scholarly journals Cell-cycle manipulation of human leukemic progenitor cells with humoral adjustment in vitro

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 179-182 ◽  
Author(s):  
CH Park
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Conditioned Medium ◽  
Culture Medium ◽  
Culture Method ◽  
Leukemic Cells ◽  
Agar Culture ◽  
Daily Feeding

Abstract The cell-cycle change of human leukemic colony-forming cells was studied using a new agar culture method featuring daily feeding of new culture medium with or without leukocyte conditioned medium (LCM). Leukemic cells could be kept out of cycle by withholding LCM from daily feeding and put back into cycle by adding LCM to the daily feeding.

Cell-cycle manipulation of human leukemic progenitor cells with humoral adjustment in vitro

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 179-182
Author(s):  
CH Park
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Conditioned Medium ◽  
Culture Medium ◽  
Culture Method ◽  
Leukemic Cells ◽  
Agar Culture ◽  
Daily Feeding

The cell-cycle change of human leukemic colony-forming cells was studied using a new agar culture method featuring daily feeding of new culture medium with or without leukocyte conditioned medium (LCM). Leukemic cells could be kept out of cycle by withholding LCM from daily feeding and put back into cycle by adding LCM to the daily feeding.

scholarly journals Immune regulation of in vitro murine megakaryocyte development. Role of T lymphocytes and Ia antigen expression.

1985 ◽  
Vol 162 (6) ◽  
pp. 2053-2067 ◽  
Author(s):  
M W Long ◽  
D N Shapiro
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
Immune Modulation ◽  
Immune Recognition ◽  
Activated T Cells ◽  
Ia Antigen ◽  
Cell Cycle Status

Mitogen-activated murine T lymphocytes or T cell hybridomas produce an activity (megakaryocyte [Mk] potentiator activity) that enhances the in vitro growth and development of Mk colonies. This activity was found in optimal concentrations (2.5%) in T cell hybridoma-conditioned medium, and was also produced by feeder layers of concanavalin A-activated T cells. A subpopulation of murine Mk progenitor cells (colony-forming units; CFU-Mk) bears the Ia antigen. Separate experiments indicated that T cell products stimulate CFU-Mk by increasing their basal levels of Ia expression as well as the frequency of cells actively synthesizing DNA. The hypothesis that the expression of this antigen was related to the cell cycle status of these progenitor cells was confirmed in studies that indicated that ablation of actively cycling cells in vivo abrogated the cytotoxic effects of anti-Ia monoclonal antibodies. The interdependence of T cell lymphokine regulation of both Ia expression and cell cycle status was also seen in in vitro experiments in which Ia+ progenitor cells were eliminated by complement-dependent cytotoxicity. The removal of Ia+ cells prevented 5-hydroxyurea-mediated inhibition of cells in S phase. We hypothesize that immune modulation of megakaryocytopoiesis occurs via soluble T cell products that augment Mk differentiation. Further, the mechanism of immune recognition/modulation may occur via Ia antigens present on the surface of these progenitor cells.

scholarly journals A liquid culture system for murine megakaryocyte progenitor cells

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 250-257
Author(s):  
T Nagasawa ◽  
M Nakazawa ◽  
T Abe
Keyword(s):  
Progenitor Cells ◽  
Conditioned Medium ◽  
Culture System ◽  
Liquid Culture ◽  
Culture Medium ◽  
Pokeweed Mitogen ◽  
Plasma Clot ◽  
Liquid Culture System ◽  
Cell Conditioned Medium ◽  
Number Of Cells

A liquid culture system is described for murine megakaryocyte progenitor cells (CFU-M) in the presence of pokeweed-mitogen-stimulated spleen-cell conditioned medium. There were dose-related responses between the number of CFU-M developed and the number of cells cultured and the dosage of conditioned medium in this liquid culture system. Murine CFU-M were abundantly cloned in this system an the plating efficiency was similar in comparison with that in a plasma clot system. The acetylcholinesterase-positive colonies (more than 4 acetylcholinesterase-positive cells) were clearly seen on day 3 of culture, and they reached a maximum (60.5 +/- 10.7/2 x 10(5) cells) on day 7 of culture. Ultrastructural analyses of megakaryocytic maturation in this system showed that a few megakaryocytes produced platelets that were released in the culture medium on day 5 of culture. This liquid culture system is suitable for the study of the dynamic process of the megakaryocyte-platelet system.

scholarly journals Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sadaf Vahdat ◽  
Sara Pahlavan ◽  
Elena Mahmoudi ◽  
Maryam Barekat ◽  
Hassan Ansari ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Large Scale ◽  
Culture Medium ◽  
Gene Expression Pattern ◽  
Extended Period ◽  
Scale Production ◽  
Chemical Screening ◽  
Wide Range ◽  
Cell Sources

Abstract Cardiovascular progenitor cells (CPCs) derived from human pluripotent stem cells (hPSCs) are proposed to be invaluable cell sources for experimental and clinical studies. This wide range of applications necessitates large-scale production of CPCs in an in vitro culture system, which enables both expansion and maintenance of these cells. In this study, we aimed to develop a defined and efficient culture medium that uses signaling factors for large-scale expansion of early CPCs, called cardiogenic mesodermal cells (CMCs), which were derived from hPSCs. Chemical screening resulted in a medium that contained a reproducible combination of three factors (A83-01, bFGF, and CHIR99021) that generated 1014 CMCs after 10 passages without the propensity for tumorigenicity. Expanded CMCs retained their gene expression pattern, chromosomal stability, and differentiation tendency through several passages and showed both the safety and possible cardio-protective potentials when transplanted into the infarcted rat myocardium. These CMCs were efficiently cryopreserved for an extended period of time. This culture medium could be used for both adherent and suspension culture conditions, for which the latter is required for large-scale CMC production. Taken together, hPSC-derived CMCs exhibited self-renewal capacity in our simple, reproducible, and defined medium. These cells might ultimately be potential, promising cell sources for cardiovascular studies.

Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1427-1434 ◽  
Author(s):  
Ivan N. Rich ◽  
Diana Worthington-White ◽  
Oliver A. Garden ◽  
Philip Musk
Keyword(s):  
Cell Cycle ◽  
Intracellular Ph ◽  
Annexin V ◽  
Leukemic Cell ◽  
Differential Sensitivity ◽  
Leukemic Cells ◽  
Hematopoietic Tissue ◽  
Leukemic Cell Lines ◽  
Targeted Inhibition

The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.

102 PROTEOMIC ANALYSIS OF CONDITIONED MEDIUM SUPPLEMENTED WITH PORCINE FOLLICULAR FLUID

2011 ◽  
Vol 23 (1) ◽  
pp. 156
Author(s):  
S. Hwang ◽  
K. B. Oh ◽  
H.-C. Lee ◽  
B.-C. Yang ◽  
D. Lim ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Follicular Fluid ◽  
Culture Medium ◽  
Cell Metabolism ◽  
Eukaryotic Cell ◽  
Amino Acid Sequences ◽  
Homology Search ◽  
Surface Binding ◽  
Culture Methods

Follicular fluid (FF) contains growth factors, electrolytes, hormones, amino acids, and unknown factors. Supplementation of porcine FF (pFF) to in vitro maturation (IVM) medium was reported to improve the oocyte maturation, monospermic fertilization and embryonic development. This study aimed at investigating whether pFF supplementation affects the characteristics of donor cells for somatic cell nuclear transfer and the proteomic composition of the culture medium. Ear fibroblast cells from an NIH major histocompatibility complex (MHC) inbred miniature pig were cultured with different culture methods: 1) DMEM + 10% FBS (FBS); 2) DMEM + 10% FBS + 10% pFF (pFF). The conditioned medium was collected at 72 h. After isoelectric focusing (IEF), the equilibrated strips were submitted to SDS-PAGE. Normalized protein spots were considered significantly different between the two groups if expression levels varied by two standard deviations. To identify the protein spots, an Ettan MALDI-TOF method was used. Upon submission of the amino acid sequences, proteins were identified by a homology search using ProteinInfo or BLAST search using the ExPASy Molecular Biology Server. The proportion of G0/G1 stage cells in the pFF group was significantly higher than the proportions in the other groups (P < 0.05). Among 42 differentially expressed spots, 36 proteins were identified in the pFF group. Some molecular functions of the spots were: catalytic or methytransferase activity, eukaryotic cell surface binding, or ferric iron binding. It can be concluded that pFF supplementation of culture medium positively affects cell-cycle synchronization and cell metabolism. Further studies are needed to analyse the function of these important cellular proteins. This work received grant support from the Agenda Program (No. PJ006688) and (No. PJ007189), Rural Development Administration, Republic of Korea.

92 Culture method for long-distance transport of bovine embryos derived from IVF before blastulation using microtubes

2019 ◽  
Vol 31 (1) ◽  
pp. 172
Author(s):  
T. Yamanouchi ◽  
H. Matsuda ◽  
K. Ogata ◽  
Y. Hashiyada
Keyword(s):  
Culture Medium ◽  
Liquid Paraffin ◽  
Calf Serum ◽  
Petri Dish ◽  
Culture Method ◽  
Optimal Number ◽  
Blastocyst Development ◽  
Long Distance

In vitro-produced (IVP) embryos are more easily damaged by cryopreservation than in vivo-derived embryos. Therefore, transportation of fresh IVP embryos in a manner that can maintain viability is necessary. This study was conducted to determine the preferable culture conditions for transport of embryos at 5 days post-insemination (dpi) in 1.5-mL microtubes. Cumulus-oocyte complexes derived from an abattoir were matured and then inseminated with frozen-thawed semen. Presumptive zygotes were cultured in mCR1aa (CR1)+5% calf serum (CS) until use. In Exp. 1, embryos with 5 blastomeres at 5 dpi were randomly assigned to 1 of 3 groups: 25mM Hepes-CR1aa (H-CR1)+5% CS or 25mM Hepes-M199 (H-M199)+5% CS in air, or CR1 in 5% CO2. Embryos were cultured in microdrops overlaid with liquid paraffin in a petri dish for 48h at 38.5°C. In Exp. 2, the optimal number of embryos to culture per microtube was assessed. Presumptive zygotes were cultured in groups of 20, 40, or 80 in 1mL of CR1 covered with liquid paraffin in microtubes in an incubator at 38.5°C in 5% CO2 until 7 dpi. For Exp. 3, culture of embryos in microtubes in a portable incubator was tested. At 5 dpi, 5-cell embryos (n=17 to 36 per microtube) were statically cultured in 1mL of CR1 or H-CR1 in microtubes in a portable incubator set at 38.5°C for 48h. The CR1 was pre-equilibrated in an incubator in 5% CO2 for 24h before use. Embryos were harvested from microtubes after 48h and were then cultured in microdrops of CR1 overlaid with liquid paraffin in a petri dish in an incubator at 38.5°C in 5% CO2 until 8 dpi. In Exp. 4, embryos (n=29 to 39 five-cell embryos per microtube) were transported in a portable incubator by land for 1000km over a period of 44h using the same conditions as in Exp. 3. Control embryos were statically cultured in microdrops of CR1 in an incubator in 5% CO2. Statistical analyses were carried out by ANOVA (Exp. 1 and 2), t-test (Exp. 3), or Fisher’s exact test (Exp. 4). In Exp. 1, there was no effect (P&gt;0.05) of culture medium on blastocyst development at 7 dpi (27.6±2.3, 25.7±7.2, and 17.3±2.9% for CR1, H-CR1, and H-M199, respectively). In Exp. 2, blastocyst development at 7 dpi was not affected (P&gt;0.05) by the number of presumptive zygotes cultured per microtube (43.6±8.3, 42.4±4.0, and 39.9±2.9% for 20, 40, and 80 presumptive zygotes, respectively). In Exp. 3, blastocyst development at 8 dpi was not affected (P&gt;0.05) by culture medium (60.7±7.4 and 53.1±4.4% for CR1 and H-CR1, respectively); however, the pH of CR1 changed from 7.5 to 8.1 at 48h after culture. In Exp. 4, blastocyst development at 8 dpi was not affected (P&gt;0.05) by transport (57.1, 64.4, and 75.5% for CR1, H-CR1, and control, respectively). These results indicate that IVP embryos harvested at 5 dpi can be transported by portable incubator with no effect on embryo development to the blastocyst stage. This work was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and the development for next-generation technology).

scholarly journals Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takafumi Matsumura ◽  
Takuya Sato ◽  
Takeru Abe ◽  
Hiroyuki Sanjo ◽  
Kumiko Katagiri ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Culture Medium ◽  
Animal Species ◽  
Culture Method ◽  
Tension Control ◽  
Significant Progress ◽  
In Vitro Spermatogenesis ◽  
Lower Oxygen ◽  
Rat Spermatogenesis

AbstractIn vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

Cell Cycle Progression and Self-Renewal Divisions of Cord Blood Derived CD34+ Cells Treated with the Polyamine Copper-Chelator Tetraethylenepentamine.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4237-4237
Author(s):  
Toni Peled ◽  
Noga R. Goudsmid ◽  
Frida Grynspan ◽  
Sophie Adi ◽  
Efrat Landau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Fluorescence Intensity ◽  
Cell Expansion ◽  
Progenitor Cell ◽  
Self Renewal ◽  
Cell Cycle Profile ◽  
Cd34 Cells ◽  
Copper Chelator

Abstract In vitro cell expansion is constrained by default pathways of commitment and differentiation resulting in limited expansion of hematopoietic stem-progenitor cells (HSPCs). Still, several ex vivo manipulations have been reported to achieve expansion of HSPCs by altering cell cycle kinetics and enhancing progression through the G1-S barrier. We have previously shown that addition of tetraethylenepentamine (TEPA), a polyamine copper chelator, to cytokine-supplemented CD34+ cell cultures modulates cytokine-driven hematopoietic cell fate in vitro, resulting in remarkable expansion of a cell population that displays phenotypic and functional characteristics of HSPCs (Exp Hematol.2004;32 (6):547–55). The objective of the present study was to evaluate the mechanism leading to expansion of early progenitor cells following short-term exposure to TEPA. To this end, cell cycle profile, tracking of proliferation history, as well as determination of actual numbers of progenitor subsets were studied. In order to follow the extent of proliferation by tracking the number of cellular divisions, freshly isolated CD34+ cells were labeled with PKH2, a membrane dye that is sequentially diluted during every cell division. Fluorescence intensities of CD34+ and that of a more immature CD34+CD38− cell subset were determined immediately after staining. The cells were then cultured in serum-containing medium and a cocktail of cytokines (SCF, TPO, IL-6, Flt3-ligand, at 50 ng/ml each and IL-3 at 20 ng/ml), with and without TEPA. Total nucleated cells (TNC), purified CD34+ cells and CD34+CD38− cells were analyzed for PKH2 fluorescence intensity during the first two weeks of culture. Cell cycle profile was detected with the DNA intercalating agent propidium iodide, which determines cellular DNA content. FACS analysis of the cultured cells as well as progenitor cell quantification by immuno-affinity purification revealed comparable expansion levels of TNC and CD34+ cells in both TEPA-treated and control cultures during the first two weeks, as previously published. Although similar CD34+ cell numbers were observed, the mean frequency of CD34+CD38− and CD34+CD38-Lin- cells within the CD34+ cell population was significantly higher in TEPA-treated cultures over the control (0.2 vs. 0.04 and 0.07 vs. 0.01, respectively; n=6, p&lt;0.05). Median PKH2 fluorescence intensity of CD34+CD38− subset was two fold higher in TEPA than in control cultures, demonstrating that early progenitor cells derived from TEPA-treated cultures consistently accomplished less proliferation cycles as compared to early progenitor cells derived from control cultures. This effect was not mirrored by a significant alteration of the cell cycle profile (Control (%): G1=26±14, S=2.6±0.1, G2=0.7±0.4; TEPA(%): G1=29±12, S=1.7±0.9, G2=0.4±0.2). Taken together, the data suggest that during cycling, the CD34+CD38− phenotype is preserved more successfully in TEPA-treated than in control cultures, suggesting retention of self-renewing potential of early progenitor cells under these culture conditions. This mechanism also supports a role for TEPA in inhibition of early progenitor cell differentiation. Ongoing work is aimed at further defining whether phenotype reversion or self-renewal (or both) lie at the foundation of TEPA-mediated progenitor cell expansion.

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