Atractylodes lancea is a perennial herbaceous plant of Asteraceae, with rhizomes for medical use. However, A. lancea plants from different habitats have great variability, and the germplasm resources of A. lancea are unclear and mixed during production. Therefore, it is urgent to protect new varieties of A. lancea. The distinctness, uniformity, and stability(DUS) testing of new plant varieties is the foundation of plant variety protection, and the DUS testing guidelines are the technical basis for variety approval agencies to conduct DUS testing. In this study, the phenotypic traits of 94 germplasm accessions of A. lancea were investigated considering the breeding and variety characteristics of A. lancea in China. The traits were classified and described, and 24 traits were preliminarily determined, including 20 basic traits that must be tested and four traits selected to be tested. The 20 basic traits included 3 quality traits, 5 false quality traits, and 12 quantitative traits, corresponding to 1 plant traits, 2 stem traits, 8 leaf traits, 6 flower traits, and 3 seed traits. The measurement ranges and coefficients of variation of eight quantitative traits were determined, on the basis of which the grading criteria and codes of the traits were determined and assigned. The guidelines has guiding significance for the trait evaluation, utilization, and breeding of new varieties of A. lancea.
Atractylodes/growth & development* ; China ; Phenotype ; Guidelines as Topic ; Plant Breeding

Mesenchymal stem cells (MSCs) have been officially approved in many countries to treat graft-versus-host disease, autoimmune disorders and those associated with tissue regeneration after hematopoietic stem cell transplantation. Studies in recent years have confirmed that MSC acts mainly through paracrine mechanism, in which extracellular vesicles secreted by MSC (MSC-EV) play a central role. MSC-EV has overwhelming advantages over MSC itself in the setting of adverse effects in clinical application, indicating that MSC-EV might take the place of its parent cells to be a potentially therapeutic tool for "cell-free therapy". The pharmaceutical properties of MSC-EV largely depend upon the practical and optimal techniques including large-scale expansion of MSC, the modification of MSC based on the indications and the in vivo dynamic features of MSC-EV, and the methods for preparing and harvesting large amounts of MSC-EV. The recent progresses on the issues above will be briefly reviewed.
Humans ; Extracellular Vesicles ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Mesenchymal Stem Cell Transplantation/methods* ; Mesenchymal Stem Cells ; Pharmaceutical Preparations

OBJECTIVE: To observe the safety of donor NK cell infusions in the settings of hematopoietic stem cell transplantation and after consolidation chemotherapy in elderly patients with acute myeloid leukemia (AML). METHODS: Forty patients with AML were included, in which 21 patients aged over 60 years were at the stage of complete remission (CR) and 19 patients that received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mononucleated cells were isolated from peripheral blood from the donors (for allo-HSCT) or healthy immediate family members (elderly AML). The cells were seeded into the flasks pre-coated with NK cell specific activators, and expanded in media containing recombinant human IL-15 and IL-2 for 14 days. The cells were transfused intravenously after the identification of quality control. Trypan blue exclusion test was used for the determination of cell viability and counting. Flow cytometry analysis was performed to assess the surface antigenic profile. Seventy-eight infusions of the cell products were received by the elderly patients with AML after consolidation chemotherapy, 11 infusions were received by the patients during allo-HSCT and 32 infusions 3 moths after transplantation. The safety of cell therapy, body temperature, blood pressure and other indexes were observe during and 48 hours after cell transfusion. Meanwhile, the occurrence and severity of acute graft-versus-host disease (GVHD) were documented. RESULTS: Flow cytometry analysis showed that the proportion of NK cells (CD3^-CD56⁺) in the mononucleated cells before culture was (14.10±4.22)% (n=121), and the proportion increased dramatically up to (87.29±8.75)% (n=121) after culture for 14 days, the number of NK cells increased to 753.47±140.13 times (n=121). The doses of the infused NK cells was (7.58±2.50)×10⁷/kg per infusion. Moderate fever occurred in three cases after multiple infusions, and the temperature restored to normal on the same day after treatment. Fever was observed in one patient after every infusion of four times in total. The temperature reached to 38.5-39.0 ℃ and returned to normal within 1-2 hours after adequate antipyretic treatment, and then there was no discomfort. No GVHD was observed in the elderly AML patients, while 6 cases that received allo-HSCT developed moderate acute GVHD, among them grade I in 5 cases and grade II in 1 case. No other severe toxicities were observed. CONCLUSION NK cell products with a high-purity could be obtained by ex vivo expansion with this protocol. The transfusion of these expanded cells is generally safe in the elderly patients with AML that have received chemotherapy or patients that received hematopoietic stem cell transplantation.
Aged ; Graft vs Host Disease/prevention & control* ; Hematopoietic Stem Cell Transplantation ; Humans ; Killer Cells, Natural ; Leukemia, Myeloid, Acute/therapy* ; Middle Aged ; Recurrence

OBJECTIVETo develop an easy method to amplify natural killer (NK) cells by using mononuclear cells in vitro, so as to lay the basis for NK cell therapy.

METHODSUmbilical cord blood from 3 healthy full-term pregnant women was collected, and the peripheral blood mononuclear cells (PBMNC) were harvested by density gradient centrifugation. Each sample of PBMNC was divided into 3 groups: CD16mAb, CD3 mAb and CD16mAb+CD3mAb- groups. The culture flasks were pre-coated with CD16, CD3 or CD3 plus CD16 mAb. The PBMNCs were cultured in serum-free media containing autologous plasma, recombinant human IL-2, IL-15 and IL-21 for 14 days under the same conditions. The total viable cell count was calculated. Flow cytometry was used to determine the ratio of CD56CD3 cells, MTT assay was used to measure the killing rate of NK cells under different effector/target ratio, by using the K562 cells as the target cells.

RESULTSAfter 14 days of culture, the total cell numbers of CD16mAb, CD3mAb and CD16mAb +CD3mAb groups increased by 45.71±5.54, 87.41±19.77 and 4.88±51.84 times, respectively, and those of CD3mAb group were significantly higher than the other 2 groups (P<0.05). The ratio of CD56CD3 cells before culture was 0.1663±0.0201, which was 0.8167±0.0500, 0.8077±0.0589 and 0.8077±0.0273 after incubation with CD16mAb, CD3mAb and CD16mAb +CD3mAb for 14 days, respectively (P>0.05). MTT test showed that the killing efficiencies were not significantly different among the 3 groups when the effector/target ratios were 1:1, 5:1 and 10:1 (P>0.05).

CONCLUSIONBy incubation with anti-CD3 monoclonal antibody, IL-2, IL-15 and IL-21, the highly purified NK cells can be obtained from mononucleated cells, thus providing a simple method for NK cell therapy.

CD3 Complex ; CD56 Antigen ; Cell Culture Techniques ; Cells, Cultured ; Female ; Humans ; Killer Cells, Natural ; Leukocytes, Mononuclear ; Pregnancy

OBJECTIVE: To investigate the effect of immune regulatory molecules TGF-β1 and IL-10 on the immunoregulatory activities of extracellular vesicles(EV) secreted from mesenchymal stem cells (MSCs). METHODS: MSC were isolated from human umbilical cord and expanded, then were treated with TGF-β1 and IL-10 for 72h, and MSC-EVs in supernatants were isolated. The total protein content of all samples was determined by BCA methed. The morphological structure was observed by transmission electron microscopy. The surface markers of MSC-EV were analyzed by flow cytometry. The apoptosis of peripheral blood mononuclear cells(PBMNC) stimulated by ConA and the proportion of CD4CD25/CD127 (Treg) cells were detected by flow cytometry after incubation with MSC-EV for 72 h. The CBA and ELISA kit were used to detect the contents of IL2, IL4, IL6, IL10, IFN-γ, TNF-α, Th17A and TGF-β1 in PBMC supernatants and MSC-EV. RESULTS: All the samples showed that the typical cup-shaped membrane-like structure was observed by transmission electron microscopy, and CD9, CD44, CD63 and CD81 expressed. After TGF-β1 treatment, the MSC-EV displayed the strongest ability to promote PBMNC apoptosis（P<0.01）, and in all the samples the proportion of Treg cells increased. MSC-EV could increase the content of IL-10 in the supernatants of PBMNC culture, the content of TGF-β1 in PBMNC supernatants after MSC treatment with TGF-β1 was lower than that in untreated group(P<0.05). The content of IL-6 in MSC-EV increased significantly after treatment with TGF-β1, and the content of TGF-β1 decreased. CONCLUSION TGF-β1 alters the immnomodulatory function of MSC-EV and its underlying mechanisms need to be clarified in further investigations.
Extracellular Vesicles ; Humans ; Interleukin-10 ; Leukocytes, Mononuclear ; Mesenchymal Stem Cells ; Transforming Growth Factor beta1

OBJECTIVETo analyze the therapeutic efficacy of haploidentical-hematopoietic stem cell transplantation (hi-HSCT) for patients with juvenile myelomonocytic leukemia (JMML).

METHODSThe engraftment of hematopoietic stem cells, incidence of graft versus host disease (GVHD), infection, relapse, and survival of 6 JMML patients received hi-HSCT were retrospectively analyzed.

RESULTSSix (6 males) JMML patients received hi-HSCT from haplo-HLA-matched related donors. The results showed that the hematopoictic stem cells in all 6 patients were grafted successfully. Two cases of JMML died of pulmenary infections, other 4 cases survive without disease. Acute GVHD occurred in 3 patients and chronic GVHD occurred in 1 patients. The overall survival, disease free survival and relapse rates were 66.7%, 66.7%, 0%, respectively.

CONCLUSIONThe hi-HSCT is an effective method for treatment of patients with JMML, but there also is a serial problems to be resolved.

OBJECTIVETo evaluate the safety and effectiveness of a novel therapeutic regimen for bronchiolitis obliterans sydrome (BOS) affter hematopoietic stem cell transplantation (HSCT).

METHODSSeven patients who had received HSCT and had been diagnosed as BOS were enrolled in this study. They received weekly intravenous injection of umbilical cord-derived mesenchymal stem cells (MSC) at a dose of 1 × 10(6)/kg for 4 weeks. Budesonide was given orally at a daily dose of 0.25 g, and salmeterol was inhaled at a dose of 4.5 µg for 3 times per day. Methylprednisolone was given at a dose of 1 mg/(kg·d) for 2 weeks when respiratory failure occured. The dose of methylprednisolone was tapered to 0.25 mg/(kg·d) after 4 weeks and was adjusted according to the occurrence and severity of chronic graft-versus-host disease (cGVHD).

RESULTSThe therapy was generally safe and no severe acute toxicity was observed. One patient died of heart failure during the treatment, the other 6 patients were alive and the pulmonary function parameters including FEV1, FEV1/FVC, PaO2 and AaDO2 were significantly improved after 6 months as compared with the baseline parameters (P < 0.05).

CONCLUSIONMSC combined with budesonide, almeterol and azithromycin has been confirmed to be generally safe and can reduce the dose of glucocorticoid in treatment of BOS after HSCT.

Azithromycin ; therapeutic use ; Bronchiolitis Obliterans ; therapy ; Budesonide ; therapeutic use ; Combined Modality Therapy ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation ; Humans ; Mesenchymal Stem Cell Transplantation ; Methylprednisolone ; administration & dosage ; therapeutic use ; Salmeterol Xinafoate ; therapeutic use

OBJECTIVETo investigate the protection of silymarin against the human mesenchymal stem cell (MSC) apoptosis induced by serum deprivation and its underlying mechanism.

METHODSHuman umbilical cord MSCs were cultured in the absence of serum, and the silymain of different concentration (1-10 µg/ml) was added into the medium. MTT test was performed to observe the cell proliferation status. After being cultured for 72 hours, the cells were collected, and flow cytometry with Annexin-V-PI double-staining was used to detect the apoptotic cells from the control and silymarin-treated groups. Furthermore, the intracellular contents of BAX and BCL-2 were detected by Western blot for exploring the potential mechanism.

RESULTSThe silymarin promoted the proliferation of human UC-MSCs in a dose-dependent manner, reaching its maximal at a dose of 5 µg/ml. Moreover, silymarin could inhibit the serum deprivation-induced apoptosis of MSCs and, the inhibitory rate reached up to 30% when it was added at a concentration of 5 µg/ml. The content of intracellular BAX was obviously elevated after serum-deprivation treatment, and this increase could be blunted by the addition of silymarin. Meanwhile, the content of BCL-2 was not obviously changed.

CONCLUSIONThe silymarin can stimulate MSC growth and inhibit the apoptosis of MSCs probably by the mitochondria pathway.

Apoptosis ; drug effects ; Cell Proliferation ; Culture Media, Serum-Free ; Humans ; Mesenchymal Stromal Cells ; drug effects ; Mitochondria ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Silymarin ; pharmacology ; Umbilical Cord ; cytology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate whether the progesterone can promote fibronection (FN) synthesis by human bone marrow mesenchymal stem cells (MSCs) and to explore the potential underlying mechanism.

METHODSThe human bone marrow MSCs were cultured in a serum-free medium with progesterone for 72 hours, the MTT test was performed to observe the proliferation status and adhension ability of the treated cells. Western blot was used to detect the content of FN in MSDs with GAPDH as the internal reference, the phosphorylation of ERK1/2, as well as the FN content in MSC treated by PD98059, a specific inhibitor of ERK1/2.

RESULTSThe progesterone at a range of certain doses not effect on the proliferation of human bone marrow MSCs. Progesterone (25 µg/L) treatment enhanced the FN expression and adherent ability of marrow MSCs. Progesterone could induce prompt phosphorylation of ERK 1/2 and its promoting effects on FN synthesis was reversed by PD98059.

CONCLUSIONThe progesterone can promote FN synthesis by human bone marrow MSCs via ERK 1/2 pathway, and it might be used to culture MSCs in serum-free medium.

Bone Marrow Cells ; Cells, Cultured ; Fibronectins ; Hematopoietic Stem Cells ; Humans ; MAP Kinase Signaling System ; Mesenchymal Stromal Cells ; Mitogen-Activated Protein Kinase 3 ; Phosphorylation ; Progesterone

OBJECTIVETo investigate the mechanisms underlying the incorporation of microparticles(MP) derived from human bone marrow mesenchymal stem cells (MSCs) into human umbilical cord endothelial cells (HUVECs).

METHODSMPs were isolated from the supernatants of MSCs which had exposed to a hypoxia/serum-deprivation condition. Electron microscope was used to identify the MPs. The surface molecule profile was evaluated with the bead-based flow cytometry technique. The expression level of the phosphatidylserine receptor (PSR) was detected by immunofluorescence cytochemistry. MPs were co-cultured with HUVECs in the presence or absence PSR-antibody, and the internalization of MPs was observed with laser scanning microscopy.

RESULTSThe MPs derived from MSCs expressed highly PS, while PSR expressed on the surface of HUVECs. The confocal result revealed that MPs could quickly be uptaken by the endothelial cells, and mainly distributed in the cytoplasm surrounding of the nuclei. The internalization of MPs reduced significantly after PSR specific blockage.

CONCLUSIONThe reaction between PS on the MP and the PSR of HUVECs plays an important role in the internalization of MSC-MPs.

Cells, Cultured ; Coculture Techniques ; Endothelial Cells ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; Umbilical Cord