IFN-γ stimulation enhances immunosuppressive capability of human umbilical cord mesenchymal stem cells / 中国实验血液学杂志

This study was objective to explore the effect of IFN-γ on immunosuppressive capability of mesenchymal stem cells (MSC) derived from umbilical cord. The immunomodulating capability of MSC was changed by stimulating cell surface receptors like Toll-like receptors (TLR). The inhibition of T-lymphocyte proliferation by MSC was tested via cell co-cultures. Further RT-PCR and ELISA were performed to examine the expression changes in gene and protein level. The results showed that the IFN-γ could promote the immunosuppressive effect of umbilical cord derived MSC. IFN-γ-stimulated MSC could suppress the proliferation of T cells more effectively. IFN-γ stimulation up-regulated the expression of immunosuppressive genes like IDO1, COX2, HLA-G, and soluble suppressive proteins such as HLA-G, KYN, IL10, PGE2 of MSC. And the immuno suppression capability of IFN-γ-stimulated MSC was 2-7 folds higher than control in MSC and lymphocyte co-culture tests. It is concluded that IFN-γ can effectively enhance the immunosuppressive capability of MSC.

Sequence analysis of a novel human leukocyte antigen allele B*5827 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular basis for a novel human leukocyte antigen (HLA) allele B*5827.</p><p><b>METHODS</b>DNA from the proband was analyzed by polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) typing. The amplified product was sequenced bidirectionally.</p><p><b>RESULTS</b>Abnormal HLA-B locus was observed and its nucleotide sequence was different from the known HLA-B allele sequences, with highest homology to HLA-B*5820 allele. It differs from HLA-B*5820 by 8 nucleotide substitutions in exon 3, i.e., nt 290 (G > C), nt 346 (T > A), nt 390 (A > C), nt 404 (G > C), nt 413 (C > G), nt 471 (A > G), nt 486 (A > G) and nt 487 (C > A), resulting in an amino acid change from ser > arg at nt 97, phe >tyr at nt 115, ser > arg at nt 130, thr > ala at nt 157 and thr > glu at nt 162. Nucleotide differences of nt 404 (G > C) and nt 413( C > G) did not change amino acid.</p><p><b>CONCLUSION</b>The sequences of the novel allele have been submitted to GenBank (access No.GU071234). A novel HLA class I allele B*5827 has been officially assigned by the WHO HLA Nomenclature Committee in Jan. 2010.</p>

Study of single cell PCR for HLA typing / 中华血液学杂志

<p><b>OBJECTIVE</b>To apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.</p><p><b>METHODS</b>Single cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.</p><p><b>RESULTS</b>Enzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.</p><p><b>CONCLUSION</b>The modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.</p>

In vitro expansion and function of cord blood megakaryocyte / 中华儿科杂志

<p><b>OBJECTIVE</b>Cord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.</p><p><b>METHODS</b>CB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.</p><p><b>RESULTS</b>Different results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.</p><p><b>CONCLUSION</b>Ex vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.</p>

Differentiating ability of non-hematopoietic adult stem cells from rat fetal blood and bone marrow in vitro / 中国实验血液学杂志

To compare the growth characteristics of non-hematopoietic adult stem cells (NASC) derived from rat fetal blood and rat bone marrow in vitro, and to study the differentiation of these stem cells into neuron-like cells in vitro, the fetal blood of pregnant rats and bone marrow of adult rats were sterilely collected; mononuclear cells (MNC) were isolated by using standard Ficoll-hypague techniques and then cultured in DMEM/LG containing 10% fetal bovine serum (FBS). The acquired NASCs were subcultured for passage. The immunophenotype of NASCs was detected by flow cytometry. The expanded NASCs were induced to differentiate into neurons-like cells by beta-mercaptoethanol (beta-ME), dimethylsulfoxide (DMSO), butylated hydroxyanisole (BHA). The specific markers of these neuron-like cells were detected by immunocytochemistry. The results showed that two kinds of subcultured NASCs showed homogeneous spindle-shaped and expressed antigens CD44 and CD54, but did not expressed CD11b and CD45. The both induced cells were similar to neuron in morphology and were positive for nestin and neuron-specific enolase (NSE), but negative for glial fibrillary acidic protein (GFAP). It is concluded that no significant difference of NASCs derived from pregnant rat fetal blood and adult rat bone marrow found in cell morphology and biological characteristics. NASCs of both origins can be induced to differentiate into neuron-like cells, so fetal blood can be regarded as another resource of NASC.

Biological characteristics of rabbit bone marrow mesenchymal stem cells and their response to different growth factors / 中国实验血液学杂志

This study was aimed to analyze the biological characteristics of rabbit bone marrow mesenchymal stem cells (rBM-MSCs) and their response to different growth factors. Rabbit BM-MSCs were separated from bone marrow mononuclear cells by using adherent cultivation. Biological characteristics were investigated by optical and electron microscopy. Immunophenotype of rBM-MSCs was measured by flow cytometry. The expression of collagen was detected by RT-PCR. Differentiation potential was identified by specific staining and RT-PCR. The response of rBM-MSCs to IL-1, 3, 8 and HGF with different concentrations were tested by MTT. The results showed that the rBM-MSCs gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology and could be cultured for over 15 passages. CD44 was highly expressed on F5 rBM-MSCs (32%) and CD45 was lowly expressed (4.7%). Type I collagen was highly expressed, while type II collagen was lowly expressed and type X collagen was not detected on rBM-MSCs using RT-PCR method. In various conditions inducting differentiation, rBM-MSCs could differentiate into the osteoblast, chondrocyte, adipocyte and neuron-like cells. The rBM-MSCs were sensitive to IL-3, even low concentration (10 ng/ml) of IL-3 could promote the proliferation of rBM-MSCs effectively (>32%, P < 0.01), whereas high concentration IL-3 inhibited it significantly. It is concluded that rabbit BM-MSCs were successfully isolated and culture-expanded. The biological characteristics of rabbit BM-MSCs are similar to those of human and rhesus BM-MSCs. IL-3 with low concentration can promote the proliferation of rBM-MSCs effectively, but high concentration of IL-3 can inhibit their proliferation.

Double chimerism in recipient by transplantation of two allogeneic MHC-mismatched mouse fetal blood units / 中国实验血液学杂志

We have constituted a mouse model for fetal blood transplantation (FBT) to cross over major histocompatibility complex (MHC) without causing serious GVHD. It seems that full matching at the MHC appears not necessary for FBT, while the nucleated cell dose is critical. Two fetal blood units were combined from different donors to increase the stem/progenitor cell dose so as to explore the possibility of MHC-mismatched allogeneic transplantation. 26 out of 40 mice in mixed FBT group survived in the observation period of 60 days after transplantation without obvious GVHD. Double chimerism was demonstrated by PCR and flow cytometric analysis; and skin transplantation test proved the induction of donor specific immune tolerance. Our data suggest that two MHC-mismatched allogeneic donor fetal blood units could simultaneously engraft and reconstitute immune and hematopoietic system in a mouse model. The result may be beneficial for the expansion of cord blood application and enables more patients to share the advantages of cord blood transplantation.

Statistical analysis of 4000 umbilical cord blood units preserved in shandong cord blood bank / 中国实验血液学杂志

The current study analyzed the data of 4 000 umbilical cord blood (UCB) units collected in Shandong Cord Blood Bank from the end of 1999 to March 2001. The averages of nucleated cells and CD34(+) cells were more than 1.2 x 10(9) and 3.9 x 10(6) per UCB unit respectively, and more than 1.5 x 10(9) nucleated cells per UCB unit were obtained in 768 UCB units. These UCB units are suitable for transplantation in patients with a body weight greater than 40 kg. The analysis of HLA gene frequency showed that A2, A24, A11, B13, B51, DR15, DR7 and DR9 are the common halotypes in Shandong population and similar to those in the other areas of China. 40% patients could search out at least 1 UCB unit with 1 mismatched HLA locus in Shandong Cord Blood Bank.

Operating Procedure of Collection, Processing and Preservation of 3000 Units Umbilical Cord Blood in Shangdong Cord Blood Bank / 中国实验血液学杂志

The experience with the umbilical cord blood (UCB) stem cells for unrelated transplantation from our 3 000 UCB storage was described. UCB, collected from closed blood bags, were mixed with hydroxyethyl starch for nucleated cell (NC) enrichment. After finishing CD34 analysis, culture of hematopoietic progenitors (CFU-GM and CFU-GEMM) assays, microbial culture, HLA Class I (A, B) serology and class II (DR) low resolution SSP typing, cord blood units are stored in the liquid nitrogen for clinical applicatoin. Cord blood contained an average of nuclear cell (NC) (1.2 +/- 0.6) x 10(9), CD34(+) cells (3.0 +/- 3.7) x 10(6), CFU-GM (1.1 +/- 0.7) x 10(6) and CFU-GEMM (1.1 +/- 1.2) x 10(6) for storage and the recovery rates were 91%, 88%, 85% and 82%, respectively. The recovery rates for red blood cell and Hb were (39 +/- 9)% and (40 +/- 8)%, respectively. The storage volume was (35.1 +/- 7.1) ml in a 50 ml storage bags. The mean time from collection to processing of 15 hours (range 4 - 24 hours) had no influence on cell viability. The cell viability before processing is more than 95% and 92% after UCB thawing. The recovery rates of NC, CD34(+) cells and CFU-GM post-thawing were 96%, 90% and 91%, respectively. There were no HIV antibody (HIVAb) positive in all of UCB units. For an incidence of processed samples, infection with syphilis, HBsAg, HBcAb, HCVAb, CMV, bacterial contamination and abnormal hemoglobin were 0.1%, 0.8%, 3.2%, 0.2%, 87.1%, 1.2% and 0.1%, respectively. More than 3 HLA loci matched can be found for random patients in our cord blood bank and 6 HLA loci matched have 5%. For transplantation with nucleated cell counts of > 2.7 x 10(7) cells/kg, our cord blood bank will be able to provide all of the umbilical cord blood stem cell samples for children and 50% of units can be used for some of adult recipients transplantation in the country. It is concluded that: (1) The large cord blood banking for 20 000 UCB storage is feasible in China. (2) Our system of whole procedure and methods is functionable for supplying qualified cord blood units in transplantation. (3) The volume for collection is critical to the yield of CD34(+) cells or hematopoietic progenitor cells, however cord blood NC is also important and proportional with CD34(+) cells. Only the units containing more than 8 x 10(8) cells and more than 60 ml of cord blood can be in the procession for storage.

Quality Control in Umbilical Cord Blood Bank / 中国实验血液学杂志

Recent clinical reports have demonstrated that the use of umbilical cord blood (UCB) opened a new source of stem cell for hematopoietic stem cell transplantation, leading to the development of cord blood banks world-wide. Prior to the large scale construction of UCB banks, quality control must be performed for health care providers and manufactures. With increasingly stringent regulatory requirement in blood industry, quality control is playing an important role in the operation of blood centers and stem cell laboratories. Reviewed the lectures in the biology of UCB and UCB banks published in recent years, our experiences were discussed in setting up Shandong blood bank to define process variables associated with the collection of UCB, to determine and optimize the procedures and materials used, to ascertain how UCB can be processed in clean room as mononucleated cell preparations, and to analyze using of long-term storage of UCB in research and clinic in the future. Our conclusions are: (1) the establishment of UCB banks for use in transplantation appears to be easy, effective and particularly suitable approach in China under cGMP conditions; (2) the procedures for volume reduction by closed and semi-automated blood processing system, SSP HLA typing, biocode and local computer net, microbiological tests and the 50 ml cryobags for storage constitute a cost efficient system for large-scale UCB banking; (3) the average of 60 ml UCB collection may contain sufficent marrow repopulating cells for children and most of adult recipients; and (4) hematopoietic stem and progenitor cells in cord blood have a more potent proliferative ability than those derived from bone marrow in cell expansion potentials.