[Factor analysis of effective platelet-producing ability of fetal liver-derived cells].

Objective: To study the different factors affecting platelet production post transplantation of hematopoietic stem cells (HSCs) isolated from different sources in order to explore novel options for treating platelet depletion following HSCs transplantation. Methods: HSCs and their downstream derivatives including myeloid and lymphoid cells (i.e., collective of mononuclear cells (MNCs)), were isolated from E14.5 fetal liver (FL) and bone marrow (BM) of 8-week-old mice by Ficoll separation technique. These cells were subsequently transplanted into the tibia bone marrow cavity of recipient mice post lethal myeloablative treatment in order to construct the FL-MNCs and BM-MNCs transplantation mouse model. Routine blood indices were examined in these recipient mice. The chimeric rate of donor cells in recipient peripheral blood cells were determined by flow cytometry. Different groups of cells involved in platelet reconstruction were analyzed. CD41+megakaryocytes were sorted from fetal liver or bone marrow using magnetic beads, which were then induced to differentiate into platelets in an in vitro assay. Quantitative RT-PCR was used to detect the expression of platelet-related genes in CD41+megakaryocytes from the two sources. Results: Both the FL-MNCs and the BM-MNCs transplantation groups resumed normal hematopoiesis at the 4th week after transplantation, and the blood cells of the recipient mice were largely replaced by the donor cells. Compared with the mice transplanted with BM-MNCs, the platelet level of mice transplanted with FL-MNCs recovered faster and were maintained at a higher level. At week 4, the PLT level of the FL-MNCs group was (1.45±0.37)×1012/L, and of the BM-MNCs group was (1.22±0.24)×1012/L, P<0.05. The FL-MNCs contain a higher proportion of hematopoietic stem cells (Lin-Sca-1+c-Kit+)(7.60%±1.40%) compared to the BM-MNCs (1.10%±0.46%), P<0.01; the proportion of the megakaryocyte progenitor cells (Lin-Sca-1-c-Kit+CD41+CD150+) and mature megakaryocyte cells (CD41+CD42b+), also differ significantly between the FL-MNCs (3.05%±0.22%, 1.60%±0.06%, respectively) and the BM-MNCs (0.15%±0.02%, 0.87%±0.11%, respectively) groups, both P<0.01. In vitro functional studies showed that FL-MNCs-CD41+megakaryocytes could produce proplatelet-like cells more quickly after induction, with proplatelet-like cells formation on day 3 and significant platelet-like particle formation on day 5, in contrast to bone marrow-derived BM-MNCs-CD41+megakaryocytes that failed to form proplatelet-like cell on day 5. In addition, FL-MNCs-CD41+cells expressed higher levels of platelet-related genes, Mpl (3.25-fold), Fog1 (3-fold), and Gata1 (1.5-fold) (P<0.05). Conclusion: Compared with the BM-MNCs group, the FL-MNCs transplantation group appears to have a more efficient platelet implantation effect in the HSCs transplantation recipient in vivo, as well as a higher platelet differentiation rate in vitro. This might be related to a higher proportion of megakaryocytes and higher expression levels of genes such as Mpl, Fog1, and Gata1 that could be important for platelet formation in FL-MNCs-CD41+cells. Further exploration of the specific functions of these genes and the characteristics of the different proportions of the donor cells will provide valuable clues for the future treatment of platelets reconstitution after HSCs transplantation clinically.

[The effect of DNA hydroxymethylase Tet2 on γ globin activation in the treatment of ß-thalassemia].

Objective: To study the function of ten-eleven translocation 2 (Tet2) in γ globin gene expression in patients with ß- thalassemia. Methods: Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line. The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit. 5hmC level of γ globin gene was quantified by sulfite sequencing. The mRNA level of Tet2, γ globin, and related transcription factors Nfe4 and Klf1 were quantified by real-time PCR. Results: Tet2 knockdown resulted in a decreased global 5hmC level from 0.14% to 0.03% as of the control group in K562 cells. The expression of γ globin was enhanced after 5-azacytidine treatment in vitro. However, γ globin mRNA level in Tet2 knockdown cells was only 55% as that in control group. The CG sites on γ globin gene were unmethylated. As Tet2 was down-regulated, the expression levels of Nfe4 and Klf1 decreased by about 80% and increased to 3.5 folds, respectively. Conclusions: Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation. Moreover, Tet2 more likely regulates γ globin expression via affecting transcription factors rather than the gene itself. Thus, Tet2 could be a potential therapeutic target for ß thalassemias.