CD133(+) cells derived from human placenta identified as initiating cells for LTC-IC colony formation / 中国实验血液学杂志

The aim of this study was to evaluate whether human placenta CD133(+) cells have an ability to reconstitute long-term hematopoiesis. Magnetic-activated cell sorting (MACS) was applied to enrich human placental CD133(+) cells. The isolated human placental CD133(+)cells of four different densities were established by limiting-dilution assay and primary fetal bone marrow stromal cells separated from bone marrow as feeder layer cells were co-cultured in long-term culture system so as to observe the incidence of long-term culture initiating-cells (LTC-IC) and their ability of proliferation and differentiation.The results showed that human placenta derived CD133(+) cells contained LTC-IC with frequency of 1/645 which have an ability to proliferate and differentiate into granulocyte/macrophage colony-forming units (CFU-GM) and mixed colony-forming units (CFU-Mix). In all LTC-IC positive wells, 71.43% form only CFU-GM and 28.57% display both CFU-GM and CFU-Mix formation. It is concluded that human placental CD133(+) cells possess LTC-IC with colony-forming capacity of hematopoietic early progenitor cells.

In vitro expansion of megakaryocyte progenitor cells from human placenta CD133+ cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expansion potential of megakaryocyte progenitor cells (MPC) from human placenta tissue CD133+ (PT-CD133+) cells.</p><p><b>METHODS</b>PT-CD133+ cells were purified from mononuclear cells (MNC) by magnetic activated cell sorting (MACS) and seeded in serum-free liquid culture medium supplemented with thrombopoietin (TPO), interleukin-3 (IL-3), and stem cell factor (SCF) to expand MPC. At day 7, 10 and 14, the total cell number was counted and the dynamic changes of CD133, CD34, and CD41 antigens expression during ex-vivo expansion were analyzed by flow cytometry (FCM). PT-CD133+ cells at different expansion time were collected and cultured in collagen semisolid medium for colony forming units-megakaryocyte (CFU-MK) assay.</p><p><b>RESULTS</b>PT-CD133+ cells could be optimally expanded at day 7 by 13 +/- 2 fold increase in serum-free liquid culture systems and the total cell number was expanded by 160 fold at day 14. With the expansion time going on, the expression of CD133, CD34 decreased and that of CD41 increased. The expanded megakaryocytes were immature and no sign of platelet formation. Both PT-CD133+ cells before and after expansion could form CFU-MK, the total number of CFU-MK peaked at day 10 of expansion by 54 +/- 10 fold increase.</p><p><b>CONCLUSION</b>Human PT-CD133+ cells have a high capacity of MPC expansion, 10 days culture could give rise to the maximum number of CFU-MK.</p>

Ex vivo expansion of megakaryocyte progenitor cells for CD133(+) cells derived from human umbilical cord blood / 中国实验血液学杂志

To study the expansion potentiality of megakaryocyte progenitor cells (MPCs) derived from human umbilical cord blood CD133(+) (UCB-CD133(+)) cells and determine the optimal harvest time. UCB-CD133(+) cells were purified from mononuclear cells (MNCs) by magnetic activated cell sorting (MACS) and seeded in serum-free liquid culture medium supplemented with thrombopoietin (TPO), interleukin-3 (IL-3), and stem cell factor (SCF) to expand MPCs. At day 0, 6, 10 and 14 of culture, the total cell number was counted and the dynamic changes of CD133, CD34, and CD41 antigen expression during ex vivo expansion were analyzed by flow cytometry (FCM). At different expansion times, the CD133(+) cells were collected and cultured in collagen semisolid medium to carry out CFU-MK colony culture. The incidence of CFU-MK was calculated and the morphology of MPCs and CFU-MK were detected by immunohistochemistry and Wright-Giemsa staining. The results showed that UCB-CD133(+) cells optimally expanded at day 7 with expansion multiple of 8.2 +/- 2.2 in serum-free liquid culture systems and the total cell number was expanded by 116-fold at day 14. At 10 days, each UCB-CD133(+) cell can form 2.5 +/- 1.0, 2.6 +/- 0.5 and 20.3 +/- 5.9 cells of CD133(+)CD41(+), CD34(+)CD41(+) and CD41(+) respectively, from which the number of CD133(+)CD41(+) and CD34(+)CD41(+) cells reach the highest. UCB-CD133(+) cells both before and after expansion could form CFU-MK, the total number of CFU-MK reached the peak from cells of 10 days expansion of UCB-CD133(+) cells and the expansion multiple of CFU-MK was 59.5 +/- 11.8. Immunohistochemical results indicated that the expanded megakaryocytic cells were immature and no sign of platelet formation. It is concluded that the human UCB-CD133(+) cells have a high ability of MPC expansion, 10 days of culture can be result in optimal expansion effect.

Phenotypic characteristics of peripheral blood lymphocytes and its clinical significance in patients with coal arsenic poisoning / 中国地方病学杂志

Objective To study the change and rule of immunological function among the patients with coal arsenic poisoning in order to provide a basis for tumor risk evaluation and monitoring.Methods Seventy patients with coal arsenic poisoning aged from 24 to 71 years old(44 men,26 women,averaging 41 years old)were divided into 4 groups including 23 cases having a course less than 10 years,21 case8 lasting for 10～19 years,20 cages for more than 20 years,6 cases of cancer,and 26 healthy normal controls.Flow cytometer(FCM)was used to analyze the frequency of CD3+(total T cell),CD3+CD4+(inducer/helper T cell),CD3+CD8+(suppressor/cytotoxic T cell),CD19+(B lymphocyte),and CD56+CD16+(natural killer cell)lymphocyte subsets in the peripheral blood of the subjects and the expression rates of lymphocytic membrane surface molecules of human leucocvte antigen (HLA)-DR,CD25,CD38 were also determined by FCM.Results The pmportions of CD3+cells in periDheral blood of less than 10 years,10～19 years,more than 20 years and cancer groups were (63.76±9.32)%。(55.63± 12.97)%,(51.00±12.23)%and(49.83±,9.89)%respectively,which were significantly lower than that in control group[(68.10±8.62)%],and there was a significant difference between different groups(F=12.862,P<0.05). In less than 10 years,10～19 years,more than 20 years and cancer groups,the proportion of CD3+CD4+cells cells was (31.35±6.62)%,(28.38±8,66)%,(24.13±6.46)%and(19.17±4.96)%respectively,which wag significantly lowerthan that in control group[(34.28±7.32)%],and significant in a-group difference was found(F=10.455, P<0.05).The percentages of CD19+cells in more than 20 yeats and cancer groups[(9.00±5.32)%,(9.00± 3.29)%]were lower than that in control group and less than 10 years group[(11.80±3.43)%,(12.35±4.53)%] (P<0.05),while no statistical difference was found between other groups.The expression rates of CD25 and CD38 in lymphocytes of cancer group[(17.96 ±4.98)%,(41.38±8.54)%]were obviously higher than those in control group[(13.10±338)%,(28.60±5.51)%]and there were statistical differences between the experimental groups(P<0.05).The expression rate of HLA-DR in 10～19 years groups[(18.20±6.25)%]was significantly higher than that in control group[(10.72±7.06)%]and less than 10 years group[(11.78±5.13)%],while it was the same in more than 20 years and cancer group[(20.30±8.01)%,(21.82±10.97)%].Conclusions Reduction of cellular immune function caused by coal arsenic poisoning may be an important mechanism of skin cancer.CelMar immune function may be used as a warning signal of skin cancerization of patients with coal arsenic poisoning.

Differentiation of Human Amniotic Mesenchymal Cells into Cardiomyocyte-like Cells in Vitro / 中国生物工程杂志

To explore the plasticity of human amniotic mesenchymal cells(hAMCs) into cardiomyocyte-like cells,hAMCs were isolated from human amnion with collagenase digestion.Phenotype of the isolated cells was analyzed by flow cytometry(FCM).hAMCs were treated with 5-azacytidine and basic fibroblast growth factor(bFGF) to investigate their ability of differentiation into cardiomyocytes.The induced differentiated cells were evaluated by immunofluorescence for desmin and ?-actin expression and by RT-PCR for Nkx2.5,GATA-4 and alpha-myosin heavy chain(?-MHC) mRNA expression.The results showed that,after primary culture,hAMCs could reach a confluence of 80% with swirl like growth at 6 days.The cells proliferated rapidly after passages with a 100% confluence at 3~4 d.hAMCs were positive expression of CD44 and vimentin,but negative for CK19.After induced differentiation at 8~10d,the differentiated cells have close-up arranged with long spindle-shape.At 2 weeks and 4 weeks,induced cells expressed ?-actinin and cardiac-specific transcription factor Nkx2.5.Expressions of GATA-4 and desmin can be detected but ?-MHC can not in the hAMCs both before and after the induction.In conclusion,hAMCs have the ability of differentiation into cardiomyocyte-like cells,which means that hAMCs can be regarded as candidate cells for cellular cardiomyoplasty(CCM).

Hematopoietic stem/progenitor cells and phenotypes of lymphocyte subpopulations in human placenta / 中华血液学杂志

<p><b>OBJECTIVE</b>To study whether human placenta contains hematopoietic stem/progenitor cells (HSPCs), and analyze phenotypes of lymphocyte subpopulations in the placenta.</p><p><b>METHODS</b>Nucleated cells from fresh human placenta were analyzed for phenotypes of HSPCs and lymphocyte subpopulations by flow cytometry (FCM). And CD(34)(+) cells were sorted from human placenta nucleated cells by FCM or MiniMACS.</p><p><b>RESULTS</b>(1) CD(34)(+) cells, CD(34)(+)/CD(38)(+) cells, and CD(34)(+)/CD(38)(-) cells from a human placenta were 8.8, 4.6 and 11.9 times higher than those from umbilical cord blood (UCB), respectively. (2) The yields and purity of CD(34)(+) cells isolated from human placenta by FCM sorting system were (63.05 +/- 10.14)% and (86.39 +/- 11.27)%, respectively. (3) Lymphocytes, T cells (CD(3)(+)/CD(2)(+)), B cells (CD(19)(+)), Th cells (CD(3)(+), CD(4)(+)), and Th/Ts ratio in the placenta tissue were apparently lower than those in the UCB, while the CD(8)(+)/CD(28)(-) T suppressor cells were higher in the placenta than in the UCB.</p><p><b>CONCLUSIONS</b>Human placenta is rich in HSPCs, and has important hematopoietic function in ontogeny. It is probable that human placenta would be graft resource for HSPCs transplantation. CD(8)(+)/CD(28)(-) T suppressor cells might play an important role in feto-maternal immunologic tolerance.</p>

Optimization of Technique Conditions for Decolorization and Extract of Exopolysaccharide by Cordyceps jiangxiensis / 微生物学通报

The technique conditions of decolonization of fermentation broth were successively optimized using single factor assay and orthogonal layout method in Cordyceps jiangxiensis. The optimal condition of decolorization was investigated to be 3g/100mL active carbon, 5 min absorption time, pH5 of fermented broth and 25℃absorption temperature. Under the optimal condition, the maximum decolorization rate of fermented broth reached 89. 6% , simultaneously 10. 7% consuming rate of exopolysaccahride was minimum. Subsequently, the extract condition of exopolysaccharide of C. jiangxiensis was further optimized by orthogonal layout design. The maximum exopolysaccharide production was 0. 38 g/L under the optimal condition, i. e. firstly fermented filtrate decolorized and deproteined was concentrated to 1/7 of its total volume, secondly concentration broth was mixed with four times its volume of absolute ethanol and stirred vigorously, lastly precipitation of exopolysaccharide proceeded at 4℃for 16 hrs and the exopolysaccharide collected by centrifugal ion and dryness.