AIM:There is no standard isolation method of mesenchymal stem cells.Percoll density gradient centrifugation is treated as the traditional method among numerous methods but it is so complicated.This study compares whole bone marrow culture to traditional Percoll density gradient centrifugation in order to establish a better method in vitro.METHODS:Experiments were conducted at Central Laboratory of Affiliated Hospital of Qingdao University Medical College from September 2006 to June 2007.Bone marrow was supported by patients who had autologous stem cell transplantation by endocrinology department for diabetes treatment.The experiments had known by the patients and authorized by the hospital ethic committee.The two different methods,whole bone marrow culture and Percoll density gradient centrifugation,were compared in the number of cell clone,cell morphology,cell superficial mark,the differentiation into fat cell.RESULTS:① The number of clone cell by whole bone marrow culture was more than by Percoll density gradient centrifugation(P

OBJECTIVE: Pancreatic stem cell transplantation is a new approach for diabetes treatment. It may overcome the shortage of human donor islets and avoid the long-term applying of immunity depressors. In this article, we review the recent progress of the sources, induced differentiation and the molecular markers of pancreatic stem cells. DATA SOURCES: A computer-based online search of PUBMED was undertaken to identify relative articles published in English from June 1997 to June 2006 with the keywords of "pancreatic, stem cell, differentiate, marker". Meanwhile, we searched Wanfang database for the related Chinese articles published between June 1997 and June 2006 with the same key words in Chinese. STUDY SELECTION: The articles were firstly selected and the quotations cited by every article were looked over. Inclusive criteria: The articles focusing on the source, induced differentiation and molecular markers of pancreatic stem cells. Exclusive criteria: Repeated experiments or Meta analysis articles. DATA EXTRACTION: According to the inclusive criteria, 31 articles were finally selected among the 82 ones collected from databases.Fifty-one old data or duplicated researches were excluded. Among the 31 selected articles, 6 presented the main conceptions, 15 were related to the sources of pancreatic stem cell and its induced differentiation, 9 were focused on the surface molecular markers and 2 discussed the major obstacles at present. DATA SYNTHESIS: Stem cells are pluripotential cells with the ability to self-renew; they are classified into two major classes: embryonic stem cell and adult stem cell. Pancreatic stem cells, belonging to the adult stem cells, could differentiate into tube cells, insulin-secreting cells, exocrine cells and other specific pancreatic tissue cells, which possess the fundamental properties of long-term proliferation and self-renewal. The studies on the sources, induced differentiation and molecular markers of pancreatic stem cells are extremely useful for the treatment of diabetes. Recent studies have shown that besides gene engineering, insulin-secreting cells are mainly generated from the differentiation of embryonic stem cells and adult stem cells such as pancreatic duct epithelial cells, nestin-expressing pancreatic stem/precursor cells, bone marrow mesenchymal stem cells and haematopoietic stem cells. The investigation on the molecular markers of pancreatic stem cell surface is essential to the basic and clinical research. PDX-1, nestin, CK-19, CK-20, Ngn3 and PGP9.5 are the major markers used in the identification, isolation and purification of pancreatic stem cells. CONCLUSION: The investigations on islet cells and pancreatic stem cells transplantations are promoting. Significant advancements in search for the sources and identification of pancreatic stem cells have been achieved. However, many obstacles should be overcome before any clinical breakthroughs can be expected. With the significant improvement in stem cell research and techniques, it is possible to obtain enough pancreatic stem cells from in vitro culture for transplantation.

BACKGROUND:At present,mesenchymal stem cells(MSCs) is mainly obtained from stem cells.But the quantity of human bone marrow mesenchymal stem cells(BMSCs) is lower.Fetal MSCs possess weak immunogenicity and good differentiation potential.OBJECTIVE:To compare the biological difference in adult and fetal BMSCs.DESIGN,TIME AND SETTING:The cytology in vitro controlled study was performed at the Central Laboratory,Hospital Affiliated to Medical College,Qingdao University from February to October 2008.MATERIALS:Three samples of adult bone marrow blood were obtained from Hospital Affiliated to Medical College,Qingdao University.Three samples of fetal bone marrows were supplied by Qingdao Municipal Hospital.METHODS:Human and fetal BMSCs were harvested in vitro by the Percoll method,and incubated in LG-DMEM containing 10% volume fraction fetal bovine serum.Till cells were 90% confluent,cells were digested and passaged.The same passage of adult and fetal BMSCs were collected,and treated with KGla suspension and DMEM containing fetal bovine serum at 37 ℃ in 0.05 volume fraction CO2 for 2 hours.Non-adherent cells were removed.At the third passage,BMSCs at a density of 1?106 were incubated.When cells were 60%-80% confluent,cells were incubated in osteoblast medium for 16 days.MAIN OUTCOME MEASURES:Cell morphological changes were measured.Growth curve was detected by MTT assay.Cell adherent rate in vitro was calculated.Alkaline phosphatase staining was used to detect the differentiational results of osteoblasts.RESULTS:Adult and fetal BMSCs showed spindle-shape,but the fetal BMSCs were small.The doubling time of them was 50 hours and 30 hours,respectively.Compared with the adult BMSCs,cell adherent rate was significantly lower in the fetal BMSCs(t=4.22,P

Bone marrow mesenchymal stem cells is a new approach for diabetes treatment, it can transdifferentiate into insulin cells in vitro, that may overcome the shortage of human donor islets, avoid the immunological rejection and moral principle problems which are involved into the research of embryonic stem cells.Studies have shown that insulin gene and its other necessary gene can introduce into primary cells or other somatic stem cells directly in diabetes mellitus patients, such as hepatic cells, intestines epidermis K cells, fibroblast cells and liver tumor cells and so on.Thereby, it can be translated into insulin-secreting cells.At present, the main study direction internationally is how to import insulin gene into target cells effectively, and make it express in a physical model.

BACKGROUND: In recent years, there are some effects on using pancreatic islet cell or pancreas transplantation in the treatment of diabetes. But there are still two major problems: lack of donor and immune rejection. Mesenchymal stem cells can be induced to islet-like cells in vitro. OBJECTIVE: To study the progress in the differentiation of insulin secretion cells induced by mesenchymal stem cells. RETRIEVAL STRATEGY: A computer-based online search of Pubmed was undertaken for the related English articles dated from August 1997 to August 2007 with the of "mesenchymal stem cells,insulin secreting cell". At the same time, we searched China Journal Full-Text Database for the related Chinese articles published between August 1997 and August 2007 with the same key words. Total retrieved 108 literatures which are firstly selected into the inclusive criteria: ①The articles focusing on the mesenchymal stem cells induced differentiation into insulin secreting cells. ②choose articles at the same field or published in authoritative magazine published in the recent time. Exclusive criteria: ①Repeated experiments or ②Meta analysis articles. LITERATURE EVALUATION: All the literatures are about the differentiation of insulin secretion cells induced by bone marrow mesenchymal stem cells. Of the 30 selected literatures, 2 are reviews and the others are clinical or basic experiment study. DATA SYNTHESIS: ①Because the pancreas is similar to the nervous system in the mechanism of development and control, now people think that nerve growth factor may be the key signal to the pancreas development. Mesenchymal stem cells have an ability of multi-differentiation; it can be induced into nestin positive cells by using epidermal growth factor and basic fibroblast growth factor. Mesenchymal stem cells will be further induced to pancreatic cells if we add conditioned medium as the appropriate micro-environment for islet cells. ②Now identification and functional study of insulin-like cells are for the following areas: observe whether there is islet-like cells group gathered by cell morphology; test insulin, glucagons and somatostatin expression after induction; test insulin and glucagons gene expression using RT-PCR method after induction; to study the function of induced pancreatic cells by using glucose-stimulated insulin release test. CONCLUSION: As the research about mesenchymal stem cells can be induced islet-like cell in vitro becoming ever-changing technology, it becomes a new program of cell therapy to diabetes in face of all problems. According to all the present experimental results, the insulin secretion of induced pancreatic islet cells is much smaller than the normal amount of insulin secretion. Therefore, how to induce efficiently with human physiology of islet cell in vitro and then transplant safely into the human body will bring a new leap of diabetes treatment.

BACKGROUND: Cells grew slowly in a low number after conventional induction. Growth factor can accelerate induction speed, but growth factor is expensive. OBJECTIVE: To verify the efficiency of differentiation of human bone marrow mesenchymal stem cells (BMSCs) into osteoblasts in vitro in a inductor without growth factor. DESIGN, TIME AND SETTING: The control cytology observation experiment was conducted from July 2007 to February 2008 at the Center Laboratory of Hospital Affiliated to Medical College of Qingdao University. MATERIALS: Bone marrow was collected from diabetic patients undergoing autologous stem cell transplantation. Informed consent was obtained from patients. METHODS: Using density gradient method, BMSCs were separate and primary cultured. At the third passage, BMSCs were incubated in 0.1 ?mol/L dexamethasone, 10 mmol/L ?-glycerine sodium, and 50 ?mol/L vitamin C. BMSCs in the control group were left intact. MAIN OUTCOME MEASURES: Morphology of BMSCs was observed under a phase contrast microscope. Morphological changes were measured by alkaline phosphatase staining and calcified nodules staining after induction. RESULTS: Under the phase contrast microscope, the third passage of BMSCs was uniform, spindle. At 7 days, cells were confluence. Some cells were overlapped over time. Mesenchymal tissues gradually piled up, with mineralization, multiple nodes. At 14 days, brown mineralized nodes were found under an inverted microscope. At 21 days, mineralization in fragmentis were formed. Von Kossa method demonstrated black calcified nodules and positive alkaline phosphatase. CONCLUSION: BMSCs can be induced into osteoblasts easily, with high and rapid proliferation.

BACKGROUND: There are no studies on differentiation of bone marrow mesenchymal stem cells (BMSCs) into neurons directly induced by neural cells. OBJECTIVE: To establish the co-culture system between BMSCs and neural cells in vitro, and to study the influence of neural cells on the differentiation of BMSCs into neuron in the co-culture system. DESIGN, TIME AND SETTING: The in vitro cytology control experiment was performed at the Central Laboratory of Hospital Affiliated to Medical College of Qingdao University from December 2006 to December 2007. MATERIALS: BMSCS were harvested from bone marrow of patients with diabetes meliuts, who underwent autologous stem cell transplantation at the Hospital Affiliated to Medical College of Qingdao University. Neural cells were collected from brain tissues of infants die of asphyxia during delivery. The third passage of neural cells was used in this study. Transwell double-deck culture dish was purchased from Corning Costar, with a pore diameter of less than 3.0 ?m. Cells could not traverse, but the medium could traverse. METHODS: Neural cells were incubated in Transwell double-deck culture dish at a density of 1?106 in the co-culture group. BMSCs were incubated in the upper layer in LG-DMEM medium for 4-5 days. BMSCs were incubated in both layers in the control group. MAIN OUTCOME MEASURES: The morphological changes of BMSCs were observed and the special markers of neurons cells in BMSCs were examined by immunofluorescence. RESULTS: BMSCs in the co-culture group grew slowly, showing radial processes and connected each other. Positive rate of neuron specific enolase was (32.7?11.5)%. BMSCs in the control group were flat and wide, and negative for neuron specific enolase. CONCLUSION: Microenvironment provided by neural cells promotes the differentiation of BMSCs into neurons.

BACKGROUND: There is no ideal method about adult bone marrow mesenchymal stem cells (BMSCs) differentiate into islet-secreting cells and appreciation in vitro at present. Transgene requests strict conditions and complex program. The induction using chemicals is a present-used method. OBJECTIVE: To investigate the culture conditions for inducing adult BMSCs into islet-like cells in vitro. DESIGN, TIME AND SETTING: The cytology in vitro experiment was performed at the Central Laboratory of Affiliated Hospital of Medical College of Qingdao University from January to October 2007. MATERIALS: Bone marrow was collected from diabetic patients undergoing autologous stem cell transplantation at Department of Endocrinology, Affiliated Hospital of Medical College of Qingdao University after signing the informed consent. Epidermal growth factor and basic fibroblast growth factor were obtained from Peprotech Asia. B27 adjunct was purchased from Gibco. Nicotinamide, 2-mercaptoethanol, glutamine and dithizone were bought from Sigma, USA. METHODS: The adult BMSCs were isolated by Percoll from adult bone marrow aspirates and cultured in LG-DMEM, were suspended by Trypsin and passaged for subsequent passages. At the third to fifth passages, BMSCs were incubated at a density of 1?108 L-1 and were induced differentiation into islet-like cells through three developmental stages. In the first stage, BMSCs were incubated in HG-DMEM supplemented with 2-mercaptoethanol, glutamine for 2 days. In the second stage, BMSCs were incubated in HG-DMEM supplemented with epidermal growth factor, basic fibroblast growth factor, B27 and glutamine for 6 days. In the third stage, BMSCs were incubated in HG-DMEM supplemented with nicotinamide and 2-mercaptoethanol for 6 days. BMSCS in the control group were only incubated in the HG-DMEM. MAIN OUTCOME MEASURES: Morphological changes in BMSCs were analyzed under a phase contrast microscopy. Duodenal Homeobox 1 (PDX-1) expression was detected during the second and differentiation stage by immunofluorescence assay. Islet ?-like cell clusters from the 3rd stage were identified by positive dithizone staining. The insulin secretions under the stimulation of high or low glucose were detected by electrochemiluminescence immunoassay. RESULTS: The undifferentiated BMSCs exhibited adherent long spindle-shaped cells. After induction, cells gradually became round and formed clusters. Cells expressed the PDX-1 gene at 8 days and formed islet-like cell clusters that exhibited positive dithizone staining at 14 days. After high and low glucose treatment, no insulin was detected in the control group; insulin content significantly increased at 14 days (t=3.638-9.387, P

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BACKGROUND:Uric acid, as an endogenous antioxidant, exhibits anti-oxidative and anti-DNA damage effects, promotes osteogenic differentiation, and therefore has been paid more attentions.
OBJECTIVE:To investigate the effects of different concentrations of uric acid on the expression of genes related to Wnt/β-catenin signaling pathways during osteogenic differentiation of human bone marrow mesenchymal stem cel s.
METHODS:Healthy adult human bone marrow mesenchymal stem cel s were in vitro isolated by adherent culture of whole bone marrow. Passage 3 bone marrow mesenchymal stem cel s were induced to differentiate into osteoblasts using different concentrations of uric acid (0, 140, 280, 560μmol/L). Alkaline phosphatase activity, cel proliferation capacity, and Wnt-3αandβ-catenin mRNA expression in the Wnt signaling pathways were detected.
RESULTS AND CONCLUSION:Alkaline phosphatase staining was positive. After treatment with uric acid, alkaline phosphatase activity and cel proliferation capacity were increased, the expression of Wnt signaling pathway-related genes Wnt-3a andβ-catenin was up-regulated in a dose-dependent manner. There were significant differences in the abovementioned indices between experimental groups and between each experimental group and control group (P<0.05). These findings suggest that uric acid up-regulates Wnt-3a/β-catenin signaling pathway and thereby promotes the osteogenic differentiation and proliferation of human bone marrow mesenchymal stem cel s in a dose-dependent manner.