[Research Advance on Role of Umbilical Cord Mesenchymal Stem Cell in Hematopoietic Stem Cells Transplantation-Review].

Hematopoietic stem cell transplantation is an important method for the treatment of hematological malignancies. The hematopoietic recovery after transplantation, graft-versus-host disease (GVHD) prevention and treatment are the key to affect transplant success. Umbilical cord mesenchymal stem cells (hUC-MSC) can improve the hematopoietic microenvironment, promote hematopoietic stem cell homing and hematopoietic recovery, support hematopoiesis. In aduition, the hUC-MSC have immune function for a variety of immune cells, can avoid and alleviate the host immune response, have a certain effect on the treatment of GVHD. This article reviews the research advances on umbilical cord mesenchymal stem cells in hematopoietic stem cell transplantation.

[Curative effect of human umbilical cord mesenchymal stem cells for treatment of acute graft-versus-host disease of children after allo-HSCT].

This study was aimed to investigate the curative effect and safety of human umbilical cord mesenchymal stem cells (hUCMSC) to treat acute graft-versus-host disease (aGVHD) of children after hematopoietic stem cell transplantation (HSCT). HUCMSC were isolated and cultured by collagenase digestion and passage culture. The 3rd to the 5th passage of hUCMSC were used for clinical treatment. Five cases of children acute leukemia achieved complete remission after chemotherapy. Two cases received HLA 3/6 loci matched haploidentical bone marrow HSCT. One case received HLA-matched sibling bone marrow and peripheral blood HSCT. One case received unrelated HLA 4/6 loci matched umbilical cord blood HSCT. One case received unrelated HLA 5/6 loci matched umbilical cord blood HSCT. The children received immunosuppressive therapy after III-IV aGVHD occurring. They received 0.5×10(6)/kg hUCMSC infusion when conventional therapy was ineffective. The results showed that 5 cases of children acute leukemia achieved hematopoietic reconstitution and developed the III-IV grade aGVHD. The five cases of children were infused with hUCMSC. The rash subsided, the liver function was normalized and the gastrointestinal symptoms were improved. The infusion-related adverse reaction did not happen. At present, the 5 children are in remission. It is concluded that allogeneic HSCT is an effective therapeutic method for children with acute leukemia. HUCMSC infusion can be safely and effectively used for the treatment of refractory aGVHD.

[Ultrastructure of human umbilical cord mesenchymal stem cells].

The purpose of this study was to observe the ultrastructure of human umbilical cord mesenchymal stem cells (hUCMSC). hUCMSC from full-term newborn umbilical cord were isolated and cultured by collagenase digestion, and then subcultured, amplification, and cell morphology was observed by microscopy. The immunophenotype and trilineage differentiation potential of hUCMSCs at passage 3 were analyzed. Transmission electron microscopy and scanning electron microscopy were used to observe the ultrastructure of hUCMSC. The results indicated that appearance of hUCMSC was spindle-shaped and polygonal, and nuclei were observed. hUCMSC expressed immunophenotype CD44, CD73, CD105, did not express CD34, CD45, CD31 and human leukocyte antigen HLA-DR. hUCMSC were capable of adipogenic, osteogenic, and cartilage differentiation; the short and thick microvilli processes were seen at the surface of hUCMSC by scanning electron microscope. Two different cell morphologies of hUCMSC were seen under transmission electron microscope, the one was a quiescent period in which a large and round or oval nucleus only one nucleolus were seen, cytoplasmic organelles were less; the other was in a relatively active period in which one or two nuclei in the same one cell were observed, the organelles were rich, structure was clear, expansion of the mitochondria was visible. It is concluded that the cells successfully isolated and cultured from umbilical cord, which possess biological characteristics of MSC and display two different states of ultrastructure.

[Culture and pluripotentiality of murine compact bone-derived mesenchymal stem cells].

This study was purposed to culture murine compact bone-derived mesenchymal stem cell (MSC) and analyze the immunological and trilineage differentiation potential. Tibia and femur were extracted. Bone marrow cells were flushed out and compact bone fragments were digested with collagenase. The digested cells were cultured in 6-well plates. The immunophenotype, immunosuppressive function and trilineage differentiation potential were analysed by flow cytometry, mixed lympocyte reaction and Oil red O, von Kossa and alcian blue straining, respectively. The results indicated that the pure compact bone MSC could be isolated with in 3 weeks. The resulting MSC had trilineage differentiation potential and immunosuppressive effect on mixed lymphocyte reaction. The count per minute (CPM) value in control group of BALB/c T cells cocultured with irradiated C57BL/6 T cells was (2.56 ± 0.31) × 10(4), while CPM values of mixed lymphocyte cocultured with C57BL/6 compact bone MSC at ratios of 100:1 and 10:1 were (0.47 ± 0.12) × 10(4) and (0.28 ± 0.09) × 10(4). The CPM value of control group was higher than those of MSC cocultured group (P < 0.001). Compact bone-MSC had an immunosuppressive effect on mixed lymphocyte reaction in a dose dependent manner. It is concluded that murine compact bone has rich MSC and the primary MSC is contaminated with less hematopoietic cells. Murine compact bone-MSC have immunosuppressive effect on mixed lymphocyte reaction and trilineage differentiation potential. Compact bone-MSC have promising experimental study value.

[Comparison of the biological characteristics of serum-free and fetal bovine serum-contained medium cultured umbilical cord-derived mesenchymal stem cells].

OBJECTIVE: To compare the differences of biological characteristics between human umbilical cord-derived mesenchymal stem cells (UC-MSCs) cultured by serum-free medium or fetal bovine serum-contained complete medium to establish a xenogeneic protein-free UC-MSCs culture system. METHODS: Healthy human umbilical cord segments were digested with collagenase. UC-MSCs were cultured by serum-free MesenCult-XF medium and FBS-based αMEM complete medium, then analyzed the morphology, immunophenotype, expansion potential, lineage differentiation potential, karyotype and immunosuppression of early passages. RESULTS: The average cell diameters of UC-MSCs in suspension cultured by serum-free medium and FBS-based medium were 26 (18 - 39) µm and 35 (20 - 61) µm, respectively. Cell expansion folds with serum free medium and FBS-based medium were (5.2 ± 0.2) and (3.5 ± 0.1) respectively, in the first five passages. The expansion potential of serum-free medium cultured UC-MSCs was significantly higher than FBS-based medium cultured ones (P < 0.05). A panel of markers CD29, CD44, CD90, CD73, CD105 and HLA-ABC expressed on human UC-MSCs. Hematopoietic lineage markers CD34, CD45 and HLA-DR were not detectable on UC-MSCs. The cpm were (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured MSCs were added to the cultures at MSCs/T cell ratios of 1:100, 1:10 and 1:5. While the cpm was (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured UC-MSCs were added to the cultures. The immunosuppressive potential of serum-free medium-cultured UC-MSCs was higher than serum-contained medium cultured ones at three different MSC/T cell ratios (P < 0.05). CONCLUSION: Compare with serum-contained medium cultured early passages of UC-MSCs, the cell diameter of serum-free medium cultured UC-MSCs was smaller with higher expansion potential. No xenogeneic proteins were presented in UC-MSCs preparations when cultured with serum-free medium. Human UC-MSCs suppressed T-cell proliferation in a dose-dependent manner. The immunosuppressive potential of serum-free medium cultured UC-MSCs was higher than FBS-based medium cultured ones.

[Actions of keratinocyte growth factor in leukemic mice allogeneic umbilical cord blood cell transplantation].

OBJECTIVE: To explore the actions of keratinocyte growth factor (KGF) in leukemic mice allogeneic umbilical cord blood cell transplantation (UCBT) and elucidate its mechanism. METHODS: Peripheral blood drawn from the litters of C57BL/6 females was used as umbilical cord blood (UCB) graft. BALB/c mice were randomly divided into 7 groups (n = 12 each). The grouping was as follows. Control group 1, inoculated with leukemia. Control group 2, inoculated with leukemia at -4 d and total body irradiation (TBI) treatment. Control group 3, TBI treatment and reconstituted with 2 × 10(6) UCB-TNCs. Control group 4, injected with PBS subcutaneously, TBI treatment and reconstituted with UCB-TNCs with platelet transfusion. Control group 5, inoculated with leukemia, injected with PBS subcutaneously, TBI treatment and reconstituted with UCB-TNCs with platelet transfusion. Experiment group 1, injected with KGF subcutaneously, TBI treatment and reconstituted with UCB-TNCs with platelet transfusion. Experiment group 2, inoculated with leukemia, injected with KGF subcutaneously, TBI treatment and reconstituted with UCB-TNCs with platelet transfusion. The survival status, pathohistological changes, splenic lymphoid cell subsets and thymic output post-UCBT were compared between groups. RESULTS: The survival time of control group 1 was (11.1 ± 1.5) days and all died of leukemia. The survival time of control group 2 was (11.5 ± 2.5) days and all died of aplasia. Five of 12 mice of control group 3 survived for 100 days and 7 mice died of visceral hemorrhage. Four of 12 mice of control group 5 survived for 100 days and 8 mice died of leukemia with a survival rate of 33.3%. Nine of 12 mice of experiment group 2 survived for 100 days and 3 mice died of leukemia with a survival rate of 75.0%. The survival was prolonged in experiment group 2 mice as compared with that of control group 5 mice (χ² = 4.996, P = 0.0254). The splenic T, NK and B cell counts in control group 4 mice at +35 d were (9.32 ± 0.48) × 106, (1.59 ± 0.11) × 106 and (18.74 ± 2.01) × 106 respectively. While in group 6 mice at +35 d were (13.20 ± 1.14) × 106, (1.75 ± 0.12) × 106 and (20.36 ± 0.86) × 106 respectively. The counts of T cell and NK cell of group 6 were higher than those of group 4 (both P < 0.05). The level of signal joint T-Cell receptor excision circles (sjTRECs) in control group 4 mice was (167 ± 17) copies per 105 cells while that of experiment group 1 mice (228 ± 24) copies per 105 cells. They were higher than that of control mice (P = 0.002). CONCLUSION: Hematopoietic stem/precursor cells are abundant in full-term murine fetal peripheral blood. The infusion of KGF reduces the post-UCBT relapse of leukemia through the enhancement of thymic output.