Amplification Of Human Bone Marrow Derived Hepatic Stem Cells In Vitro For Clinic Application

At present, for end stage liver disease (such as liver cirrhosis, etc.), the best treatment is liver transplant. However, because of the lack of donor as well as the rejection, that in the clinical application is limited. In contrast, stem cell transplantation is not only the wide variety of sources, but also less traumatic. The immunogenicity of stem cells is lower than its rejection in liver transplant. Stem cell transplantation not only can be used for end stage liver disease, but also can be used for the treatment of severe liver disease. Differentiation of bone marrow cells can form liver-specific stem cell lines, known as bone marrow-derived liver stem cells (BMDLSC). The treatment of liver diseases has provided new ideas and new tools. Although the characteristics of liver stem cell surface markers has not been found, Lagasse's study has found that c-kit+Sca+Lin- cells have relatively strong ability to differentiate into liver cells in the tyrosine mice, then Tang et al has noted that the c-kit+Lin-(CD117) is a group of rich BMDLSC bone marrow stem cells in the research of radioactive liver damage repair.Our preliminary studies have confirmed that CD117-positive bone marrow cells and CD184-positive cells have a strong the potential of liver stem cells. As same as other types of bone marrow stem cells, c-kit+Lin-cells are in a limited number, and it is difficult to meet the needs of future applications. In this study, human bone marrow is separated in the way of discontinuous Percoll density gradient centrifugation. After the separation the interface layer of cells are detected for the proportion of CD117-positive cells and the proportion of CD184-positive cells to determine the highest proportion of cell populations in the way of flow cytometry. Then from fresh bone marrow, components rich in CD117-positive cells and CD184-positive cells for post-test are got. Then the cells in vitro are cultured for 0 days,7 days, and 14 days. The culture medium programs are high glucose DMEM medium with 10%autologous serum system in and serum-free high glucose DMEM medium system by adding different concentrations of promoting hepatocyte growth factor (HGPF), growth hormone thrombopoietin (TPO) and interleukin 3 (IL-3), that is, to explore the best culture system separately. Cultured in the best medium system, amplification times of cell mass positive for CD117-positive cells and CD 184 cells in the number of changes are detected by flow cytometry and c-Met RNA expression changes in real-time quantitative PCR. To conclude process program for the future with human autologous bone marrow-derived liver stem cells cloning in vitro for clinical application.[Materials and Methods]1. Isolate bone marrow-derived liver stem cells with the combination of density gradient centrifugation and flow cytometry1.1 Percoll discontinuous density gradient separation of bone marrow cell massFrom the orthopedic surgery adult bone marrow 2～5ml is collected, using percoll as the separation medium divided into 60%(1.077 g/ml),50%(1.067 g/ml), 40%(1.056 g/ml),30%(1.043 g/ml) 4 density gradient. Four nucleated cell components are isolated.1.2 Flow cytometry in various cellular components for CD117-positive cells percentage and CD184-positive cells percentageAfter cell counting, take 1×106 cells placed in 1.5ml EP tube, and adjust that to 100μl volume. In dark environment, the tubes are marked with PE directly labeled anti-human CD117 antibody and APC directly labeled anti-human CD 184 antibodies directly. Then resuspend cells to 300μl-on testing, and record ratio of CD117-positive cells and CD184-positive cell.2. Protocol for cloning human bone marrow-derived liver stem cells in vitro2.1 Isolate cells rich in CD 117+cell populations with the combination of density gradient centrifugation and flow cytometryCollect human bone marrow 10～20ml. Use percoll as the separation medium divided into 50%(1.067 g/ml),40%(1.056 g/ml) two kinds of concentration(filtration sterilization) and bottom-placed in centrifuge tubes according to the density decrease. The pretreatment of bone marrow cell suspension are slowly added to the stratified fluid.400g centrifugal force, centrifugal 25～30 minutes, the density of cell layer are showed. Specimens from the highest percentage of CD117+ cells (Percoll concentration of 50%and 40%between the cell layer) layer of cells as the purpose of cell groups.2.2 In vitro amplification for enriched CD117+human autologous bone marrow-derived liver stem cells2.2.1 determine the density of cell cultureBone marrow cell suspension is centrifuged by density gradient centrifugation. The specimens between Percoll concentration of 50%and 40%of the layer of cells are adjusted cell concentration to 6.25×105/ml,1.25×106/ml,2.50×106/ml,5.00×106/ml,1.00×107/ml,2.00×107/ml five groups of cell concentration, in 24-well cell culturing for 14 days and culture medium DMEM medium is high glucose, containing 10%autologous serum,2mg/ml injection cefmenoxime hydrochloride. Analysis of cell counts respectively.2.2.2 To determine the best system for cell amplificationMedium is DMEM medium (high glucose,2mg/ml injection cefmenoxime hydrochloride). It is added injection of hepatocyte growth promote factor (10μg/ml, 20μg/ml,40μg/ml,80μg/ml), injection of promoting platelet growth factor (25ng/ml, 50ng/ml, 100ng/ml,200ng/ml), interleukin-3 (5ng/ml, lOng/ml,20ng/ml,40ng/ml), autologous serum (10%,0%), that is, a total of 128 combinations. Bone marrow cell suspension is centrifuged by density gradient centrifugation, the specimens between Percoll concentration of 50%and 40%of the layer of cells between the groups are collected, in 24-well are randomly divided into 138 by the best cell density (cell concentration of 2.50×106/ml) and are added the combination of the above-mentioned medium, a total of 128 holes. Another 10 are added the random medium mix, as the count of the control hole. All of these are cultured for 14 days, according to the growth of passage. Cells quantities of 7 days 14 days are recorded.2.3 Detection after cloned by best amplified systemConfigure the best culture system:DMEM medium (containing 10%autologous serum,40μg/mL injection of hepatocyte growth factor,50ng/ml injection TPO, 10ng/ml interleukin-3,2 mg/ml injectable cephalosporin A hydrochloric acid oxime).2.3.1 Detection of the surface marker of cells CD117, CD184 before and after cells cultured by the optimal cloning system. Bone marrow of eight cases are collected from patients with cirrhosis of the liver and isolated by density gradient centrifugation. The specimens between Percoll concentration of 50%and 40%of the layer of cells are collected and cultured at 37℃,5%CO2 incubation, every 3 to 4 days to replace 1/3 volume of culture medium, according to the growth of passage. Specimens are collected respectively before the culture and cultivate the 7th day, the 14th days. The environment of 100ul is added 20ul PE labeled anti-human CD117 antibody, as well as direct 20ul APC labeled anti-human CD 184 antibodies directly per 1×106 cells, incubated for 20 minutes,600g centrifuge 3 minutes, and is supernatant with 1×PBS army uniform cells,600g centrifugal force, centrifuge 3 minutes, washing three times. Observe and calculate the best media system by flow cytometry respectively, when culture for 7 days,14 days, for the cell components in CD117-positive cells and CD184-positive cells amplification.3. change of c-Met RNA expression from human bone marrow-derived liver stem cells after amplification in vitro3.1 GroupingCloning 0 days (negative control), cloning of 7 days, cloning by 14 days and liver tissue (positive control)3.2 Primer design and synthesisSearch for the target gene mRNA sequences on GenBank. Design specific primers in the CDS area. Primers are designed by software name:Primer express 2.0 software, sequence is as follows:Sequence Name:H-HGFR (amplified fragment length 101 bp)Forward Primer:5'-GCT GGA ACA CCC AGA TTG TTT C-3'Reverse Primer:5'-CGA CAA CTA GAG CCA TGT TGA TG-3'Sequence Name:H-β-actin (amplified fragment length 106bp)Forward Primer:5'-GCA TGG GTC AGA AGG ATT CCT-3'Reverse Primer:5'-TCG TCC CAG TTG GTG ACG AT-3'Total cellular RNA extraction, reverse transcription reaction (reaction conditions: 37℃1h, and then 95℃3 min), fluorescence quantitative PCR reaction (reaction conditions:93℃3 minutes, then 93℃30 Miao,55℃45 seconds,72℃45 seconds, a total of 40 cycles).3.3 Calculate the ratio of the cell groups HGFR/c-Met mRNA expression levels of genes to H-β-actinAfter completion of the reaction, data are analyzed by automated computer. The results of copy number, named with B, ie the B= copy number/μl cDNA. Taking into account the various differences in the concentration of the sample total RNA ultimately results is in the following conversion formula:A= B1 (target gene)÷B2 (restricted reference materials genes), A is ratio of compared with HGFR/c-Met mRNA expression level of H-βgene to restricted reference materials-actin.4. Statistical AnalysisAll data show in X±S.Application of SPSS 13.0 statistical analysis software for statistical analysis. For differences in the proportion of cells in each layer, because of the homogeneity of variance test missing, between groups are compared with Dunnett T3. When determine the best system of cell proliferation, the 256 holes are divided into four groups analyzed (the first 7 days in serum-free,7 days plus 10% serum, serum-free 14 days,14 days plus 10%serum), in orthogonal design. Test the four effects of HGPF, TPO, IL-3, autologous serum. There no interaction effect. independent ANOVA HGPF, TPO, IL-3 effect on cell number.between groups are compared with a Benforroni method, respectively. Analysis day 7 and day 14 of autologous serum on cell count by t test. For the best culture medium for the case system, homogeneity of variance, the day 0 group and the group on day 7 of amplification compared with single-sample t test. Day 7 group and day 14 group of amplification compared with the two independent sample t test. For the cells c-Met mRNA expression level differences in testing methods varied by completely randomized design and testing of the comparative rank (Kruskal-Wallis H test). With P<0.05 indicated statistically significant differences.[Results]1. In density gradient centrifugation cells are isolated to four cell components, cell mass between Percoll 40～50%gradient fractions of cells have the highest proportion of CD117-positive cells and of CD184-positive cells, that are 2.11±0.49%,5.71±1.04%.2. It is best for the effect of high glucose DMEM with 10%self blood serum group added 40μg/ml HGPF,50 ng/ml TPO,10 ng/ml IL-3. The quantity of CD117(+) cells and CD184(+) cells increased for 6.55 and 6.20 times respectively at 7th day and 11.62 and 20.57 times respectively at 14th day.3. With the restricted reference materials H-P-actin gene ratio, with the best culture medium system, expansion of 0 days,7 days,14 days and normal liver tissue is amplified c-Met RNA expression levels of c-Met/H-β-actin are 0.016±0.005,1.873±1.954,0.340±0.113 and 0.140±0.086. The first 7 days'the expression level compared with other groups have a significant difference.[Conclusion]1. After human bone marrow cells isolated by Percoll density gradient centrifugation, it is located between 40%and 50%Percoll layer for the highest percentage of the group of CD117-positive and CD184-positive cells.2. It is a viable program that high glucose DMEM medium system contain 10% autologous serum, added HGPF 40μg/ml, TPO 50 ng/ml, IL-3 10 ng/ml for cloning of bone marrow-derived liver stem cells; By the above cloning programs, It is a viable program that high glucose DMEM medium system contain 10%autologous serum, added HGPF 40μg/ml, TPO 50 ng/ml, IL-3 10 ng/ml for cloning of bone marrow-derived liver stem cells; By the above cloning programs, The quantity of CD117(+) cells and CD184(+) cells increased for 6.55 and 6.20 times respectively at 7th day and 11.62 and 20.57 times respectively at 14th day. Using this clone program can reduce the volume of bone marrow.3. After BMDLSC is cloned by this program for 0,7 and 14 days, c-Met RNA expression level First increases and then decreases, that is 0.016±0.005,1.873±1.954 and 0.340±0.113 respectively. It is suggested that differentiation exits and amplification time should not be too long.