Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits.     

3-羟基丁酸与3-羟基己酸共聚酯在心血管组织工程中的应用

为了研究可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯 (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)的血管内生物相容性, 采用脱细胞羊肺动脉为支架, 以PHBHHx涂层, 构建复合补片(Hybrid patch), 植入New Zealand兔腹主动脉内(12只), 以脱细胞未涂层羊肺动脉片(Uncoated patch)做为对照(12只)。分别于术后第1、4和12周处死动物, 取出移植补片进行组织学、免疫荧光染色、扫描电镜和钙含量测定。结果表明: hybrid patch管腔面光滑无血栓, 内膜增生适度, 再细胞化完全; 免疫荧光染色检测, 新生内膜组织中类内皮细胞呈CD31阳性反应, 单层连续排列, 间质细胞呈现SMA阳性反应; 钙含量测定, hybrid patch明显低于uncoated patch(P<0.05)。由此认为: PHBHHx的血管内生物相容性满意, 是心血管组织工程较为理想的腔内涂层材料。     

The human natural killer cytotoxic cell line NK-92, once armed with a murine CD16 receptor,represents a convenient cellular tool for the screening of mouse mAbs according to their ADCC potential

To take advantage of the large number of well-characterized mouse immunoglobulins (IgGs) for the study of antibody-dependent cell-mediated cytotoxicity (ADCC) in human cells, we armed human cytotoxic lymphocytes with a mouse receptor for the Fc portion of IgG antibodies. The human ΝΚ−92 natural killer cell line was transduced with a mouse receptor gene (mCD16), which was stably expressed on the cell surface (referred to as NK-92^mCD16). When tested against a B-lymphoblastoid cell line (BLCL) coated with mouse anti-CD20 IgG1, IgG2a or IgG2b monoclonal antibodies (mAbs), the newly expressed mouse Fc receptor enabled the NK-92^mCD16 cells to kill the BLCL by ADCC. Next, using the NK-92^mCD16 we compared mouse mAbs directed at B lineage specific CD antigens for their ability to induce ADCC against human Epstein-Barr virus- infected B lymphoblastoid (for anti-CD19, -CD20 and -CD21) or against myeloma (for anti-CD38 and –CD138) target cells. Our results demonstrated that the “NK-92^mCD16 assay” allows convenient and sensitive discrimination of mouse mAbs for their ability to mediate ADCC in a human cellular system. In addition, our results provide examples of dissociation between opsonization and target cell killing through ADCC. These “murinized” human effector cells thus represent a convenient cellular tool for the study of ADCC.     

3-羟基丁酸与3-羟基己酸共聚酯在心血管组织工程中的应用

为了研究可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯 (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)的血管内生物相容性, 采用脱细胞羊肺动脉为支架, 以PHBHHx涂层, 构建复合补片(Hybrid patch), 植入New Zealand兔腹主动脉内(12只), 以脱细胞未涂层羊肺动脉片(Uncoated patch)做为对照(12只)。分别于术后第1、4和12周处死动物, 取出移植补片进行组织学、免疫荧光染色、扫描电镜和钙含量测定。结果表明: hybrid patch管腔面光滑无血栓, 内膜增生适度, 再细胞化完全; 免疫荧光染色检测, 新生内膜组织中类内皮细胞呈CD31阳性反应, 单层连续排列, 间质细胞呈现SMA阳性反应; 钙含量测定, hybrid patch明显低于uncoated patch(P<0.05)。由此认为: PHBHHx的血管内生物相容性满意, 是心血管组织工程较为理想的腔内涂层材料。     

Third-party umbilical cord blood–derived regulatory T cells prevent xenogenic graft-versus-host disease

Background aimsNaturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1–5% of total nucleated cells in cord blood (CB) (<3 × 10⁶ cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed.MethodsSeveral methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model.ResultsWith the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⁺ cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo–expanded Treg were CD4⁺25⁺FOXP3⁺127^lo and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4⁺25⁻ cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro.ConclusionsIn our NOD-SCID IL-2Rγ^null xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo–expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.     

Enhanced MHC I antigen expression on tumour target cells is inversely correlated to lysis by allogenic but not by xenogenic NK cells

Relationship between the levels of MHC class 1 antigen expressed on tumour cells and their susceptibility to allogenic and xenogenic NK cells was investigated. Mouse and human natural killer-resistance inducing factor (NK-RIF) preparations were used for augmenting/inducing MHC 1 antigen expression on murine YAC and human K562 tumour cells, respectively YAC cells with augmented MHC I antigen expression became relatively resistant to lysis by murine NK cells but not to rat NK cells. Similarly, induction of MHC I antigens on K562 cells reduced their susceptibility to human NK cells but not to monkey NK cells. These results indicate that the inverse correlation of MHC I antigen expression and NK susceptibility does not hold true for xenogenic pairs of NK effector and target cells.     

Effects of human adipose-derived stem cells on the viability of rabbit random pattern flaps

Background aimsFlap necrosis is the most commonly encountered outcome influencing the effect of operations in clinical practice. The advent of cytotherapy and regenerative medicine with stem cells, especially adipose-derived stem cell therapy, appears to be a promising approach in providing multi-lineage differentiating cells. However, autologous stem cells are limited in both quantity and quality in aging individuals. Hence, xenogenic stem cell therapy was used in this study.MethodsRandom pattern flaps (6 cm × 2 cm) were prepared in a rabbit model transplanted either with 4 × 10⁵ human adipose-derived stem cells at five sites or equal volumes of Dulbecco's modified Eagle's medium. At 7 days after operation, the viability of the flaps from both groups was evaluated. We determined the numbers of locally infiltrating T cells, whereas the CD4/CD8 ratio, interferon, interleukin (IL)-2, IL-4 and IL-10 in the serum were determined to evaluate the immunological response of the rabbit. Moreover, Dil labeling was administrated to trace the homing of the transplanted stem cells.ResultsBoth the survival areas and the capillary number of the flaps that were injected with human adipose-derived stem cells significantly increased as compared with the control group (P < 0.05). Additionally, no significant difference in the immune response was detected between the groups. Dil-labeled stem cells were found to participate in the formation of tubular structures, which were further shown to be CD31+, although not predominantly.ConclusionsHuman adipose-derived stem cells could be used therapeutically to improve the viability of random pattern flaps without detection of serious immune rejection of stem cells.