Establishment of a platelet production model by bone marrow cavity transplantation of mouse primary megakaryocytes / 中华血液学杂志

Objective: To establish an intramedullary transplantation model of primary megakaryocytes to evaluate the platelet-producing capacity of megakaryocytes and explore the underlying regulatory mechanisms. Methods: Donor megakaryocytes from GFP-transgenic mice bone marrow were enriched by magnetic beads. The platelet-producing model was established by intramedullary injection to recipient mice that underwent half-lethal dose irradiation 1 week in advance. Donor-derived megakaryocytes and platelets were detected by immunofluorescence staining and flow cytometry. Results: The proportion of megakaryocytes in the enriched sample for transplantation was 40 to 50 times higher than that in conventional bone marrow. After intramedullary transplantation, donor-derived megakaryocytes successfully implanted in the medullary cavity of the recipient and produce platelets, which showed similar expression of surface markers and morphology to recipient-derived platelets. Conclusion: We successfully established an in vivo platelet-producing model of primary megakaryocytes using magnetic-bead enrichment and intramedullary injection, which objectively reflects the platelet-producing capacity of megakaryocytes in the bone marrow.

Role of BET Bromodomain in Hematopoietic Differentiation from hESCs / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the role of bromodomain and extra terminal (BET) bromodomain in hematopoietic differentiation from human enbryonic stem cells (hESC).</p><p><b>METHODS</b>The effect of BET hematopoietic inhibitor I-BET151 on hematopoietic differentiation from hESC was detected by using a monolayer hematopoietic defferentiation model, immunofluorescence, flow cytometry and real-time PCR; moreover the role of I-BET151 in process of hematopoietic differentiation was explored by adding I-BET151 in different differentiation stages.</p><p><b>RESULTS</b>The analysis results of immunofluorescence, flow cytometry and real-time PCR showed that I-BET 151 significantly inhibited the generation of CD43 positive hematopoietic stem and progenitor cells (HSPCs). It was found that the addition of I-BET 151 in different stages, including APLNR lateral plate mesoderm production, CD34CD31 hemogenic endothelium (HEP) generation and endothelial-to-hematopoietic transition, significantly suppressed the generation of CD43 positive hematopoietic progenitor cells.</p><p><b>CONCLUSION</b>I-BET 151 inhibites hematopoietic differentiation from hESCs at several stages, suggesting that the BET bromodomain plays important roles in multiple stages of hematopoietic differentiation from hESCs.</p>

DMSO Promotes Hematopoietic Differentiation of Human Embryonic Stem Cells in vitro / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the role of dimethyl sulfoxide (DMSO) in the hematopoietic differentiation of human embryonic stem cells (hESCs).</p><p><b>METHODS</b>The role of DMSO in hematopoietic differentiation of hESC was investigated by using a established stepwise hematopoietic differentiation system from hESC, immunofluorescouse assay and flow cytometry. Furthermore, the window phase of DMSO action was explored by adding it to the different stage of hematopoietic differentiation.</p><p><b>RESULTS</b>Immunofluorescence and flow cytometry analysis showed that DMSO significantly promoted the generation of CD43hematopoietic progenitor cells (HPC). The flow cytometry demonstrated that DMSO profoundly promoted the generation of APLNRlateral plate mesoderm cells and CD31CD34hemogenic endothelium progenitors (HEP). The addition of DMSO in the window phase of lateral plate mesoderm cell generation could markedly improve the generation of hematopoietic progenitor cells.</p><p><b>CONCLUSION</b>DMSO promotes hematopoietic differentiation of hESC through enhancing the generation of APLNR positive lateral plate mesoderm cells. The addition of DMSO in the window phase of lateral plate mesoderm cell generation can significantly improve the generation of hematopoietic progenitor cells.</p>

Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1 / 中国实验血液学杂志

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.