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The ultrastructural characteristics of apoptosis have been described microscopically for four decades. Alterations of nuclei, apoptotic bodies, cytoplasm, and some organelles have been illustrated and investigated during apoptosis. The successive changes of cellular components corresponding with differentiation of apoptotic cells are illustrated in the present review, based on ultrastructural observation of leukemia cells of patients in our routine clinic work by transmission electron microscopy. Most electron micrographs demonstrated that membranous components of nuclear envelop, rough endoplasmic reticulum and Golgi apparatus, and mitochondria were degenerated step by step during apoptosis. The successive images suggested that the endoplasmic reticulum and Golgi apparatus were transferred to cell surface from cytoplasm and participated in formation of apoptotic bodies in apoptosis, although relevant clinical data and more experimental evidence were needed for restraining of leukemia cases from diagnostic work randomly in recent decades. [ABSTRACT FROM PUBLISHER]

Megakaryocytes (MKs) build characteristic structures to produce platelets in a series of steps. Although mechanisms of demarcation membrane system (DMS) and open canalicular system transformation have been proposed based on experimental studies in recent decades, the related evidence is lacking in human cells in vivo. The present review describes and discusses the development of MKs, transformation of DMS, and the release and maturation of proplatelets based on our observation of human MKs in vivo and bone marrow biopsy by light microscope and transmission electron microscope. Four stages were subdivided from megakaryoblasts to matured cells; presumption of DMS transformation from endoplasmic reticulum and Golgi apparatus were evidenced in contrast to another presumption of DMS transformation from plasma membrane in this review. Effectors of interaction between hematopoietic cells, the sucking and shearing force of sinus blood flow on movement of MKs, and release of proplatelets were emphasized. Additionally, the mechanism of secondary splitting of proplatelets in circulation was demonstrated ultrastructurally. These findings and conceptions might significantly promote our understanding of the mechanism of platelet production in human in vivo cells. [ABSTRACT FROM AUTHOR]

Objective To analyze the differences in biological functions between bone marrow(BM)-derived CD106 + mesenchymal stem cells(MSCs)and the CD106 - subgroup. Methods The MSCs from normal BM were isolated and expanded.The subgroups of CD106 + and CD106 - MSCs were sorted.The cell proliferation and adhesion functions,chemotactic activities,adipogenic and osteogenic potentials,senescence,and senescence protein 21(p21)were detected.The capacity of translocation into nucleus of nuclear factor-kappa B(NF-κB)when stimulated by tumor necrosis factor(TNF-α)was measured. Results The proliferative ability was higher in CD106 + MSCs than that in CD106 - MSCs.In 48 hours,the value of optical density(OD)was significantly higher in CD106 + MSCs than that in CD106 - subgroup(1.004±0.028 vs. 0.659±0.023, t =3.946, P =0.0225).In 72 hours,this phenomenon was even more pronounced(2.574±0.089 vs. 1.590±0.074, t =11.240, P =0.0000).The adhesive capacity of CD106 + MSCs was significantly stronger than that of CD106 - subgroup(0.648±0.018 vs. 0.418±0.023, t =7.869, P =0.0002).Besides,the metastasis ability of CD106 + MSCs were significantly stronger than that of CD106 - subgroup(114.500±4.481 vs. 71.000±4.435, t =6.900, P =0.0005).The CD106 + MSCs had signifcnatly lower proportions of senescent cells.The expression of aging protein p21 in CD106 + MSCs was significantly lower than that in CD106 - MSCs [(17.560±1.421)% vs. (45.800±2.569)%, t =9.618, P =0.0000].Furthermore,there were no visible pigmenting cells after β-galactosidase staining in CD106 + MSCs subgroup.However,in CD106 - MSCs,some colored green cells were detected.The rate of NF-κB translocation into nucleus after stimulated by TNF-α was significantly higher in CD106 + MSCs than CD106 - MSCs [(37.780±3.268)% vs. (7.30±1.25)%, t =8.713, P =0.0001]. Conclusion Bone marrow-derived CD106 + MSCs possess more powerful biological functions than CD106 - MSCs.

Objective: To investigate the Raman spectral characteristics of leukemia cells from 4 patients with acute promyelocytic leukemia (M 3 ) and 3 patients with acute monoblastic leukemia (M 5 ), establish a novel Raman label-free method to distinguish 2 kinds of acute myeloid leukemia cells so as to provide basis for clinical research., Methods: Leukemia cells were collected from bone marrow of above-mentioned patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 30-50 cells from each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA) and cluster analysis, and the spectra of leukemia cells from 7 patients were analyzed and classified. Characteristics of Raman spectra were analyzed combining with ultrastructure of leukemia cells., Results: There were significant differences between Raman spectra of 2 kinds of leukemia cells. Compared with acute monoblastic leukemia cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks in 622, 643, 757, 852, 1003, 1033, 1117, 1157, 1173, 1208, 1340, 1551, 1581 cm -1 . The diagnostic models established by PCA-DFA and cluster analysis could successfully classify these Raman spectra of different samples with a high accuracy of 100% (233/233). The model was evaluated by "Leave-one-out" cross-validation and reached a high accuracy of 97% (226/233)., Conclusion: The level of macromolecules of M 3 cells is higher than that of M 5 . The diagnostic models established by PCA-DFA can classify these Raman spectra of different cells with a high accuracy. Raman spectra shows consistent result with ultrastructure by TEM.

Objective: To analyze and evaluate the application of spinning disk confocal microscopy and imaging analysis software in movement and phagocytosis of neutrophils., Methods: Neutrophils were isolated from bone marrow by centrifugation on discontinuous Percoll gradient, and then were stained with PE Gr-1 antibody and mixed with FITC-labeled Zymosan A bioparticles. Multichannel time-lapse videos were captured by using the spinning disk confocal microscopy. The result was analyzed by using volocity and ImageJ software, the parameters associated with movement and phagocytosis of neutrophils were analyzed, including morphological changes, cell tracking, pseudopod dynamics, binding and phagocytosis index., Results: Most neutrophils would be polarized in response to Zymosan particles during a short time. Binding and phagocytosis process occured in forty minutes., Conclusion: A method of precisely quantifying the movement and phagocytosis of neutrophils using microscopic imaging and imaging analysis technique has been set up successfully. Using this method, biological activity and function of neutrophils can be evaluated visually and rapidly. The physiologically rapid response to Zymosan particles can be applied to the neutrophils function research in the future.

The genome instability and tumorigenicity of induced pluripotent stem cells (iPSC) hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells (CB MNC), to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre-stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV (spleen focus forming virus) promoter under the hypoxic condition (3%), CB MNC could be mostly efficiently reprogrammed with the efficiency 0.12%. Furthermore, the results showed that erythroblasts (CD36(+)CD71(+)CD235a(low)) were the cells that are reprogrammed with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.

Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.