted phase contrast microscope at 5, 7, and 10 days after continuous mechanical stretch. And the expressions of mRNA for myogenic differentiation antigen (MyoD), Myogenin, Desmin, and myosin heavy chain (MyHC) were detected by RT-PCR and real-time fluorescent quantitative PCR (QRT-PCR), respectively. Results The mechanical stretch could promote the al igned fusion and increase the number of myotubes. Indeed the multilayer myotubes arranged more closely in group B at 7 days. At the same group, as the time went on, the mRNA expressions of MyoD gradually decl ined in each group. There were significant differences in mRNA expressions of MyoD between 5 days and 7, 10 days (P lt; 0.05). The mRNA expressions of Myogenin, Desmin, and MyHC were highest at 7 days. There were significant differences between different time points (P lt; 0.05), except the mRNA expression of Desmin of group B between 7 and 10 days (P gt; 0.05). At the same time, with the increase of frequency, the highest mRNA expressions of MyoD, Myogenin, Desmin, and MyHC were in group B. There were significant differences at the same time between group B and the other groups (P lt; 0.05), except mRNA expression of Desmin at 5 days between groups B and C, and mRNA expression of MyHC at 10 days between groups A and B (P gt; 0.05). Conclusion Low frequency (0.25 Hz) and suitable time (7 days) periodic mechanical stretch is beneficial to the differentiation of the multilayer myotubes cultured in the groove casts of Sylgard 184, but as the stretch time goes on the aging of myotubes will be accelerated.
Objective Bone marrow mesenchymal stem cells (BMSCs) are multi potent and thus are able to differentiate into a number of different cell types under certain culture condition. However, the effect of age on the differentiation remains unknown. To explore the effect of the microenvironment formed by Schwann cells (SCs) on BMSCs differentiation into neurons and ol igodendrocytes in rats at different ages in vitro. Methods SCs were extracted and purified from the distal sciatic nerves of neonatal Wistar rats. BMSCs were isolated from bone marrow of Wistar rats (aged 1 month, 6 months, and 12 months, respectively) and cultured in vitro. The cells were identified by immunofluorescent staining. The BMSCs at passage 2 were labeled by PKH26 and cocultured with SCs at passage 3 in equal proportions in two layer Petri dish. According to the BMSCs from the rats at different ages, experiment was divided into 3 groups: SCs were cocultured with 1-month-old rat BMSCs (group A), 6-month-old rat BMSCs (group B), and 12-month-old rat BMSCs (group C), respectively. The morphological changes of cocultured BMSCs were observed by inverted phase contrast microscope, the expressions of neuron-specific enolase (NSE) and myel in basic protein (MBP) in the cocultured BMSCs were tested by immunofluorescent staining, and the expression of neuregul in 1 (NRG1) was detected by ELISA method. Results SCs and BMSCs were isolated and cultured successfully. The identification of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD29, CD44, and CD90. At 7 days after coculture, the BMSCs in group A began retraction, and became round or tapered with the processes and had a nerve cells or ol igodendrocytes-l ike morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. Immunofluorescent staining showed that the positive expression rates of NSE in groups A, B, and C were 22.39% ± 2.86%, 12.89% ± 1.78%, and 2.69% ± 0.80%, respectively, and the positive expression rates of MBP in groups A, B, and C were 16.13% ± 2.39%, 6.33% ± 1.40%, and 0.92% ± 0.17%, respectively. There were significant differences in terms of NSE and MBP positive expression rates among 3 groups (P lt; 0.05). ELISA analysis showed that NRG1 in the supernatant of group A was increased after coculture in a time-dependent manner. At 6, 9, and 12 days of coculture, NRG1 content was higher in group A than in groups B and C, and in group B than in group C, showing significant differences (P lt; 0.05). Conclusion The microenvironment formed by SCs can promote BMSCs differentiation into neurons and ol igodendrocytes, but the differentiation capabil ity of BMSCs decreases with aging, and the variety of growth factors secreted by SCs is l ikely important factors that induce the differentiation of BMSCs into neurons and ol igodendrocytes.
Objective The bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into insul in-producing cells (IPCs) in vitro. However, low differentiation efficiency and poor maturity are the main obstacles. To investigate the feasibil ity of BMSCs differentiation into IPCs in diabetic pancreatic microenvironment of pigs. Methods BMSCs were isolated and purified from the bone marrow of a 4-week-old male pig. Fifteen female pigs (aged 8 to 10 weeks, weighing 8 to 10 kg) were randomly divided into 3 groups: normal control group (group A, n=5), diabetic control group (group B, n=5), and BMSCs transplanted group (group C, n=5). The pigs of groups B and C were treated by auris vein injections of styeptozocin and alloxan for 3 days to induce diabetes mell itus (DM) model, whose blood glucose level 2 days all greater than 17 mmol/L was successful DM model. A total of 1.1 mL of the 3rd passage BMSCs labeled with enhanced green fluorescent protein (EGFP), with cell density of 5 × 107/ mL, were injected into subcapsular pancreas of group C at multi ple points, normal saline at the same dosage into those of groups A and B. After 30 days of monitoring blood glucose, the histological analysis of islet number and size were done; the immunofluorescence staining was used to detect the protein expression of insul in in the new-formed islets. The EGFP+ cells were collected from the sections using laser-capture microdissection; RT-PCR was used to detect insulin mRNA and pancreatic and duodenal homeobox factor 1 (PDX1) mRNA expressions from EGFP+ cells, and the insul in and sexdetermining region of the Y chromosome (SRY) genes were detected by fluorescence in situ hybridization (FISH). Results The blood glucose level decreased significantly in group C when compared with that in group B from 18 days and gradually decreased with time (P lt; 0.05). The histological observation showed that the number of islets was increased significantly in group C when compared with that in group B (10.9 ± 2.2 vs. 4.6 ± 1.4, P lt; 0.05), and there was no significant difference when compared with that in group A (10.9 ± 2.2 vs.12.6 ± 2.6, P gt; 0.05). The size of new-formed islets in group C was significantly smaller than that in group A [(47.2 ± 19.6) μm vs. (119.6 ± 27.7) μm, P lt; 0.05]. The immunofluorescence staining showed that new-formed islets of group C expressed insulin protein. RT-PCR showed that the microdissected EGFP+ cells of group C expressed insulin mRNA and PDX-1 mRNA. FISH showed that the new-formed islet cells of group C contained SRY gene in Y chromosome and insulin double positive cells. Conclusion BMSCs can differentiate into IPCs in diabetic pancreatic microenvironment of pigs.