Effect of human liver-derived stem cells in prevention and treatment of alcoholic fatty liver disease in mice / 临床肝胆病杂志

ObjectiveTo investigate the effect of intraperitoneal transplantation of human liver-derived stem cells in the prevention and treatment of alcoholic fatty liver disease in mice. Methods A total of 30 male C57BL/6 mice were randomly divided into blank control group (group N), model control group (group M), and stem cell transplantation group (group S). The mice in group N were fed a normal diet, and those in the other two groups were fed Lieber-DeCarli alcohol liquid diet; at the same time, the mice in group S were given intraperitoneal transplantation of human liver-derived stem cells twice a week. After six weeks of intervention, body weight and liver index were measured, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were also measured. The levels of TG and non-esterified fatty acid (NEFA) in the liver were measured, and liver pathological examination and oil red O staining of the liver were performed. One-way ANOVA was used for comparison of continuous data between multiple groups, and the SNK-q test was used for further comparison between two groups. Results There were significant differences in the serum levels of ALT, AST, TG, TC, and HDL-C and the content of TG and NEFA in the liver between the three groups (F=66.94, 7.15, 8.02, 18.64, 386, 2314 and 3049, all P＜0.05), Compared with group N, group M showed significant increases in levels of ALT, AST, and TG in serum and levels of TG and NEFA in liver tissue (all P＜0.05). Group S had significantly lower levels of ALT, AST, and TG in serum and levels of TG and NEFA in liver tissue than group M (all P＜0.05). Liver HE staining and oil red O staining showed that group S had a significantly lower degree of liver steatosis than group M. ConclusionIntraperitoneal transplantation of human liver-derived stem cells has a marked effect in the prevention and treatment of alcoholic fatty liver disease in mice.

Effect of human liver-derived stem cells in prevention and treatment of alcoholic fatty liver disease in mice / 临床肝胆病杂志

ObjectiveTo investigate the effect of intraperitoneal transplantation of human liver-derived stem cells in the prevention and treatment of alcoholic fatty liver disease in mice. Methods A total of 30 male C57BL/6 mice were randomly divided into blank control group (group N), model control group (group M), and stem cell transplantation group (group S). The mice in group N were fed a normal diet, and those in the other two groups were fed Lieber-DeCarli alcohol liquid diet; at the same time, the mice in group S were given intraperitoneal transplantation of human liver-derived stem cells twice a week. After six weeks of intervention, body weight and liver index were measured, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were also measured. The levels of TG and non-esterified fatty acid (NEFA) in the liver were measured, and liver pathological examination and oil red O staining of the liver were performed. One-way ANOVA was used for comparison of continuous data between multiple groups, and the SNK-q test was used for further comparison between two groups. Results There were significant differences in the serum levels of ALT, AST, TG, TC, and HDL-C and the content of TG and NEFA in the liver between the three groups (F=66.94, 7.15, 8.02, 18.64, 386, 2314 and 3049, all P＜0.05), Compared with group N, group M showed significant increases in levels of ALT, AST, and TG in serum and levels of TG and NEFA in liver tissue (all P＜0.05). Group S had significantly lower levels of ALT, AST, and TG in serum and levels of TG and NEFA in liver tissue than group M (all P＜0.05). Liver HE staining and oil red O staining showed that group S had a significantly lower degree of liver steatosis than group M. ConclusionIntraperitoneal transplantation of human liver-derived stem cells has a marked effect in the prevention and treatment of alcoholic fatty liver disease in mice.

The role of poloxamer 188 for cord blood mononuclear cells into megakaryocytes cultivation and induction in three-dimensional WAVE Bioreactor / 中华血液学杂志

Objective: To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC). Methods: The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14. Results: In the two-dimensional (2D) culture, CD41(+), CD41(+)/CD61(+), CD61(+) megakaryocytic numbers increased significantly after adding P188 (all P<0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group (P<0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P>0.05). Conclusion: 3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.

The role of poloxamer 188 for cord blood mononuclear cells into megakaryocytes cultivation and induction in three-dimensional WAVE Bioreactor / 中华血液学杂志

Objective@#To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC).@*Methods@#The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14.@*Results@#In the two-dimensional (2D) culture, CD41+, CD41+/CD61+, CD61+ megakaryocytic numbers increased significantly after adding P188 (all P<0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group (P<0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P>0.05).@*Conclusion@#3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.

Mononuclear cells of umbilical cord blood differentiation to granulocyte cell in vitro / 中华血液学杂志

Objective@#To explore an optimal method for granulocyte cell production from umbilical cord blood mononuclear cells.@*Methods@#Erythrocytes were precipitated by hydroxyethyl starch. Mononuclear cells were isolated through Ficoll density gradient centrifugation. Different media, additives and cultivation model were chosen for granulocyte induction. Cell morphology was observed by microscopy, and cell phenotype was detected by flow cytometry. The CD18 expression of granulocytes was tested by immunofluorescence assay, and phagocytosis test was executed as well.@*Results@#Compared to fetal bovine serum (FBS) treatment group, cell viability, counts and differentiation rate of granulocytes induced by X-VIVOTM 15 combined with TPO, SCF, G-CSF but without FBS were superior. And X-VIVOTM15 medium was better than SCGM medium at effectiveness and cost. Using two-stage mode of hematopoietic stem cell expansion followed by granulocyte induction with X-VIVOTM15 combining TPO, SCF and G-CSF, cell proliferation was nearly 132 times at day 21. Flow cytometry showed that the differentiation was lagged in 2-stage mode than in direct induction mode, CD15 expression was (69.60± 1.06) % vs (97.73±0.39) %; Wright-Giemsa staining demonstrated mature granulocytes; immunofluorescence showed the expression of lysosomal proteins CD18. A strong phagocytic function of mature granulocytes was demonstrated by phagotrophic efficiency of (51.43±0.05) %. And granulocyte had chemotaxis ability under the role of chemotactic factor IL-8.@*Conclusion@#Optimized culture media and cultivation mode are achieved for functional granulocytes induction in vitro.

Protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A-induced acute liver injury in mice / 中华肝脏病杂志

Objective@#To investigate the protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A (ConA)-induced acute liver injury in mice.@*Methods@#A total of 88 male C57BL/6 mice were randomly divided into normal control group (group C), ConA model group (group M), and human liver-derived stem cells (HYX1)+ConA group (group E); according to the interval between phosphate buffer/HYX1 injection and ConA injection, Groups M and E were further divided into 3-hour groups (M1 and E1 groups), 6-hour groups (M2 and E2 groups), 12-hour groups (M3 and E3 groups), 24-hour groups (M4 and E4 groups), and 48-hour groups (M5 and E5 groups). The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and total bilirubin (TBil) in peripheral blood were measured, liver tissue sections were used to observe pathological changes, and the Ishak score for liver inflammation was determined. The independent samples t-test was used for comparison between groups, and P < 0.05 was considered statistically significant.@*Results@#The levels of ALT, AST, and TBil in group C were (36.25±1.16) U/L, (120.20±5.77) U/L, and (2.20±0.23) μmol/L, respectively; the levels of ALT, AST, and TBil and Ishak score were (8 721.23±837.39) U/L, (8 110.31±290.10) U/L, (8.41±0.10) μmol/L, and (13.32±1.30), respectively, in group M1, (8 334.31±666.50) U/L, (7 560.20±760.34) U/L, (10.40±0.80) μmol/L, and (12.67±0.81), respectively, in group M2, (8 960.75±551.93) U/L, (8 535.62±675.14) U/L, (10.95±1.43) μmol/L, and (14.57±0.65), respectively, in group M3, (8 618.57±886.40) U/L, (11 440.54 ± 1 327.86) U/L, (13.30±1.86) μmol/L, and (13.21±1.06), respectively, in group M4, and (10 170.13±1 112.37) U/L, (11 470.56±1 108.40) U/L, (12.75±1.55) μmol/L, and (15.07±1.58), respectively, in group M5. The levels of ALT, AST, and TBil and Ishak score were (1 016.35±163.47) U/L, (952.30±103.91) U/L, (7.77±0.62) μmol/L, and (3.50±0.21), respectively, in group E1, (42.10±6.20) U/L, (126.72±13.33) U/L, (3.41±0.53) μmol/L, and (2.01±0.40), respectively, in group E2, (44.21±4.30) U/L, (216.71±35.88) U/L, (3.47±0.44) μmol/L, and (2.13±0.25), respectively, in group E3, (2 909.69±212.14) U/L, (2 988.43±333.70) U/L, (7.03±0.93) μmol/L, and (4.70±0.50), respectively, in group E4, and (7 874.26±799.60) U/L, (10 940.54±947.35) U/L, (10.53±1.09) μmol/L, and (8.60±0.83), respectively, in group E5. Groups M1-M5 had significantly higher levels of ALT, AST, and TBil than group C (all P < 0.01), and groups M1-M4 had significantly higher levels of AST and ALT than groups E1-E4 (all P < 0.01), while there were no significant differences in the levels of AST and ALT between groups M5 and E5 (both P > 0.05). The pathological sections of liver tissue showed that compared with group M, group E had significant reductions in the degree of necrosis and Ishak score (both P < 0.05).@*Conclusion@#Intraperitoneal transplantation of human liver-derived stem cells has a protective effect against ConA-induced acute liver injury in mice, and the injection at 6 and 12 hours in advance has the best protective effect.

MiR-223-3p modulates megakaryocyte polyploidization by targeting MYH10 / 军事医学

Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.

MiR-223-3p modulates megakaryocyte polyploidization by targeting MYH10 / 军事医学

Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.

Establishment of human gastric cancer model in normal immune mice based on micro-carrier 6 / 中国肿瘤临床

Objective:To establish a mouse model of gastric cancer by inoculating MKN45 cells into mice with normal immune function utilizing microcarrier technology. Methods:A total of 60 male C57BL/6 mice were randomly divided into three groups, namely, 2D, con-trol, and 3D groups, according to the coculture system of MKN45 and microcarrier. The mouse models of gastric carcinoma were estab-lished by hypodermic injection. The time of tumorigenesis, rate of tumor formation, and pathological features were observed in each group. Results:In the 3D group, the time of tumor formation was short, whereas the rate of tumor formation was high (80%). No de-tectable tumor formations were observed in the 2D and control groups. HE and immunohistochemical staining of the transplantation tumor model showed evident characteristics of human gastric cancer. Conclusion:A human gastric cancer model in normal immune mice was successfully established. The onset and development mechanism of gastric cancer could be more effectively investigated in mice with normal immune function through this model. Moreover, a more valuable and new animal model for the research and devel-opment of anticancer drug was established.

NOTCH signaling of the vascular niche prompts differentiation of functional HSCs from hPSCs / 军事医学

Objective To generate hemogenic endothelial cells(HECs)from human induced pluripotent stem cells (hiPSCs)in vitro in order to learn more about the mechanism by which the vascular niche affects HECs production and self -renewal.Methods hiPSCs with reporter gene runx1c were differentiated to hematopoietic cells by spinEB method.The CD34 positive cells were sorted by magnetic-activated cell sorting(MACS)at day 10 after hematopoietic differentiation. Afterwards,these CD34 positive cells were co-cultured with DLL4 overexpressed vascular niche cells VeraVec to further differentiate to HECs.The HECs derived from the hiPSCs were characterized by FACS.Results We first established an hiPSCs single cell culture method for spinEB differentiation.Single cell cultured hiPSCs with reporter gene runx 1c were differentiated to form embryonic bodies(EBs)by spinEB method.The HECs were enriched from the day 10.Meanwhile, we cultured the E4ORF1 transfected human umbilical vein endothelial cell(HUVEC)line(VeraVec)and examined the expression of NOTCH signaling pathway related genes.According to the results, VeraVec had a high expression level of NOTCH ligand DLL4 at both mRNA and protein levels.And the CD34 positive HECs were co-cultured with DLL4 overexpressed VeraVec cells,which promoted the expression of tdTomato during hematopoitic differentiation and increased HSCs production.Conclusion A method of inducing hiPSCs differentiation by spinEB has been established, which can enrich HECs.This model can be applied to study the mechanism by which the vascular niche promotes hematopoietic differentiation from hPSCs.The generated functional HSCs are of great social and military values for HSCs transplantation and battlefield radiation injury treatment.

Specific cytotoxicity of natural killer cells against hepatocellular carcinoma and potential mechanism / 军事医学

Objective To evaluate the cytotoxicity of natural killer(NK)-92 cell lines against various human hepatocellular carcinoma cells(HCCs)and to explore the potential mechanism.Methods We established a culture method of NK-92 cell lines in vitro.Lactate debydrogenase(LDH)cytotoxicity assays and cytokine release assays were performed to determine whether NK-92 cell lines could recognize and kill HCCs in vitro.At the same time,Nu/Nu mices were housed. Subcutaneous(sc)xenografts HepG2 models of human hepatocellular carcinoma were established.1×107NK-92 cells were intravenously(iv)injected through the tail vein on days 2,9,16,23 while the control group was injected with PBS in the same way.Tumor size, tumor volume, tumor mass and mouse survival status were closely observed in experimental and control groups.Mice were euthanized when tumor-bearing time reached 28 days.Xenograft tissues were taken for general observation.Sections were cut and processed for HE staining and immunofluorescence staining.The expression of glypican-3(GPC3)protein in xenografts tissue was clearly defined.Results NK-92 cell lines that were chronically cultured in vitro and maintained typical phenotypic characteristics of NK cells with good cellular activity.Enhanced cytotoxicity and IFN-γ production of NK-92 cell lines were identified by LDH and ELISA,indicating that NK-92 cell lines could recognize and kill different kinds of HCCs.In addition,NK-92 cell lines efficiently suppressed the growth of HCC xenografts in vivo.Tumor volume in experimental group was significantly reduced compared with control group and there was low a GPC 3 expression in experimental group through immunofluorescence and immunohistochemistry results, pointing to the possibility that the cytotoxicity of NK cells was correlated with GPC3 +HCCs.Conclusion NK cells provide a promising means of therapeutic intervention for HCCs.NK-92 cell lines could eliminate HCC cells in vitro and in vivo.The cytotoxicity of NK-92 cell lines may work by killing the GPC3-positive cells in the liver cancer tissue.In addition to the anti-tumor effect, NK cells also have cytotoxicity on pathogens such as bacteria and viruses.

The induction and cryopreservation of erythroid progenitor cells derived from umbilical cord blood mononuclear cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).</p><p><b>METHODS</b>UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.</p><p><b>RESULTS</b>With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.</p><p><b>CONCLUSIONS</b>This non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.</p>