Objective To investigate the effect of combined delivery of hepatocyte growth factor (HGF) and insulinlike growth factor-1 (IGF-1) on the development of bone mesenchymal stem cells (BMSCs) differentiation by expression of GATA-4，and to supply some evidence for clinical BMSCs transplantation therapy. Methods BMSCs were isolated from the femurs and tibias of the randomly assigned rabbits and cocultured with myocytes in a ratio of 1∶1. Myocytes were obtained from neonatal rabbits ventricles. 150 ng/ml HGF and 200 ng/ml IGF-1 were added into 4 culture bottles of 8 bottles and the other 4 bottles were not. After BMSCs were cocultured with myocytes for 1 day, 3 days, 1 week, and till 6 weeks， differentiated BMSCs were targeted and microdissected with a laser capture microdissection system, and then ribonucleic acid (RNA) was extracted and isolated. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcriptionpolymerase chain reaction (RT-PCR). And expression of GATA-4 in BMSCs was detected by semiquantitative RT-PCR. Results Before coculturing, the BMSCs were negative for α-actinin and exhibited a nucleus with many nucleoli. After coculture with myocytes, some BMSCs became αactininpositive and showed a cardiomyocytelike ultrastructure, including sarcomeres, endoplasmic reticulum, and mitochondria. BMSCs cocultured with myocytes expressed cardiac transcription factor GATA-4. IGF-1 and HGF delivery can significantly increased expression of GATA-4 for the differentiated BMSCs as compared with cells of no delivery of HGF and IGF-1. The expression level of GATA-4 in captured BMSCs began to increase at the 1st day, reach the peak at the 2nd week and kept high expression level after the 2nd week. Conclusion BMSCs can transdifferentiate into cells with a cardiac phenotype when cocultured with myocytes. Differentiated myocytes express cardiac transcription factors GATA-4. Administration of HGF and IGF-1 promoted the development of BMSCs transdifferentiate into cardiac phenotype, which is associated with the increase in expression level of GATA-4.
【Abstract】 Objective To investigate the expression of connexin 40 (Cx40) and hyperpolarization-activated cycl icnucleotide-gated cation channel 4 (HCN4) in rat bone marrow mesenchymal stem cells (BMSCs) cocultured with the sinoatrialnode (SAN) tissues in vitro, so as to evaluate the possibil ity of BMSCs differentiation into SAN cells. Methods BMSCs wereisolated from Sprague Dawley rats (aged 4-6 weeks, male or female) by the adhesive method and cultured; BMSCs at the 3rdpassage were marked with carboxyfluorescein succinimidyl ester, and then were incubated on 6-well culture plate; cell climingsl ices were prepared at the same time. SAN tissue was taken and cut into 0.3 cm × 0.3 cm mass, and then placed into 4℃ PBSsolution. The SAN tissue mass was cocultured with marked BMSCs at the 3rd passage for 3 weeks as the experimental group, andBMSCs at 3rd passage were cultured alone for 1 week as the control group. At 1, 2, and 3 weeks after coculture, the mean integratedabsorbance (MIA) values of Cx40 and HCN4 were measured by Image pro plus 5.0 through the method of immunohistochemistry,and the mRNA expressions of Cx40 and HCN4 were identified by real-time fluorescent quantitative PCR. Results TheMIA values of Cx40 and HCN4 in the experimental group were higher than that in the control group, showing significantdifferences (P lt; 0.01). In the experimental group, the expressions of Cx40 and HCN4 increased gradually with time. The longerthe culture time was, the higher the expressions of Cx40 and HCN4 were, showing significant differences (P lt; 0.05). The mRNAexpressions of Cx40 and HCN4 in the experimental group were significantly higher than those in the control group (P lt; 0.01); inthe experimental group, the mRNA expressions of Cx40 and HCN4 increased gradually with time, showing significant differencesbetween different time points (P lt; 0.05). Conclusion The expressions of Cx40 and HCN4 increase obviously after coculturingBMSCs with SAN tissue, indicating that BMSCs could differentiate into SAN cells by coculturing with SAN tissue in vitro.
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.
【Abstract】 Objective To explore an effective method to cultivate esophageal mucosa epithel ial cells (EMECs)of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Methods Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. Results The primary culture of canine EMECs arranged l ike slabstone. Immunohistochemical staining of CK-19 of the2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. E MECs werepolygon in shape and arranged l ike slabstone, and formed a single layer on the surface of SIS. The cells were contact ed closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, andshowed laminate arrangement. Conclusion With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6 %FBS is a simple and feasible method. SIS shows good biocompatibil ity and can be used as a good scaffold material in th e tissue engineered esophagus.
Objective To evaluate the effect of Schwann cell (SC) on the proliferation of marrow mesenchymal stem cells (MSCs) and provide evidence for application of SC in construction of the tissue engineered vessels.Methods SC and MSCs were harvested from SD rats(weight 40 g). SC were verified immunohstochemically by the S-100 staining, and MSCs were verified by CD 44, CD 105, CD 34 and CD 45. The 3rd passages of both the cells were cocultured in the Transwell system and were amounted by the 3H-TDR integration technique at 1, 3, 5 and 7 days,respectively. The results were expressed by the CPM(counts per minute, CPM) values. However, MSCs on both the layers were served as the controls. The Westernblot was performed to assess the expression of the vascular endothelial growth factor (VEGF), its receptor Flk-1, and the associated receptor neuropilin 1(NRP-1) in SC, the trial cells, and the controls. Results SC had a spindle shape in the flasks, and more than 90% of SC had a positive reaction for the S-100 staining.MSCs expressed CD44 and CD105, and had a negativesignal in CD 34 and CD 45. The CPM values of MSCs in the trial groups were 2 411.00±270.84，3 016.17±241.57，6 570.83±2 848.27 and 6 375.8±1 431.28at 1, 3, 5 and 7 days, respectively. They were significantly higher in their values than the control group (2 142.17±531.63，2 603.33±389.64，2 707.50±328.55，2 389.00±908.01), especially at 5 days （P＜0.05）. The Western blot indicated that VEGF was expressedobviously in both the SC group and the cocultured MSCs grou,p and was less visible in the control cells. The expressions of Flk-1 and NRP-1 inthe cocultured MSCs were much ber than in the controls. Conclusion SC can significantly promote the proliferation of MSCs when they are cocultured. The peak time of the proliferation effect appeared at 5 days. This effect may be triggered by the up-regulation of VEGF in MSCs, which also leads to the upregulation of Flk-1 and NRP-1 .
Organoids are three-dimensional structures formed by self-organizing growth of cells in vitro, which own many structures and functions similar with those of corresponding in vivo organs. Although the organoid culture technologies are rapidly developed and the original cells are abundant, the organoid cultured by current technologies are rather different with the real organs, which limits their application. The major challenges of organoid cultures are the immature tissue structure and restricted growth, both of which are caused by poor functional vasculature. Therefore, how to develop the vascularization of organoids has become an urgent problem. We presently reviewed the progresses on the original cells of organoids and the current methods to develop organoids vascularization, which provide clues to solve the above-mentioned problems.