Effect of Rheb1 in the Development of Mouse Megakaryocyte-Erythroid Progenitor Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Rheb1 in the development of mouse megakaryocyte-erythroid progenitor cells and its related mechanism.@*METHODS@#Rheb1 was specifically knocked-out in the hematopoietic system of Vav1-Cre;Rheb1fl/fl mice(Rheb1Δ/Δ mice). Flow cytometry was used to detect the percentage of red blood cells in peripheral blood and erythroid cells in bone marrow in Vav1-Cre;Rheb1fl/fl mice and control mice. The CFC assay was used to detect the differentiation ability of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Real-time fluorescence quantification PCR was used to detect the relative expression of PU.1,GATA-1,GATA-2,CEBPα and CEBPβ of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Rapamycin was added to the culture medium, and it was used to detect the changes in cloning ability of megakaryocyte-erythroid progenitor cells from wild-type mice in vitro.@*RESULTS@#After Rheb1 was knocked out, the development and stress response ability of megakaryocyte-erythroid progenitor cells in mice were weaken and the differentiation ability of megakaryocyte-erythroid progenitor cells in vitro was weaken. Moreover, the expression of GATA-1 of megakaryocyte-erythroid progenitor cells was decreased. Further, rapamycin could inhibit the differentiative capacity of megakaryocyte-erythroid progenitor cells in vitro.@*CONCLUSION@#Rheb1 can regulate the development of megakaryocyte-erythroid progenitor cells probably through the mTOR signaling pathway in mice.

Optimization of Mouse Model of Aplastic Anemia Induced by Pan T Lymphocytes Combined with Irradiation / 中国实验血液学杂志

METHODS@#To establish the acquired aplastic anemia mouse model through the X-ray irradiation in combination with lymphocytes injection. AA Group: the purified Pan T lymphocytes from the spleen of C57BL/6J mice were enriched and injected to the mice through tail vein(5×10@*RESULTS@#Compared with 4, 5 Gy irradiated mice in AA groups, the survival time of 3 Gy irradiated AA groups was significantly prolonged. 3, 4 and 5 Gy X-ray irradiation combined with Pan T lymphocyte injection could successfully induced severe reduction of red blood cells, blood neutrophils, and platelets, severe reduction of bone marrow nucleated cells, severe bone marrow hematopoietic failure, and the significant expansion of T lymphocytes ratio in the bone marrow. CD4@*CONCLUSION@#3, 4 and 5 Gy X-ray irradiation combined with 5×10

Role of Rictor in Hematopoietic Stem Cells during Fetal Liver Hematopoiesis / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Rictor on the hematopoiesis of fetal liver by specific knock-out of Rictor in hematopoietic cells of Vav-Cre mice.@*METHODS@#E12.5 0.08ee fetal liver cells from the experimental group Vav-Cre; Rictor embryos and control group Rictor or Rictor embryos were transplanted to recipients respectively to observe the effect of Rictor on reconstitution ability of hematopoietic stem cells. In the meantime, E14.5 0, 10, 20, 40, 60, 80 sorted hematopoietic stem cells from the Vav-Cre; Rictor fetal liver of experimental group and Rictor or Rictor fetal liver of control group were transplanted in to recipients to analyze the numbers of functional hematopoietic stem cells after Rictor was knocked-out. Furthermore, the self-renewal capacity was investigated by secondary transplantation of BM cells from primary recipients that had been successfully repopulated with E12.5 fetal liver-derived cells and by cell cycle analysis.@*RESULTS@#All the recipients receiving E12.5 Rictor or Rictor cells were repopulated (8/8, from 2 independent experiments) with an average chimerism of 77.2%±11.1% at 4 months post-transplantation, which resulted in 57 LT-RU per FL. In comparison, 8 out of 8 recipients receiving Vav-Cre; Rictor cells were repopulated with significantly reduced chimerism (37.0%±16.3%) (P＜0.01), which was equivalent to 8 LT-RU per FL. The limiting dilution transplantation experiment showed that there was one functional hematopoietic stem cell out of 17 sorted SLAM cells in the control group, and one functional hematopoietic stem cell out of 39 sorted SLAM cells in the experimental group. The secondary transplantation experiments showed that 2 out of 4 recipients were reconstructed in the control group after 1 month, and 0 was reconstructed in the experimental group by transplanting 4×10 donor cells respectively. What＇s more, the percentage of S/G/M cells in the experimental group increased when compared with controls.@*CONCLUSION@#In the process of fetal liver hematopoiesis, the specifically knocking-out the Rictor in hematopoietic system can lead to defect of reconstitution ability, decrease of the functional hematopoietic stem cell numbers and reduction of self-renewal ability of hematopoietic stem cells.

Effect of PDK1 on Neutrophil Differentiation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To knock out PDK1 by using Vav-Cre and observe the effects of PDK1 knock out on the ratio, number and differentiation of neutrophil.</p><p><b>METHODS</b>The PDK1 expression level of Vav-Cre;PDK1mouse in the bone marrow cells was analyzed by RT-PCR. The effect of PDK1 on hematopoietic progenitor was observed by CFU-C assay, and the effect of PDK1 on the ratio and number of neutrophil was detected by flow cytometric analysis.</p><p><b>RESULTS</b>The RNA expression level of bone marrow cells of Vav-Cre;PDK1mouse was dramatically lower than that of the control mouse. The number of functional GMP was lower in Vav-Cre;PDK1mouse in contrast to controls. The percentage and number of neutrophil were lower, but the percentage of premyelocyte/myelocytes was more than 2 times in Vav-Cre;PDK1group compared with control groups.</p><p><b>CONCLUSION</b>PDK1 affects the process of the differention of hematopoietic stem cells to GMP, the neutrophil differention and maturation.</p>

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells / 中国医学科学杂志(英文版)

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells (iPSCs) derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX (F IX) gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay. Results The cell line bore a missense mutation in the 6coding exon (c.676 C>T) of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% (10/45) and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Effect of PDK1 on Notch1-Induced Mouse T-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the role of PDK1 in T-ALL development through establishing the Notch1-induced T-ALL mouse model by using Mx1-cre; LoxP system to knock-out PDK1.</p><p><b>METHODS</b>Cell cycle and apoptosis of leukemic cells were detected by flow cytometry, and relative expression of tumor-related genes and transcription factors of leukemic cells were determined by quantitative real-time PCR.</p><p><b>RESULTS</b>Notch1-induced T-ALL mouse model with inducible knock-out of PDK1 was established successfully. Compared to T-ALL control mouse model, PDK1 knock-out mice showed a significant longer survival time (P<0.01). There was no difference of cell cycle between control and PDK1 knock-out mice, and the apoptosis rate of leukemic cells in PDK1 knock-out mice was higher than that of control mice (P<0.001). PDK1 knock-out resulted in decreased expression of tumor-related genes and transcription factors, such as c-Myc and NF-κB (P<0.01), and increased expression level of P53 (P<0.01).</p><p><b>CONCLUSION</b>PDK1 knock-out can inhibit the development of T-ALL, and its mechanism may be the leukemia progression inhibited by regulating the apoptosis and expression of multiple related genes and transcription factors.</p>

ADAR1 Knockout Inhibits Notch1-induced T-ALL in Mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of ADAR1 on the occurrence and development of mouse T cell acute lymphoblastic leukemia (T-ALL).</p><p><b>METHODS</b>Lck-Cre; ADAR1lox/lox mice were generated through interbreeding. The lineage-cells of Lck-Cre; ADAR1lox/lox mice and the control were enriched respectively by the means of MACS, and the lin- cells were transfected with retrovirus carrying MSCV-ICN1-IRES-GFP fusion gene. Then the transfection efficiency was detected by the means of FACS, and the same number of GFP+ cells were transplanted into lethally irradiated recipient mice to observe the survival of mice in 2 recipient group after transplantation.</p><p><b>RESULTS</b>T cell-specific knockout ADAR1 mice were generated, and Notch1-induced T-ALL mouse model was established successfully. The leukemia with T-ALL characteristics occured in the mice of control group, but did not in the ADAR1 kmockout mice after transplantation.</p><p><b>CONCLUSIONS</b>ADAR1 plays a key role in the incidence and development of Notch1-induced T-ALL.</p>

Role of Rheb in Human Acute Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of Rheb (mTOR activator) in AML development by measuring Rheb expression in bone marrow of adult AML patients and in AML cell line HL-60.</p><p><b>METHODS</b>Real-time PCR assay was used to measure the Rheb mRNA expression in 27 AML patients and 29 ITP patients as control. The relationship between Rheb mRNA expression and age, AML subtype, fusion gene, splenomegaly, hepatomegaly and survival of AML patients was analyzed and compared. In addition, HL-60 cell line over-expressing Rheb was established, and the HL-60 cells and HL-60 cells with overexpression of Rheb were treated with Ara-C of different concentrations, the proliferation level was detected by CCK-8 method, and the IC50 was calculated.</p><p><b>RESULTS</b>The mRNA level of Rheb in AML patients was similar to that in ITP patients (control). Interestingly, higher expression of Rheb was associated with better survival and was sensitive to Ara-C treatment. However, the expression level of Rheb was not associated with age, AML subtype, fusion gene, and hepatomegaly of patients. Lower expression level of Rheb was associated with splenomegaly. In vitro analysis of HL-60 line indicated that overexpression of Rheb could increased the cell sensitivity to Ara-C treatment (IC50=0.54 µmol/L) and caused HL-60 cell apoptosis.</p><p><b>CONCLUSION</b>The lower Rheb expression is a poor prognostic indicator for AML patients, which is associated with AML splenomegaly, the patients and HL-60 cells with low expression of Rheb are insensitive to Ara-C treatment.</p>

Effect of TEB-415, a Derivative of Imatinib, on Multiple Myeloma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the growth inhibitory effect of Imatinib derivative TEB-415 on various multiple myeloma (MM) cell lines, such as U226, H929, RPMI8226, MM1R and MM1S.</p><p><b>METHODS</b>TEB-415, a derivative of Imatinib was synthesized by modifying the chemical structure of Imatinib. MM cell lines (U226, H929, RPMI8226, MM1R and MM1S) were treated with TEB-415, Imatini and Bortezomib of various concentrations. Cells were grown for 72 hours and the growth rate was measured by CCK-8 method, cell morphology was observed and the IC50 was calculated.</p><p><b>RESULTS</b>TEB-415 could inhibit H929 and RPMI8226 growth significantly. When the concentration of TEB-415 was <0.1 nmol/L, >50% H929 cells died. The IC50 of Imatinib was 0.123 mol/L while the IC50 of Bortezomib was 0.03 nmol/L. In RPMI8226 cell line, when the concentration of TEB-415 was 11.9 mol/L, more than 50% of cells died. In contrast, when RPMI8266 were treated with Imatinib of the concentration of 12.8 mol/L, cells grew normally.</p><p><b>CONCLUSION</b>In comparison to Imatinib, TEB-415, a derivative of Imatinib, can kill H929 MM cells much effectively, its effecacy is only inferior to Bortezomib. RPMI8226, an MM cell line is insensitive to Imatinib, but still sensitive to TEB-415 and its growth can be inhibited by TEB-415.</p>

Ex vivo Culture System of Single Human Hematopoietic Stem Cell Used to Screan the Small Molecular Compounds / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore an efficient, stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells (hHSC).</p><p><b>METHODS</b>By using combination of flow cytometry with study results of surface markers on hHSC, and optimation of sorting process for further studying the effect of small molecular compounds on stem property of hHSC, the single hHSC was treated with published small molecular compounds such as SR1 and UM171 which possess the expansion effect. After treating with hHSC for 14 d, the flow cytometric analysis of cell phenotypes and cell morphologic observation were performed, at the same time the hematopoietic function of cultured hHSC was verified by colony-forming cell (CFC) test and cobblestone area forming cell (CAFC) test.</p><p><b>RESULTS</b>The effects of SR1 and UM171 and their compositions in multi-cell culture were consistent with the published data, therefore the useful concentration of compounds were obtained. The results of multiparameter sorting of single cell (CD34+ CD38- CD45RA- CD90+ CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis was in accordance with flow cytometric results. In addition, CFC test and CAFC test revealed that the colony-forming ability in treated group was significantly higher than that in control group (P<0.05).</p><p><b>CONCLUSION</b>The rapid, efficient stably amplified and short-time culture system for single hHSC and method for varifying the effect of small molecular compounds are established, which provides platform for screening small molecular compounds and lays the foundation for further study of hHSC expansion.</p>

Role of NF-κB inhibitor in Acute Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of NF-κB inhibitor in occurence and development of AML.</p><p><b>METHODS</b>AML and normal bone marrow samples were collected from 8 AML patients and 8 normal persons. The expression of NF-κB signaling pathway genes was detected by NF-κB PCR array. Then, AML mouse model was constructed to test the role of NF-κB inhibitor in AML.</p><p><b>RESULTS</b>The NF-κB signal pathway was activated in AML patients. The up-regulated genes, EDARADD, TNFSF14, could activate the NF-κB signal pathway, IL6 could regulate the inflammatory signal. The down-regulated genes, TNFRSF 10B, TNFRSF1A, could lead to cell apoptosis. the AML mouse model was constructed successfully. Then administration of NF-κB inhibitor reduced the inhibition of leukemia niche to the normal hematopoietic stem cells (HSCs), promoted the HSC to enter into cell cycle.</p><p><b>CONCLUSION</b>The NF-κB signal pathway is activated in AML cells. AML mouse model is constructed successfully. NF-κB inhibitor has a potential to treat AML and promotes the HSC to enrter into cell cycle.</p>

Effects of catalase on the engraftment of human hematopoietic stem cells in NOD/SCID mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of catalase (CAT) on engraftment of human hematopoietic stem cells (HSC) by co-transplanting umbilical cord-derived mesenchymal stem cells (UC-MSC) with over-expressed CAT and human HSC into NOD/SCID mice.</p><p><b>METHODS</b>The UC-MSC cultured in vitro were transfected by the retrovirus containing green fluorescent protein (GFP) and GFP-CAT genes respectively. MSC-GFP and MSC-GFP-CAT cell lines were sorted by flow cytometry. Co-culture and co-transplant experiments were performed to detect the effects of CAT on expansion and engraftment of human HSC.</p><p><b>RESULTS</b>The percentage of GFP(+) cells were approximately 97.6% and 96.8% after sorting. The mRNA expression of CAT in MSC-GFP-CAT was 23.9-fold higher than that in UC-MSC. The activity of CAT in UC-MSC, MSC-GFP, MSC-GFP-CAT cells were 19.5, 20.3 and 74.1 Unit respectively. There was no significant differences in the percentage of CD34(+) cells between 3 groups in co-culture experiment. And the percentage of human CD45(+) cells in NOD/SCID mice were (3.22 ± 3.1)%, (4.26 ± 3.56)% and (7.37 ± 4.51)% respectively.</p><p><b>CONCLUSION</b>MSC-GFP-CAT significantly improves the engraftment of human HSC in NOD/SCID mice, whereas co-culture with the MSC-GFP-CAT can not promote the expansion of HSC in vitro.</p>

Macrophage inflammatory protein-1α promotes the growth of acute myeloid leukemia cells / 中国实验血液学杂志

<p><b>UNLABELLED</b>BACKGROWND: Macrophage inflammatory protein-1α (MIP-l α/CCL3) belongs to the C-C chemokine family (CCL3), which can be secreted by macrophages, other types of hematopoietic cells and bone marrow stromal cells. Higher levels of MIP-1α were found to be associated with several kinds of hematologic malignancies, including multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Moreover, MIP-1α has been reported to be an adverse prognostic factor for CLL. However, the impact of MIP-1α on acute myeloid leukemia (AML) has been poorly investigated.</p><p><b>OBJECTIVE</b>To investigate the influence of MIP-1α on proliferction of AML cells.</p><p><b>METHODS</b>Using MLL-AF9 induced AML mouse model, the expression of MIP-1α was measured by real time quantitative RT-PCR. AML cell proliferation was examined by cell counting and colony forming assay (CFC). The influence of blocking the MIP-1α action on the growth and pathogenic ability of AML cells was explored by using the small molecule antagonist for interfering interaction of MIP-1α with its receptor CCR1.</p><p><b>RESULTS</b>The MIP-1α could promote the proliferation and colony formation of AML cells, the blocking MIP-1a could inhibit the growth of AML cells and delay onset of AML.</p><p><b>CONCLUSION</b>The MIP-1a promotes the occurence and progression of AML, therefore blocking the MIP-1α signal pathway may be served as a strategy to inhibit the growth of AML cells, and MIP-1α can be a potential target for treatment of AML.</p>

Applications of Microscopic Imaging and Analysis Technology in Studies of Neutrophil Movement and Phagocytosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze and evaluate the application of spinning disk confocal microscopy and imaging analysis software in movement and phagocytosis of neutrophils.</p><p><b>METHODS</b>Neutrophils were isolated from bone marrow by centrifugation on discontinuous Percoll gradient, and then were stained with PE Gr-1 antibody and mixed with FITC-labeled Zymosan A bioparticles. Multichannel time-lapse videos were captured by using the spinning disk confocal microscopy. The result was analyzed by using volocity and ImageJ software, the parameters associated with movement and phagocytosis of neutrophils were analyzed, including morphological changes, cell tracking, pseudopod dynamics, binding and phagocytosis index.</p><p><b>RESULTS</b>Most neutrophils would be polarized in response to Zymosan particles during a short time. Binding and phagocytosis process occured in forty minutes.</p><p><b>CONCLUSION</b>A method of precisely quantifying the movement and phagocytosis of neutrophils using microscopic imaging and imaging analysis technique has been set up successfully. Using this method, biological activity and function of neutrophils can be evaluated visually and rapidly. The physiologically rapid response to Zymosan particles can be applied to the neutrophils function research in the future.</p>

Establishment of Integration-Free Human Induced Pluripotent Stem Cells from A Patient with Primary Myelofibrosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish the primary myelofibrosis (PMF)-induced pluripotent stem cell line (iPSC) by means of iPSC techinique so as to provide a experimental model for studying the blood disease mechanisms.</p><p><b>METHODS</b>Induced pluripotent stem cells were generated from mononuclear cells isolated from a PMF patient with JAK2(V617F) mutation by using episomal vectors.</p><p><b>RESULTS</b>PMF-derived iPSC was established from the patient with JAK2(V617F) gene mutation. The PMF-iPSC could be stably passaged, highly expressed pluripotent genes as human embryonic stem (ES) cells, and were able to form teratoma in NOD/SCID mice in vivo. H & E staining of the teratoma showed the presence of tissue type derived from all three embryonic germ layers. Sanger sequencing confirmed that PMF-derived iPSC carried different allele burdens of JAK2(V617F) gene mutation.</p><p><b>CONCLUSION</b>The interation-free iPSC from primary myelofibrosis patient in vitro has been established. This PMF-derived iPSC line provides a valuable tool for studying the pathogenesis, screening of chimical drugs and realizing the standard therapy of PMF.</p>

Commonly used cre transgenic mice and their applications in hematopoietic system / 中国实验血液学杂志

Cre-lox recombination system consists of two elements: Cre recombinase enzyme and lox sites. Cre recombinase can recombine the lox site sequences by specifically detecting and cutting them. The direction and position of lox sites determine the functional effects of Cre enzyme such as deletion, inversion or chromosomal translocation. The hematopoietic system of mouse consists of multi-lineages and various developmental stage hematopoietic cells that are differentiated from hematopoietic stem cells (hematopoietic stem cells, HSC). The hematopoietic stem cells are maintained in the bone marrow microenvironment (niche). Currently, a variety of floxed conditional-knockout mice, recognized by Cre-lox recombination system, are used for the study of the hematopoietic system. This review summarizes the commonly used Cre transgenic mice and their applications in the study of hematopoietic system.

Anti-mouse CD122 antibody promotes the hematopoietic repopulating capacity of cord blood CD34⁺ cells in NOD/SCID mice / 中国实验血液学杂志

The study was aimed to investigate the effect of anti-mouse CD122 antibody on the hematopoietic repopulating capacity of cord blood CD34⁺ cells in a humanized murine model-non obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After sublethal irradiation with γ-ray, NOD/SCID mice were intraperitoneally injected with 200 µg mouse isotype control antibody or anti-mouse CD122 antibody. Human cord blood CD34⁺ cells or phosphate-buffered saline (PBS) were injected via the tail vein at 6-8 hours later. Cohort of the mice injected with anti-mice CD122 antibody or control antibody alone were sacrificed at different time point (at week 2, 3, and 4 weeks) after the injection, and the percentage of NK cells in the peripheral blood was analyzed by flow cytometry. To evaluate the effect of anti-mouse CD122 antibody on the repopulating capacity of cord blood CD34⁺ cells in the recipient mice, phenotype analysis was performed in the bone marrow at 6 and 8 weeks after the transplantation. The results showed that the proportion of NK cells in the peripheral blood were (4.6 ± 0.6)% and (5.7 ± 1.7)% at week 2 and 3 after anti-CD122 antibody injection respectively,which decreased by 60%, compared with the mice injected with isotype control antibody. After 6 and 8 weeks of cord blood CD34⁺ cell transplantation,the percentage of human CD45⁺ in the bone marrow of the recipient mice treated with anti-mice CD122 antibody was (63.0 ± 12.2)% and (53.2 ± 16.3)%,respectively,which were dramatically higher than that in the mice treated with isotype control antibody (7.7 ± 3.6)% and (6.1 ± 2.4)%. Moreover,at 8 weeks after transplantation,human CD34⁺ cells appeared significantly in the recipients treated with anti-CD122 antibody. It is concluded that the anti-mouse CD122 antibody enhances the hematopoietic repopulating capacity of cord blood CD34⁺ cells in the NOD/SCID mice through decreasing the proportion of NK cells.

Knockdown of Puma protects cord blood CD34(+) cells against γ- irradiation / 中国实验血液学杂志

Puma (P53 upregulated modulator of apoptosis) is a BCL-2 homology 3 (BH3)-only BCL-1 family member and a critical mediator of P53-dependent and -independent apoptosis. Puma plays an essential role in the apoptosis of hematopoietic stem cells exposed to irradiation without an increased risk of malignancies. This study was purposed to develop an effective lentiviral vector to target Puma in human hematopoietic cells and to investigate the effect of Puma gene knockdown on the biological function of human cord blood CD34(+) cells. SF-LV-shPuma-EGFP and control vectors were constructed, and packaged with the pSPAX2/pMD2.G packaging plasmids via 293T cells to produce pseudo-type lentiviruses. SF-LV-shPuma-EGFP or control lentiviruses were harvested within 72 hours after transfection and then were used to transduce human cord blood CD34(+) cells. GFP(+) transduced cells were sorted by flow cytometry (FCM) for subsequent studies. Semi-quantitative real time RT PCR, Western blot, FCM with Annexin V-PE/7-AAD double staining, Ki67 staining, colony forming cell assay (CFC), CCK-8 assay and BrdU incorporation were performed to determine the expression of Puma and its effect on the cord blood CD34(+) cells. The results showed that Puma was significantly knocked down in cord blood CD34(+) cells and the low expression of Puma conferred a radio-protective effect on the cord blood CD34(+) cells. This effect was achieved through reduced apoptosis and sustained quiescence after irradiation due to Puma knockdown. It is concluded that knockdown of puma gene in CD34(+) hematopoietic stem cells of human cord blood possesses the radioprotective effect, maintains the cells in silence targeting Puma in human hematopoietic cells may have a similar effect with that on mouse hematopoietic cells as previously shown, and our lentiviral targeting system for Puma provides a valuable tool for future translational studies with human cells.

Expression of CD48 as a live marker to distinguish division of hematopoietic stem cells / 中国实验血液学杂志

Hematopoietic stem cells are capable of self-renewal or differentiation when they divide. Three types of cell divisions exist. A dividing stem cell may generate 2 new stem cells (symmetrical renewal division), or 2 differentiating cells (symmetrical differentiation division), or 1 cell of each type (asymmetrical division). This study was aimed to explore an efficient and stable method to distinguish the way of cell division in hematopoietic stem cells. Previous studies showed that the distribution of Numb in a cell could be used to distinguish the type of cell division in various kinds of cells. Therefore, the distribution of Numb protein was detected by immunofluorescence in mitotic CD48(-)CD150(+)LSK cells of mice exploring the relationship between Numb protein and centrosomes. Since CD48 positive marks the HSC that have lost the ability to reconstitute the blood system in mice, CD48 marker could be used to distinguish cell fate decision between self-renewal and differentiation as a living marker. In this study, the CD48(-)CD150(+)LSK cells were sorted from bone marrow cells of mice and the cells were directly labeled with Alexa Fluor (AF) 488-conjugated anti-CD48 antibody in living cultures. After 3 days, the percentage of AF488(+) cells was evaluated under microscope and by FACS. Then colony forming cell assay (CFC) was performed and the ability of cell proliferation were compared between AF488(+) and AF488(-) cells. The results showed that Numb could be used to distinguish different cell division types of hematopoietic stem cells, which was symmetrically or asymmetrically segregated in mitotic CD48(-)CD150(+)LSK cells. The self-labeled fluorochrome could be detected both by FACS as well as microscope. There were about 40% AF488(+) cells after 3 day-cultures in medium titrated with self-labeled AF 488-conjugated anti-CD48 antibody, and the results were consistent between confocal fluorescence microscopy and flow cytometry analysis. The colony forming ability of AF488(+) cells was significantly higher than that of AF488(-) cells (P < 0.05). The proliferation ability of AF488(-) cells was also significantly higher than AF488(+) cells (P < 0.05). It is concluded that the expression of CD48 can distinguish cell division of hematopoietic stem cells and can be used as a live marker for the loss of stemness. In comparison with the Numb protein staining, this method can be used in living cells, thus provides greater convenience for subsequent cell culture studies and cell transplantation experiments.

An improved method for generating integration-free human induced pluripotent stem cells / 中国实验血液学杂志

The genome instability and tumorigenicity of induced pluripotent stem cells (iPSC) hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells (CB MNC), to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre-stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV (spleen focus forming virus) promoter under the hypoxic condition (3%), CB MNC could be mostly efficiently reprogrammed with the efficiency 0.12%. Furthermore, the results showed that erythroblasts (CD36(+)CD71(+)CD235a(low)) were the cells that are reprogrammed with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.