Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.     

Stimulatory activity of PHA-LCM for normal human hemopoietic progenitors and leukemic blast cell precursors: Separation by isoelectric focusing

Medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes growth of human hemopoietic progenitors (CFU-GEMM, BFU-E, CFU-C) and precursors of leukemic blast cells. PHA-LCM was separated by isoelectric focusing and each fraction tested with nonadherent cells of normal individuals as well as blast cells from two patients with acute myelogenous leukemia. Activity profiles for CFU-GEMM, BFU-E and CFU-C ranged from pH 5.0–6.5. The profile for activity stimulatory for leukemic blast cells was broader and ranged from pH 5.5–7.5. Although some overlap was observed, the main peaks of stimulatory activity for normally differentiating progenitors and precursors of leukemic blast cells were separable with respect to their isoelectric point.     

Permissive role of interleukin 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture

We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.     

Recombinant murine granulocyte-macrophage (GM) colony-stimulating factor supports formation of GM and multipotential blast cell colonies in culture: comparison with the effects of interleukin-3

We studied the effects of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) on murine hemopoiesis in methylcellulose culture. The GM-CSF was purified from cultures of Saccharomyces cerevisiae transfected with a cloned murine GM-CSF cDNA. In cultures of spleen cells from normal mice, only granulocyte-macrophage (GM) colonies were supported by GM-CSF. Blast cell colonies were the predominant type in cultures of spleen cells from 5-fluorouracil (5-FU)-treated mice. Dose-response studies revealed that maximal GM and blast cell colony formation is achieved with 100 U/ml GM-CSF. Blast cell colonies revealed variable but high replating efficiencies, and the secondary colonies included multilineage colonies. Serial replating of washed blast cell colonies in cultures with GM-CSF provided evidence for the direct effects of GM-CSF on the proliferation of multipotential blast cells. A combination of GM-CSF and interleukin-3 (IL-3) did not increase the number of blast cell colonies over the level supported by IL-3. This observation indicates that the progenitors for blast cell colonies that responded to GM-CSF are a subpopulation of multipotential progenitors that are supported by IL-3. Cytological studies of colonies derived from GM-CSF and/or IL-3 suggest that the eosinophilopoietic ability of murine GM-CSF is less than that of IL-3.     

Proliferative activity and the rate of differentiation of rat bone marrow cells in normal conditions and in early periods of acute radiation sickness

The autoradiographic method was used to study proliferative activity of cells of a myeloid and erythroid compartments of the bone marrow of intact and irradiated (a single exposure of 10 Gy) rats. The proliferative activity and differentiation rate of cells decreased at early times after irradiation. It was shown that giant neutrophils had heavy tracer nuclei, were formed from myeloblasts, being at the most active phase of the S stage at the time of irradiation, and had a very low differentiation rate.     

Evidence for genetic differentiation and variable recombination rates among Dutch populations of the opportunistic human pathogen Aspergillus fumigatus

As the frequency of antifungal drug resistance continues to increase, understanding the genetic structure of fungal populations, where resistant isolates have emerged and spread, is of major importance. Aspergillus fumigatus is an ubiquitously distributed fungus and the primary causative agent of invasive aspergillosis (IA), a potentially lethal infection in immunocompromised individuals. In the last few years, an increasing number of A. fumigatus isolates has evolved resistance to triazoles, the primary drugs for treating IA infections. In most isolates, this multiple-triazole-resistance (MTR) phenotype is caused by mutations in the cyp51A gene, which encodes the protein targeted by the triazoles. We investigated the genetic differentiation and reproductive mode of A. fumigatus in the Netherlands, the country where the MTR phenotype probably originated, to determine their role in facilitating the emergence and distribution of resistance genotypes. Using 20 genome-wide neutral markers, we genotyped 255 Dutch isolates including 25 isolates with the MTR phenotype. In contrast to previous reports, our results show that Dutch A. fumigatus genotypes are genetically differentiated into five distinct populations. Four of the five populations show significant linkage disequilibrium, indicative of an asexual reproductive mode, whereas the fifth population is in linkage equilibrium, indicative of a sexual reproductive mode. Notably, the observed genetic differentiation among Dutch isolates does not correlate with geography, although all isolates with the MTR phenotype nest within a single, predominantly asexual, population. These results suggest that both reproductive mode and genetic differentiation contribute to the structure of Dutch A. fumigatus populations and are probably shaping the evolutionary dynamics of drug resistance in this potentially deadly pathogen.     

Proliferation and differentiation in culture of mast cell progenitors derived from mast cell-deficient mice of genotype W/Wv

Mice of genotype W/Wv have less than 1% of normal mast cells in the skin, stomach, and cecum. In order to further clarify the mechanism of this deficiency, we studied committed mast cell progenitors and multipotent progenitors, which are capable of mast cell differentiation in clonal culture. The relative concentration of mast cell progenitors in the bone marrow, spleen, and peripheral blood of W/Wv mice was similar to that of +/+ mice. However, the cellularity of the marrows of W/Wv mice was 54% of that of their normal littermates. Identification of mast cells was established by metachromatic staining with toluidine blue, transmission electron microscopy, and demonstration of membrane receptors for immunoglobulin E. The time course of colony formation and the morphology of W/Wv mast cell colonies in culture was identical to that of normal littermates. The percentages of mast cells in individual multi-lineage colonies were extremely variable. The histamine content of mast cells derived from W/Wv mice was similar to that of mast cells from +/+ mice. These studies demonstrated the normal capacity for differentiation and proliferation in culture of mast cell progenitors from W/Wv mice.     

Sexual differentiation of brain and behavior in the zebra finch: Critical periods for effects of early estrogen treatment

In order to determine the critical period(s) during which estrogen alters sexually dimorphic behavior and neuroanatomy in zebra finches (Poephila guttata), nestlings were injected daily 20 μg estradiol benzoate (EB) during posthatching week 1, week 2, week 3, or weeks 1, 2, and 3. At 7 months of age, birds were implanted with testosterone propionate and tested with female partners for singing, dancing, and copulatory mounting. Brains were subsequently processed for morphometry, and the volumes of the song system nuclei HVC, area X, and RA and the soma sizes and densities of neurons in RA were determined. Males given EB during week 1 failed to mount. Females given EB during week 1 were fully masculinized with respect to dancing and RA neuron soma size and density, and were partially masculinized with respect to song nuclei volumes and singing. Treatment beginning after week 1 was ineffective or less effective for all measures. Only for RA neuron measures was treatment for all three weeks more effective than week 1 treatment. Thus the first post-hatching week is the most influential period of those tested for effects of exogenous estrogen on sexual differentiation in this species, and is a period during which both masculinization of females and demasculinization of males is possible. 1994 John Wiley & Sons, Inc.     

Terminal differentiation of hemopoietic cell clones cultured in tridimensional collagen matrix: in situ cell morphology and enzyme histochemistry analysis

Collagen, a major component of the extracellular matrix in vivo, has been used as a tridimensional gel matrix for cultured hemopoietic clones. Its resemblance to the natural matrix produced by cells makes it ideal for studies on proliferation and differentiation of hemopoietic lineages. Every lineage, including granulocytes (basophilic, eosinophilic and neutrophilic polymorphs) monocyte-macrophages, megakaryocytes, erythroid and lymphoid lineages could be grown using a standardized collagen medium, provided that specific stimulators were added in the culture. Clones were scored on either live or fixed cultures. Compared to other gel substrates, collagen matrix proved superior for cell proliferation and maturation. Additional advantages (in situ clonal analysis by histological staining, enzyme cytochemistry), and other possibilities of the method are reported and discussed. The system offers great potential for cellular immunology, hematology and molecular biology with peculiar reference to differentiation of normal hemopoietic cells, viral transformation and leukemogenesis in vitro. These applications are reviewed.     

Differential kinetics for induction of interleukin-6 mRNA expression in murine peritoneal macrophages: Evidence for calcium-dependent and independent-signalling pathways

It is presently unclear what role elevations in intracellular calcium concentration ([Ca²⁺]_i) play in the control of monokine secretion, or whether such alterations underlie the ability of physiologic stimuli to induce production of these important signalling molecules. To address these issues, we have performed experiments in murine peritoneal macrophages to determine whether lipopolysaccharide (LPS) or interferon gamma (IFN-γ) initiate production of the proinflammatory monokine interleukin 6 (IL-6) concomitant with elevations in [Ca²⁺]_i and with kinetics similar to that seen with known Ca²⁺ mobilizing agents. Alterations in [Ca²⁺]_i after treatment with LPS, IFN-γ, platelet activating factor (PAF), or thapsigargin were measured by fluorimetric methods. These effects were compared with the ability of each to induce IL-6 mRNA expression as measured by semiquantitative reverse-transcribed polymerase chain reactions. We report that neither LPS nor IFN-γ elicited detectable elevations in [Ca²⁺]_i but that both up-regulated expression of IL-6 mRNA expression within 60 min. In contrast, experiments using either thapsigargin or PAF showed rapid and dramatic elevations in [Ca²⁺]_i with marked increases in IL-6 mRNA expression, as quickly as 15 min after initial exposure. Elevations in mRNA encoding IL-6 by thapsigargin and PAF were found to occur in a dose-dependent manner, mirroring their ability to elicit elevations in [Ca²⁺]_i. These data demonstrate that LPS and IFN-γ induce IL-6 message expression by means of Ca²⁺-independent signalling pathways. Furthermore, Ca²⁺-mobilizing agents that evoke monokine message expression do so far more rapidly than do LPS or IFN-γ. Taken in concert, these data are consistent with the hypothesis that multiple signalling pathways exist by which production of proinflammatory monokines are initiated. J. Cell. Physiol. 177:232–240, 1998. © 1998 Wiley-Liss, Inc.     

Hairy cell leukemia: kinetics and intercellular relationships of cells in leukemic colonies grown in a semisolid medium

We examined cellular relationships and cytokinetics of hairy cells in colonies formed in an in vitro cloning system. Cells in small colonies and at the periphery of larger ones were separated by wide, irregular intercellular spaces. Cells in the core of large colonies were elaborately intertwined by their cytoplasmic processes and so densely packed that their intercellularity was greatly reduced. Cell labeling with 3H-thymidine revealed indices ranging from less than 1% to 35%. The combination of autoradiography with a stain for tartrate-resistant acid phosphatase showed that the enzyme was not expressed by cells in S-phase. The cellular relationships of hairy cells in colonies grown in this system closely mimic those of their in vivo counterparts in the spleen and bone marrow.     

In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme

The sensory hair cells and supporting cells of the organ of Corti are generated by a precise program of coordinated cell division and differentiation. Since no regeneration occurs in the mature organ of Corti, loss of hair cells leads to deafness. To investigate the molecular basis of hair cell differentiation and their lack of regeneration, we have established a dissociated cell culture system in which sensory hair cells and supporting cells can be generated from mitotic precursors. By incorporating a Math1-GFP transgene expressed exclusively in hair cells, we have used this system to characterize the conditions required for the growth and differentiation of hair cells in culture. These conditions include a requirement for epidermal growth factor, as well as the presence of periotic mesenchymal cells. Lastly, we show that early postnatal cochlear tissue also contains cells that can divide and generate new sensory hair cells in vitro.     

Evidence for an early G1 ionic event necessary for cell cycle progression and survival in the MCF-7 human breast carcinoma cell line

The mechanism of the G0/G1 arrest and inhibition of proliferation by quinidine, a potassium channel blocker, was investigated in a tissue culture cell line, MCF-7, derived from a human breast carcinoma. The earliest measurable effect of quinidine on the cell cycle was a decrease in the fraction of cells in S phase at 12 hr, followed by the accumulation of cells in G1/G0 phases at 30 hr. Arrest and release of the cell cycle established quinidine as a cell synchronization agent, with a site of arrest in early G1 preceding the lovastatin G1 arrest site by 5–6 hr. There was a close correspondence among the concentration-dependent arrest by quinidine in G1, depolarization of the membrane potential, and the inhibition of ATP-sensitive potassium currents, supporting a model in which hyperpolarization of the membrane potential and progression through G1 are functionally linked. Furthermore, the G1 arrest by quinidine was overcome by valinomycin, a potassium ionophore that hyperpolarized the membrane potential in the presence of quinidine. With sustained exposure of MCF-7 cells to quinidine, expression of the Ki67 antigen, a marker for cells in cycle, decreased, and apoptotic and necrotic cell death ensued. We conclude that MCF-7 cells that fail to progress through the quinidine-arrest site in G1 die. J. Cell. Physiol. 176:456-464, 1998. © 1998 Wiley-Liss, Inc.     

Use of G1.2/G2.2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture

The expanded application of commercial bovine IVM, IVF, and IVC systems is dependent on the ability to produce embryos in culture that are capable of producing normal pregnancies. Because serum containing culture systems can induce neonatal and fetal problems there exists a definite need for a serum-free culture system that produces viable blastocysts. This study demonstrated that the physiological sequential media system G1.2/G2.2 could produce bovine blastocysts at rates equivalent to co-culture. Additionally, these blastocysts had equivalent or increased cell numbers and inner cell mass development. Blastocysts produced in the G1.2/G2.2 culture system produced pregnancies following both fresh transfer and cryopreservation at equivalent rates to co-culture. Finally, this study demonstrated that the media system G1.2/G2.2 could be used in a commercial OPU transfer program without any loss in the numbers of blastocysts produced or the numbers of pregnancies resulting following transfer from either fresh or cryopreserved blastocysts.     

Impact of medium volume and oxygen concentration in the incubator on pericellular oxygen concentration and differentiation of murine chondrogenic cell culture

Previous studies have demonstrated that oxygen environment is an important determinate factor of cell phenotypes and differentiation, although factors which affect pericellular oxygen concentration (POC) in murine chondrogenic cell culture remain unidentified. Oxygen concentrations in vivo were measured in rabbit musculoskeletal tissues, which were by far hypoxic compared to 20% O₂ (ranging from 2.29 ± 1.16 to 4.36 ± 0.51%). Oxygen concentrations in murine chondrogenic cell (C3H10T1/2) culture medium were monitored in different oxygen concentrations (20% or 5%) in the incubator and in different medium volumes (3,700 or 7,400 μl) within 25-cm² flasks. Chondrogenic differentiation was assessed by glycosaminoglycan production with quantitative evaluation of Alcian blue staining in 12-well culture dishes. Expression of chondrogenic genes, aggrecan, and type II collagen α1, was examined by quantitative real-time polymerase chain reaction. Oxygen concentrations in medium decreased accordingly with the depth from medium surface, and POC at Day 6 was 18.99 ± 0.81% in 3,700-μl medium (1,480-μm depth) and 13.26 ± 0.23% in 7,400-μl medium (2,960-μm depth) at 20% O₂ in the incubator, which was 4.96 ± 0.08% (1,480-μm depth) and 2.83 ± 0.42% (2,960-μm depth) at 5% O₂, respectively. The differences of POC compared by medium volume were statistically significant (p = 0.0003 at 20% and p = 0.001 at 5%). Glycosaminoglycan production and aggrecan gene expression were most promoted when cultured in moderately low POC, 1,000 μl (2,960-μm depth) at 20% O₂ and 500 μl (1,480-μm depth) at 5% O₂ in 12-well culture dishes. We demonstrate that medium volume and oxygen concentration in the incubator affect not only POC but also chondrogenic differentiation.     

Comparison of human-induced pluripotent stem cells and mesenchymal stem cell differentiation potential to insulin producing cells in 2D and 3D culture systems in vitro

Diabetes is one of the most common diseases in the world that is chronic, progressive, and costly, and causes many complications. Common drug therapies are not able to cure it, and pancreas transplantation is not responsive to the high number of patients. The production of the insulin producing cells (IPCs) from the stem cells in the laboratory and their transplantation to the patient's body is one of the most promising new approaches. In this study, the differentiation potential of the induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) into IPCs was compared to each other while cultured on poly(lactic-co-glycolic) acid (PLGA)/polyethylene glycol (PEG) nanofibrous scaffold as a 3D substrate and tissue culture polystyrene (TCPS) as a 2D substrate. Although the expression level of the insulin, Glut2 and pdx-1 genes in stem cells cultured on 3D substrate was significantly higher than the stem cells cultured on 2D substrate, the highest expression level of these genes was detected in the iPSCs cultured on PLGA-PEG. Insulin and C-peptide secretions from differentiated cells were also investigated and the results showed that secretions in cultured iPSCs on the PLGA-PEG were significantly higher than cultured iPSCs on the TCPS and cultured MSCs on both PLGA-PEG and TCPS. In addition, insulin protein was also expressed in the cultured iPSCs on the PLGA-PEG significantly higher than cultured MSCs on the PLGA-PEG. It can be concluded that differentiation potential of iPSCs into IPCs is significantly higher than human MSCs at both 2D and 3D culture systems.     

Different culture conditions used for arresting the G0/G1 phase of the cell cycle in goldfish (Carassius auratus) caudal fin-derived fibroblasts

One of the most important factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We investigated the effects of serum starvation, culturing to confluence and roscovitine treatment on the cell cycle synchronization of goldfish caudal fin-derived fibroblasts by flow cytometric analysis. The results show that culturing the cells to confluence (85.5%) and roscovitine treatment (82.71%) yield a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than serum starvation (62.85%). Different concentrations of roscovitine (5, 10, or 15 μM) induce cell cycle arrest at the G0/G1 phase.