miR-146b facilitates the differentiation of induced pluripotent stem cells into neuron-like cells / 中国组织工程研究

BACKGROUND: To facilitate the neuronal differentiation of induced pluripotent stem cells (iPSCs) is of great benefit for the repair of nerve injury and nerve regeneration. OBJECTIVE: To investigate the expression of miR-146b in iPSCs differentiation, and its effects on the neural differentiation of iPSCs. METHODS: After 7-day neural induction of mouse iPSCs, the expressions of miR-146b and pluripotent stem cell markers were detected by real-time PCR. Then, we generated miR-146b overexpressing iPSCs followed by the neural induction. The expression of stem cell and neuroectoderm markers were examined by cell immunofluorescence and real-time PCR respectively. Meanwhile, the expression of Tuj-1, a nerve cell marker, was also detected. RESULTS AND CONCLUSION: Compared with the normal iPSCs, the expressions of pluripotent stem cell markers Nanog, Oct4 and Sox2 were significantly down-regulated in iPSCs undergoing 7-day neural induction, while the level of miR-146b was increased obviously. In miR-146b-overexpressing iPSCs, we found that the expression of Oct4 was substantially decreased, while there were no statistical changes in the expression of Nanog and Sox2. Meanwhile, during the neural differentiation, miR-146b overexpression significantly down-regulated the expression of Nanog, Oct4 and Sox2, and up-regulated the expression of neuroectoderm markers, Nestin, Sox1 and Pax6. After 18 days of neural induction, the miR-146b overexpression significantly increased the number of Tuj-1-positive cells. Taken together, miR-146b plays a vital role in facilitating the differentiation of iPSCs into neuron-like cells.

Establishment of nonmyeloablative allogeneic stem cell transplantation model in H-2 haploidentical mice and its related study / 中国实验血液学杂志

To explore the feasibility of nonmyeloablative conditioning regimens, hematopoietic reconstitution, chimera level and the occurrence of GVHD after nonmyeloablative allogeneic stem cell transplantation in H-2 haploidentical mice, CB6F1 mice were used as the recipient and were divided into 3 groups, mice were pretreated five days before transplantation. Group A was pretreated with myeloablative conditioning regimens (TBI with 10.5 Gy), group B was pretreated by TBI (2 Gy) + Ara-C + Cy and group C-TBI (2 Gy) + Ara-C + CY + Flu, respectively. For all recipient mice, the prevention of GVHD was not given, and 2 x 10(7) bone marrow cells mixed 1 x 10(7) spleen cells from C57BL/6 mice were injected through tail vein on day 0, and then hematopoietic recovery, engraftment and GVHD of recipients were observed. The results of chimera detection after transplantation showed that the engraftment of group A remained full donor chimerism, and engraftments of group B and group C were associated with mixed chimerism or full donor chimerism, but the chimerism of group B remained below 80% and tended to decrease after 50 days whereas chimerism of group C was above 80% (chimerism close to or being full donor type) and preserved even after 50 days. GVHD occurred in all the recipient mice due to that prevention was not given, wherein the occurrence and death rate of GVHD in group A was obviously higher than that of group B and group C (P <0.01), but there was no statistical difference between group B and group C. In conclusion, the nonmyeloablative conditioning regimens mainly based on fludarabine can form stable and lasting engraftment in the body of recipients. The mixed chimerism established in recipients induce tolerance of transplantation and decrease or avoid the occurrence of GVHD.

Cyclosporine A and mycophenolate mofetile as prophylactics for GVHD in a murine model of H-2 haploidentical nonmyeloablative bone marrow transplantation / 中国实验血液学杂志

In order to research the prophylactic effect of cyclosporine A (CSA) and mycophenolate mofetile (MMF) on GVHD in mice with H-2 haploidentical nonmyeloablative bone marrow transplantation, a murine model was established by using of C57BL/6J mouse as donor and (C57BL/6J x BALB/C) F(1) mouse as the recipient. The recipient mice were given CSA + MTX or CSA + MMF for prophylaxis of acute GVHD (aGVHD). The survival rate, hematopoietic recovery, and morbidity and mortality of aGVHD were observed for 50 days after transplantation. The results showed that typical aGVHD developed in the transplanted mice without prophylactic treatment during 22 to 25 days after transplantation. The morbidity of aGVHD was 75% (15/20), 40% (8/20) and 30% (6/20) and mortality was 100% (15/15), 62.5% (5/8) and 50% (3/6) respectively in unprophylactic group (control), CSA + MTX and CSA + MMF groups. In conclusion, CSA and MTX reduce the morbidity and mortality of aGVHD in mice with haploidentical nonmyeloablative bone marrow transplantation, and the effect of CSA + MMF is better than that of CSA + MTX.

Development of methodology for isolation and culture of mesenchymal stem cells derived from mouse skeletal muscle / 中国实验血液学杂志

To address the question whether there exists mesenchymal stem cells in adult mouse skeletal muscle, mononuclear cells from muscle were obtained by digestion and density gradient centrifugation and plated in alpha-MEM/F12 medium containing 10% fetal bovine serum. Cell biological properties including morphology, cytochemistry, growth pattern and phenotypes as well were evaluated. Likewise, the osteogenesis of cultured cells was also observed. The results showed that adherent cells homogenous in shape proliferated quickly in the culture system. The phenotypes of the cells were unique, which were positive for CD29 and Sca-1, and negative for CD34 and CD45. Cytochemistry evaluation showed that they were homogeneously positive for acid alpha-naphthl acetate esterase (ANAE) and acid phosphatase (ACP), and negative for alkaline phosphatase (ALP) and that, around 5% of them were positive for glycogen (periodic acid-Schiff reaction, PAS). Cells became ALP-positive after the induction by ascorbic acid, beta-phosphoglycerol and dexamethasone. It is concluded that mesenchymal stem cells exist in murine skeletal muscle and compose the complex heterogenous population of stem cells in muscle.

The experimental study on idiotypic DNA vaccine against human B-cell lymphoma to induce antitumor immune response / 中国实验血液学杂志

The purpose of this study was to evaluate whether the DNA vaccine containing idiotypic gene fragment of human B-cell lymphoma cell line Namalwa could elicit the specific anti-idiotypic immune response in vivo. The candidate gene fragment of the lymphoma cell, variable region of heavy chain (VH) of the membranous immunoglobulin, was amplified using Ig superfamily primers by means of RT-PCR. Also, the intact cDNA of murine monocyte chemoattractant protein (MCP-3) was cloned and used as the adjuvant molecular. The two gene fragments of VH and MCP-3 were fused together by 8aa linker peptide with recombinant PCR. Subsequently, the fusion gene fragment was cloned into eukaryonic expression vector pcDNA3.1 to construct DNA vaccine plasmid. Prior to the immunization, the transient transfection coupled with RT-PCR was performed to prove that the recombinant plasmid could express in eukaryonic cells in right way. Then two groups of mice were immunized by intramuscular injection with DNA vaccine and mock plasmid pcDNA3.1 respectively. Three times of injection were performed with 100 micro g plasmid respectively at the beginning of the experiment and 2, 4 weeks after the first injection for all the groups. FACS analysis was chosen to detect the antibodies recognizing lymphoma cells at different time following vaccination. The results demonstrated that specific anti-idiotypic antibody could be detected in the group of DNA vaccine immunized mice as early as eight weeks after the first immunization. Further test demonstrated that the anti-idiotypic antibody could maintain for at least twenty weeks with high titer. Anti-idiotypic antibodies were elicited in three of five mice of the DNA vaccine immunized group. The Abs of DNA vaccine immunized mice could only recognize Namalwa cell line instead of another unrelated human cell line A549. There is no cellular response detected in the DNA vaccine immunized mice. It is concluded that the DNA vaccine containing fused MCP3-VH sequence could elicit specific anti-idiotypic antibody against B-cell lymphoma in vivo and could be used in further study of DNA vaccine against B-cell lymphoma. The results would provide the basis for further studies and optimization of this therapeutic strategy on patients with B-lymphoproliferative disease.