Optimal in vitro culture conditions for murine predominant immature CD8a+ dendritic cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The prospects of using immature CD8a(+) dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers of predominant DC2 from murine bone marrow cells.</p><p><b>METHODS</b>Three groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without endogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Flt3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml Flt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without additional cytokines. All cells were cultured at 37 degrees C under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies.</p><p><b>RESULTS</b>The cytokine group exhibited higher proportions of typical immature CD8a(+) DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P<0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P<0.05).</p><p><b>CONCLUSIONS</b>Culture in the presence of 5 ng/ml GM-CSF, 25 ng/ml Flt3L, 20 ng/ml IL-4 and 100 ng/ml SCF can rapidly induce large quantities of predominant immature CD8a(+) DC from murine bone marrow cells. Therefore, these represent optimal culture conditions for preparing murine immature DC2 in vitro.</p>

Establishment and characterization of two new human embryonic stem cell lines, SYSU-1 and SYSU-2 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stem cell lines are highly warranted.</p><p><b>METHODS</b>Surplus embryos (blastocysts) from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation.</p><p><b>RESULTS</b>Two human embryonic stem cell lines (SYSU-1 and SYSU-2) were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA-4, Tra-1-60, Oct-4, Nanog and Rex-1. They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo.</p><p><b>CONCLUSION</b>Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.</p>

Association between the beta 2 adrenergic receptor polymorphism and blood pressure in YueXi population / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the association between Arg16Gly polymorphism of beta(2)-adrenergic receptor (ADRB2) gene and blood pressure levels.</p><p><b>METHODS</b>A total of 487 hypertensive individuals were recruited from YueXi county of Anhui province. 672 patients' parents and siblings were also invited to take part in the study and used as genomic control. Blood pressure was measured and a standardized questionnaire regarding social-economic characteristics, general health status, occupational history and life-style and dietary factors was administered for each participant. The ADRB2 Arg16Gly polymorphism was genotyped by using a PCR-Restriction Fragment Length Polymorphism (RFLP) method. The association between the ADRB2 polymorphism and hypertension in hypertensive adults was determined by utilizing a family-based association test analysis.</p><p><b>RESULTS</b>In this study population, carriers of the ADRB2 Arg16 allele had lower systolic (P < 0.01) and diastolic (P < 0.01) blood pressure, suggesting that the genetic effect on blood pressure was more likely to fit an additive model.</p><p><b>CONCLUSION</b>Our results suggest a probable association between Arg16Gly polymorphism of ADRB2 gene and hypertension.</p>

Effect of rat mesenchymal stem cells on hematopoietic reconstitution after allogeneic co-transplantation with bone marrow / 中国实验血液学杂志

To investigate effects of rat bone marrow mesenchymal stem cells (rBMMSC) on hematopoiesis after allo-hematopoietic stem cell transplantation (HSCT), allogeneic BMT model from Fischer 344 rats (RT-1Al) to Wistar rats (RT-1Au) was established; effects of MSCs on hematopoietic reconstitution were studied by survival rate, peripheral blood counts, histological analysis and FACS at day 30 after transplantation. The results showed that (1) MSCs from donor Fisher344 could survive in recipient irradiated by lethal dose and could be found in the thymus, spleen and bone marrow of the recipient at 30 days after cotransplantation with BM by measuring EGFP gene. (2) Cotransplanation of MSCs and BM improved hematopoietic reconstitution. Lymphocyte and platelet counts of peripheral blood in cotransplantation group were higher than those in the control group. Active hematopoiesis and increase of bone marrow nucleated cells were observed in cotransplantation group. MSCs significantly enhanced hematopoiesis of B lymphocyte and megakaryocytopoietic lineages by FACS analysis. It is concluded that (1) MSCs of Fisher344 can be found in the thymus, spleen, bone marrow of the recipients at 30 days after cotransplantion by measuring EGFP gene. (2) hematopoietic reconstitution is significantly enhanced by MSCs cotransplanted with BM.

Generation of CD34+/Sca-1+ cells from mouse embryonic stem cells with two-step differentiation in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>Embryonic stem cells (ESCs) are derived from totipotent cells of early embryo and they are potential to differentiate to any kind of cells of tissues in the body. Some reports showed that ESCs had broad capabilities of differentiating to variety of hematopotietic cells, such as erythroid, granulocyte/macrophage, megakaryocyte, mast and lymphocyte precursors. However, it is very difficult to control the phase of differentiation for ESCs in vitro. There is few report about hematopotietic stem cells (HSCs) from ESCs. Therefore, this research was designed to establish a culture system for generation of CD(34)(+)/Sca-1(+) HSC from ESC in vitro.</p><p><b>METHODS</b>Single mouse E 14.1 cells were suspended in methylcellulose medium, containing 40 ng/ml stem cell factor (SCF) and 20 ng/ml vascular endothelial growth factor (VEGF) and incubated at 37 degrees C with 5% CO2. In order to ensure the viability of the primary differentiation cultures over an extended period of time, the cultures were fed on day 7 with a dilute methylcellulose medium containing VEGF, SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (EPO), which promoted their primary differentiation into embryoid bodies (EBs) with more CD(34)(+)/Sca-1(+) cells. Then, EBs with peak level of CD(34)(+)/Sca-1(+) cells were dispersed into single cells and replanted either in methylcellulose medium or in bone marrow stromal cells differentiation system containing 15% fetal bovine serum (FBS), 160 ng/ml SCF, 20 ng/ml VEGF, 30 ng/ml IL-3, 30 ng/ml IL-6, 3 U/ml EPO and 20% BIT for HSC into second-step differentiation. The HSCs were characterized by flow cytometric analysis, colonogenic cell assay and Wright-Giemsa stains.</p><p><b>RESULTS</b>VEGF had the strongest stimulatory effect on the enhancement of the CD(34)(+)/Sca-1(+) cells population when combined with SCF, IL-3, IL-6 and EPO. It could markedly accelerate mouse E14.1 cells to differentiate into EB with more CD(34)(+)/Sca-1(+) cells. Cell cytometric analysis showed CD(34)(+)/Sca-1(+) cells were up to (1.91 +/- 0.40)% by day 5 and (8.11 +/- 1.17)% by day 8, and the peak level of CD(34)(+)/Sca-1(+) cells was (13.72 +/- 1.92)% by day 12. However, CD(34)(+)/Sca-1(+) cells could not increase in number with the prolongation of differentiation. So renewal single cells suspension from EB by day 12 was dispersed into the second step differentiation. The results showed that HSC was slowly generated with a few hematopoietic colony formations in methylcellulose medium differentiation system. CD(34)(+)/Sca-1(+) cells got (2.74 +/- 0.80)% by day 5 and (11.37 +/- 1.84)% by day 10, and apex percentage of CD(34)(+)/Sca-1(+) cells was about (20.52 +/- 2.78)% by day 14. However, EBs generated quickly for HSC with increased hematopoietic cell population by co-culture on bone marrow stromal cells feeder. Flow cytometric analysis showed that the percentages of CD(34)(+)/Sca-1(+) cells was (7.33 +/- 1.61)% by day 5, (13.28 +/- 2.59)% by day 8, and (20.81 +/- 3.19)% by day 10. EB cells were induced after 12 days to reach the peak level of (34.60 +/- 3.71)%. Hematopoietic colony formation unit (CFU) analysis showed that CFU was sufficient from cells on bone marrow stromal cells differentiation system in the second step compared to that in methylcellulose medium differentiation system, and Wright-Giemsa stain could confirm its characteristics of hematopoietic progenitors.</p><p><b>CONCLUSION</b>Using two-step differentiation, the investigators got a good way to control the phase of differentiation from ESC to HSC. The bone marrow stromal cell differentiation system combining with VEGF, SCF, IL-3, IL-6 and EPO was an optimal system for the generation of HSC with CD(34)(+)/Sca-1(+) surface marker from ESC differentiated in vitro. This study demonstrated that these cells could form more hemopoietic colonies.</p>

Expression of killer cell inhibitor receptors on immunocompetent cells with relation to graft-versus-host disease after hematopoietic stem cell transplantation / 中国实验血液学杂志

The study was aimed at the exploration of relationship between T cells expressing killer cell inhibitor receptors (KIR, CD158 and CD94) and graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation. The expression rates of CD158a, CD158b and CD94 on T cells and NK cell were detected by flow cytometry and donor/recipient HLA-Cw was analyzed using PCR after peripheral blood stem cell transplantation (PBSCT) and umbilical cord blood transplantation (UCBT). After both PBSCT and UCBT, the rates of CD3(+)CD158a(+) and CD3(+)CD158b(+) T cells increased, especially the rate of CD8(+)CD158b(+) T cells. In both acute and chronic GVHD groups, the rate of CD3(+)CD158b(+) T cells increased, especially in acute GVHD. The CD94 mainly expressed on CD3(+)CD8(+) T cells. The percentage of the expression of CD94 on CD4(+) and CD8(+) cells after UCBT and PBSCT increased significantly. The expression of KIR in GVHD (early stage of transplantation) increased but the expression of KIR in chronic GVHD (advanced stage of transplantation) decreased. Five patients who HLA-Cw matched had no severe GVHD. In four patients who underwent allo-PBSCT and UCBT from related HLA-matched donors, only 2 patients had no aGVHD. Four patients underwent transplantation from unrelated HLA-matched donors had GVHD. These observations suggested that there is some relationship between GVHD and KIR expression on T cells. CD158b might be an inhibitory molecule of T cell activated at early stage after transplantation. Understanding the mechanism of GVHD with the expression of KIR on T cells, especially those binding the HLA-Cw might shed light on the establishment of the specific immunotolerance for the prevention of GVHD. To pay attention to HLA-Cw typing is very important to reduce GVHD and increase GVL effect in related or unrelated HLA-matched transplantation.

Establishment of a murine model for allogeneic umbilical cord blood transplantation / 中国实验血液学杂志

This study was undertaken to establish a murine model for unrelated allogeneic umbilical cord blood transplantation (UCBT). The characteristics and percentage of hematopoietic stem/progenitor cells between near-term fetal and neonatal murine peripheral blood (FNPB) and bone marrow (BM) were evaluated by flow cytometry and semisolid methylcellulose culture. BABL/c (H-2(d)) recipient mice conditioned with high dose CTX were transplanted with FNPB form C57BL/6 (H-2(b)) mice and the survival rate, hematopoietic and immunological reconstruction, graft versus host disease (GVHD) and engraftment level were observed. The results showed that the numbers of day 14 CFU-GM and CFU-GEMM in FNPB (176.40 +/- 78.39)% and (141.40 +/- 56.57)%, respectively were much higher than those in BM (75.20 +/- 26.41)% and (68.80 +/- 23.95)%, respectively. Moreover the percentage of Sca-1(+) CD34(+) cell subsets in FNPB (3.63 +/- 1.13)% was also higher than that in BM (1.41 +/- 0.8 7)%. FNPB transplantation improved survival rate and reconstituted hematopoietic and immune function in recipients. There was no evidence of GVHD. Chimeric analysis showed that the proportion of donor cells in BM of recipients was 27.94% at 21 days after transplantation. It was concluded that FNPB contains more hematopoietic stem and progenitor cells with high expansion ability and weak allogeneic immunity, which was similar to human UCB. The murine model for allogeneic UCBT (C57BL/6-->BALB/c) was established successfully.

Osteogenic and neurogenic differentiation of human yolk sacm esenchymal stem cells / 中国病理生理杂志

AIM: To purify human yolk sac mesenchymal ste m cells (hYS-MSC) and investigate its osteogenic and neurogenic differentiation potentials. METHODS: hYS-MSC were separated from yolk sac and purified via p assage culture. The karyotype of hYS-MSCs was analyzed via G-banded characterist ics. Flow cytometric analysis was used to determine the cell cycle and phenotype of hYS-MSC. The AKP expression of hYS-MSC was also tested. Osteogenic different iation of hYS-MSCs was induced by 10 -8mol/L dexamethasone, 10 mmol/L ?-gl ycerophosphate and 50 mg/L vitamin C. Alizarin red S stain was used for identifi cation of mineralization. ?-mecaptoethanol or salviae miltiorrhizae were used t o induce neurogenic differentiation of hYS-MSCs. The expressions of NSE, NF and GFAP were identified by immunohistochemical method. RESULTS: hYS-MSCs could be purified at passages 2 or 3. The cell cycle analysis suggested that hYS-MSCs showed strong proliferational potentials by which the cells kept normal diploid karyotype during the in vitro cultur e. Flow cytometry showed the phenotype of purified hYS-MSCs was uniformly positi ve for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD3 4, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Osteogenic d ifferentiation was appeared after induction of osteogenic differentiation. hYS-M SCs, which were of spindle shape, uniform in size, were induced to pleomorphism osteoblast-like cells which expressed high level of AKP. Aggregates or nodules w ere formed at day 7 and calcium accumulation was detected by alizarin red S stai ning on day 10 or day 14. Neurogenic differentiation of hYS-MSCs was induced by ?-mecaptoethanol or salviae miltiorrhizae. NSE, NF or GFAP positive cells were detected by immunohistochemical staining. CONCLUSIONS: hYS-MSCs have strong proliferation potential and th e normal diploid karyotype is kept during the in vitro culture. The phenoty pe of hYS-MSCs is coincident with adult hMSCs. hYS-MSCs could be induced to dif ferent iate into osteogenic or neurogenic cells.