scholarly journals Effects of C-Mpl Ligand on Cytoplasmic Maturation of Murine Megakaryocytes and on Platelet Production

1998 ◽  
Vol 46 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Yoji Ishida ◽  
Toshiharu Ito ◽  
Shin-ichiro Kuriya
Keyword(s):  
Progenitor Cells ◽  
Biological Effects ◽  
Primary Factor ◽  
Serum Free ◽  
Platelet Production ◽  
Nuclear Development ◽  
Cytoplasmic Maturation ◽  
Serum Free Conditions ◽  
Ache Expression

To test the hypothesis that the c-mpl ligand is not a primary factor in thromb-ocytopoiesis, we investigated the biological effects of recombinant human (rh) c-mpl ligand on differentiation of murine progenitor cells and on maturation of the cultured murine megakaryocytes under serum-free conditions on the basis of ploidy distribution, megakaryocyte/platelet-specific surface antigen CD 61 [glycoprotein (GP) IIIa], and cytoplasmic acetylcholinesterase (AchE) expression in vitro. In addition, we studied the effect of c-mpl ligand on proplatelet formation (PPF) by murine mature megakaryocytes. AchE was less strongly expressed in cultured megakaryocytic cells stimulated by c-mpl ligand than in those stimulated by recombinant murine (rm) IL-3 + rh IL-6 during the differentiation of progenitor cells. Less CD 61 was expressed by c-mpl ligand during both the differentiation of progenitor cells and the maturation of megakaryocytes compared with that by rm IL-3 + rh IL-6. Endomitosis, however, was more stimulated by c-mpl ligand than by rm IL-3 + rh IL-6 under both conditions. Furthermore, PPF of mature megakaryocytes was not stimulated by c-mpl ligand. These results indicate that c-mpl ligand stimulates the nuclear development of megakaryocytic cells but that it does not stimulate cytoplasmic maturation and PPF as much as IL-6. These data strongly suggest that c-mpl ligand is not a primary factor in platelet production.

scholarly journals Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells

2020 ◽  
Vol 90 ◽  
pp. 52-64.e11 ◽  
Author(s):  
Hanyang Lin ◽  
Jackie E. Damen ◽  
Marta A. Walasek ◽  
Stephen J. Szilvassy ◽  
Ali G. Turhan ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Serum Free

scholarly journals Defined, serum-free conditions for in vitro culture of primary human T-ALL blasts

Leukemia ◽  
2012 ◽  
Vol 27 (6) ◽  
pp. 1437-1440 ◽  
Author(s):  
A J Yost ◽  
O O Shevchuk ◽  
R Gooch ◽  
S Gusscott ◽  
M J You ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Serum Free ◽  
Serum Free Conditions

scholarly journals Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1867-1871 ◽  
Author(s):  
AR Migliaccio ◽  
M Bruno ◽  
G Migliaccio
Keyword(s):  
Direct Action ◽  
Erythroid Progenitors ◽  
Serum Free ◽  
Gm Csf ◽  
Plastic Surface ◽  
Blast Cells ◽  
Fetal Bovine ◽  
Serum Free Conditions ◽  
Stimulating Factor

Abstract The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.

FDA-Approved Pharmacological Agents, Romiplostium and Oprelvekin, Synergistically Promote Megakaryocytic Differentiation From Human iPSCs In a Chemically Defined System

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1208-1208
Author(s):  
Yanfeng Liu ◽  
Yongxing Gao ◽  
Sid Shah ◽  
Lewis Becker ◽  
Linzhao Cheng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Patient Specific ◽  
Hematopoietic Progenitor ◽  
Free System ◽  
Serum Free ◽  
Megakaryocytic Differentiation

Abstract Platelets, anucleate cells derived from megakaryocytes (MKs) that are generated within the bone marrow, play an important role in the process of physiological hemostasis and in vascular repair. Low platelets in the blood stream result in bleeding risk in thrombocytopenic patients with liver failure, leukemia, or undergoing chemotherapy. Platelet transfusions remain the mainstay of treatment and require a constant supply of platelets. Because platelets from donor blood have a short life-span (only few days in storage), platelets are always in a short supply. In vitro generation of MKs and platelets from human induced pluripotent stem cells (hiPSCs) would provide a patient-specific renewable cell source of MKs and platelets to treat thrombocytopenic patients at risk of hemorrhage. We derived integration-free hiPSCs from peripheral blood cells of more than 20 individuals, and examined two methods of in vitro differentiation into MKs: i) co-culture on 10T1/2 cells or OP9 cells first developed by Takayama et al. (2010), and ii) a feeder-free and serum-free system by first forming embryoid bodies (EBs) in a chemically defined condition, similar to the recently published method of Pick et al. (2013). Although both methods gave rise with similar efficiency to CD41a+CD42a+ MKs with large cell size and high-ploidy DNA, we chose to focus on the feeder-free system that began with EB formation with centrifugal aggregation of hiPSCs (spin-EBs) because it is cheaper, faster, easier to scale up, and represents a chemically defined system. To investigate the effect of growth factors on hiPSC differentiation to MKs, we modified the spin-EB system to three steps: i) mesoderm induction and hematopoietic commitment in the presence of BMP4, VEGF, bFGF and SCF (day 0 to day 11), ii) hematopoietic progenitor and MK differentiation by adding TPO (day 11 to 14), and iii) MK maturation (day 14 to 19). To assess whether the FDA-approved pharmacological agent, Romiplostium (Nplate®, TPO analog), has a similar effect to TPO on MK differentiation from hiPSCs, we isolated hematopoietic progenitor cells at day 14, and differentiated them into MKs with Romiplostium or TPO. Our data demonstrated that Romiplostium (50 ng/ml) gave a 3-fold increase of CD41a+CD42a+ MKs, with similar dose-dependent kinetics as TPO. IL-11 has also been reported to enhance MK development. To test whether FDA-approved pharmacological IL-11, Oprelvekin (Neumega®), further stimulated MK differentiation from hiPSCs, we cultured hematopoietic progenitor cells from day 14 in the presence of Romiplostium and Oprelvekin for 5 days. Our data showed that Romiplostium and Oprelvekin synergistically promote megakaryocytic differentiation. In the presence of Romiplostium, 60 to 95 % of cells were CD41a+CD42a+ MKs. Addition of Oprelvekin significantly increased the number of CD41a+CD42a+ MKs, but not the percentage of CD41a+CD42a+ MKs, suggesting that Oprelvekin enhanced a proliferation of MK progenitors. So far, 10 hiPSC lines from several individuals have been tested using the combination of Romiplostium and Oprelvekin in the feeder-free and serum-free differentiation condition. We are currently investigating if the MKs and platelets generated by this defined and scalable system are as fully functional as those generated from bone marrow CD34+ cells from healthy donors. * The first three authors contributed equally; This study is supported in part by an NIH grant U01 HL-107446 and 2012-MSCRFII-0124 (to ZZ Wang). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality

2020 ◽  
Vol 21 (15) ◽  
pp. 5340
Author(s):  
Yulia N. Cajas ◽  
Karina Cañón-Beltrán ◽  
Magdalena Ladrón de Guevara ◽  
María G. Millán de la Blanca ◽  
Priscila Ramos-Ibeas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Biological Effects ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Cytoplasmic Maturation

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 μM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 μM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

190 EFFICIENT GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM PORCINE ADIPOSE-DERIVED STEM CELLS WITH A FEEDER-INDEPENDENT AND SERUM-FREE SYSTEM

2014 ◽  
Vol 26 (1) ◽  
pp. 209
Author(s):  
Y. Zhang ◽  
C. Wei ◽  
P.-F. Zhang ◽  
X. Li ◽  
Y.-S. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Adipose Derived Stem Cells ◽  
Free System ◽  
Serum Free ◽  
Induced Pluripotent ◽  
Serum Free Conditions

Somatic cells could be directly reprogrammed into stem state by ectopic expression of transcription factors, which share similar features of embryonic stem cells (ESC). Induced pluripotent stem cells (iPSC) possess promising application in producing genetically modified animals, whereas the generation of porcine offspring from iPSC is still difficult and controversial, and new materials are needed. In this study, we report the generation of iPSC from porcine adipose-derived stem cells (pADSC) using drug-inducible expression of defined human factors (Oct4, Sox2, Klf4, and c-Myc) and ‘2i’ plus leukemia inhibitory factor (LIF) culture system. pADSC were isolated from subcutaneous adipose tissue of a 28-day-old Danish Landrace, and subsequently characterised by high proliferation rate at low passages, long period passaging without significant replication senescence, mesenchymal stem cell-specific surface markers expression, including CD29 (0.995 ± 0.0577), CD44 (0.999 ± 0.0333), and CD90 (0.994 ± 0.0333), together with successful adipogenic and osteogenic differentiation ability in vitro. The reprogramming of iPSC from pADSC was evidently more efficient than the process from adult fibroblasts (P < 0.01), both of which were carried out under feeder-independent and serum-free conditions, and this may be due to the higher demethylation level of genomic DNA in pADSC. Two lines of porcine iPSC with naïve-like state were finally obtained through feeder-independent and serum-free conditions. The successful reprogramming of iPSC was demonstrated by short cell cycle interval, alkaline phosphatase (AP) staining positive, expression of stemness-related proteins including OCT-4, SOX2, NANOG, SSEA3, and SSEA4. Full reprogramming of iPSC was evaluated by the significant up-regulation of LIN28, ESRRB, UTF1, and DPPA5. Naïve-like state of porcine iPSC was further confirmed by the striking resemblance to naïve mESC, single-cell dissociation, LIF-dependency, up-regulation of STELLA and ERAS, and little translation of TRA-1-60 and TRA-1-81. In addition, porcine naïve-like iPSC possessed normal karyotypes, and could differentiate into cell types of all three germ layers in vitro and in vivo. Furthermore, in vivo studies to determine the capacity of these cells to integrate into the inner cell mass of blastocysts are still being undertaken for validation. Together, our study provided an efficient method to derive porcine naïve-like iPSC from pADSC, which may be useful for the production of living offspring. Y. Zhang and C. Wei contributed equally to this work. Y.-H. Zhang is the corresponding author. This work was supported by the National Natural Science Foundation Program 31272442.

Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures

1995 ◽  
Vol 108 (1) ◽  
pp. 105-114 ◽  
Author(s):  
P. Chen ◽  
S. Vukicevic ◽  
T.K. Sampath ◽  
F.P. Luyten
Keyword(s):  
Bone Morphogenetic Protein ◽  
Chondrocyte Differentiation ◽  
Mrna Levels ◽  
Embryonic Chick ◽  
Serum Free ◽  
Chondrocyte Maturation ◽  
Morphogenetic Protein ◽  
Osteogenic Protein ◽  
Serum Free Conditions

We examined the effect of recombinant human osteogenic protein-1 (OP-1, or bone morphogenetic protein-7), a member of the bone morphogenetic protein family, on growth and maturation of day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently (10-50 ng/ml) promoted chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increased mRNA levels and protein synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promoted cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-beta 1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro.

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