High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

[Detection of hematopoietic chimera by real-time fluorescent quantitative PCR with erythrocyte Kidd blood group gene].

This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.

[Quantitative chimerism analysis of regulatory T cell subsets based on immunomagnetic sorting].

The aim of study was to explore the feasibility of quantitative chimerism analysis of regulatory T (Treg) cells using immune sorting coupling short tandem repeat (STR) method. 14 sets of artificial chimera samples were prepared by mixed lymphocytes according to different proportion. The CD4(+)CD25(+) Treg cells were harvested by negative and positive selection of immunomagnetic beads, then the STR polymorphisms of 16 loci in sorted Treg cells was analyzed. The results showed that the DNA amount extracted from sorted Treg cells was fit for STR detection. All STR alleles specific for recipient or donor could be detected and the quantitative results were consistent with theoretic values in over 10% recipient chimeras. But only partial recipient alleles could be detected and the quantitative results were different from theoretic values in less then 1% recipient chimeras. It is concluded that a quantitative chimerism analysis of Treg cell based on immune sorting is established. The sensitivity and accuracy for chimera detection are 1% to 10%, and this method can be used to monitoring hematopoietic chimerism following allogeneic hematopoietic stem cell transplantation.

[Probability of high resolution full match for human leukocyte antigen loci in unrelated donors and recipients with low resolution match].

This study was aimed to analyze the possibility of high resolution matching for human leukocyte antigen (HLA) loci in unrelated donor-recipient pair with low resolution match in HLA-A, -B, -DRB1 loci. Samples were genotyped for HLA-A, -B, -C, -DRB1 and -DQB1 by polymerase chain reaction sequence based typing (PCR-SBT). The results showed that the total number of patients and the donors were 166 and 274. 97 (58.43%) patients were matched for 1 donor and 47 (28.31%) patients were matched for 2 donors at low resolution level; among 274 donor-recipient pairs, HLA-A, -B, -C, -DRB1 and -DQB1 loci matching for 6/10, 7/10, 8/10, 9/10 and 10/10 were 32 (11.68%), 54 (19.71%), 62 (22.63%), 49 (17.88%) and 48 (17.52%) respectively; there were mismatch in HLA-A, -B, -C, -DRB1 and -DQB1 loci, and the most mismatch was in HLA-C locus. The number of alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci were 23, 46, 21, 30 and 17 respectively in the donors. The alleles number HLA-A, -B, -C, -DRB1 and -DQB1 loci were 20, 40, 22, 29 and 16 respectively in the patients; the haplotype number of HLA loci were 311 in the donors and 224 in the patients. The high frequency of haplotype was A*02:07-B*46:01-C*01:02-DRB1*09:01:02-DQB1*03:03 (5.63% and 6.88%). It is concluded that the probability of high resolution mismatch of HLA loci is high in unrelated donor-recipient pairs with low resolution match in HLA-A, -B, -DRB1 loci.

[Construction and identification of recombinant retroviral vector containing human homeobox gene HoxA10 and its stably packaged cell lines].

This study was purposed to construct the recombinant retroviral vector containing human homeobox gene hoxA10 and to establish the packaging cell lines which stably produce viruses. The whole coding region of hoxA10 gene was amplified by PCR and inserted into the retroviral vector MSCVneo. The recombinant vector was identified by DNA sequencing. The recombinant and control retroviral vectors were transfected into the packaging cell line PT67 by liposome Lipofectamine(TM) 2000. These cell lines stably producing retrovirus were isolated following G418 selection. The viral suspension was harvested and the viral titer was determined by NIH3T3 cells. The results showed that the recombinant retroviral vector was proved to encode hoxA10 genes by sequencing. The cell lines efficiently producing virus were screened by G418 and designated as PT67/MSCVneo and PT67/MSCVneo-hoxA10. The titers of them were 5 x 10(5) CFU/ml and 4 x 10(4) CFU/ml respectively. It is concluded that the recombinant retroviral vector containing homeobox gene hoxA10 and the stably packaging cell lines which efficiently and correctly produce viruses are successfully constructed, which provides a basis for further exploration of the hoxA10 gene function in the regulation of hematopoiesis.

[Molecular genetic analysis of rare cisAB variants in Chinese population.].

We investigated the molecular genetic basis of rare cisAB variants at the ABO locus in Chinese population. Serological techniques were performed to characterize erythrocyte phenotype of 12 discrepant samples and 116 randomly selected samples. Mutations of complete exon 6 and 7 including flanking intron in the ABO genes were screened by PCR and directly sequencing. The haplotypes of diverse genotypes were also analyzed by cloning sequencing. Twelve samples were identified as AweakB or ABweak phenotype by serological technology, and were also identified as cisAB variants including four genotypes by directly sequencing. Two cisAB alleles were found by haplotype sequencing. One allele was cisAB01, in which four nucleotide acid alterations were observed (467C>T and 803G>C in exon 7, 163T>C and 179C>T in intron 6); the other allele maintained a nucleotide acid of A101 allele (803G) compared with B101 allele, which resulted in a polypep-tide containing 176G, 235S, 266M, and 268G four amino acids. This is a novel allele, which has been named cisAB05 by Blood Group Antigen Gene Mutation Database. According to systematically investigation of the molecular genetic basis of the cisAB variants in Chinese population, we found a novel cisAB05 allele and presumed that the cisAB01 allele is derived from homologues exchange of A101-B101 combination.

[Identification of the hemolytic transfusion reaction caused by lewis antibody using serological and molecular biological methods].

To analyse the reason for one case of hemolytic transfusion reaction, antibodies in a patient's serum were identified using panel cells and Le (a-b-) phenotype cells, patient phenotype was identified by using anti-Le(a) and anti-Le(b) blood grouping reagents and the entire coding region of FUT3 gene was amplified by PCR and sequenced directly. The results showed that both IgM anti-Le(a) and anti-Le(b) antibodies were detected in patient's serum. Red cells was typed as Le (a-b-) phenotype and the FUT3 genotype was homozygote for non-functional le(59, 508) alleles. In conclusion, anti-Le(b) antibody can result in hemolytic transfusion reaction, FUT3 gene is homozygous for le(59, 508) allele resulting in Le (a-b-) phenotype.

[Effect of mesenchymal stem/progenitor cells on differentiation of cord blood CD34+ cells towards megakaryocytes].

AIM: In order to investigate the effect of mesenchymal stem/progenitor cells on proliferation and differentiation towards megakaryocytes of CD34+ cells from human umbilical cord blood in vitro. METHODS: After mesenchymal stem/progenitor cells were advancely planted in DMEM medium and grown up to 80%, then the CD34+ cells were added to culture with mesenchymal stem/ progenitor cells or without mesenchymal stem/progenitor cells in DMEM for 14 days with TPO + IL-3 + SCF, TPO + IL-3 + SCF + IL-11 respectively. After cultured for 14 days, mononuclear cells (MNCs) were counted by automatic cell analyzer. The number of CD41+ cells and platelets were detected by flow cytometry. Platelets function were assessed through platelet aggregation test which was induced by thrombin. RESULTS: As compared with the control group, the number of MNCs of co-culture system was not increased significantly (P > 0.05), but the number of CD4+ cells and platelets were increased significantly (P < 0.05). The platelets were aggregated by thrombin induced which could be seen in microscope or flow cytometry. CONCLUSION: It is concluded that mesenchymal stem/progenitor cells may be promoted to induce the cord blood CD34+ cells to differentiate towards megakaryocyte in the culture medium.

[Human cord blood adult stem cells differentiate into hepatocyte-like cells in vitro].

The aim of this study was to investigate the feasibility of the human cord blood adult stem cells (ASCs) to differentiate into hepatocytes in vitro induced by combined stimulation with hepatocyte growth factor (HGF), stem cell factor (SCF) and leukemia inhibitory factor (LIF). The adult stem cells were obtained through density gradient centrifugation and magnetic activated cell sorting (MACS). The adult stem cells were cultured in DMEM with HGF (10 ng/ml)+SCF (10 ng/ml)+LIF (10 ng/ml) in induced group I. In induced group II the enriched cells were cultured in DMEM with SCF (10 ng/ml)+LIF (10 ng/ml) and the undifferentiated cells acted as the control group without the factors. The morphology of cells was observed by the inverted phase contrast microscopy; the expression of albumin (Alb), human hepatocyte cytokeratin (CK18) and alpha-fetoprotein (AFP) were detected by immunofluorescence, immunohistochemistry and RT-PCR assay in the 21-day culture. Alb secreted by hepatocytes in the medium was determined by radioimmunoassay (RIA) at day 7, 14, 21, 23 and 25. The results showed that the shapes of ASCs changed and their sizes and number increased in the course of culture in group I. After being induced for three weeks, the cells turned round and resembled hepatocyte-like cells. The mRNA for Alb could be detected by RT-PCR in the differentiated adult stem cells in group I, and the mRNA for AFP was poorly detected by RT-PCR at day 21. Alb and CK18 were positive through immunofluorescence and immunohistochemistry at day 21, compared with group II and the control group. In group I, Alb in the medium significantly increased, compared with control group, and reached the highest level at day 21, then decreased at day 23. It is concluded that under some definite inducing conditions, human cord blood adult stem cells can differentiate into hepatocyte-like cells and HGF plays a critical role during the course.

[Analysis of natural killer cell immunoglobulin-like receptor gene family in HLA-identical sibling].

The aim of study was to analyze natural killer immunoglobulin (Ig)-like receptor (KIR) gene content in HLA-identical sibling and to investigate the possibility of their KIR match. Samples were genotyped for HLA by Luminex method and polymerase chain reaction sequence based typing, the KIR gene was detected by polymerase chain reaction sequence-specific primers. The results showed that 17 KIR genes could be observed in the 27 pairs HLA-A, -B, -Cw and -DRB1 locus identical sibling samples. All individuals contained KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2.; 20 different KIR genotypes and 12 haplotypes have been found, the most common KIR genotypes was 2,2 with frequency 29.6% and KIR haplotype was 2 with frequency 53.0%. The A KIR haplotype was the most prevalent with frequency 67.2%; 12 pairs (44.4%) HLA identical sibling donor-recipients showed KIR match in genotype and haplotype, 13 pairs (48.1%) with one KIR haplotype mismatch and 2 pairs (7.4%) with two KIR haplotype mismatch; 1 pair was matched between donor KIR2DL1 and patient HLA-Cw (Lys80) ligand, 17 pairs were matched between KIR2DL2/KIR2DL3 and HLA-Cw (Asn80) ligand, 5 pairs were matched between KIR3DL1 and HLA-Bw4 ligand. It is suggested that the probability of KIR mismatch is high in HLA-identical sibling.

A multi-Chinese blood center study testing serologic-negative donor samples for hepatitis C virus and human immunodeficiency virus with nucleic acid testing.

BACKGROUND: A multi-blood center study was conducted to evaluate a human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) multiplex nucleic acid testing (NAT) donor screening test and to determine the residual risk for HIV-1 and HCV infection. STUDY DESIGN AND METHODS: A commercially available HIV-1 and HCV assay (Procleix, Chiron Corp.) was used for simultaneous detection of HIV-1 RNA and HCV RNA on 89,647 unlinked donor samples. NAT was performed with pools of 16 samples that had passed all routine screening tests. Single-donor NAT was performed for samples that had been disqualified by any reactive screening test result(s). Anti-HCV (Ortho third-generation HCV enzyme immunoassay [EIA]), alanine aminotransferase, and HCV NAT (Roche COBAS Amplicor HCV test) confirmatory tests were used for HCV EIA-nonreactive, HCV NAT-reactive samples. RESULTS: Three HCV NAT yield cases and no HIV-1 yield cases were detected. The yield rate for HCV NAT was 3.4 per 10(5) (95 percent confidence interval [CI], 0.7-9.8). The estimated incidence rate for HCV is 24.2 per 100,000 person-years (95% CI, 3.4-88.0). If minipool NAT is added to routine donor screening, the residual risk for HCV is estimated to be reduced to 1 in 20.4x10(4) (95% CI, 1 in 5.2x10(4)-1 in 165.5x10(4)). CONCLUSION: The residual risk for transfusion-transmitted HCV infection is still relatively high in China. Incorporating NAT technology into blood donor screening would be estimated to reduce the residual risk of HCV infections eightfold over current EIA screening.

[The genetic polymorphism of cytokine genes in Zhejiang Han individuals].

In order to investigate the genetic polymorphism of 13 cytokines genes in Han individuals from Zhejiang. The polymerase chain reaction-sequence specific primers (PCR-SSP) method was used to determine genetic polymorphisms in 100 unrelated healthy Han individuals from Zhejiang province. We found that: (1) The frequency of the TGFbeta (G codon 25) allele is 1.00 and no TGFbeta (C codon 25) allele is found. (2) The frequencies of IL-1alpha(C-889), IL-1beta(C +3962), IL-4 (T-1098), IL-6 (G-174), IL-6 (G nt565), IL-10 (A-1082) and TNFalpha (G-238) alleles are more than 0.95; while the frequencies of IL-1alpha (T-889), IL-1alpha (T +3962), IL-4 (G-1098), IL-6 (A-174), IL-6 (A nt565), IL-10 (G-1082), TNFalpha (A-238) alleles are less than 0.05. (3) The haplotype frequency of the high-producer-type GCC in IL-10 gene is 0.05, while the low-producer-type ATA is 0.735. The haplotype frequency of the high-producer-type TG in the TGFbeta gene is 0.49, while the low-producer-type CC is 0. The haplotype frequency of the high-producer-types AG and AA in the TNFalpha gene is 0.055, while of the low-producer-types GG and GA is 0.945. The results showed that the genetic polymorphism of cytokine genes in Han individuals from Zhejiang Province is distinctive.

[Formation of platelets from cord blood CD34+ cells-derived megakaryocytes induced by S-nitrosoglutathione].

To investigate the effect of S-nitrosoglutathione (GSNO), a nitric oxide donor, on platelet production from megakaryocytes differentiated from cord blood CD34(+) cells in vitro, the CD34 (+) cells from eight fresh umbilical cord blood samples by a high-gradient magnetic cell sorting (MACS) system were cultured in serum-free medium for 14 d with thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and rHuGM-CSF 20 ng/ml. Then, CD61 (+) cells were purified by MACS system from these CD34 (+) cells, and were cultured in serum-free medium supplemented with TPO 50 ng/ml, IL-3 10 ng/ml and SCF 50 ng/ml in the presence (treatment group) and absence (control group) of GSNO for 30 min or 2 h. Platelet-sized particles were counted by flow cytometry; megakaryocyte structure was detected by scanning electron microscope. Aggregation of the thrombin-induced platelet particle was observed under inversion microscope. cGMP was assessed by commercial ELISA kit. The results showed that, compared with the control group, the number of platelet-sized particles significantly increased (P<0.05) in the treatment group, in which megakaryocytes presented significant pseudopod formation and extensive membrane blebbing. The platelet particle aggregation could be observed under microscope after thrombin induction. cGMP activity was significantly increased after treatment with GSNO (P<0.05). These results propose that GSNO can facilitate platelet production from megakaryocyte, and it may be partly through cGMP pathway.

[Effect of angiotensin II on differentiation of umbilical cord blood CD34+ cells into megakaryocytes].

This study was aimed to investigate the effect of angiotensin II on differentiation of cord blood CD34+ cells into megakaryocytes in vitro. The CD34+ cells from eight fresh umbilical cord blood samples sorted by a high-gradient magnetic cell sorting system (MACS) were cultured in serum-free culture medium containing thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and different concentrations of angiotensin II (0, 50, 100, 1000 microg/ml) for 14 days. Mononuclear cells (MNC) were counted by automatic cell analyzer. Cultured CD41+ cell and platelet counts in cultured system, and cell cycle were analyzed by flow cytometry. CD41 specific monoclonal antibody staining was observed by immunofluorescence microscopy. The results showed that as compared with the control group, the number of MNC not increased significantly (P > 0.05), but the number of CD41+ cells and platelets increased significantly in treatment group (P < 0.05). Cell cycle analysis revealed that the amounts of 4N cells increased and apoptosis cells obviously existed in treatment group (P < 0.05). After fluorescence staining, more CD41+ cells of different sizes were observed by means of fluorescence microscopy in both groups. It is concluded that angiotensin II can induce the cord blood CD34+ cells to differentiate towards megakaryocyte, and enhance the function of megakaryocyte to produce platelet.

[FUT3 gene polymorphism associated with Lewis blood group in Chinese Zhejiang population].

To investigate the alpha-1, 3/4-fucosyltransferase gene (FUT3) polymorphism associated with Lewis blood group in Zhejiang population, the Lewis phenotypes of 183 random samples from Chinese blood donors in Zhejiang province were identified by standard serological techniques. The entire coding region of FUT3 gene were amplified by PCR from genomic DNA of 39 Lewis negative and 9 Lewis positive phenotype samples and sequenced directly. The haplotypes of FUT3 allele were identified by TOPO cloning sequencing method. The results showed that the frequency of true Le (a-b-) phenotype in Zhejiang population was 10.4% according to serological and molecular biological methods. Five nucleotide acid variant sites (59T > G, 202T > C, 314C > T, 508G > A and 1067T > A) were detected in all 48 sequencing samples. Besides the wild type Le allele, 2 common (le(59, 1067) and le(59, 508) and 3 rare non-functional le alleles (le(59), le(1067) and le(202, 314) were found in this population. In conclusion, the polymorphism of non-functional FUT3 allele was found to be relatively variable in Chinese Zhejiang population.

[Establishment of delta block matching technique].

To establish delta block HLA-matching technique, DNA was extracted from whole blood by salting-out method, delta block was amplified by polymerase chain reaction (PCR), and PCR product was detected by GeneScan. The results showed that delta block had polymorphism in 104 samples without sibship of the Han people from Zhejiang province. The range of DNA fragment length was 81-393 bp and could be divided into 4 groups: 81-118 bp, 140-175 bp, 217-301 bp, 340-393 bp. The numbers of DNA fragments were 6-32. It is concluded that the method of delta block matching is reliable and can be applied to select donors for the patients to be transplanted. It is the first time to get delta block data of the Han people in China.

[Establishment of beta block matching technique].

The purpose of this study was to establish beta block matching technique. DNA was extracted from whole blood by salting-out method, beta block matching was performed by PCR and GeneScan technique. The results showed that the length of fragments amplificated in 100 samples was different and the range of them was 91-197 bp. Amplification fragments could be divided into four regions: 91-93, 105-113, 125-139 and 177-197 bp respectively. 91 bp DNA fragments could be found in all of samples. The numbers of DNA fragments with different length have been shown high polymorphism and they focused on the range of seven to twenty four. In conclusion, the beta block matching technique is reliable and applicable to the selection of hematopoietic stem cell transplantation donors.

Significance of the signal-to-cutoff ratios of anti-hepatitis C virus enzyme immunoassays in screening of Chinese blood donors.

BACKGROUND: The correlation between signal-to-cutoff (S/CO) ratios of a second-generation hepatitis C virus (HCV) enzyme immunoassay (EIA; Abbott) and a third-generation HCV enzyme-linked immunosorbent assay (ELISA; Ortho) and confirmed HCV infection has been reported. The utility of the values for the Chinese anti-HCV EIA kits, however, has not been studied in evaluating test results in Chinese blood donors. STUDY DESIGN AND METHODS: A total of 156 donor samples repeat reactive for anti-HCV at routine screening from five representative regions of China were retested for anti-HCV by the Ortho third-generation HCV ELISA and six Chinese EIA kits and for HCV RNA by a human immunodeficiency virus-1 and HCV assay (Procleix, Chiron Corp.). The HCV RNA-nonreactive samples were further tested for anti-HCV by a third-generation recombinant immunoblot assay RIBA (Chiron Corp.). The positive result by either nucleic acid amplification test or RIBA was interpreted as confirmed HCV infection. RESULTS: The confirmed HCV prevalence rate in donors in five representative regions obtained in this study was 0.20 percent (77/37,900) in 2004. All seven anti-HCV EIA kits had a significant correlation between S/CO ratios and confirmed HCV infection. The threshold S/CO ratios, which predicted more than 95 percent of confirmed HCV infections for the Ortho, SABC, BGI-GBI, InTec, GWK, KHB, and WANTAI kits, were 3.8, 6.0, 7.0, 8.6, 10.0, 10.0, and 14.0, respectively. CONCLUSIONS: Anti-HCV EIA kits commonly used in Chinese donors screening demonstrate good correlation between S/CO ratios and the confirmed infection. For the Ortho third-generation HCV ELISA, the S/CO ratio of 3.8 determined by the US Centers for Disease Control and Prevention is applicable to Chinese blood donors. The Chinese domestic EIA kits evaluated show a diverse range of threshold S/CO ratios.

Processing and cryopreservation for 1963 units of human umbilical cord blood.

The study was aimed to establish a standard procedure for human umbilical cord blood bank. The hematopoietic nucleated cells in cord blood were processed by using sedimentation and centrifugation method. After finishing CD34(+) cell counting, hematopoietic progenitor cell assay, microbial culture, infectious disease test and HLA typing, cord blood units were stored in the liquid nitrogen for further application. The results showed that nucleated cells of cord blood were (10.94 +/- 2.74) x 10(8) per unit; recovery rate of nucleated cells was (79.82 +/- 17.76)%. CD34(+) cells in cord blood were counted as (51.62 +/- 30.53) x 10(5) per unit. Eight units of cord blood were thawed after two years of cryopreservation, the recovery rate of nucleated cells, CD34(+) cells and CFU-GM were (91.4 +/- 6.0)%, (84.6 +/- 20.0)% and (85.8 +/- 14.9)% respectively. It is suggested that the methods and procedure reported for processing and cryopreservation of hematopoietic stem/progenitor cells in the human umbilical cord blood is effective.

[Effect of GM-CSF on expansion and differentiation of CD34+ megakaryocyte progenitor cells from cord blood in vitro].

To study the effect of GM-CSF on in vitro expansion of megakaryocyte progenitor cells from cord blood, CD34(+) cells isolated by magnetic cell sorting system (MACS) were cultured in serum-free medium containing TPO, IL-3, SCF and with or without various concentrations of GM-CSF (5, 20, 100 ng/ml). The numbers of MNC, proportion of CD34(+)CD41(+) cells and CFU-MK were measured at 6, 10 and 14 days. The results showed that the expansion of MNC and proportion of CD41(+) cells was accelerated distinctly by various concentrations of GM-CSF after 14 days, while 20 and 100 ng/ml GM-CSF exhibited higher expansion effect than that of 5 ng/ml. TPO + IL-3 + SCF with 5 ng/ml or 20 ng/ml GM-CSF could stimulate the formation of CFU-MK, while TPO + IL-3 + SCF with 100 ng/ml GM-CSF could inhibit it. It is concluded that GM-CSF can accelerate the expansion of megakaryocyte progenitor cells from CD34(+) cells in cord blood in the serum-free medium containing TPO + IL-3 + SCF.