The effects of the Smad3- knockout on the hematopoiesis of mouse were investigated in this work. Five pairs of wild type and Smad3- null mice were studied. White blood cell(WBC), red blood cell(RBC) and platelet (PLT) counting of peripheral blood cells were performed with blood obtained from tails. And white blood cells were classified by their morphology. Bone marrow nucleated cells (BMNCs) were counted and classified. The CFU-GM, BFU-E, CFU-GEMM yields were measured in each pair of mice. CFU-S yield of each mouse was measured by injecting bone marrow cells into lethally irradiated 8-10 weeks old wild type female mice. And the pathomorphism of their bone marrows, spleens and livers were observed. As a result, WBC and PLT of Smad3- null mice were significantly higher than those in wild type mice. Smad3- null mice had much more proportion of granulocytes in classification. There wasn't any difference in RBC counting and BFU-E measurement. The yield of CFU-GM increased, while the yields of CFU-GEMM and CFU-S markedly reduced. Bone marrows are actively proliferative, with granulocytosis. The granulocyte/erythrocyte ratio increased. There were no obviously alterative in spleen and liver. Thus Smad3- knockout results in a decreased number of stem and progenitor cells. Moreover hematopoietic differentiation is abnormal with a tendency to forming more granulocytes and platelets. The effect of Smad3 on hematopoiesis is correlative to that of TGF-beta.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Erythrocytes ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; metabolism ; Granulocytes ; cytology ; metabolism ; Hematopoiesis ; genetics ; Mice ; Mice, Knockout ; Myeloid Progenitor Cells ; cytology ; metabolism ; Smad3 Protein ; genetics

Glioma is one of the most common primary tumors in the human brain with poor prognosis. The local and systemic immunosuppressive environment created by glioma cells enables them to evade immunosurveillance. Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppression system. They are a heterogeneous cell population composed of early myeloid progenitor cells and precursor cells. Although the cells are diverse in phenotypes and functions, they all have strong immunosuppressive functions. MDSCs are extensively infiltrated into tumor tissues and play an important role in the glioma immunosuppressive microenvironment, which also hinders the immunotherapeutic effects of glioma. This article will review the phenotypic characteristics of MDSCs in the glioma microenvironment and their role in the progression of glioma. It is of positive significance to better understand the pathogenesis of glioma and explore effective comprehensive treatments.
Glioma ; pathology ; Humans ; Immune Tolerance ; Myeloid-Derived Suppressor Cells ; cytology ; Tumor Microenvironment

This study was aimed to compare partial biological characteristics of bone marrow mesenchymal stem cells (BMMSCs) in AML patients, AML patients with complete remission (CR) and non-leukemia patients. The bone marrow (BM) MSCs were divided into 3 groups: group of MSCs from AML patients, group of MSCs from AML patients with CR, group of MSCs from non-leukemia patients. The morphologic features of MSCs were observed by light microscopy; CFU-F numbers of MSCs were counted after Wright-Giemsa staining in situ; the fusion times of MSCs were determined; the growth curves of MSCs were drawn by counting cell numbers; the immunophenotypes and cell cycle of MSCs were detected by flow cytometry; the DI values of MSCs were calculated. The results showed that the morphologic features of MSCs in 3 groups did not display difference; there was significant difference (p < 0.01) of CFU-F numbers in 3 groups, while CFU-F number of MSCs in AML group was minimal; there was significant difference of MSC fusion time in 3 groups, while fusion time of MSCs in AML group was most long; the growth curves of MSCs in 3 groups were similar; MSCs in 3 groups highly expressed CD105 and CD106, but not expressed CD45; the cell distribution ratios at phase of G(0)/G(1) for MSCs in 3 groups were 89.9 +/- 4.0%, 90.2 +/- 3.0% and 91.0 +/- 3.0% respectively; the DI values of MSCs in 3 groups were between 0.9 and 1.1. It is concluded that no significant difference of biological characteristics of the second generations of MSCs is found between those in leukemia and non-leukemia patients.
Bone Marrow Cells ; cytology ; Cell Cycle ; Cell Fusion ; Cell Proliferation ; DNA ; genetics ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; genetics ; immunology ; pathology ; Mesenchymal Stromal Cells ; cytology

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.
Apoptosis ; physiology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; T-Lymphocytes ; cytology ; Tumor Cells, Cultured ; U937 Cells

OBJECTIVETo explore the autophagy activity of CD34+ cells in bone marrow of MDS patients and its clinical significance.

METHODSThe activity of autophagy in bone marrow CD34+ cells from 20 MDS patients, 20 non-malignant anemia patients and 5 AML patients admitted in our hospital from October 2012 to March 2014 was detected by flow cytometry (FCM).

RESULTSThe autophagy activity in low risk MDS patients and non-malignant anemia patients were both significantly higher than that in both high risk MDS and AML patients (P<0.05), and more interestingly, the autophagy activity in MDS negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r=-0.877) .

CONCLUSIONThe autophagy activity CD34+ cells in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores, indicating that the decrease of autophagy activity maybe accelerate the genesis and development of MDS and relate with the prognosis of MDS patients.

Antigens, CD34 ; metabolism ; Autophagy ; Bone Marrow Cells ; cytology ; pathology ; Flow Cytometry ; Humans ; Leukemia, Myeloid, Acute ; pathology ; Myelodysplastic Syndromes ; pathology ; Prognosis

To detect the characteristics of "pre-ALIP" and to investigate their relevance with the development of acute myeloid leukemia (AML) by computer image procession technology, bone marrow (BM) was collected by aspiration/trephine biopsy from AML patients during the complete remission (CR). BM sections were stained by HGF (haematoxylin-Giemsa-acid fuchsin) and photographed by optical microscope imaging system. 4 kinds of computer image segmentation technologies were compared to select the best one for detecting the localization and quantitation of the precursor cells. Planimetry was combined with morphology to segment bone trabeculae. The number of single and double-cluster precursor cells and their distance from bone trabeculae was detected with Euclidean distance change method in BM images of AML patients, and compared with the normal controls. Moreover, the morphological characteristics of "pre-ALIP" were investigated, and the correlation with the development of AML was analyzed. The results showed that the computer image segmentation method based on morphology could identify the precursor cells and bone trabeculae more exactly in BM image, as compared with the methods of 8-Sobel operater. Canny operator and watershed algorithm. Bone trabeculae could be segmented with combinative methods of morphology and planimetry. The number of single precursor cells (19.27 ± 11.60)/mm(2) and double-cluster precursor cells (1.77 ± 1.76)/mm(2) in CR group were higher than that in normal controls (p < 0.05). The distance of single precursor cells from bone trabeculae in CR group were closer to bone trabeculae than that in controls [(230.12 ± 97.68) µm vs (260.92 ± 99.88 µm)] (p < 0.05), but the distance of double-cluster precursor cells from bone trabeculae in AML patients was (274.56 ± 139.48) µm, which showed no statistically significant different from controls (p > 0.05), while the double-cluster precursor cells showed the tendency of migrating to the intermediate zone of bone trabeculae compared with the single precursor cells in CR group (p < 0.05). It is concluded that the structure of "pre-ALIP" in BM tissue exists before the occurrence of ALIP. The characteristics of "pre-ALIP" are single and double-cluster precursor cells with abnormal localization or quantitation, which showed correlation with the development of AML.
Adolescent ; Adult ; Aged ; Bone Marrow Cells ; cytology ; pathology ; Female ; Humans ; Leukemia, Myeloid, Acute ; pathology ; Male ; Middle Aged ; Young Adult

Acute myelogenous leukemia (AML) cells are organized in a hierarchical fashion, with only the most primitive rare population (leukemia stem cell, LSC) of AML cells capable of maintaining the leukemic clone. A broad range of studies has indicated that AML results from mutations at the level of the stem cells of AML cells. The changes of cellular and molecular features in these malignant stem cells determine the features of leukemic clone and give rise to different subtypes of AML. LSCs share some similar characteristics with normal hematopoietic stem cells (HSC) including the ability to self-renew, and also have the potential of limited differentiation. LSCs, also have some features that are not found in normal HSC. LSCs have unique phenotype such as CD90-, CD117- and CD123+. Tumor-suppressor protein-death associated protein kinase and interferon regulatory factor 1 were overexpressed in LSCs, but not in normal HSC. Due to a predominantly G0 cell-cycle status, LSCs may not be responsive to conventional chemotherapeutic agents, compared with leukemia blasts. It is proposed that surviving LSCs are a major contributing factor to leukemic relapse. Although LSC population is likely to be drug-resistant, quiescent LSCs are preferentially susceptible to apoptosis induction while sparing normal HSC, with the appropriate stimulus such as proteasome inhibitor MG-132. This article reviewed the data emerging from the study of LSCs, and elucidated the distinct cellular and molecular characteristics of the LSC population, which may shed new light on AML therapy and leukemogenesis study.
Cell Count ; Cell Cycle ; Humans ; Leukemia, Myeloid, Acute ; pathology ; Neoplastic Stem Cells ; cytology ; Oligonucleotide Array Sequence Analysis

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME) and are critical for cancer initiation and progression. MicroRNAs (miRNAs) could notably influence the phenotype of TAMs through various targets and signal pathways during cancer progression due to their post-transcriptional regulation. In this review, we discuss mainly the regulatory function of miRNAs on macrophage differentiation, functional polarization, and cellular crosstalk. Firstly, during the generation process, miRNAs take part in the differentiation from myeloid cells to mature macrophages, and this maturation process directly influences their recruitment into the TME, attracted by tumor cells. Secondly, macrophages in the TME can be either tumor-promoting or tumor-suppressing, depending on their functional polarization. Large numbers of miRNAs can influence the polarization of macrophages, which is crucial for tumor progression, including tumor cell invasion, intravasation, extravasation, and premetastatic site formation. Thirdly, crosstalk between tumor cells and macrophages is essential for TME formation and tumor progression, and miRNAs can be the mediator of communication in different forms, especially when encapsulated in microvesicles or exosomes. We also assess the potential value of certain macrophage-related miRNAs (MRMs) as diagnostic and prognostic markers, and discuss the possible development of MRM-based therapies.
Cell Communication ; Cell Differentiation ; Cell Polarity ; Humans ; Macrophages/physiology* ; MicroRNAs/physiology* ; Myeloid Cells/cytology* ; Neoplasms/therapy* ; Tumor Microenvironment

OBJECTIVETo observe the viability and function of human bone marrow stem cell-derived hepatocytes following cryopreservation in vitro.

METHODSHuman bone marrow cells were induced to differentiate into hepatocytes in the presence of multiple factors. Mature hepatocytes were cryopreserved in 90% FBS and 10% DMSO (Group A), 10% FBS, 30% glycerol and 60% conditioned medium (Group B), and 10% FBS, 10% DMSO, and 80% UW solution (Group C). The cells were thawed after 4 weeks, and the cell viability and the albumin level were determined.

RESULTSThe human bone marrow derived hepatocytes maintained functional morphology after thawing. The viabilities in Group A, B and C were (60.0 +/- 3.3)%, (91.0 +/- 2.6)%, and (89.0 +/- 1.4)%, respectively. After culturing for 24 h, the albumin levels in Group A, B and C were (0.210 +/- 0.005) g/L, (0.340 +/- 0.020) g/L and (0.330 +/- 0.030) g/L, respectively.

CONCLUSIONSHuman bone marrow stem cell-derived hepatocytes can maintain the viability and function after cryopreservation. These cells may contribute to an important source of hepatocytes for clinical hepatocyte transplantation and artificial liver support system.

Adult ; Bone Marrow Cells ; cytology ; physiology ; Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; drug effects ; Cells, Cultured ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Hepatocytes ; cytology ; physiology ; transplantation ; Humans ; Myeloid Progenitor Cells ; cytology ; physiology

ZCH-2B8a (IgG2a) is a novel monoclonal antibody (McAb) generated in laboratory of Children Hospital of Medical College, Zhejiang University recently using human myeloblastic leukemia cell line KG1a as immunogen. This antibody has been submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. The aim of this study was to investigate the reactivity of this antibody on normal blood cells and malignant cell lines and to explore its possible application in clinical practice. The multi-parameter flow cytometry was used to analyze the expression pattern of 2B8a antigen in triplicate on normal blood components including T cells, B cells, natural killers (NK), neutrophils, monocytes, dendritic cells (DC), red blood cells (RBC), platelets (Plt), hematopoietic stem/progenitor cells derived from either bone marrow or G-CSF mobilized peripheral blood CD34(+) cells and malignant cell lines including 14 hematopoietic, 5 neuroblastoma, 1 colon cancer and 1 amniotic epithelium cell lines. The amount of positive cells > or = 20% was considered as positivity. The results showed that 2B8a antibody reacted to 3/3 specimens of blood B cells with a positive rate of 26.29% and 2/3 specimens of monocytes with an average positive rate of 59.84%. 2B8a was weakly reactive to neutrophils (23.72%) and negative for T cells, NK, DC, RBC and Plt. The antibody reacted to all 3 marrow CD34(+) cells with an average positive rate of 39.33% while it was negative for G-CSF-mobilized CD34(+) peripheral blood stem/progenitor cells (PBSC, 1.25%). Cell line analysis showed that the antibody notably reacted to three out of 4 cell lines (Raji, SMS-SB, Nalm-6 and Nall-1) with the positive rates of 98.78%, 98.61%, 94.93% respectively and weakly to one of them with 5.68% in B lineage cell lines and monoblastic cell line (U937, 67.78%) while it was only weakly positive or negative for other myeloid leukemia cell lines including Meg01 (33.40%), HL-60 (29.70%), K562 (28.19%), KG1a (16.23%) and HEL92.1.7 (8.02%). Among 4 T lineage leukemia, 5 neuroblastoma and 1 colon cancer cell lines tested, only Molt-3 was found weakly positive (31.40%) for 2B8a, while the remaining 3 T cell lines (Molt4, JM and CCRF-CEM), 5 neuroblastoma cell lines (LA-N1, KCNR, BE, SK-N-SH, SK-N-AS) and the colon cancer cell line (HR8348) tested were negative. An amniotic epithelium cell line (FL) was showed positive for the antibody (45.03%). It is concluded that 2B8a antibody primarily reacts to B lineage and monocytic lineage cells which may bear the diagnostic and therapeutic applications among different types of hematopoietic malignancies.
Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; Antigen-Antibody Reactions ; Antigens, Neoplasm ; analysis ; immunology ; Antigens, Surface ; immunology ; B-Lymphocytes ; cytology ; immunology ; HLA Antigens ; immunology ; Hematopoietic Stem Cells ; cytology ; immunology ; Hematopoietic System ; cytology ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; Tumor Cells, Cultured