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.

Differentiation potential of CD41⁺ cells derived from the mouse aorta-gonad-mesonephros region, yolk sac and embryonic circulating blood / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the differentiation ability difference of hematopoietic, mesenchymal and endothelial potential between CD41⁺ cells derived from the mouse aorta-gonadmesonephros (AGM) region, yolk sac (YS) and embryonic circulating blood (CB).</p><p><b>METHODS</b>CD41⁺ cells were sorted from AGM, YS and CB. The CD45 and c-kit expression were studied in CD41⁺ cells by flow cytometry. IL-3 and bone morphogenetic protein 4 (BMP-4) treatment together with semi solid culture were used to assess hematopoietic potential difference of CD41⁺ cells. Immunofluorescence staining of α-SMA was used to assess mesenchymal potential difference. The endothelial cell induction system was used to assess endothelial potential difference.</p><p><b>RESULTS</b>The proportions of CD45+ cells in CD41⁺ population were 51.9% (AGM), 45.8% (YS) and 22.2% (CB), respectively, while those of c-kit⁺ cells were 40.0% (AGM), 39.6% (YS) and 36.2% (CB), respectively. After stimulated by IL-3 factor, the number of total colonies increased in all three groups-derived CD41⁺ cells compared to that of unstimulated group[(14.1±1.9) vs (1.2±0.2), (32.4±1.1) vs (18.4±2.2) and (41.8±0.9) vs (10.4±1.8)], (P<0.01). After stimulated by BMP-4 factor, compared to unstimulated group, CFU-Mix colony number in CD41⁺ cells from AGM region and YS were significantly decreased[(0.5±0.6) vs (3.2±0.8), (1.3±0.7) vs (7.4±1.7)](P<0.01), but there was no difference in CB group[(2.5±0.5) vs (3.9±1.5)](P>0.01). The mesenchymal marker α-SMA was highly expressed in CD41⁺ cells from AGM region and YS, but lowly expressed in CD41⁺ cells from CB.</p><p><b>CONCLUSION</b>There are some differences between CD41⁺ cells in AGM region, YS and CB on hematopoietic cell surface marker expression, hematopoietic colony formation with IL-3 and BMP-4 stimulation.</p>

Application of melanocytes and bone marrow mesenchymal stem cells in tissue engineered skin construction / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the method of constructing tissue-engineered skin using melanocytes and bone marrow mesenchymal stem cells (BMSCs) in vivo.</p><p><b>METHODS</b>Melanocytes were isolated from human foreskin. BMSCs were isolated from human bone marrow. Both of them were co-cultured at a ratio of 1:10, and then were implanted into the collagen membrane to construct the tissue-engineered skin, which was applied for wound repair in nude mice. The effectiveness of wound repair and the distribution of melanocytes were evaluated by morphological observation, in vivo 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) fluorescent staining tracing, HE staining, S-100 immunohistochemistry, and transmission electron microscopy.</p><p><b>RESULTS</b>The wounds were satisfactorily repaired among the nude mice. The melanocytes were distributed in the skin with normal structure, as confirmed by DAPI fluorescent staining tracing, HE staining, S-100 immunohistochemistry, and transmission electron microscopy.</p><p><b>CONCLUSION</b>Melanocytes and BMSCs, after proper in vitro culture at an appropriate ratio, can construct the tissue-engineered skin with I type collagen membrane.</p>

Erythropoietin gene-modified conditioned medium of human mesenchymal cells promotes hematopoietic development from human embryonic stem cells / 中国实验血液学杂志

The study was aimed to investigate the effect of deriving hematopoietic cells from human embryonic stem cells (hESCs) by the erythropoietin gene-modified conditioned medium of human mesenchymal cells. The mesenchymal stem cells (MSCs) steadily expressing EPO were established by lentiviral system. The expression of exogenous EPO was detected by RT-PCR and Western blot. After suspension culture, hESCs developed into embryonic bodies (EBs). Then the EB cells were cultured in conditional medium. The hESCs-derived hematopoietic cells were analyzed by immunofluorescence, CFU assay and RT-PCR. The results indicated that the exogenous EPO successfully expressed in the EPO transfected MSCs (EPO/MSCs). The supernatant from EPO/MSCs increased CD34(+) cell population and the expression of globin, and enhanced colony forming unit incidence. These effects were obviously higher than that of control. It is concluded that the EPO gene-modified conditioned medium of human mesenchymal cells can induce the hESCs to differentiate into hematopoietic cells.

Induction of hepatic specification of human adipose-derived stem cells (hADSCs) in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro.</p><p><b>METHODS</b>hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined.</p><p><b>RESULTS</b>The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production.</p><p><b>CONCLUSION</b>hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.</p>

Effect of doxorubicin induced GM-CSF gene expression regulated by Egr-1 promoter on recovery of hematopoietic function in bearing-melanoma mice / 中国实验血液学杂志

This study was purposed to investigate the effect of hematopoietic growth factor expression regulated by Egr-1 promoter on the recovery of hematopoietic function in bearing-melanoma mice after chemotherapy with doxorubicin (ADM). The human GM-CSF cDNA and enhanced green fluorescence protein (GFP) cDNA were linked together with internal ribozyme entry site (IRES) and then were inserted into the expression vector pCI-neo under control of the Egr-1 promoter (Egr-EG). This vector was transduced into human bone marrow stromal cell lines HFCL by lipofectamine and was transfused in severe combined immunodeficiency (SCID) mice. The experimental mice were randomly divided into 4 groups (6 mice in each group): (1) HFCL/EG + ADM group in which the HFCL/EG cells were transplanted intravenously in SCID mice bearing melanoma, ADM was given intraperitoneally after 3 days; (2) HFCL + ADM group in which the HFCL cells were transplanted intravenously, ADM was given intraperitoneally after 3 days; (3) HPCL/EG group in which HFCL/EG cells were transplanted alone; (4) HFCL group in which HFCL cells were transplanted alone. The dynamic change of peripheral blood picture was assayed by hemocytometer; the eGFP(+) human stromal cells were detected by flow cytometry; the expression of GM-CSF mRNA and protein were determined by RT-PCR and Western blot respectively. The results indicated that as compared with HFCL/EG and HFCL groups, the leukocyte count in HFCL/EG + ADM group decreased, but decrease level was weaker than that in HFCL + ADM group, meanwhile the recovery of leukocyte count was earlier than that in HFCL + ADM group. The CFU-GM amount between 4 groups showed no significant difference. The detection results showed that the inhibitory rate of tumor was related to chemotherapy, but not to expression of exogenous gene; the eGFP(+) stromal cells existed in bone marrow of mice treated with ADM. The RT-PCR and Western blot assays revealed enhancement of GM-CSF mRNA and protein. It is concluded that the ADM-inducible GM-CSF gene therapy regulated by Egr-1 promoter may promote the hematopoietic recovery after chemotherapy.

Regulatory effect of Egr-1 promoter sequences induced by doxorubicin in transcriptional targeting on expression of GM-CSF gene / 中国实验血液学杂志

In order to explore the regulatory effects of Egr-1 promoter sequences induced by doxorubicin (ADM) in transcriptional targeting on the expression of hematopoietic growth factor genes. The human GM-CSF cDNA and enhanced green fluorescent protein (eGFP) cDNA were linked together with internal ribozyme entry site (IRES) and then inserted into the expression vector pCIneo under control of the Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transfected cell clones (HFCL/EG) were selected by the addition of G418. The cells were exposed to the clinically important anticancer agent doxorubicin. The activity of eGFP in HFCL/EG cells was detected by flow cytometry. The amounts of GM-CSF in HFCL/EG postchemotherapy were confirmed with ELISA. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM derived from cord blood was also studied. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production following exposure to ADM was examined. The results indicated that the activity of eGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to ADM increased as compared to non-ADM group. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM was significantly higher than that of non-ADM group. N-acetylcysteine significantly decreased the concentration of GM-CSF produced by HFCL/EG treated with ADM. It is concluded that these in vitro data provide an experimental basis for the use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from ADM-injury.

Experimental study of transplanting human mesenchymal stem cells via portal vein to treat acute liver injury of mice induced with acetaminophen / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the effects of human mesenchymal stem cells (hMSCs) transplantation via portal vein to treat acute liver injury in mice induced with acetaminophen.</p><p><b>METHODS</b>A model of acute liver injury was established by acetaminophen gavage with a dose of 500 mg/kg. Twenty severe combined immune deficient mice (SCID mice) were randomly divided into 2 groups; one with hMSCs transplantation via their portal veins, the other group served as controls and only saline was infused into their veins. Liver function tests, fluorescein staining and reticular fiber staining of liver histological preparations and fluorescence- and light-microscopy were applied to observe the biochemical and pathological changes in the mice before and after the transplantation of hMSCs.</p><p><b>RESULTS</b>Liver function of the hMSCs group was significantly better than that of the controls (P less than 0.05). Fluorescence microscopy revealed that the hMSCs appeared in the areas of the periportal veins at first and then extended to the central vein areas; the reticular fiber staining indicated that hMSCs could repair the architecture of the hepatic acini. No prominent fibrosis and pseudolobules were found.</p><p><b>CONCLUSIONS</b>hMSCs transplantation via portal vein to SCID mice with acute liver injury induced by acetaminophen can improve their liver function effectively; hMSCs growth in their livers and acinus reconstruction can be affected. We think it is a good method to treat acute liver injury.</p>

FRIL maintains quiescence of hematopoietic stem cells through regulation of cell cycle related factors / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of Flt3 receptor-interacting lectin (FRIL) maintains quiescence of hematopoietic stem cells (HSCs) in vitro.</p><p><b>METHODS</b>Cord blood CD34+ cells were cultured in suspension medium supplemented with or without FRIL and FL. Cells were collected at different time points and the expression of some cell cycle regulators, especially those involved in G0/G1 phase regulation were detected on mRNA and protein level.</p><p><b>RESULTS</b>The expressions of G0/G1 phase related cyclins or CDKs were undetectable in the newly isolated CD34+ cells, expressions of Cyclin D3, CDK6 and P27 were the lowest in FRIL cultured group after 3d's culture (FRIL group: 483 +/- 63, 553 +/- 39, 0.312 +/- 0.030; FL group: 2437 +/- 52, 3209 +/- 98, 0.787 +/- 0.024; BLANK: 914 +/- 105, 1497 +/- 55, 0.616 +/- 0.029, respectively), but the expression of P53 was the highest in FRIL group (FRIL group: 4.476 +/- 0.159; FL group: 0.581 +/- 0.099, BLANK: 2.167 +/- 0.114). The expression of positive regulators of cell cycle in FRIL group were the same as that of FL group and blank group or lower.</p><p><b>CONCLUSION</b>FRIL preserves HSCs effectively in vitro through the mechanisms of down-regulation of cyclin D3 and CDK6 and activation of P53. P27 is mostly involved in the differentiation of HSCs.</p>

Differentiation of rat bone marrow derived Thy-1+ beta2M- cells into hepatocytes / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the differentiation of bone marrow derived Thy-1(+)beta(2)M(-) cells (BDTC) into mature and functional liver cells and its mechanism.</p><p><b>METHODS</b>BDTC were cocultured with allyl alcohol (AA)-injured hepatocytes and cultured alone in conditional medium containing HGF and bFGF, respectively. BDTC morphologic transformation was observed with phase-contrast and electron-microscopy. Hepatocyte-specific gene expression in cultured BDTC was identified by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Indocyanine green (ICG) ingestion/excretion and urea, albumin production were carried out to evaluate hepatocyte-related function.</p><p><b>RESULTS</b>Some BDTC derived hepatocyte-like cells with high nuclear to cytoplasmic ratio containing mono- or multi-nuclei and abundant mitochondria, endoplasmic reticulum and glycogenic granules appeared after 7-day culture in both the two culture systems. These cells expressed hepatocyte-specific genes (AFP, OV-6, CK18, etc.), and possessed functions of ICG uptake, albumin production and ammonium metabolism.</p><p><b>CONCLUSION</b>Rat BDTCs could differentiate into mature and functional liver cells in special stimulation systems. Moreover, these differentiations were realized by "transdifferentiation", and might dispense with "cell fusion".</p>

Sustaining effect of gene-transferring hepatic stellate cell strain CFSC/HGF on hepatocytes development / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the sustaining effects of gene-transferring hepatic stellate cell strain CFSC/HGF on the development of hepatocytes.</p><p><b>METHODS</b>A CFSC/HGF strain, expressing HGF steadily and effectively was established by recombined retroviral vector pMSCV-HGF infection. Morphology and ultra structure of hepatocytes, albumin and urea production, as well as ICG uptake and excretion were studied continuously following the hepatocytes cultured on the CFSC/HGF feeder layers. Parallel group of collagen-dependent hepatocytes culturing and hepatocytes culturing on CFSC were also conducted. Semi-quantitative RT-PCR analysis was made to evaluate the expression of HGF receptor c-Met.</p><p><b>RESULTS</b>The hepatocytes cocultured on CFSC/HGF feeder layers had a higher survival rate, and the functions of albumin secretion and urea syntheses and ICG uptake and excretion, were superior to the other two culture methods. The result of RT-PCR indicated that the c-Met expressed on the CFSC/HGF coculturing hepatocytes was up-regulated 2.23 times.</p><p><b>CONCLUSION</b>Gene-transferring hepatic stellate cell strain CFSC/HGF exhibited a remarkable sustaining effect on the hepatocytes development. The up-regulation of c-Met expressed on the surfaces of the hepatocytes induced by CFSC/HGF might play some part in this function.</p>

Regulatory role of HTm4 gene in hematopoietic cell cycle / 生理学报

Cell cycle progression is tightly regulated in hematopoietic stem cells. The cycle state decides cells' fates, which includes self-renewal, proliferation and differentiation. Proper cell cycle regulation is a pivotal element for the maintenance of hematopoiesis homeostasis. HTm4 is a newly identified specific cell cycle regulator of the hematopoietic cell. Through interacting with KAP-CDK2 complex, it arrests cells in G(0)/G(1) phase. K562 is a human chronic myelogenous leukemia cell; it could be induced to megakaryoblast by phorbol 12-myristate 13-acetate (PMA). Such differentiation must be associated with cell cycle change. To further clarify HTm4's function in hematopoietic cell cycle regulation, K562 cells were treated with PMA. Cell cycle change was analysed using flow cytometric system. And during the induction process gene expression of HTm4 as well as CycleE and CDK2, which are responsible for G(1) to S transition, were analysed using semi-quantitative RT-PCR. The C-terminal domain of HTm4 protein has been shown to be important for HTm4's binding with KAP-CDK2 complex. To determine its impact on HTm4's function, HTm4 and C-terminal truncated HTm4 (HTm4-ct) were transfected into K562 cells using Tet-Off regulation expression system. Their influence on cell cycle was observed. The results showed that PMA induced both expansion and differentiation of K562 cells as measured by cell number count and NBT staining respectively. During PMA treatment, G(0)/G(1) cell proportion and HTm4 expression displayed coordinated change, which suggested that HTm4 might drive K562 cells out of cell cycle but was not involved in the quiescence maintenance. Additionally, transfection of HTm4 caused G(0)/G(1) arrest in K562 cells, while transfection of HTm4-ct did not. It is therefore suggested that the C-terminal domain is important for the function of HTm4 in cell cycle regulation.

Hope to the cancer therapy: cancer stem cell / 中国医学科学院学报

Cancer stem cells are defined as rare cells in cancer tissues with indefinite potential for self-renewal that drives tumorigenesis. It was first extensively documented for leukaemia and multiple myeloma. It has also been found in solid cancers such as human breast cancer and nervous system tumors. Studies of cancer stem cell biology and mechanisms of tumorigenesis are lending insight into the origins of cancer and will ultimately yield new approaches to fight cancer.

In vitro differentiation of human bone marrow-derived mesenchymal stem cells into blood vessel endothelial cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study differentiation of human bone marrow-derived mesenchymal stem cells (BDMSC) into blood vessel endothelial cells for ideal cell origin of complex organ tissue engineering vascularization and injured tissue repairing by cell transplantation.</p><p><b>METHOD</b>After different days of induction with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in 3D fibrin-gels and matrigel, BDMSC and angiogenesis were determined by the utilization of morphological observation, tissue section and CD34, CD31, vascular endothelial growth factor receptor-1 (VEGFR-1, Flt-1), VEGFR-2 (Flk-1), and vWF that were special for blood vessel endothelial cells.</p><p><b>RESULT</b>After 3D-cultured and induced with VEGF and bFGF in vitro in fibrin-gels and matrigel for 3-21 days, BDMSC expressed CD34, CD31, Flt-1, Flk-1, and vWF came into vessel-like configuration.</p><p><b>CONCLUSION</b>VEGF, bFGF as well as Flt-1 and Flk-1, expressed by BDMSC, may form a feasible microenviroment after induction and play an important role during processes of blood vessel endothelial cell differentiation and vessel-like configuration forming of BDMSC. Mesenchymal stem cells may be applied to tissue engineering vascularization and injured tissue repairing by cell transplantation.</p>

Autologus bone marrow-derived mesenchymal stem cells intracoronary delivery after acute myocardial infarction in miniature pig / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the possibility that using intracoronary delivery of autologus bone marrow-derived mesenchymal stem cells (MSCs) to improve the cardiac function after acute myocardial infarction (AMI) in miniature pig.</p><p><b>METHODS</b>MSCs were cultured in Dulbecco's modified Eagle's medium-F12 (DMEM/F12) medium. AMI model was made by blocking the blood stream of the first diagonal branch in miniature pig, and released the branch after 90 minutes. After 10-14 days, (4-6) x 10(7) culture-expanded autologus 4', 6-diamidino-2-phenylindole (DAPI)-labelled MSCs were transplanted into each host heart's AMI area through intracoronary way. Ultrasonic cardiography (UCG) was performed to observe the left ventricular function at 3 months after transplantation. The cellular transplanted hearts were harvested and investigated by immunohistochemical analysis.</p><p><b>RESULTS</b>Left ventricular function of the MSCs group was improved significantly 3 months later compared with the control group [(54.65 +/- 3.39) vs (43.98 +/- 4.21)%, (P < 0.01)]. Exogenous MSCs survived and site-differentiated into cardiomyocytes in infracted hearts.</p><p><b>CONCLUSION</b>MSCs can play a benificial role to repair damaged heart. Heart function can be improved after MSCs transplantation in porcine myocardial infarction model.</p>

Prediction of acute GVHD after umbilical cord blood transplantation by HLA three-dimensional structure matching / 中国实验血液学杂志

To explore a method of predicting acute graft versus host disease (aGVHD) after unrelated cord blood transplantation (UCBT), the HLA-A, -B, -DRB1 molecular three-dimensional structures in 25 patients with blood disorder who underwent UCBT and their donors were modeled by using molecular modeling technique. First, full amino acid sequences of each HLA antigen from HLA data banks were loaded down, and then amino acid sequence of extracellular antigen binding region was chosen. Third step, SPDBV software of SWISS-MODEL server was used to modeling the three-dimensional structures of each different allele of HLA-A, -B and -DRB1 between patients and donors and the parameter "root mean square deviation" (RMSD) was used to indicate the structure differences. Last, RMSD of each different HLA allele of each donor-patient pair were added together to get total RMSD. The 25 patients were divided into 3 groups: the first group did not develop aGVHD; the second group developed aGVHD graded I-II and the third group developed aGVHD graded III-IV. The results showed that in the 25 patients divided into three groups, 8 patients in the first group did not develop aGVHD (32%); 13 patients in the second group developed grade I-II of aGVHD (52%) and 4 patients in the third group developed aGVHD III-IV (16%). The total RMSDs of each group were 0.24 +/- 0.15, 0.25 +/- 0.14 and 0.47 +/- 0.22 respectively. The total RMSD of the third group was significantly higher than that of the other two groups. In conclusion, utilization of modeling HLA molecular three-dimension can predict the severe aGVHD after UCBT quickly, simply and accurately. It provides scientific basis in choosing a optimal cord blood donor to avoid severe aGVHD for physicians and the cord blood banks. And it is instructive too to direct the application of immunosuppressive agents after transplantation in clinic.

The maintenance of cord blood CD34+ progenitor cells with plant lectin FRIL in vitro and the expression of related cell cycle modulator HTm4 and HTm4S / 生理学报

Ex vivo maintainance of human stem cells is crucial for many clinical applications. Current culture conditions provide some level support but cytokines induce most quiescent stem cells to proliferate and differentiate. Better control of primitive cells is needed to extend the time and range of manipulation of such cells. A recently identified plant lectin Flt3 receptor-interacting lectin (FRIL) present may a special ability to preserve primitive CB progenitors for extended periods in culture without exogenous cytokines. But the mechanisms of FRIL preserving quiescent primitive cells are still unknown. Recently a novel protein HTm4 and its alternatively spliced variant HTm4S, which serve as hematopoietic cell cycle regulators, have been identified. In this report we studied the effect of FRIL on the in vitro maintenance of quiescent human cord blood stem cells and the expression of the novel hematopoietic cell cycle regulator HTm4 and HTm4S in progenitor cells cultured in FRIL. We analyzed the proliferation and the HPP-CFC proportion of CD34(+) cells treated with FRIL. The human HTm4 and HTm4S mRNA expression was detected by semi-quantitative RT-PCR, and the cell cycle status of CB CD34(+) cells was analyzed by FACS. The results showed that incubation of CD34(+) cells in FRIL resulted in a low proliferation of progenitor cells and fewer cycling cells, but FRIL selectively maintained a higher number of primitive cells with proliferative potential in suspension culture. CB CD34(+) cells cultured in FRIL showed significant diversity in the expression of HTm4 and HTm4S during 0~14 d. On d 0, HTm4 was detected at high level, downregulated on d 1, but upregulated during d 3 to d 14, and reaching the highest level on d 7. But the expression levels of HTm4S changed little in the cells cultured in FRIL except the obviously increased expression on d 7. Exogenous expression showed that HTm4 was localized around the karyon while HTm4S scatted in the cytoplasm, respectively, which may be responsible for their difference in function. Thus, FRIL can preserve quiescent primitive CD34(+), and FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications. In other words, HTm4 and HTm4S may play a crucial role in the cell cycle modulation of CD34(+) progenitor cells maintained with FRIL in vitro.

Immune response of dendritic cells acquiring antigens from apoptotic cholangiocarcinoma cells induced by mitomycin / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor immune response of dendritic cells (DCs) acquiring antigens from apoptotic cholangiocarcinoma cells and their therapeutic effects on cholangiocarcinoma cells.</p><p><b>METHODS</b>DCs from human peripheral blood monocytes which acquired antigen capturing and processing capacity, characteristics of maturation, were established in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Then cholangiocarcinoma cells were induced to apoptosis with mitomycin. The three groups included (1) coculture of DCs, apoptotic cancer cells and T cells, (2) coculture of DCs, necrotic cancer cells and T cells, (3) coculture of DCs, cultured cancer cells and T cells. After 7 days, DCs and T cells were riched separately to perform anti-tumor cells test and immune response test.</p><p><b>RESULTS</b>these cells had typical dendritic cell morphology, expressed high levels of CD1a and B7, acquired antigen from apoptotic cells caused by mitomycin and could stimulate T cells to inhibit, even kill cholangiocarcinoma cells.</p><p><b>CONCLUSIONS</b>The DCs from peripheral blood monocytes induced by GM-CSF and IL-4 can efficiently present antigen derived from apoptotic cells caused by mitomycin, and stimulate T cells activity obviously. It maybe become an effective therapy for tumor.</p>

Identification of differentially expressed genes in Lin-CD34- and Lin-CD34+ cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify genes that differentially expressed in Lin(-)CD(34)(-) and Lin(-)CD(34)(+) cells.</p><p><b>METHODS</b>With Lin(-)CD(34)(-) cells as tester and Lin(-)CD(34)(+) cells as driver, cDNA subtractive library for Lin(-)CD(34)(-) cells was constructed using suppression subtractive hybridization technique. Part of clones in the library were sequenced and the homologue analysis was conducted against the DNA database in GenBank.</p><p><b>RESULTS</b>593 clones containing an average of 300 - 500 bp insert were identified. Of them, 53 randomly selected ESTs were sequenced. Homologue analysis revealed that 37 ESTs represented 10 known genes, and the other 16 ESTs represented 4 novel sequences.</p><p><b>CONCLUSION</b>Part of specifically expressed genes in Lin(-)CD(34)(-) cells were identified, which maybe related to Lin(-)CD(34)(-) cells' specific characteristics.</p>

Effect of Tpo and/or IL-11 gene modified stromal cells on the expansion of CD34+ CD38- hematopoietic primitive progenitor cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Tpo and/or IL-11 gene modified stromal cells on the expansion of CD(34)(+) hematopoietic stem/progenitor cells in cord blood.</p><p><b>METHODS</b>Retroviral vectors containing Tpo or IL-11 gene were constructed and used to transfect the stromal cell line HFCL. Tpo and/or IL-11 mRNA was assayed by Northern blot. Non-modified stromal cells were used, CD(34)(+) hematopoietic stem/progenitor cells from cord blood were expanded on gene-modified stromal cells for 7 days. The phenotype of CD(34)(+)CD(38)(-) primitive progenitors was detected by flow cytometry.</p><p><b>RESULTS</b>HFCL expressed Tpo and/or IL-11 mRNA after transfected by the retroviral vectors. The percentages of CD(34)(+)CD(38)(-) primitive progenitors in the cultures of Tpo, IL-11 and Tpo + IL-11 modified HFCL were (1.8 +/- 0.24)%, (1.62 +/- 0.23)%, and (2.45 +/- 0.28)%, respectively, which were higher than that in the control [(0.8 +/- 0.23)%].</p><p><b>CONCLUSION</b>The stromal cells modified by Tpo and/or IL-11 gene were able to enhance ex vivo expansion of CD(34)(+) and CD(34)(+)CD(38)(-) hematopoietic stem/progenitor cells from cord blood.</p>