Umbilical cord blood-derived mesenchymal stem cells ameliorate graft-versus-host disease following allogeneic hematopoietic stem cell transplantation through multiple immunoregulations / 华中科技大学学报（医学）（英德文版）

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.

Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation / 华中科技大学学报（医学）（英德文版）

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

Exogenous VEGF promotes hematopoietic stem cell mobilization / 中国实验血液学杂志

This study was aimed to investigate the effect of exogenous VEGF on hematopoietic stem cell mobilization and immune system. The C57BL/6J mice were randomly divided into the normal control group, VEGF short-term group (5 d) and VEGF long-term group (27 d). Mice in the experimental group were injected ip with VEGF (100 ng/d); mice in control group were injected ip with PBS. The white blood cell (WBC) count and the ratio of lymphocyte in the peripheral blood at different time point were assayed by hemacytometer. The percentage of hematopoietic stem cell (HSC), lymphocyte subgroup, regulatory T cell (Treg), myeloid-derived suppressor cells (MDSC) in the peripheral blood and spleen of different groups were detected by flow cytometry. The morphological changes of spleen and spleen index of mice in the control and long-term group were observed by microscopy. The results showed that the absolute number of WBC in the peripheral blood of mice significantly increased after injection of VEGF, and the peak value was at day 3. The percentage of Lin(-)Sca-1(+)CD117(+) cells in the peripheral blood and spleen of the long-term group were significantly higher than that in the normal control group (P < 0.05). The spleen of the mice in VEGF long-term group was larger than that of the control group, the spleen index also increased (P < 0.05), and remarkable extramedullary hematopoietic signs were found in the HE stained sections. There was no significant change in the total ratio of lymphocytes in the peripheral blood after injection, but the percentage of CD3(+) cells and the CD3(+)/B220(+) ratio in the long-term group deceased; the percentages of Treg and Gr-1(+)CD11b(+) MDSC in the experimental groups increased (P < 0.05), which more significantly increased in the long-term group than that in the short-term group (P < 0.05). It is concluded that the exogenous VEGF promotes hematopoietic stem cell mobilization, and at same time up-regulates the many kinds of suppressive immune cell levels which leads to changes of immuno-function.

Modified method for whole bone marrow adherent culture of human bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate a more convenient and efficient method to cultivate the human bone marrow mesenchymal stem cells by means of natural erythrocyte sedimentation principle, based on the whole bone marrow adherent method. The bone marrow was cultured with a six-well plate instead of the flasks.Firstly, the bone marrow specimen was cultivated with the MSC complete medium for 48 h, then the upper RBC-free supernatant layer was drawn and placed into the new wells to isolate MSC. Inverted microscope was used to observe the cell morphology and to record the adherent time of first cell passage, first passaging time. The traditional whole bone marrow adherent method was used as the control. The cell cycle and cell surface markers were detected by flow cytometry,and the differentiative capacity of MSC into osteocyte and adipocyte was identified by alkaline phosphatase kit and oil red O, respectively. Besides, the proliferative curve of P1,P3,P5 of BMSC was depicted by counting method. The results showed that MSC cultured by the modified method highly expressed CD90, CD105, CD13, CD44 and lowly expressed CD14, CD45, CD34. Concerning the cell cycle feature, it was found that most of the cells were in G0/G1 phase (88.76%) , followed by G2/M phase (3.04%) and S phase (8.2%), which was in accordance with stem cell cycle characteristics. The proliferative curve showed a typical "S" type, and both the oil red O and alkaline phosphatase staining of MSC were positive. Compared with the traditional method, the modified method had the advantage of high adherence rate (P = 0.0001) and shorter passaging time for the first passage (P = 0.001), with the statistically significant difference. It is concluded that there is a large number of adherent, active and suspended MSC in the RBC-free supernatant layer after the culture of bone marrow for 48 h. Isolating MSC by the modified method is more convenient and efficient than the traditional whole bone marrow adherent method.

Amplification of functional myeloid-derived suppressor cells during stem cell mobilization induced by granulocyte colony-stimulation-factor / 华中科技大学学报（医学）（英德文版）

The effects of granulocyte colony-stimulation-factor (G-CSF) on stem cell mobilization and its impact on the amplification of myeloid-derived suppressor cells (MDSCs) of donor mice were examined. A mouse model of stem cell mobilization was established by consecutive subcutaneous injection of 100 μg/kg G-CSF for 5 days. The blood from the donor mice was routinely examined during mobilization. Stem cells and MDSCs were analyzed by flow cytometry. The immunosuppressive molecules derived from MDSCs in serum and spleen, including hydrogen dioxide (H2O2) and nitric oxide (NO), and the activity of nitric oxide synthase (NOS) were determined during the mobilization. Apoptosis of T lymphocytes was assessed by using Annexin-V/PI. During stem cell mobilization, the number of lymphocytes and white blood cells in the peripheral blood was increased, and peaked on the 4th day. The number of stem cells in G-CSF-treated mice was significantly greater than that in controls (P<0.01). The expansions of MSDCs were also observed after G-CSF mobilization, with a more notable rate of growth in the peripheral blood than in the spleen. The activity of NOS and the production of NO were increased in the donor mice, and the serum H2O2 levels were approximately 4-fold greater than the controls. Consequently, apoptosis of T lymphocytes was increased and showed a positive correlation with the elevated percentage of MDSCs. It was concluded that G-CSF could provide sufficient peripheral blood stem cells for transplantation. Exogenous administration of G-CSF caused the accumulation of MDSCs in the peripheral blood and the spleen, which could lead to apoptosis of T lymphocytes and may offer a new strategy for the prevention and treatment of graft versus host disease.

Analysis of seizure risk factors after allogeneic hematopoietic stem cell transplantation: a 8 case report and literature review / 华中科技大学学报（医学）（英德文版）

The clinical characteristics of patients with seizures after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were analyzed. A total of 8 cases of seizures after allo-HSCT were investigated. Clinical data of these cases were studied retrospectively. Of 159 cases subjected to allo-HSCT, seizure occurred in 8 cases during 29-760 days after transplantation, median survival time was 46 days, and there were 6 cases of tonic-clonic seizure. The incidence of seizure after matched unrelated HSCT was higher than that after related HSCT (P=0.017). Of 7 cases treated with cyclosporine A (CsA), 4 cases obtained high blood levels of CsA. In addition, hyponatremia was diagnosed in 5 cases. Abnormal electroencephalogram and brain MRI findings were found in some cases. During 20 days after seizure, 2 cases died due to infection and graft-versus-host disease (GVHD), respectively. It was suggested that multiple factors are associated with seizures after allo-HSCT. Rapid identification and correction of the causative factors are very important to prevent permanent central nervous system damage and reduce the mortality.

Analysis of seizure risk factors after allogeneic hematopoietic stem cell transplantation: A 8 case report and literature review / 华中科技大学学报（医学）（英德文版）

The clinical characteristics of patients with seizures after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were analyzed. A total of 8 cases of seizures after allo-HSCT were investigated. Clinical data of these cases were studied retrospectively. Of 159 cases subjected to allo-HSCT, seizure occurred in 8 cases during 29-760 days after transplantation, median survival time was 46 days, and there were 6 cases of tonic-clonic seizure. The incidence of seizure after matched unrelated HSCT was higher than that after related HSCT (P=0.017). Of 7 cases treated with cyclosporine A (CsA), 4 cases obtained high blood levels of CsA. In addition, hyponatremia was diagnosed in 5 cases. Abnormal electroencephalogram and brain MRI findings were found in some cases. During 20 days after seizure, 2 cases died due to infection and graft-versus-host disease (GVHD), respectively. It was suggested that multiple factors are associated with seizures after allo-HSCT. Rapid identification and correction of the causative factors are very important to prevent permanent central nervous system damage and reduce the mortality.

Amplification of functional myeloid-derived suppressor cells during stem cell mobilization induced by granulocyte colony-stimulation-factor / 华中科技大学学报（医学）（英德文版）

The effects of granulocyte colony-stimulation-factor (G-CSF) on stem cell mobilization and its impact on the amplification of myeloid-derived suppressor cells (MDSCs) of donor mice were examined. A mouse model of stem cell mobilization was established by consecutive subcutaneous injection of 100 μg/kg G-CSF for 5 days. The blood from the donor mice was routinely examined during mobilization. Stem cells and MDSCs were analyzed by flow cytometry. The immunosuppressive molecules derived from MDSCs in serum and spleen, including hydrogen dioxide (H2O2) and nitric oxide (NO), and the activity of nitric oxide synthase (NOS) were determined during the mobilization. Apoptosis of T lymphocytes was assessed by using Annexin-V/PI. During stem cell mobilization, the number of lymphocytes and white blood cells in the peripheral blood was increased, and peaked on the 4th day. The number of stem cells in G-CSF-treated mice was significantly greater than that in controls (P<0.01). The expansions of MSDCs were also observed after G-CSF mobilization, with a more notable rate of growth in the peripheral blood than in the spleen. The activity of NOS and the production of NO were increased in the donor mice, and the serum H2O2 levels were approximately 4-fold greater than the controls. Consequently, apoptosis of T lymphocytes was increased and showed a positive correlation with the elevated percentage of MDSCs. It was concluded that G-CSF could provide sufficient peripheral blood stem cells for transplantation. Exogenous administration of G-CSF caused the accumulation of MDSCs in the peripheral blood and the spleen, which could lead to apoptosis of T lymphocytes and may offer a new strategy for the prevention and treatment of graft versus host disease.

Infusions of recipient-derived cytokine-induced killer cells of donor origin eradicated residual disease in a relapsed leukemia patient after allo-hematopoietic stem cell transplantation / 中华医学杂志(英文版)

A female patient diagnosed with acute myelocytic leukemia M5a (AML-M5a) relapsed 986 days after her allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an unrelated male donor with matched human leukocyte antigen (HLA). Three re-induction chemotherapies were administered, and partial remission was achieved. The patient was given repetitive infusion of cytokine-induced killer (CIK) cells expanded from recipient peripheral mononuclear cells of full donor chimerism due to loss of contact of quondam donor for donor lymphocyte infusion (DLI) and rejection of second transplantation. The patient achieved complete cytogenetical remission. This strategy might overcome the obstacle of donor unavailability and present an appealing new therapeutic alternative to donor-recruited adoptive immunotherapy for relapsed disease at post-transplantation.

Effects of adenovirus mediated vascular endothelial growth factor gene transfer on reconstitution of hematopoiesis in post-bone marrow transplantation mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Bone marrow transplantation (BMT) conditioning procedure is considered as the cause of damage to bone marrow microvasculature and the delay of hematopoiesis recovery. However, hematopoiesis regulation post BMT by vascular endothelial growth factor (VEGF) has not yet been studied. In this study, adenovirus were used to investigate the effects of VEGF gene transfer on preventing damages to bone marrow microenvironment and its promotion of hematopoiesis in post-BMT mice.</p><p><b>METHODS</b>Recombinant adenovirus (Ad)-enhanced green fluorescent protein (EGFP)/hVEGF165 was injected via tail vein into BALB/c mice undergoing syngeneic BMT. During the different phases post BMT, the distribution of adenovirus and the plasma levels of hVEGF were measured as well as the numbers of white blood cells (WBC), platelet (PLT) and red blood cells (RBC) in peripheral blood. At the same time, the mice were injected with Chinese ink via tail vein, following which the tibias were separated and were used for analysis of bone marrow microvasculature surface area and cellularity.</p><p><b>RESULTS</b>Significant expression of EGFP and hVEGF was observed in multiple organs at different phases post BMT, and the plasma level of hVEGF was up to (866.67 +/- 97.13) pg/ml. The recovery of WBC, PLT and RBC of the group treated with recombinant adenovirus Ad-EGFP/hVEGF165 were significantly more rapid than those of other BMT groups (P < 0.05, respectively). At the 20th day post BMT, the percentage of bone marrow microvasculature surface area in group treated with VEGF [(61.2 +/- 4.0)%] returned to normal level [(62.0 +/- 5.0)%, P > 0.05]. The restoration of hematopoiesis was retarded more than that of microvasculature. The cellularity of bone marrow in each group was still lower than that of normal control [(62.3 +/- 4.0)%, P < 0.05] at the 30th day post BMT, but the percentage in group treated with VEGF at the 20th and 30th days post BMT [(46.5 +/- 5.0)% and (55.1 +/- 4.5)%] exceeded those of other BMT groups (P < 0.05, respectively).</p><p><b>CONCLUSION</b>VEGF gene transfer mediated by adenovirus may protect the hematopoietic microenvironment to promote the restoration of hematopoiesis in post-BMT mice.</p>

Expression of BLM mRNA in six tumor cell strains / 中国实验血液学杂志

The mutations of BLM gene may result in Bloom's syndrome which includes immunodeficiency, predisposition to malignant tumors and so on, and enhances sister chromati exchange (SCE), DNA replication failure, genome instability, and increases cancer susceptibility. This study was aimed to investigate the variability of mRNA expression level and cDNA structure of BLM gene in tumor cell strains so as to look for a new cancerogenic mechanism and to find a new therapeutic target. The expression level of mRNA and the structure of cDNA of BLM gene in six tumor cell strains and the normal human bone marrow mononuclear cells were detected with RT-PCR and DNA sequencing was performed. The results indicated that these tumors cells expressed BLM mRNA higher than the normal human bone marrow mononuclear cells (P < 0.01), but no cDNA sequence abnormality of BLM gene in these tumors cells was observed. It is concluded that the increase of expressing level of BLM mRNA may play an important role in the development of these tumors.

Recombinant adenovirus mediated hVEGF165 gene transfer promotes recovery of hematopoiesis in post-BMT mice / 中国实验血液学杂志

To investigate the effects of vascular endothelial growth factor (VEGF) on the recovery of hematopoiesis in post-BMT mice, the recombinant adenovirus Ad-EGFP/hVEGF(165) was injected into syngeneic BMT BALB/c mice via the tail vein. At day 10, 20, 30 after BMT, the in vivo expression of hVEGF(165) was measured. At the same different time points, the numbers of WBC, Plt, RBC in peripheral blood and MNC in bone marrow were counted. By the way, the bone marrow MNCs at day 30 post-BMT were used for further CFU-S assay. The results indicated that a long-term expression of hVEGF(165) in plasma and different organs was successfully mediated by recombinant adenovirus. At each time point of post-BMT, the numbers of WBC, Plt, RBC as well as bone marrow MNC observed in the group treated with recombinant adenovirus Ad-EGFP/hVEGF(165) were lower than those of the normal control group, but were higher than those in other testing groups (P < 0.05). The number of CFU-S (21.4 +/- 2.67) formed by bone marrow MNC at day 30 after BMT reached to the normal level (19.50 +/- 2.46) (P > 0.05), which was much higher than that in other groups (P < 0.05). It is concluded that hVEGF(165) gene transfer mediated by recombinant adenovirus plays a role of promoting the recovery of hematopoiesis in post-BMT mice.

Distribution and efficiency of recombinant adenovirus mediated hVEGF165 gene transfer in bone marrow transplanted mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the rapid construction of enhanced green fluorescent protein (EGFP) labeled recombinant adenovirus containing hVEGF165 and its distribution and efficiency in bone marrow transplanted mice.</p><p><b>METHODS</b>The recombinant adenovirus Ad-EGFP/hVEGF165 was rapidly constructed by using AdEasy system based on the homologous recombination in bacteria, and its property was studied in vitro. Then 3x10(8) PFU adenovirus was injected into BALB/c mice via the tail vein accepted syngeneic bone marrow transplantation. The in vivo distribution of adenovirus and plasma levels of VEGF were measured at different phases.</p><p><b>RESULTS</b>The adenovirus Ad-EGFP/hVEGF165 was quickly constructed by homologous recombination in bacteria using AdEasy system. The purified particles were homogenous hexagon with titers between 10(10) PFU/ml and 10(11) PFU/ml. The Hela cells infected with Ad-EGFP/hVEGF165 did not show cytopathic effects after several times passages. Under the fluorescent microscope, EGFP was revealed in the heart, lung, liver, spleen, kidney and intestine of mice at different phases. RT-PCR and immunohistochemistry showed hVEGF165 expressed significantly. No obvious damages were observed in different organs by HE staining. The plasma level of hVEGF165 was up to 866.67+/-97.13 pg/ml.</p><p><b>CONCLUSION</b>These results suggested that the construction of adenovirus vector by homologous recombination in bacteria was an efficient and time-saving method, and high titer adenovirus could successfully mediate the safe and stable expression of hVEGF165 in post bone marrow transplanted mice. All these would make further gene therapy in bone marrow transplantation possible.</p>

Study on the expression of hematopoietic growth factor gene in human umbilical vein endothelial cells using gene chip / 中国实验血液学杂志

In order to detect the hematopoietic growth factor gene expressed in human umbilical vein endothelial cells using gene chip, human umbilical vein endothelial cells (ECV304) were cultured in vitro and divided into VEGF group and control group in same medium. 50 ng/ml hVEGF165 was added in the VEGF group. After culture for 24 hours all cells were collected for total RNA extraction. Then, cDNAs were marked with Cy3 and Cy5 for control group and VEGF group, respectively, and hybridized with gene chip. Hybridization signals were collected and analyzed following scanning by laser co-focal microscopy. The results showed that a large number of hematopoietic growth factor and receptor genes (Epo/R, GM-CSF/R, G-CSF/R, LIF, IL-3, TPO, Flt-3, SCF) were expressed in both groups, while many other growth factors (VEGF, IGF2, PDGFA, PDGFB, TGFbeta1) and receptors (neuropilin-1, neuropilin-2, TGFbeta-R1)were expressed. The differentially expressed genes amounted to 24. It is concluded that many hematopoietic growth factors and receptors expressed by hUVECs could be analyzed in a short period by using gene chip, which provides a powerful method for further studies on characteristics of vascular endothelial cells.

The growth characteristics of mesenchymal stem/progenitor cells in human umbilical cord blood / 中国实验血液学杂志

This study was done for investigating the frequency and proliferative feature of mesenchymal stem/progenitor cells (MSPC) in human umbilical cord blood (CB) and for searching a new seed cell for tissue engineering. Mononuclear cells was separated by Ficoll-Hypaque from cord blood and suspended in DMEM culture medium supplemented by 2% fetal bovine serum. The adherent CB cells were cultured and expanded at same medium. The results showed that the frequency of CB-MSPC was 0.5 x 10(-6) [(0.2 - 0.8) x 10(-6)]. The CB-MSPC showed a fibroblast-like morphology and retained their morphological feature at least after 20 sub-passages, and could extensively be expanded by about 1.3 x 10(7) times as much. The conclusion is that low serum DMEM culture could maintain the proliferation and differentiation potential of CB-MSPC. CB-MSPC might be a favorable seed cell for tissue engineering and regeneration.

A new method for construction of EGFP-labled recombinant adenovirus containing hVEGF(165) and its property in vitro / 中国实验血液学杂志

By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labled recombinant adenovirus vector containing hVEGF(165) was generated quickly and its property was studied in vitro. First, hVEGF(165) coding sequence was subcloned into the shuttle plasmid pAdTrack-CMV, then linearized and cotransferred with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ(5183). After positive kanamycin-resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis with PacI and transfected into HEK 293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF(165). The further amplified recombinant adenoviruses were purified by CsCl banding at 32,000 rpm for 18 to 24 hours. Electron microscopy and PCR were performed for testing the recombinant adenovirus. The results showed that the purified particles were homogenous hexagon with a high titer up to 2 x 10(12)pfu/ml. An amplified band of 540 bp fragment demonstrated the successful insert of hVEGF(165). Under fluorescence microscopy, the expression of EGFP was easily detected in HEK 293 and other target cells. The maximal stimulating effect on the proliferation of hUVEC was obtained when the given multiplicity of infection (MOI) of Ad-EGFP/hVEGF(165) was 100. The rate of EGFP positive mouse bone marrow mononuclear cells analysed by flow cytometry was 27.3% after 24 hour-incubation with Ad-EGFP/hVEGF(165) (MOI = 100), and the expression of hVEGF(165) protein in the conditioned medium was 1385 +/- 332 pg/10(6) cells. It is concluded that the construction of adenovirus vector by homologous recombination in bacteria using AdEasy system can be quickly and easily performed, and the prepared high titer of Ad-EGFP/hVEGF(165) is an efficient helpful vector to transfer genes into target cells, all of which make the further in vivo experiments with VEGF(165) possible.