Effects of long-term subculture on biological characteristics of bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND:There is still controversy whether human bone marrow mesenchymal stem cells can maintain their biological characteristics,energy metabolism patterns,and multidirectional differentiation potential after long-term expression in vitro.Further comprehensive and systematic research is needed. OBJECTIVE:To evaluate the effects of long-term expansion in vitro on the biological characteristics of mesenchymal stem cells. METHODS:Human bone marrow mesenchymal stem cells cultured to passage 5,10,and 15 in vitro.MTT assay was used to detect cell proliferation ability.Flow cytometry was used to detect cell cycle.The multi-differentiation potential of mesenchymal stem cells was detected by inducing to adipogenic,osteogenic and chondrogenic differentiation.Cell migration and invasion abilities were detected by scratch test and Transwell assay.The mitochondrial oxidative phosphorylation and glycolysis function were analyzed using energy metabolism analyzer.The cell senescence was detected by senescence-associated β-galactosidase staining.The expression levels of p21,p16,and p53 proteins were detected by western blot assay. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells at passages 5,10,and 15 grew adherently;the volume of passage 15 mesenchymal stem cells increased and its proliferation ability decreased;the percentage of S-phase cells decreased(P<0.05).With the increase of culture passages,the migration and invasion abilities decreased gradually(P<0.05).There was no significant difference in the differentiation potential,demonstrated by adipogenic,osteogenic and chondrogenesis induction.The ability of oxidative phosphorylation of mitochondria and glycolysis decreased gradually(P<0.05).The number of senescence-associated β-galactosidase-positive cells increased with the increase of passages(P<0.05),and the expression of senescence protein p21,p16,and p53 increased gradually(P<0.05).The results indicated that the biological characterization of mesenchymal stem cells changed after long-term in vitro expansion.Mesenchymal stem cells cultured over 10 passages may have a reduced activity due to increasing senescence.Therefore,bone marrow mesenchymal stem cells cultured less than 10 passages are suitable for clinical research/therapy.

Design and experiment of online monitoring system for long-term culture of embryo / 生物医学工程学杂志

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for

Extended culture up to the blastocyst stage: a strategy to avoid multiple pregnancies in assisted reproductive technologies

The aim of this study was to review the experience and outcomes of assisted reproduction cycles with embryos grown up to day 5 of development, comparing different parameters according to the ages of the patients. We retrospectively studied 1,874 assisted reproduction cycles where embryo culture was extended up to the fifth or sixth day of development. All IVF and ICSI cycles were included, comparing, according to patient age, the following rates: blastocyst formation, pregnancy, implantation and abortion. As control, we analyzed cycles with donated oocytes from young donors (OD). The number of embryos reaching the blastocyst stage is similar in all groups of patients. Only the OD group was different in terms of blastocyst formation, pregnancy and implantation rates. Patients over 39 years of age had an abortion rate of 59.1 percent, which is significantly higher than the other groups. Extended embryo culture up to the blastocyst stage can be implemented in programs of assisted reproduction in order to increase the pregnancy rate. The potential of blastocyst implantation is high, allowing us to transfer fewer embryos and reduce the probability of multiple pregnancies.

Long-term culture of utricular sensory epithelial cell of rats and expression of the hair cell characteristic markers / 临床耳鼻咽喉头颈外科杂志

Objective:To investigate the generative or regenerative hair cell and studes of molecular and genetic in the inner ear,the long-term culture systems of utricular sensory epithelial cell of the rats were established.Method:Utricular sensory epithelial of postnatal day 1 rats was isolated by mechanical dissociation.The explants were digested by thermolysin,then transfrred to an aliquot containing trypsin and collagenase for incubation to harvest the pure utricular sensory epithelial cell. USEC wes cultured in Dulbecco modified Eagle medium(DMEM) and passaged.USEC of the 25 passages observed by inverted microscope and ultrastrctural examination with transmission electron microscope.Immunocytochemical method with cytokeratin 18 ,vimentin,Brn3.a and Calretinin; reverse transcription PCR with mRNA of AchRa9 and Myosin Ⅶa.The markers of hair cells were used identify the characteriztion of USEC of the 25 passages.Result:USEC have been cultured for more than 6 months and passaged 25 passages.USEC of the 25 passages showed a large,flat,polygonal epichlial morphotype with big,round neuclei.The cells grew into monolayer,cobblestone-likeappearance and showed Dome formation. The cells expressed cytokeratin 18 , not expressed vimentin,has rich microvilli and complex tight junction,which indicated the epithlial origination of USEC.Coexpressed of the hair cell characteristic markers Brn3.a,Calretinin and mRNA of AchRa9,Myosin Ⅶa was identified the culture cell that may represent rat progenitor hair cell.Conclusion:The long-term culture systems of utricular sensory epithelial cell of the rats were successfully established. The long-term culture USEC coexpressed the characteristic markers of the hair cell ,which identified the culture cell may represent progenitor cell of rats hair cell .It may provide valuable cell sources for in-depth investigation the mechanisms of hair cell generation or regeneration and studes of molecular and genetic in the inner ear.

Comparison of Hematopoietic Stem Cell Activity in Normal Bone Marrow, in vivo G-CSF Stimulated Bone Marrow, and G-CSF-Mobilized Peripheral Blood / 대한수혈학회지

BACKGROUND: Classically, bone marrow (BM) has been the sole source of hematopoietic stem cell transplantation, but limitations of conventional bone marrow transplantation have stimulated a search for alternative sources of stem cells. METHODS: We compared hematopoietic stem cell activity of normal bone marrow (BM), in vivo G-CSF-stimulated bone marrow (G-CSF BM), and G-CSF-mobilized peripheral blood (G-CSF PB) by immunophenotyping, clonogeneicity, and long-term culture-initiating cell (LTC-IC) analysis. RESLUTS: The average numbers of CD34+/HLA-DR- cells after CD34+ cells isolation from each stem cell source were 59.64 +/- 8.70%, 91.39 +/- 1.98%, and 95.75 +/- 2.08% in normal BM, G-CSF BM, and G-CSF PB, respectively (normal BM vs. G-CSF BM, normal BM vs. G-CSF PB, p<0.0001). And the average numbers of CD34+/CD38- cells were 66.23 +/- 9.33%, 95.08+/- 2.09%, and 91.76 +/- 4.59% in normal BM, G-CSF BM, and G-CSF PB, respectively (normal BM vs. G-CSF BM, normal BM vs. G-CSF PB, p<0.0001). The numbers of CFU-GM was significantly higher in G-CSF PB (53.2 +/- 4.05) and G-CSF BM (52.5 +/- 3.63) than that of normal BM (31.3+/- 5.50) (p<0.0001). Also the numbers of CFU-GEMM and CFU-Mk were also significantly higher in G-CSF PB (110.3 +/- 8.79 and 13.3 +/- 1.49) and G-CSF BM (109.7 +/- 10.78 and 11.2 +/- 1.69) than that of normal BM (48.8 +/- 1.48 and 8.5 +/- 1.72) (p<0.05). Comparison of LTC-IC in the three sources of stem cells showed that G-CSF PB and G-CSF BM were superior to normal BM at five weeks of culture (p<0.05). CONCLUSIONS: These data suggest that the amount of both early progenitor cells and late progenitor cells in G-CSF PB and G-CSF BM are higher than that of normal BM. And our results further support that the higher stem cell transplantation using G-CSF-mobilized PB and in vivo G-CSF-stimulated BM can lead to more rapid and sustained engraftment even in cases of high risk of rejection.

Immunophenotypic Analysis of Long-term Culture-Initiating Cells in Long-term Liquid Culture / 대한소아혈액종양학회지

Purpose: Long-term culture- initiating cells(LTC-IC) are stem cells that have the capacities of long-term engraftment and helping to establish hematopoietic microenvironment. For evaluation of the LTC-IC, we measured the counts and function with multidimentional flowcytometry in long-term culture media. METHODS: Samples were obtained from umbilical cord blood, leukapheresis products and bone marrow(BM). LTC-IC were counted with flowcytometric analysis using anti- CD34, anti-CD38, and anti-HLA-DR antibodies at 0, 5, and 8 weeks. Cell adhesion molecule related with stem cell were evaluated with flowcytometric analysis also using anti-VCAM-1(CD106) and anti-VLA-4(CD49d) at 0 and 8 weeks. RESULTS: The proportion of CD34+/CD38- cell from fractionated mononuclear cells at 0 week were 0.46%, 0.044%, and 0.038% for BM, leukapheresis products, and umbilical cord blood respectively and then rapidly decreased at 5 weeks, but still persisted at 8 weeks in all three groups. The proportion of CD34+/HLA-DR- cells was the same tendency to CD34+/CD38-. VCAM+ expression rate from fractionated CD34+ cells at 0 and 8 weeks were 67.3% and 40.2% for BM and 64.1% 44.2% for umbilical cord blood but it was very low 31.2% and 5.1% for leukapheresis products. VLA-4+ expression rate for fractionated CD34+ cells at 0 and 8 weeks were similar tendency to VCAM+ cells. CONCLUSION: This study suggest that the count of LTC-IC decreased with time but still persisted until 8 weeks. Umbilical cord blood including BM help to establish the hematopoietic microenvironments.