Study on hematopoiesis reconstitution by co-transplant of human bone marrow mesenchymal stem cells and umbilical cord blood CD34(+) cells at different ratios in NOD/SCID mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of co-transplant of human bone marrow mesenchymal stem cells (BMMSCs) and umbilical cord blood (UCB) CD34(+) cells on hematopoiesis reconstruction in NOD/SCID mice and to investigate the optimal proportion between the two kind of cells.</p><p><b>METHODS</b>Female NOD/SCID mice were sublethally irradiated by (60)Co gamma-ray and transplanted with BMMSCs and different ratios of UCB CD34(+) cells. From day +3 till day +42 after transplantation, 20 microl peripheral blood (PB) was collected from the retro-orbital plexus of mice weekly, and the variations of WBC and PLT were counted. Mice were sacrificed 42 days after transplantation, and human CD45 positive (huCD45(+)) cells in PB, BM, and spleen were detected by flow cytometry.</p><p><b>RESULTS</b>Compared with transplant of UCB CD34(+) cells alone, co-transplantation of BMMSCs and UCB CD34(+)cells at ratios of 1:1, 5:1 and 10:1, (1) significantly mitigated the decrease range (P < 0.01) and led to the recovery of WBC and platelet in PB one week earlier (P < 0.05), and the difference among the three groups was not statistically significant (P > 0.05); (2) significantly enhanced hematopoietic stem cells (PB, BM and spleen cells) engraftment in recipient mice, and the effect was most pronounced at the ratio of 10:1. huCD45(+) cells in PB, BM and spleen were increased by (2.75 +/- 0.63), (3.51 +/- 0.86) and (5.18 +/- 0.57) fold, respectively (P < 0.01).</p><p><b>CONCLUSION</b>The optimal hematopoiesis reconstruction is achieved by co-transplant of UCB CD34(+) cells and BMMSCs at a ratio of 1:10.</p>

Establishment of an xenogeneic acute graft-versus-host disease model in NOD/SCID mice by engraftment of G-CSF mobilized human mononuclear cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish an xenogeneic acute graft-versus-host disease model by engraftment of G-CSF mobilized human mononuclear cells into NOD/SCID mice.</p><p><b>METHODS</b>Mobilized human peripheral blood mononuclear cells (PBMNCs) were transplanted into sublethally irradiated NOD/SCID mice. After transplantation, complete blood count, huCD45+ cells and other phenotype human lymphocytes were determined weekly. Mice were sacrificed, and their tissues were examined histopathologically and immunophenotypically. Genomic DNA was also prepared for detecting human beta-globin DNA sequence and endogenous mouse RAPSYN gene.</p><p><b>RESULTS</b>The human CD45+ cells in the mice appeared 1 week after transplantation. Its percentage was increased with an acute X-GVHD syndrome characterized by rapid and severe weight loss and pancytopenia. Both the specific DNAs of human beta-globin DNA gene and the murine RAPSYN gene were detected in the hu-NOD/SCID chimeras; The survival rate was 14% at 6 weeks posttransplantation. The engrafted human cells consisted mainly of CD3+ T lymphocytes but CD4/CD8 ratios seemed inverted in the chimeras. The xenogeneic graft versus host reaction was heterogeneous in different organs mainly with human lymphocytes infiltration and the liver and lungs were the critical organs.</p><p><b>CONCLUSION</b>Mobilized peripheral blood mononuclear cells are capable of engrafting in irradiated NOD/SCID mice with induced acute X-GVHD syndrome. The liver and the lungs are the critical organs. This is a good model for investigating the effects of human cells in inducing acute graft versus host disease in animal and for testing effective intervention methodology.</p>

Effect of murine mesenchymal stem cells (mMSC) on the allogeneic immuno-response of syngeneic and allogeneic spleen cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of murine mesenchymal stem cells (mMSC) on immunoproliferative response of spleen cells.</p><p><b>METHODS</b>Mitogen (Con A, 20 microg/ml) or irradiated (20 Gy) allogeneic spleen cells (from BALB/c or C57BL/6 mouse depending on the responder cells) were used as stimulators. Proliferations of the responder cells were determined with MTT on day 3 after culture at 37 degrees C, 5% CO2 humidified atmosphere. The ratios of CD4+/CD8+ and CD4+CD25+ cells were analyzed with FACS assay, and the levels of cytokines in supernatants with ELISA.</p><p><b>RESULTS</b>1) mMSC inhibited the response of both syngeneic and allogeneic splenic cells to ConA. At the ratio of mMSC to splenic cells being 1: 1, the inhibition rate reached 84.21%. With the ratio decreasing, the inhibition rate decreased. 2) mMSC inhibited the response of both syngeneic and allogeneic splenic cells to alloantigen. When the ratio of mMSC to responder cells was 1: 10, the inhibition rate was as high as 88.07%. 3) mMSC could increase the ratio of CD4+/ CD8+ T cells and the percentage of CD4+ CD25+ cells in splenic cells. These abilities were in a dose-dependent manner and non-MHC antigen restricted. 4) mMSC decreased interleukin (IL) -2, interferon (IFN)-gamma, while increased TGF-beta1 and IL-4 in the co-culture system.</p><p><b>CONCLUSION</b>mMSC can suppress proliferative response of splenic cells to mitogen and alloantigen, increase the ratio of CD4+/CD8+ and the proportion of CD4+ CD25+ in T cells, decrease the secretion of proinflammatory cytokines and increase the anti inflammatory cytokines in a dose-dependent and non-MHC antigen restricted manner.</p>

Enrichment and biological characteristics of murine mesenchymal stem cells / 中国实验血液学杂志

The study was aimed to isolate and establish mesenchymal stem cell line from adult murine bone marrow as well as to identify its biological characteristics and differentiation potential. Bone marrow cells (BMCs) were collected by flushing the femurs and tibias of 4 - 5-week-old male C57BL/6 mice, and were inoculated at a concentration of 1 x 10(6)/cm(2). mMSCs were isolated, enriched and expanded by using bone marrow adherant culture and monoclonal culture. The characteristics of the cells, such as morphology, growth pattern, cell cycle, phenotype, karyotype and multipotent differentiation potential, cytogenetic stability and tumorigenesis were determined. The results indicated that the cell population consisted of spindle- and star-shaped cells, they were highly positive for CD29, CD44, Sca-1, MHC-I, moderate positive for CD13, CD90.2 and negative for CD117, CD45, Flk-1 and MHC-II. mMSCs could be induced to differentiate into adipocytes, osteoblast cells and chondrocytes. It is concluded that mMSC can be isolted, expanded and enriched by using bone marrow adhcrent culture and monoclonal culture. No tumor formations are observed for 3 months in nude mice after subcutaneous injection. mMSCs retain their properties after at least 30 passages in culture as well as from frozen stocks.

Regulation mechamion of mesenchymal stem cells to immunoeffector cells--review / 中国实验血液学杂志

Mesenchymal stem cells have two main properties: self renewal and the ability to differentiate multiple lineage. Because MSCs exhibit low immunogenicity and demonstrate significant suppressive activity in cell cultures containing alloreactive T cells, they play an important role in transplantation immunology, but the exact mechanism remains unknown. This article focuses on the immunoregulatory feature of MSCs to immunoeffector cells, such as T cells, B cells, and NK cells. The role of MSC in transplantation immunoregulation, regulatory mechanism of MSC in cellular immunity (direct contact of cells with cells, apoptosis and immunoregulation of MSC on lymphocytes), immunoregulation of MSC on DC and NK cells were reviewed.

Effects of human mesenchymal stem cells and fibroblastoid cell line as feeder layers on expansion of umbilical cord blood CD34(+) cells in vitro / 中国实验血液学杂志

To investigate the effects of human mesenchymal stem cells (MSC) and human fibroblastoid cell line (HFCL) as feeder layer on expansion of umbilical cord blood CD34(+) cells in vitro, (60)Co gamma-ray irradiated MSC and HFCL were used as feeder layer to expand cord blood CD34(+) cells in culture. The efficiencies of MSC and HFCL on expansion of CD34(+) cells in culture with or without cytokines were compared. The results showed that no matter whether cytokines (rhFL, rhSCF, rhTPO) were added, the proliferation of nucleated cells after expansion for 12 days in HFCL group was statistically higher than that in MSC group, i.e. with cytokines (9797 +/- 361)% vs (7061 +/- 418)%; without cytokines (5305 +/- 354)% vs (1992 +/- 247)%, when the cell numbers at day 0 was accounted as 100%), P < 0.01. The proliferation of propagated CD34(+) cells between MSC group and HFCL without addition of cytokines was not statistically different (820 +/- 191)% vs (825 +/- 305)%, P > 0.05. However, in the presence of cytokines, the propagating rate of MSC group was lower than that of HFCL group (939 +/- 212)% vs (1617 +/- 222)%, P < 0.01. MSC was better than HFCL in maintaining the LTC-IC of UCB CD34(+) cells, i.e. the number of CFU-GM colonies in the fifth week was (129.95 +/- 8.73) /10(5) seeded cells vs (89.81 +/- 10.29) colonies/10(5) cells, P < 0.05; with addition of cytokines, the effect was more obvious, i.e. the number of CFU-GM colonies in the fifth week (192.93 +/- 4.95)/10(5) seeded cells vs (90.47 +/- 14.28) colonies/10(5) seeded cells, P < 0.01. MSC mixed with a certain proportion of HFCL facilitated maintaining the LTC-IC of UCB CD34(+) cells. When the proportion was 4:1, the number of CFU-GM colonies was the highest (186.89 +/- 11.11)/10(5) seeded cells, which was higher than that of both 3:2 group [(138.92 +/- 14.84) colonies/10(5) seeded cells] and MSC only group, i.e. (64.63 +/- 6.11) colonies/10(5) seeded cells, both P < 0.01. It is concluded that HFCL is better than MSC in maintaining the expansion of CD34(+) cells and cytokines can enhance this effect, while MSC are stronger than HFCL in maintaining the LTC-IC of UCB CD34(+) cells in vitro. MSC with addition of a certain proportion of HFCL can significantly enhance the efficiency of CD34(+) cell expansion.