| Objective To investigate the biocharacteristics of mesenchymal stem cells (MSCs) derived from the tissue of human umbilical cord. To explore the effect of umbilical cord mesenchymal stem cells (UCMSCs) on participating in micro-autografts overlaid with allograft skin for repairing rabbit's skin defect wound, so as to support the clinical use of MSCs. To observe growth and migration of UCMSCs on polycarbonate membrane with different pore size and explore the criteria of selecting optimal Transwell insert for indirect co-culture for inducing UCMSCs differentiation. Human epidermal stem cells (ESCs) were isolated and co-cultured with UCMSCs on two sides of polycarbonate membrane, and UCMSCs were differentiated into epidermal lineage. All of above aim at providing experimental evidence for using UCMSCs as tissue engineering skin seed cells.Methods 1 Get healthy full-term cesarean umbilical cord and isolate MSCs by two methods of enzymatic digestion and explant culture. Observe the morphology of UCMSCs by inverted microscope and HE staining. Observe the ultrastructure of UCMSCs by transmission electron microscopy. By MTT assay, detect 1,3, 5-generation cell proliferation situation and draw the growth curve. By flow cytometry, detect the cell-cycle of 2,4-generation and identify the specific surface markers of UCMSCs. Identify the multi-differentiation potentials of UCMSCs by UCMSCs differentiations into osteocytes and adipocytes.2 Ad.GFP were transfected into the UCMSCs by recombinant adenovirus vector. 48h later, GFP expression in UCMSCs were observed by inverted fluorescence microscope and GFP genetically modified UCMSCs were injected subcutaneously on the auricles of rabbits. On days 3,7,14 and 21 after injection, the specimens were observed by inverted fluorescence microscope to identify the survival of UCMSCs. Eight adult Japanese Large-Ear Rabbits were randomly divided into 4 groups (n=2) for operations. Acute full-thickness skin defects were reproduced on backs of two rabbits in every group at the same time as a wound model (2 skin defect wounds per rabbit), followed by covering with equal size allograft skin plus micro-autografts, and then injecting underneath with 2 different agents respectively, including UCMSCs suspension (A group, n=8) and PBS (B group, n=8). Wound condition was observed on days 14 and 21 after operation. Wound healing rate was determined on day 21 postoperation. HE staining of the specimen was performed at 21d postoperation.3 Epiderm was obtained by digesting human foreskin with DispaseⅡand it was dissociated into single cells with trypsin and EDTA. These single epidermis cells were inoculated onto human collagenⅣcoated flasks and cultured at 37℃in a hum-idified atmosphere containing 5% CO2. The nonadherent cells were rinsed off 10-15 minutes after inoculation. The adherent cells were observed under phase contrast microscope, and they were identified with fluorescent immunocytochemical method.4 After treated with mitomycin C, UCMSCs were seeded on porous membranes of 6-well-dish Transwell inserts with different pore sizes as 0.4μm,3.0μm, and 8.0μm respectively. The cells on the bottom of each porous membrane were observed and counted after cultured for 7 days, being followed by the calculation of migration ratio. The growth and migration of UCMSCs on porous membranes were also surveyed under scanning electron microscope.5 Mitomycin C-treated ESCs were seeded and cultured on an inverted Transwell cell culture insert for six-well plates (24mm diameter with 0.4μm pores) for 1 day. The following day, UCMSCs were seeded inside of the insert. After the first 2 days, the medium was refreshed every other day. Co-culture these UCMSCs with ESCs on two sides of polycarbonate membrane for 10 days to differentiating UCMSCs into epidermal lineage. CK19, P63 andβ1-integrin expression in UCMSCs were measured by immunohistochemical staining, Western blotting, and Real Time-PCR respectively. Results 1. Fibroblastic-like cells were isolated from wharton's jelly of umbilical cord by two methods. The cells were spindle or polygonal, rendering the swirling hyperplasia, and nucleus were large, irregular, prominent nucleoli, few cytoplasm, many rough endoplasmic reticulums and mitochondria. By MTT test, similar features of cells in growth curve were detected, after 1day-incubation, the cells were added into the logarithmic phase.Generations were contacted inhibited by the different concentrationsct inhibition after 7days,. More than 80% of the cells were detected in a quiescent phase(GO-G1) of cell-cycle. The cells expressed CD44, CD29, CD105, CD73, HLA-I, did not express CD34, CD31, HLA-DR, CD45. Fluorescent immunocytochemical test showed that the cells had a significant expression of CD90 and CD44, did not express CD31 and CD45. In the conditions of vitro specific induced, these cells can differentiate into adipose cells and osteoblasts, indicating that the cells have multi-differentiation potential.2 At 48h after Ad.GFP were transfected into the UCMSCs by recombinant adenovirus vector, good GFP expression in UCMSCs were observed by inverted fluorescence microscope. On days 3,7,14 and 21 after GFP genetically modified UCMSCs injection, GFP expression cells were found in frozen sections of all specimens by inverted fluorescence microscope.3d,7d-GFP expression cells were clear, and 14d,21d-GFP expression cells were darker. Wound healing rate was higher in A group than that in B group at 21d postoperation (P<0.05). There were lots of irregular collagen fibers, fibroblasts and small vessels beneath the epidermis by HE staining of specimen observation. Some rete pegs could be found in epidermis of A group, while epidermis' basal part of B group was smooth, which was loosely connected with tissues beneath.3 The rapidly adherent cells were observed to form colonies 24 hours after inoculation. ESCs rendered a swirling proliferation, when they were growing and fusing together. HE staining showed that the cells were polygonal and the nucleuses were circular. Immunocytochemistry showed that the adherent cells were positive for CK19, P63 andβ1 intergrin. 4 Migration ratios of UCMSCs on 0.4μm,3.0μm, and 8.0μm pore sizes membranes were 0,1.8% and 8.0% respectively. The migration ratio of cells on 0.4μm pore size membrane was statistically different from that of the other two pore sizes groups (P<0.01). It was observed that a small portion of cells were growing on the bottoms of membranes and moving through the pores, except that there were no cells moved through 0.4μm pore size membrane.5 After co-culturing UCMSCs with ESCs on two sides of polycarbonate membrane for 10 days, UCMSCs were polygonal similar to ESCs, while cells in the control group still presented fibroblast-like shape. The results of immunofluorescence and Western blotting showed that CK19 and P63 were positive. However, they were negative in the control group.Investigated by Real-Time PCR, transcripts for CK19 and P63 were present with a significantly higher amounts compared to control group cells (P< 0.01). Expression ofβ1integrin did not decrease obviously. In flow cytometer test, positive ratio of CK19 in novel Transwell co-culturing was higher than that in traditional Transwell co-culturing (P< 0.01).Conclusion UCMSCs, which could be obtained by two methods of enzymatic digestion and explant culture, were powerful in self-renewal and proliferation.The results of cellular markers, biocharacteristics and multi-differentiation potentials corresponded with the standards of MSCs. UCMSCs could promote the growth of micro-autografts and wound healing. Enzymatic digestion combined with collagenⅣadherence could isolate ESCs rapidly. UCMSCs can not migrate through the 0.4μm polycarbonate membrane. Thus, both sides of 0.4μm pore size polycarbonate membrane may be used for close indirect co-culture. UCMSCs could differentiate into epidermal-like cells more efficiently by this method. All of above proved that UCMSCs could be the ideal seed cells for tissue engineering skin. |
