Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform.</p><p><b>METHODS</b>A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility.</p><p><b>RESULTS</b>Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform.</p><p><b>CONCLUSIONS</b>We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies.</p>

Detecting the spectrum of multigene mutations in non-small cell lung cancer by Snapshot assay / 癌症

As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.

Impact of smoking status and pathologic type on epidermal growth factor receptor mutations in lung cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Epidermal growth factor receptor (EGFR) mutations in lung carcinomas can make the disease more responsive to the treatment with tyrosine kinase inhibitors. We aimed to evaluate the prevalence of EGFR mutations in a large series of lung carcinomas.</p><p><b>METHODS</b>We examined 1195 consecutive lung cancer patients for EGFR mutations in exons 18, 19, and 21 using direct sequencing of polymerase chain reaction products. A detailed smoking history was obtained. Patients were categorized as never smokers (< 100 lifetime cigarettes), former smokers (quit > 1 year ago), or current smokers (quit < 1 year ago).</p><p><b>RESULTS</b>There were EGFR mutations in 9 (4.5%) of 201 squamous carcinomas, in 1 (2%) of 50 large cell carcinomas, and in 1 (2.3%) of 44 small cell carcinomas that were investigated. Three hundred and twenty-seven mutations were found in the series of 858 adenocarcinomas (38.1%). Among 858 lung adenocarcinomas, we detected EGFR mutations in 250 (48.6%) of 514 never smokers, 39 (33.9%) of 115 former smokers, and 38 (16.6%) of 229 current smokers. Significantly fewer EGFR mutations were found in people who smoked for more than 15 pack-years (P = 0.0002) or stopped smoking less than 15 years ago (P = 0.033) compared with individuals who never smoked.</p><p><b>CONCLUSIONS</b>Adenocarcinoma is the most frequent EGFR mutation pathologic type in lung cancer. The likelihood of EGFR mutations in exons 18, 19 and 21 decreases as the number of pack-years increases. Mutations were less common in people who smoked for more than 15 pack-years or who stopped smoking cigarettes less than 15 years ago. These data can assist clinicians in assessing the likelihood of exons 18, 19, or 21 EGFR mutations in Chinese patients with lung cancer when mutational analysis is not feasible.</p>

Mutation of plakophilin-2 gene in arrhythmogenic right ventricular cardiomyopathy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Arrhythmogenic right ventricular cardiomyopathy (ARVC) is one of the leading causes of sudden cardiac death. Recent studies have shown that ARVC, which is an inheritable genetic change, results from mutations in genes encoding desmosomal proteins. Plakophilin-2 is an important component of the desmosome. Because the full range of genetic variations related to ARVC is unknown and no related studies of the Chinese population have been reported, we aimed to investigate the genetic variation of plakophilin-2 in ARVC patients from the Southern Region of China.</p><p><b>METHODS</b>Genomic DNA was isolated from peripheral blood samples of all 34 ARVC patients, who were screened through a clinical evaluation. They were used to detect variations in the sequences of the plakophilin-2 genes by polymerase chain reaction amplification in combination with direct sequencing.</p><p><b>RESULTS</b>In exon-1 of the plakophilin-2 gene, a deletion mutation (c.145_148 del GACA) was found in one family pedigree. The mutation was also found in exon-2, 4, and 11 of the plakophilin-2 gene. The QT interval dispersion of the ECG was considerably longer in the mutation group than in the non-mutation group of ARVC patients, and this result was statistically significant (P < 0.05).</p><p><b>CONCLUSION</b>We discovered a plakophilin-2 mutation that prolongs the QT interval dispersion in the southern Chinese ARVC population.</p>

Effect of intra-bone marrow infusion of allogeneic mesenchymal stem cells on reconstruction of marrow mesenchymal stem cells in rat HSCT models / 中国实验血液学杂志

This study was aimed to investigate the effect of intra-bone marrow (IBM) injection of allogeneic mesenchymal stem cells (MSCs) on reconstruction of bone marrow MSCs (BM-MSCs) in rats that received hematopoietic stem cell transplantation (HSCT), and to detect the donor MSCs in the hosts for clarifying the effect mechanism of donor MSCs. Wistar female rats conditioned with lethal dose 60Co gamma-ray irradiation were co-transplanted with F344 female fetal and neonatal peripheral blood (FNPB) and BrdU-labeled MSCs separated from bone marrow mononuclear cells of F344 male rats. The donor MSCs were infused by IBM injection in bilateral tibia or intravenous injection (IV), while the FNPB were all via IBM route. The survival rate, engraftment level of HSCs and recovery of BM-MSCs of recipients were monitored. The ratio of BrdU-labeled MSCs in recipient rats was calculated by immunofluorescence assay (IFA) and the Y chromosomes were examined by PCR. The results showed that the recipient rats of the two co-transplantation groups were all alive at day 60 after transplantation. There was no significant difference between these two groups on the survival rates or the engraftment levels of HSCs, but each of them was much better than that of the FNPB group. At day 30 after transplantation, the proliferation ability of recipients' BM-MSCs was still below normal, while that of the FNPB (IBM)+MSC (IBM) group was the best of all the experiment groups (p<0.01). At 60 days, the donor MSCs coexisted with host MSCs in only a few recipient rats examined by IFA, while the Y chromosomes could be detected in all the recipient rats in the two cotransplantation groups. It is concluded that the infusion of allogeneic MSCs can accelerate the recovery of HSCT recipients' BM-MSCs. The IBM route is safe and more effective than intravenous infusion.

The cytotoxicity of CIK/NK cells stimulated by K562-DC fusion vaccines in NOD/SCID mice model for human erythroleukemia / 中华血液学杂志

<p><b>OBJECTIVES</b>To study the in vivo efficacy and the safety of cord blood derived CIK/NK cells stimulated by K562-dendritic cells (DC) fusion vaccines in NOD/SCID mice model for human erythroleukemia.</p><p><b>METHODS</b>DC and CIK /NK cells were both derived from cord blood mononuclear cells. DC were fused with inactivated K562 leukemia cell by PEG to produce K562-DC fusion vaccines. K562-DC fusion vaccines were co-cultured with CIK/NK cells to prepare K562-DC fusion vaccine stimulated CIK/NK cells. NOD/SCID mice were inoculated with 1 x 10(6) K562 cells. 24 hours later, 1 x 10(7) vaccines stimulated CIK/ NK cells and 1 x 10(7) CIK/NK cells were transfused into the NOD/SCID mice. NOD/SCID mice without inoculation of K562 cells were used as control group. CD13 and CD56 positive cells were assayed by flow cytometry.</p><p><b>RESULTS</b>All the leukemia NOD/SCID mice without therapy died within 39 days, tumor was found in 5 of 8 mice. One of 8 leukemia mice treated with K562-DC fusion vaccines stimulated CIK/NK cells died at the 65th day, the anti-tumor response rate was 87.5%. Two of the leukemia mice treated with CIK/NK cells died at the 56th and 65th day respectively, the anti-tumor response rate was 75%. There was no significant difference in survival time between these two groups, and both survivals were longer than that of the control group. There was no significant difference in CD13 positive cells in the survival mice between these two groups, and both of that were less than that of the control mice. There was no significant difference in CD56 positive cells between the two treated groups and the control group.</p><p><b>CONCLUSIONS</b>Cord blood derived CIK/ NK cells stimulated by inactivated tumor cells retain the cytotoxicity and do not develop tumor in vivo.</p>

Isolation and culture of human embryonic AGM derived HSPCs in hematopoietic culture systems created by AGM stromal cells / 中国实验血液学杂志

This study was purposed to isolate human embryonic AGM derived HSPCs and investigate the effect of AGM stromal cells on AGM-derived HSPCs. Immunohistochemical sections of human AGM tissue were investigated for CD34, Flk-1 and VEGF expression. Human AGM-derived single cells were isolated and seeded onto pre-treated feeder of human AGM stromal cells (hAGMS3 and hAGMS4) by direct contact and non-contact co-culture in Transwell culture system. Growth characteristics of HSPCs with cobblestone area-forming cells (CAFCs) were observed and number of cobblestone area (CA) was counted. Indirect immunofluorescent assay was used to detect CD34 and Flk-1 expression on the surface of suspended cells as well as CAFCs in contact co-culture system. The cells after culture for 2 weeks were collected from both contact and non-contact co-culture systems for CFU assay. The result showed that hematopoietic cells in AGM tissue expressed CD34 and Flk-1. Both of the hematopoietic culture systems could produce CFCs. Nevertheless, direct contact co-culture produced CD34(+)Flk-1(+) CAFC and more CFUs than those from indirect non-contact culture (hAGMS3 system: 1647 +/- 194 vs 389 +/- 31, p < 0.05; hAGMS4 system: 1586 +/- 75 vs 432 +/- 35, p < 0.05). It is concluded that there were CD34(+)Flk-1(+) HSCs in human embryonic AGM region. The hematopoietic co-culture systems composed of AGM-derived HSPCs and AGM stromal cells are successfully established, both direct contact and Transwell non-contact co-culture can expand AGM-derived definitive HSPCs. Cell-cell contact between AGM-derived HSPCs and AGM stromal cells are of most importance to maintain and expand AGM-HSPCs.

BMP-4 and VEGF promote development of hematopoietic stem cells during the embryoid body formation from embryonic stem cells / 中国实验血液学杂志

The objective of this study was to explore the effects of BMP-4 and VEGF on the development of primary hematopoietic stem cells during the differentiation of embryonic stem cells (ESCs) into embryoid body (EB). Murine E14 ESCs were seeded into semisolid methylcellulose-based medium for EB formation. According to added or not cytokines, experiments were divided into: (1) group of spontaneous differentiation without cytokine as control; (2) group of BMP-4 in different concentrations (0, 5, 15, 25 and 50 ng/ml); (3) group of BMP-4 combined with VEGF; (4) group of VEGF alone. EBs were collected on days 3, 6, 9, 12, 15, and the proportion of Flk-1(+) cells were assayed by flow cytometry. The results showed that in the different BMP-4 concentration groups, the proportions of Flk-1(+) cells were significantly different, and it reached the peak values in 25 ng/ml BMP-4 group as 6.51 +/- 1.02% at day 3 and 7.70 +/- 1.12% at day 6 respectively, which were statistically higher than those in control group without-BMP-4 and in 5 ng/ml BMP-4 group (p < 0.05). When BMP-4 was used in combination with VEGF, Flk-1(+) cells went to peak proportion value at day 9 as 27.53 +/- 8.14%, which was statistically higher than that in spontaneous differentiation group as 8.77 +/- 2.35% (p < 0.05) and VEGF treatment group as 11.21 +/- 2.23% (p < 0.05). It is concluded that BMP-4 in combination with VEGF can promote Flk-1(+) cells genesis during EB formation in vitro, which provides experimental evidence for researches on directed differentiation of ESCs into hematopoietic stem cells simulating the microenvironment in vivo.

Plasmid-mediated miRNA-1-2 specifically inhibits Hand2 protein expression in H9C2 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector for miRNA-1-2 that can be expressed in rat H9C2 cardiomyocytes.</p><p><b>METHODS</b>The precursor miRNA (pre-miRNA) DNA template for miRNA-1-2 was designed and generated by PCR amplification. The DNA template was inserted into the hairpin RNA expression vector pSilence-4.1-neo and identified by DNA sequencing analysis. The recombinant plasmid DNA was then transfected into H9C2 cells via Lipofectamine, and the green fluorescence protein expression vector pEGFP-N3 served as the transfection marker. Twenty-four hours after transfection, the total cellular RNA was extracted using TRIzol reagent, and thermoscript reverse transcriptase (RT)-PCR was performed to determine miRNA-1-2 precursor expression.</p><p><b>RESULTS AND CONCLUSION</b>DNA sequencing indicated that the miR-1-2 expression plasmid was correctly constructed. The precursor miRNA-1-2 was successfully expressed in the H9C2 cells, and the expression of Hand2 protein could be efficiently inhibited by miRNA-1.</p>

Experiment study of efficacy on hematopoietic reconstitution and GVHD prophylaxis after mesenchymal cell infused by intra-bone marrow cavity or intravenous in rat BMT models / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the in vivo distribution of mesenchymal stem cells (MSCs) after administrated by intra-bone marrow (IBM) or intravenous (i.v.), and compare the effects on hematopoiesis reconstitution and GVHD in rat BMT models.</p><p><b>METHODS</b>(1) MSCs from male Wistar rats marked with CFSE were injected into the bone marrow cavity (IBM) or the vein (i.v.) of recipient rats, and observed the distribution of MSCs in vivo. (2) Allogeneic BMT model of Fischer344 rats (RT1A(1)) to Wistar rats (RT1A(u)) was established. The recipient rats were exposed to 8 Gy of gamma irradiation 1 day before transplantation. The 6 groups were (1) IBM group [IBM-injection of MSCs + IV-injection of bone marrow cells (BMC)]; (2) IV group (i.v.-injection of MSCs (i.v.) + i.v.-injection of BMC); (3) BMT group (only i.v.-injection of BMC); (4) MSCs control group (only i.v.-injection of MSC); (5) normal control group and (6) irradiation control group.</p><p><b>RESULTS</b>(1) After i.v.-injection, large numbers of the MSCs lodged in lungs while small numbers in the peripheral blood, liver, thymus and spleen, and a few marked MSCs could be seen in bone marrow. After IBM injection, most cells distributed in long bones and those lungs were less than that in i.v. group. (2) Co-transplantation of MSCs (IBM/IV) could accelerate the recovery of hematopoiesis, including the recovery of WBC, hemoglobin and platelet, and in IBM-injection was more effective in the recovery of hematopoiesis than that in i.v. group. (3) Incidence rate of GVHD in BMT group was 42% (3/7), and no GVHD occurred in co-transplantation groups. (4) Recovery of CFU-Mix and CFU-MSCs could be seen at 21st and 30th day after transplantation in co-transplantation groups, and IBM-injection was more effective than i.v.-injection.</p><p><b>CONCLUSION</b>(1) IBM-injection results in most MSCs distributed in long bones. (2) MSCs improve the survival rate after BMT. (3) Co-transplantation of MSCs accelerates the recovery of hematopoiesis and reduces the morbidity of GVHD. (4) MSC promotes reconstitution of hematopoietic cells and bone marrow MSCs in recipient rates and the effects of MSCs administrated via IBM is more effective than via i.v.</p>

Comparison of efficacies of allogeneic hematopoietic stem cell transplantations between different routes of administration in mice / 中国实验血液学杂志

When hematopoietic stem cells (HSCs) were administrated by intravenous infusion (IV), most of them were trapped in some nonhematopoietic organs as like lungs that had abundant blood capillaries. Only a small fraction of injected cells could home to the bone marrow, which reduced the engraftment of HSCs. The purpose of intra-bone marrow (IBM) transplantation was to facilitate the homing of HSCs directly. Based on the established murine model for allogeneic umbilical cord blood transplantation (UCBT) by IBM injection, the objective of this study was to compare the distribution of fetal and neonatal peripheral blood (FNPB) mononuclear cells (MNC) in vivo and the efficacy of HSCT by different routes of administration in mice. BALB/c recipient mice exposed to sublethal dose 60Co gamma-ray were transplanted with FNPBMNCs from C57BL/6 mice. Recipient mice were divided into six groups at random: unilateral-IBM group; bilateral-IBM group; IV group; bilateral-IBM + IV group; irradiated control group and normal group. The distribution of CFSE-labeled FNPBMNCs in the recipients was observed in frozen sections of different organs or by flow cytometry. The survival rate, engraftment level, recovery of hematopoietic function and GVHD of recipient mice were studied. The results showed that infused by IBM route, FNPBMNCs mainly accumulated in the bone marrow (BM) cavity of the injected side tibia. Some of them could enter the BM of noninjected bones via blood circulation and few were trapped in the lung. Though same amount of FNPBMNCs were injected into recipient mice of unilateral and bilateral-IBM group, less cells could leak into peripheral blood or other tissues when transplanted by bilateral-IBM route. Therefore, in term of accelerating hemopoietic recovery, the injection of IBM route was better than IV route, especially bilateral IBM injection of HSCs, which neared the normal level of peripheral blood cells and colony-forming units of bone marrow nucleated cells at day 21 after transplantation, followed by unilateral-IBM group and bilateral-IBM + IV group. The percentages of H-2Db cell subsets in the three IBM groups were much higher than that in IV group. There was no significant difference of the engraftment level in the injected side tibia between the unilateral and bilateral-IBM group. When secondary transplantation was performed, the engraftment level in bilateral-IBM group was still much higher than that in IV group. At day 90, the survival rates of IBM groups were all > or = 80%, while that of IV group was only 50%. It is concluded that bilateral-IBM route can facilitate the homing of more HSCs, accelerate the engraftment of HSCs and hematopoietic reconstitution, which promoted the efficacy of IBM-HSCT.

MicroRNAs can be expressed in cardiomyocyte-like cells differentiated from human mesenchymal stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of representative heart-specific primary microRNAs (pri-miRNAs) in the cardiomyocyte-like cells differentiated from human mesenchymal stem cells (hMSCs).</p><p><b>METHODS</b>The phenotype of hMSCs isolated was identified by flow cytometry using monoclonal antibodies against FITC-conjugated CD29, CD34, and CD11b. The third-passage hMSCs were induced to differentiate into cardiomyocyte-like cells by 5-azacytidine and indirect coculture with neonatal rat myocytes, respectively. Immunocytochemical analysis was performed to detect the expression of the cardiac-specific proteins, namely cardiac troponin I (cTnI) and sarcomeric alpha-actinin, in the cardiomyocyte-like cells differentiated from hMSCs. RT-PCR and DNA sequencing were used to identify the expression of the 5 representative heart-specific pri-miRNAs.</p><p><b>RESULTS</b>High hMSC marker CD29 expression rate (98.87%) and low hematopoietic cell markers CD34 (5%) and CD11b (0.4%) expression rates were identified in the hMSCs isolated. cTnI and sarcomeric alpha-actinin expression occurred in the hMSCs following induction with the 2 differentiation-inducing methods. miRNA-143 and -181 expressions were induced in the hMSCs by 5-azacytidine and miRNA-143, -181, -206, and -208 expressions were induced by indirect coculture with neonatal rat myocytes, but pri-miRNA-1-2 expression failed to be induced by these two induction methods.</p><p><b>CONCLUSION</b>Expressions of the representative heart-specific pri-miRNAs in different patterns can be induced in cardiomyocyte-like cells differentiated from hMSCs by 5-azacytidine and indirect coculture with neonatal rat myocytes.</p>

Expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros region / 中国实验血液学杂志

The objective of this study was to investigate the expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros (AGM) region. Stromal cells derived from human AGM region (hAGM S1-S5) and fibroblasts derived from human fetal trunk (hFT) were cultured in vitro. RT-PCR was used to analyze the expression of BMP-4 in hAGM S1-S5 stromal cells at mRNA level. And BMP-4 level was detected in the supernatant of hAGM S1-S5 stromal cells by ELISA assay. hFT cells were used as control group. The results showed that the heterogenous hAGM S1-S5 stromal cells displyed shapes of fibroblast-like and endothelial-like cells. hAGM S1-S5 stromal cells expressed BMP-4 mRNA, but fetal trunk fibroblasts (hFT) did not express BMP-4 mRNA. In the supernatant of hAGM S1-S5 cells, BMP-4 could be detected by ELISA assay ana its levels were statistically higher than that in hFT group (p < 0.05), while there was no significant difference between groups of hAGM S1-S5 (p > 0.05). It is concluded that human AGM-derived stromal cells in vitro express BMP-4, and the establishment of a new culture system based on the feeder cells of AGM stroma would promote the differention of embryonic stem cells into hematopoietic stem cells at a high proportion.

Supportive effects of stromal cells from human embryonic aorta-gonad-mesonephros region on umbilical cord blood CD34+ cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the supportive effects of stromal cells from human aorta-gonad-mesonephros (AGM) region on umbilical cord blood CD34+ cells.</p><p><b>METHODS</b>Stromal cells derived from human AGM region (hAGMS1-S5) and fetal trunk fibroblasts (hFf) were cultured on the bottom of 24-well plates as feeder cells. CD34+ cells positively selected from human umbilical cord blood through immunomagnetic beads selection method, were seeded into 24-well plates, and co-cultured for 28d. The number of total nucleated cells (TNC), CD34+ cells, CD34+ CD38- cells, and CFC were counted every week.</p><p><b>RESULTS</b>Stromal cells from human AGM region significantly supported proliferation of the TNC, CD34+ cells, CD34+ CD38- cells and CFC, when compared with hFT and controls without feeder cells (P < 0.05). The TNC increased (25.13 +/- 4.83)-fold (peak value) at day 21 in group of co-culture with AGM stromal cells. CD34 and CD34+ CD38- cells increased (2.68 +/- 0.51)- and (2. 38 +/- 0.45)-fold respectively at day 14 of co-culture. In colony analysis, HPP-CFU increased (2.62 +/- 0.85)-fold at day 14 of co-culture. The supportive effects of human AGM S1-S5 were significantly different, hAGM S3 and S4 were better than hAGM SI, S2, and S5 (P < 0.05).</p><p><b>CONCLUSIONS</b>Human AGM stromal cells S1-S5 could support the maintenance and expansion of umbilical cord blood CD34+ cells in vitro. hAGMS3, S4 cell had better effects on maintaining HSC activity, which would provide model cells and basic data for researches on hematopoiesis mechanism and hematopoietic differentiation of embryonic stem cells.</p>

Screening and cloning the genes differentially expressed in human embryonic AGM-derived stromal cells / 中国实验血液学杂志

To screen and separate the genes differentially expressed in human embryonic aorta-gonad-mesonephros (AGM)-derived stromal cells, a subtracted library was generated through the suppression subtractive hybridization using the cDNA of human embryonic AGM-derived stromal cells as target and human fetal liver (FL)-derived stromal cells as drivers. Then a high though screening technique, gene chip, was used to screen the differentially expressed genes in the established subtractive library. Approximately 18 of the resulting subtracted cDNA clones were partially sequenced and analyzed by blastn in the GenBank database. The results showed that 211 Clones were selected and identified from the established subtractive library, the positive ratio was amount to 76.4%. 18 over-expressed genes were screened by gene chip with more than a 5-fold difference expression levels between AGM and FL-derived stromal cells, and were selected to sequence, results of sequencing indicated that the 18 sequences was compared to known sequences in the GenBank database, and among the sequenced clones, 14 sequences were considered as part of the known genes, and 4 sequences representing previously unknown genes. The known genes were reported to involve the regulation of cell migration, cell differentiation, cell proliferation, cell cycle, signal transduction, and angiogenesis. Most of these genes have not been reported to relate to the haematogenesis in ontogeny. It is concluded that many genes both known and unknown are differentially expressed in human embryonic aorta-gonad-mesonephros-derived stromal cells. Discovery of these genes provides a solid foundation to elucidate the mechanism of haematogenesis in ontogeny.

Supportive effects of human aorta-gonad-mesonephros-derived stromal cells on umbilical cord blood LTC-IC / 中国实验血液学杂志

The objective of this study was to explore the supportive effects of human aorta-gonad-mesonephros (AGM)-derived stromal cells on human umbilical cord blood long-term culture-initiating cells (LTC-IC). A co-culture system was established with human AGM stromal cells and umbilical cord blood CD34(+) cells. Different stromal cells derived from human AGM region (hAGM S1-S5) were plated on 24-well plates as feeder cells. CD34(+) cells were positively selected from human umbilical cord blood through immunomagnetic bead selection method, seeded on the feeder cells, and co-cultured for 8 weeks. The hematopoietic cells were collected at 5, 6, 7 and 8 weeks for CFC analysis. Frequencies of LTC-IC in umbilical cord blood CD34(+) cells after co-culture with AGM stromal cells were detected through limiting dilute analysis (LDA). The results showed that there was no any hematopoietic CFC in the feeder cell-free culture system after 5 weeks of co-culture. However, in AGM feeder cells culture systems, there were still CFCs after 5 weeks of co-culture, which indicated that human AGM stromal cells could maintain LTC-IC in vitro. In groups of hAGM feeders, hAGMS3 and S4 had better supportive effects than other AGM groups (P < 0.05). The absolute number of LTC-IC in hAGM S3 and S4 culture systems got expansion up to (176 +/- 46)% and (187 +/- 52)% respectively without significant difference between hAGMS3 and S4 (P > 0.05). It is concluded that human AGM stromal cells S1-S5 support the maintenance of umbilical blood LTC-IC in vitro, while hAGMS3 and S4 cells have better effects on maintaining LTC-IC and expansion of LTC-IC.

Hematopoietic growth factors expressed in human aorta-gonad-mesonephros (AGM)-derived stromal cells in vitro / 中国实验血液学杂志

This study was aimed to investigate the hematopoietic growth factors expressed in human aorta-gonad-mesonephros (AGM)-derived stromal cells in vitro in order to provide the basic data for elucidating the role of AGM -derived-stromal cells in embryo-hematopoiesis and its hematopoietic suppoitive effect. RT-PCR was used to analyze the expression of IL-6, SCF, Flt3-L, oncostatin M (OSM), IL-3, TPO, M-CSF and LIF in human aorta-gonad-mesonephros-derived stromal cells (hAGMS1-S5) at mRNA level. IL-6, SCF and Flt3-L levels were detected in the supernatant of hAGMS1-S5 stromal cells by ELISA assay. Umbilical cord blood CD34(+) cells were cocultured with hAGMS1-S5 feeder cells, and hematopoietic cells were collected at day 14 for colony analysis in methylcellulose semisolid medium. The results showed that human aorta-gonad-mesonephros-derived stromal cells S1-S5 expressed IL-6, SCF, Flt3-L and OSM mRNA, but did not express IL-3, TPO, M-CSF and LIF mRNA. In the supernatant of hAGMS1-S5 cells, IL-6, SCF and Flt3-L could be detected by ELISA assay at different levels, while there was no significant difference between groups of hAGMS1-S5 (P > 0.05). When cocultured with umbilical cord blood CD34(+) cells, hAGMS1-S5 could support the expansion of CFU-GM, BFU-E, and CFU-Mix. The supportive effects of hAGM S1-S5 were significantly different (P < 0.05), hAGM S3 and S4 were better than hAGM S1, S2, and S5. It is concluded that detection of hematopoietic growth factors expressed in human aorta-gonad-mesonephros-derived stromal cells provided a solid foundation to elucidate the mechanism of hematopoiesis and the hematopoietic supportive effect of these stromal cells.

Effects of human umbilical cord blood mesenchymal stem cells on the expansion of CD34+ cells from umbilical cord blood / 中华儿科杂志

<p><b>OBJECTIVE</b>The previous studies indicated that mesenchymal stem cells (MSCs) either from umbilical cord blood (UCB) or from bone marrow (BM) had the same biological characteristics and the function of secreting hematopoietic growth factors (HGFs). The present study aimed to understand the effects of human UCB MSCs on the expansion of CD(34)(+) cells from UCB.</p><p><b>METHODS</b>1. Human UCB CD(34)(+) cells were incubated in the system containing UCB MSCs, HGFs and serum free medium. 2. The surface markers (CD(34)(+), CD(34)(+)CD(38)(-), CD(34)(+)CD(3)(+), CD(34)(+)CD(19)(+), CD(34)(+)CD(33)(+), CD(34)(+)CD(41a)(+)) on expanded UCB cells were examined by flow cytometry on the 6th and 12th days. 3. The expanded and unexpanded cells were cultured in semi-solid culturing system and checked for colony forming units of granulocyte and macrophage (CFU-GM), erythroid burst-forming unit (BFU-E), colony forming units of granulocyte- erythrocyte-megakaryocyte-macrophage (CFU-Mix) and colony forming units of high-proliferative potential (CFU-HPP).</p><p><b>RESULTS</b>1. The expansion folds of CD(34)(+)CD(38)(-) cells from UCB MSCs + HGFs groups on the 6th and 12th days were 159.43 and 436.68, respectively. Interestingly, the percentage of CD(34)(+)CD(38)(-) cells declined in HGFs group after expanding for 12 days, but it rose to 9.98% in the UCB MSCs + HGFs group. 2. Colony forming capacity of expanded UCB cells showed that the folds of CFU-Mix and CFU-HPP of UCB MSCs + HGFs group increased from day 6 to day 12, but the folds decreased in the HGFs group. 3. From day 0 to day 12, CD(34)(+)CD(33)(+) cells and CD(34)(+)CD(41a)(+) cells were amplified gradually, but CD(34)(+)CD(19)(+) and CD(34)(+)CD(3)(+) cells decreased gradually, and in UCB MSCs + HGFs group this phenomenon was more significant than that in HGFs group.</p><p><b>CONCLUSION</b>1. UCB MSCs containing system not only has the ability to expand the primitive HSCs but also has the ability to sustain the proliferation of HSCs. 2. UCB MSCs containing system amplified mainly myeloid and megakaryocytoid progenitor subsets. These may have clinical significance in reducing infection and hemorrhage.</p>

Mesenchymal stem cells from human cord blood promote engraftment of human umbilical cord blood-derived CD34+ cells in NOD/SCID mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore whether the co-transplantation of mesenchymal stem cells (MSC) from human umbilical cord blood (UCB) with UCB-derived CD34(+) cells in NOD/SCID mice could promote engraftment and accelerate hematopoiesis recovery.</p><p><b>METHODS</b>After sublethal irradiated ((60)Co 2.5 Gy), NOD/SCID mice received within 24 hours UCB CD34(+) cells (1 x 10(5) per mouse for low dosage group or 1 x 10(6) per mouse for high dosage group) with or without human UCB-derived MSC (1 x 10(6) per mouse) transplantation by lateral tail vein injection. Peripheral blood cells of transplanted mice were measured for white blood cell count, hemoglobin and platelet count at 10th, 20th, 30th, 40th and 56th day. At the end of 8th week after transplantation, all the alive mice were sacrificed and human derived CD45(+), CD45(+)CD3(+), CD45(+)CD19(+), CD45(+)CD33(+) cells in the bone marrow (BM) were assayed by flow cytometry.</p><p><b>RESULTS</b>(1) In the low dosage group, co-transplantation of MSC significantly raised the engraftment rate (26.02% vs 16.52%) (P < 0.05). (2) The survival rate in high dosage group was 80% for co-transplantation mice and 70% for CD34(+) cells alone transplantation mice. The survival rate in low dosage group was 70% for co-transplantation mice and 50% in CD34(+) cells transplantation mice. (3) In both dosages groups co-transplantation accelerated the hematopoiesis recovery. (4) At the end of 8 weeks after transplantation, in low dosage group, CD45(+)CD33(+) and CD45(+)CD19(+) cells were more in co-transplantation mice than in CD34(+) cells alone transplantation mice, but in high dosage group, the percentage of these two kinds of cells had no difference. In both dosage groups the percentage of CD45(+)CD41a(+) cells were higher in co-transplantation than in transplantation alone mice. CD45(+)CD3(+) cells were low in all groups.</p><p><b>CONCLUSIONS</b>(1) In low dosage transplantation, human UCB MSC could promote human CD34(+) cells engraftment in transplanted mice. (2) Co-transplantation of human UCB MSC and human UCB CD34(+) cells could significantly promote the hematopoiesis reconstitution and improve the survival rate of NOD/SCID mice. (3) MSC could promote human UCB CD34(+) cells to differentiated into B-lymphocytes, granulocyte and megakaryocyte in vivo.</p>

Cloning of differentially displayed cDNAs involved in NaF-treated osteoblasts / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the effects of excessive fluoride on the gene expression of rat osteoblasts.</p><p><b>METHODS</b>Rat osteoblasts were treated with 2.0 mmol/L of sodium fluoride (NaF) for two weeks in vitro, and difference in the gene expression between the NaF-treated and normal osteoblasts was compared with mRNA differential display (DD-PCR) technique.</p><p><b>RESULTS</b>Among the six differentially expressed gene fragments which had been cloned, expression of the ribosomal protein L5 gene, ATPase Na(+)K(+) transporting beta polypeptide 3 gene, karyopherin alpha 2 gene and cis-Golgi body p28 gene was lower and expression of ubiquitous-conjugating enzyme E2D 3 gene and a newly-discovered gene fragment in this study showed up-regulated in the NaF-treated osteoblasts of the rats.</p><p><b>CONCLUSIONS</b>Expression of genes changed in the osteoblasts after treatment with fluoride for two weeks and most of them associated with synthesis, transportation and processing of protein. It suggested that excessive fluoride could affect the protein synthesis in osteoblasts by changing the expression of the related genes. A novel gene related to excessive fluoride exposure was also found.</p>