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BACKGROUND:Infrapatelar fat pad is often partialy resected in the knee surgery, which can be used as an important source of adipose-derived mesenchymal stem cels. OBJECTIVE: To explore the strategies of isolation, culture, and identification of adipose-derived mesenchymal stem cels from the infrapatelar fat pad and to detect the expression of cel surface markers of human adipose-derived stem cels. METHODS: Infrapatelar fat pad was obtained from patients undergoing knee arthroscopy surgery, and attached cels were obtained from adipose tissue by using colagenase I. Cels were cultured in 10% low-sugar DMEM. Stem cels proliferation was detected by means of MTT and then, cel growth curve was made. The obtained cels were induced and differentiated into adipocytes and osteocytes. Expressions of cel surface markers CD29 and CD44 were detected. RESULTS AND CONCLUSION:A few of attached cels were observed after cultured 24 hours. Cels proliferated faster and exhibited spindle shape after 1 week. Cel adherence and proliferation were speeded up after subculture. Growth curve of cels exhibited that the passages 5 and 2 cels had higher reproductive activity than passage 8 cels. The obtained cels can be induced and differentiated into adipocytes and osteocytes. Results from flow cytometry showed that 96.8% passage 5 cels expressed CD29 and 97.6% expressed CD44. These findings indicate that high-purity adipose-derived mesenchymal stem cels with high reproductive ability are easy to be isolated from the infrapatelar fat pad, which may be a kind of ideal seed cels for cartilage tissue engineering.
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BACKGROUND:EpCAMhighCD44+ colon cancer stem cels have been isolated in recent years, and most of them are related to the study of isolating tumor stem cels in colorectal cancer cel lines. There are less reports on the function of colon cancer stem cels. OBJECTIVE:To isolate, screen and culture colon cancer stem cels from colorectal carcinoma tissues and to explore the protein expression and biological characteristics of colon cancer stem cels. METHODS: The primary cels were isolated from the colon cancer tissues. EpCAMhighCD44+ cels were detected and sorted by flow cytometry. Then, these cels were cultured in serum-free medium. After the cels were replanted subcutaneously into the mice, we observed the daily performance and tumor growth in the mice. When the tumor diameter was above 1 cm, tumor tissues were taken for immunohistochemical testing. The growth of EpCAMhighCD44+ cels cultured in the serum-free medium was observed; expressions of neutral epithelial mucin and CK-20 in EpCAMhighCD44+ cels were detected. RESULTS AND CONCLUSION: EpCAMhighCD44+ colon cancer stem cels were detected in the colon cancer tissues, and the proportion of EpCAMhighCD44+ cels in primary colon cancer cels was 1.7%-38%. After 24 hours of serum-free culture, there were a few of smal suspended cel spheres which were incompact. After 21 days, dense cel spheres with uniform size were found. The formation of transplanted tumor was found in the nude mice. Hematoxylin-eosin staining showed the transplanted tumor had the typical characteristics of colon cancer cels. The expression of neutral epithelial mucin and CK-20 was observed in al the transplanted tumors. Additionaly, the proliferation of EpCAMhighCD44+ cels could be promoted by 5-fluorouracil (10, 1, and 0.1 PPC) with time. These findings indicate that EpCAMhighCD44+ colon cancer stem cels in the colon cancer have a certain ability of proliferation, regeneration, differentiation, tumor formation and chemotherapy resistance.
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BACKGROUND:Rabbit bone marrow mesenchymal stem cels as a kind of adult stem cels with strong proliferation and multilineage differentiation potential exhibit a tremendous application potential in tissue engineering and biological therapy. OBJECTIVE:To in vitro culture, proliferate and identify rabbit bone marrow mesenchymal stem cels and to observe cel biological characteristics. MEHTODS:Bone marrow of rabbits was extracted under sterile conditions to separate bone marrow mesenchymal stem cels using the whole bone marrow adherence method and Percol density gradient centrifugation method. Afterwards, the cels were purified and proliferated using differential adherence method. Morphology and growth pattern of cels were observed under microscope, and expression of cel surface antigen markers was detected by flow cytometry. RESULTS AND CONCLUSION:Rabbit bone marrow mesenchymal stem cels presented with short adherent time and fast growth. After passage and purification, impurities cel counts were decreased. Primary cels presented with triangular, fusiform and spindly shapes. Passage 5 cels with single shape showed the typical polar swirling growth, and could not express CD34 and CD45, but expressed CD29 and CD44. These findings indicate that the cels cultured using the whole bone marrow adherence method and Percol density gradient centrifugation method possess stem cel characteristics in morphology, surface markers and multilineage differentiation, which have been identified as bone marrow mesenchymal stem cels by flow cytometry.
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BACKGROUND:Placenta is a valuable source of mesenchymal stem cels for stem cel therapy and future application in the field of regenerative medicine. However, conventional methods cannot acquire a large amount of purified human placenta-derived mesenchymal stem cels. Here, we present a new method for isolating human placenta-derived mesenchymal stem cels suitable for banking strategies and for future clinical applications. OBJECTIVE:To analyze the biological characteristics of human placenta-derived mesenchymal stem cels cultured by tissue dissociating and colagenase digestion. METHODS: Human placenta-derived mesenchymal stem cels were obtained from human placenta by tissue dissociating and colagenase digestion method. Immunophenotype was analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated byin vitro adipogenic, osteogenic and chondrogenic induction as wel. RESULTS AND CONCLUSION:Human placenta-derived mesenchymal stem cels could be passaged stablyin vitro. Furthermore, the cels expressed CD73, CD90, CD105, but were negative for the markers of CD11b, CD19, CD34, CD45, and HLA-DR. Human placenta-derived mesenchymal stem cels proliferated actively and began to grow logarithmicaly at days 3-5 folowed by a plateau period at day 6. In addition, the isolated cels could be induced into adipocytes, osteocytes, chondrocytesin vitro. In a word, the results of this study demonstrated that the tissue dissociating and colagenase digestion method is an efficient method for obtaining a large amount of human placenta-derived mesenchymal stem cels that can be stably cultured in vitro and have strong proliferative ability.
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BACKGROUND:With the development of fundamental research and clinical research on stem cels, the quantity demanded for stem cels is on the increase, but the relatively out-dated cultivating technology of stem cels restrict the development of the research on stem cels. OBJECTIVE: To review the improvement and innovation of stem cel culture techniques both at home and abroad. METHDOS: In the title and abstract, “stem cels, induced pluripotent stem cels, cultivation, efficiency, reprogramming” in Chinese and English chosen as search terms were utilized to search for the papers related to stem cel culture techniques published from January 2005 to November 2014 in CNKI, VIP, Wanfang and PubMed databases, respectively. In the same field, articles published lately in the authoritative journals were preferred. Totaly 61 articles were searched initialy, and only 39 articles were included in result analysis. RESULTS AND CONCLUSION: The development and improvement of stem cel culture techniques effectively increase the culturing quantity of stem cels, improve the development of basic and clinical studies on stem cels. With the further innovation and breakthrough of stem cel culture techniques, it wil be possible to efficiently culture high-quality stem cels, and the stem cel transplantation tests for human diseases wil be promoted greatly.
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BACKGROUND:Mesenchymal stem cel s can differentiate into nerve cel s by chemical induction or co-culture method, but whether mesenchymal stem cel s co-cultured with Schwann cel s differentiate into neuronal-like cel s or Schwann-like cel s is stil controversial. OBJECTIVE:To explore the inductive role of Schwann cel s derived from rats in the differentiation of human umbilical cord mesenchymal stem cel s. METHODS:Cocultures of human umbilical cord mesenchymal stem cel s (1×109/L) and Schwann cel s (1×109/L) from neonatal rats were performed using transwel culture dishes. After 2 weeks of cocultures, morphology of the cultured human umbilical cord mesenchymal stem cel s was observed, and the phenotypic changes of cel s were also detected with immumocytochemistry techniques. RESULTS AND CONCLUSION:After 2 weeks of cocultures, some differentiated cel s showed neuron-like morphology, and expressed nestin, NF-200 andβ-III-tubulin, but did not express Schwann cel special marker S100 and oligodendrocytes special marker MAB1580. These findings indicate that human umbilical cord mesenchymal stem cel s can transdifferentiate into neuronal-like cel s by cocultures with rat’s Schwann cel s.
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BACKGROUND:Mesenchymal stem cel s as potential seeded cel s have been widely used in tissue engineering and clinic therapy;thus, the precise, safe, effective isolation of mesenchymal stem cel s is the particular important premise to build culture system. OBJECTIVE:To review the methods of isolating mesenchymal stem cel s and to compare the merit and demerit of different methods, thereby providing theoretical basis for safe and high-effective isolation of mesenchymal stem cel s. METHODS:A computer-based online research of CNKI and PubMed databases was performed to col ect articles, which included reviews, clinical trials and experiments, published between 1965 and 2014 with the key words of“mesenchymal stem cel s (MSCs), isolation methods”in Chinese and English. A total of 52 articles were included according inclusion and exclusion criteria RESULTS AND CONCLUSION:(1) The whole bone marrow culture method can derive a mass of mesenchymal stem cel s, which need to be purified. (2) The density gradient centrifugation method which uses the media with the density of 1.073 g/mL can be used to harvest more purified cel s. (3) The tissue digestion method is suitable for digestion and isolation of adipose tissue and umbilical cord tissue. Type II col agenase digestion is better, but they are both limited by a high demand for operative techniques. (4) Immunomagnetic bead separation is appropriate to study the biological characteristics of a kind of subpopulation of mesenchymal stem cel s which express special surface markers. (5) The combination method is also an optimal way. (6) Some new methods limited by few dates require further studies.
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BACKGROUND:The reports on bone morphogenetic protein-7 as a stimulating factor to induce osteogenic are relatively rare. OBJECTIVE:To study the expression of alkaline phosphatase of periosteal cel s after induced by bone morphogenetic protein-7 in vitro. METHODS:Periosteal cel s were obtained from adult tibial periosteum, and then the periosteal cel s were cultured by routine method in vitro. The cel s were divided into experimental group and control group, and then cultured with bone morphogenetic protein-7 plus osteoblast culture adjuvants and simple osteoblast culture adjuvants, respectively. The phase contrast microscope was used to observe the morphology and ultrastructure of periosteal cel s. Each group was observed at 7, 14 and 21 days, and three samples were observed at each time point. Alkaline phosphatase kit was used to detect the expression of osteoblast-specific markers alkaline phosphatase. RESULTS AND CONCLUSION:After cultured for 7 days, the proliferation of periosteal cel s in the experimental group and the control group was increased obviously, and the expression of alkaline phosphatase was detected but less. The cel s were spindle in shape, while the expression of alkaline phosphatase in the experimental group was higher than that in the control group. After cultured for 14 days, the proliferation of periosteal cel s in the experimental group and the control group was increased obviously, the cel morphology was changed from spindle-shaped to wide spindle-shaped, and the expression of alkaline phosphatase in the experimental group was increased significantly when compared with the control group. After cultured for 21 days, the proliferation of periosteal cel s was detected in the experimental group and the control group, and the proliferation in the experimental group was more significant than that in the control group, the cel morphology was wide spindle-shaped, and the number of alkaline phosphatase in the experimental group was higher than that in the control group. Statistical analysis showed that the positive rate of osteogenic markers alkaline phosphatase of bone morphogenetic protein-7 induced periosteal cel s in the experimental group was higher than that in the control group (Posteogenic and regeneration ability, the bone morphogenetic protein-7 could induce periosteal cel s, promote the expression of alkaline phosphatase, and could induce the periosteal cel s to transform into osteoblasts.
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BACKGROUND:There are various methods for the treatment of osteonecrosis of femoral head, but there is no satisfactory method to promote the repair of osteonecrosis of femoral head. In recent years, bone marrow mesenchymal stem cel transplantation for the treatment of osteonecrosis of femoral head has achieved certain effect. OBJECTIVE:To review the application progress and problems of bone marrow mesenchymal stem cel transplantation for the treatment of osteonecrosis of femoral head. METHODS:A computer-based online search was performed in PubMed database, Wanfang database and CNKI database for the related articles from 1999 to 2012. The articles on the isolation, culture, differentiation, labeling and in vivo tracing of bone marrow mesenchymal stem cel s were selected, as wel as the basic and clinical researches on bone marrow mesenchymal stem cel transplantation for the treatment of osteonecrosis of femoral head. A total of 39 articles were included for review. RESUTLS AND CONCLUSION:At present, the method for the isolation of bone marrow mesenchymal stem cel s includes adherence screening method, density gradient centrifugation, flow cytometry separation and magnetic activated cel sorting method;the commonly used method for cel labeling and tracing includes isotope tracing method, antigen labeling method, antigen labeling, fluorescent labeling and MRI contrast enhancer labeling method. The method for the treatment of osteonecrosis of femoral head with bone marrow mesenchymal stem cel s includes pith dril ing decompression combined with bone marrow mesenchymal stem cel injection and transplantation, intervention plus bone marrow mesenchymal stem cel transplantation, gene transfection combined with bone marrow mesenchymal stem cel transplantation and tissue engineering technology of bone marrow mesenchymal stem cel s. Although, the research on the bone marrow mesenchymal stem cel transplantation for the treatment of osteonecrosis of femoral head has achieved great progress, there are stil problems needed to be further solved.
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BACKGROUND: Chinese medicine Gukang prescription has a clear effect on clinical treatment of osteoporosis, but the therapeutic pathway is stil unclear. OBJECTIVE:To investigate the effects of Chinese medicine Gukang on the expression of receptor activator of nuclear factor kappa B ligand and osteoprotegerin by regulating core binding factor alpha 1 expression to control the growth and development of osteoblasts. METHODS:Sprague-Dawley neonatal rats within 24 hours after delivery were used for the separation and culture of osteoblasts. Adult Sprague-Dawley rats were used to prepare drug-containing serum, and then divided into two groups randomly:normal control group and Gukang group. Rats in the normal control and Gukang groups were intragastrical y administrated with extract of Gukang prescription and normal saline based on rat’s body surface area, for 1 consecutive week. Two hours after the last administration, blood samples were taken from the heart. Then the serum was col ected. Osteoblasts at passage 3 were confirmed with alkaline phosphatase assay and digested. After counting and planting, al osteoblasts were divided into two groups and treated with col ected serum for 72 hours. Proliferative rate of osteoblasts was detected by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Secretion of alkaline phosphatase was detected by using enzyme linked immunosorbent assay and corrected with the corresponding absorbance value. mRNA expression of core binding factor alpha 1, receptor activator of nuclear factor kappa B ligand and osteoprotegerin were detected by using reverse transcription-PCR in al groups. RESULTS AND CONCLUSION:mRNA expression of osteoprotegerin and core binding factor alpha 1 in the Gukang group was significantly higher than that in the normal control group, but protein and mRNA expression of receptor activator of nuclear factor kappa B ligand were dramatical y lower in the Gukang group compared with the normal control group (Pcore binding factor alpha 1, thereby adjusting the expression of receptor activator of nuclear factor kappa B ligand and osteoprotegerin, which may be one of the mechanisms underlying Gukang treatment for osteoporosis.
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10.3969/j.issn.2095-4344.2013.23.005
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10.3969/j.issn.2095-4344.2013.23.014
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10.3969/j.issn.2095-4344.2013.23.012
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10.3969/j.issn.2095-4344.2013.23.017