Signal requirements for growth and differentiation of activated murine B lymphocytes

Mouse B lymphocytes were stimulated at high cell concentrations with goat anti-IgM antibodies, which leads to the induction of B cell proliferation without the addition of any growth factors. After 48 hr, blast cells were purified and cultured at low cell concentrations. Proliferation and differentiation of purified B lymphocyte blasts is then dependent on the addition of either mitogens (e.g., LPS) or certain lymphokines derived from activated T cells or macrophages. One such lymphokine was isolated from supernatants of various activated T cells and characterized by gel filtration as a material with an apparent m.w. of 40,000 to 50,000, similar to BCGF II. It supports the proliferation of the B cell blasts and induces their differentiation into plaque-forming cells. Lymphokines such as BCGF I, interleukin 2, and BCDF gamma could neither maintain growth nor induce differentiation of B lymphocytes preactivated by goat anti-IgM.     

Differentiation of human pre-adipocytes by recombinant adiponectin

Obesity has become a global health problem and it is linked to a higher risk of diseases and metabolic disorders such as diabetes, cardiovascular disease, and cancer. The adipose tissue plays an important role in monitoring and controlling whole-body metabolism by secreting a variety of bioactive molecules such as adiponectin. Deficiencies of this hormone can cause type II diabetes and cardiovascular disease both in mice and in humans. Therefore, adiponectin is an attractive molecule to use in human therapy, particularly in a recombinant form. The source of recombinant adiponectin could be the expression of full-length adiponectin either in Escherichia coli, or in baculovirus. In this work we express and purify human adiponectin in both systems. The adiponectin produced by baculovirus was found to be 10 times more active as far as oligomerization and human pre-adipocyte differentiation are concerned, when compared with adiponectin produced by E. coli. We presume that adiponectin expressed in baculovirus has post-translation modifications not made by bacteria which may be responsible for these differences in activity. This renders adiponectin produced by baculovirus a better candidate for the treatment of type II diabetes and cardiovascular disease.     

Normal human serum stimulates murine erythroid precursor growth in in vitro culture

When introduced into cultures of murine CFU-E human sera inhibited the formation of erythroid colonies. However, after absorbtion on murine cells and heating all tested sera became stimulatory. Crude sera were separated into two fractions by DEAE chromatography: the first fraction was stimulatory. The second was toxic but the toxicity could be eliminated by heating; the fraction then became slightly stimulatory. Attempts at characterizing the molecular weight of the stimulatory activity led to variable results, suggesting either that the stimulatory activity(ies) could polymerize or be fixed on different serum proteins. All sera from 11 different anemic patients were also shown to be stimulatory.     

Biochemical differentiation of a murine ganglioneuroblastoma in tissue culture

A cell line derived from a murine ganglioneuroblastoma has been established in tissue culture. The highly refractile cells have round bodies and develop small processes. Their division time is 36 h under the conditions used. When the cells are grown to high densities, the acetyl cholinesterase activity increases four-fold, and is not affected by nerve growth factor. The behavior of this cell line was compared with C-1300, a murine neuroblastoma that has been cultured in many laboratories. These studies reveal three common features of the two tumors: (1) morphologic appearance in vitro does not correlate with appearance in vivo; (2) both tumors retain the ability to differentiate under appropriate conditions; and (3) biochemical differentiation can be expressed independent of morphology.     

Neurogenic differentiation of murine and human adipose-derived stromal cells

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1-3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.     

Serum-free, defined medium for the growth and differentiation of murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells are frequently employed to study both cell growth and erythroid differentiation. Although these cells are easily cultured and induced to differentiate, they are routinely maintained in a medium that contains 10%-15% fetal bovine serum. Because of the variability between different lots and the cost of serum, it was desirable to define a serum-free medium in which to culture MEL cells. In the present work, a totally serum-free, defined medium is described that supports both normal cell growth and dimethyl sulfoxide induced differentiation in the two MEL cell lines examined (DS-19 and 270). A variety of hormones and biological compounds are examined in this medium to determine their effects on growth and differentiation. This medium does not support the growth of the mouse hepatoma cell line.     

Enhanced differentiation of human dopaminergic neuronal cell model for preclinical translational research in Parkinson's disease

Human-derived neuronal cell lines are progressively being utilized in understanding neurobiology and preclinical translational research as they are biologically more relevant than rodent-derived cells lines. The Lund human mesencephalic (LUHMES) cell line comprises human neuronal cells that can be differentiated to post-mitotic neurons and is increasingly being used as an in vitro model for various neurodegenerative diseases. A previously published 2-step differentiation procedure leads to the generation of post-mitotic neurons within 5-days, but only a small proportion (10%) of the total cell population tests positive for tyrosine hydroxylase (TH). Here we report on a novel differentiation protocol that we optimized by using a cocktail of neurotrophic factors, pleiotropic cytokines, and antioxidants to effectively generate proportionately more dopaminergic neurons within the same time period. Visualization and quantification of TH-positive cells revealed that under our new protocol, 25% of the total cell population expressed markers of dopaminergic neurons with the TH-positive neuron count peaking on day 5. These neurons showed spontaneous electrical activity and responded to known Parkinsonian toxins as expected by showing decreased cell viability and dopamine uptake and a concomitant increase in apoptotic cell death. Together, our results outline an improved method for generating a higher proportion of dopaminergic neurons, thus making these cells an ideal neuronal culture model of Parkinson's disease (PD) for translational research.     

Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.     

K-Cl cotransporter gene expression during human and murine erythroid differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

Cryopreserved human adipogenic-differentiated pre-adipocytes: a potential new source for adipose tissue regeneration

BACKGROUND: Previously, we have shown that in vitro adipogenic differentiation of pre-adipocytes before implantation can enhance in vivo adipose tissue formation. For large-scale adipose tissue engineering or repeat procedures, cryopreservation of fat grafts has been commonly used in recent years. However, the feasibility of cryopreservation of adipogenic differentiated pre-adipocytes has not been investigated. METHODS: To examine the impact of cryopreservation on the adipogenic functions of adipogenic-differentiated pre-adipocytes, freeze-thawed adipocytes were compared with fresh differentiated adipocytes in vitro and in vivo. Adipogenic function was assessed by Oil red O staining, ELISA analysis of leptin secretion and RT-PCR of adipogenic-related genes. After transplantation, adipose tissue formation was assessed by histomorphologic and volumetric analysis. RESULTS: Freeze-thawed adipocytes constantly showed typical adipogenic functions in terms of lipid content, leptin secretion and adipogenic gene expression, as well as good viability. Importantly, implants derived from freeze-thawed adipocytes were successfully developed to adipose tissue and newly formed adipose tissues were similar to those developed from fresh differentiated adipocytes, based on histomorphologic and volumetric analysis. In addition, CD34-positive endothelial cells were detected in implants. These results demonstrate that the specific characters of adipogenic-differentiated pre-adipocytes are successfully conserved after cryopreservation without any significant alteration. DISCUSSION: Cryopreservation of adipogenic-differentiated pre-adipocytes is a feasible method and extends their clinical use in adipose tissue-engineering applications and transplantation.     

Chemically-defined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture

The effect on tissue differentiation and growth in vitro of certain of the factors implicated in collagen synthesis (ascorbic acid, α-ketoglutarate and oxygen) and the influence of hydrocortisone was studied using organ cultures of fetal mouse mandible as a mixed epithelial and connective tissue system. Using serum-free Waymouth’s MB 752/1 chemically-defined medium, addition of high levels of ascorbic acid (300 μg per ml), hydrocortisone (1 μg per ml) and oxygen (95%) enhanced differentiation in a number of tissues, in particular skin and appendages, tooth germs and bone, while osteoid and dentine production were noticeably promoted. It is suggested that an essential aspect of media design for organ culture involves the incorporation of collagen-promoting factors to the in vitro environment particularly with regard to the controlling role implicated for collagen in a variety of biological processes. Some of the work reported here was undertaken while A. H. Melcher was a member of the Department of Dental Science, Royal College of Surgeons of England, London, England.     

Sustainable malaria control: transdisciplinary approaches for translational applications

Background

Intravenous (IV) artesunate is the treatment of choice for severe malaria. In Europe, however, no GMP-manufactured product is available and treatment data in European travellers are scarce. Fortunately, artesunate became available in the Netherlands and Belgium through a named patient programme. This is the largest case series of artesunate treated patients with severe malaria in Europe.

Methods

Hospitalized patients treated with IV artesunate between November 2007 and December 2010 in the Netherlands and Belgium were retrospectively evaluated. Patient characteristics, treatment and clinical outcome were recorded on a standardized form and mortality, parasite clearance times and the occurrence of adverse events were evaluated.

Results

Of the 68 treated patients, including 55 with severe malaria, two patients died (2/55 = 3.6%). The mean time to 50% parasite clearance (PCT50), 90% and 99% were 4.4 hours (3.9 - 5.2), 14.8 hours (13.0 - 17.2), and 29.5 hours (25.9 - 34.4) respectively. Artesunate was well tolerated. However, an unusual form of haemolytic anaemia was observed in seven patients. The relationship with artesunate remains uncertain.

Conclusions

Data from the named patient programme demonstrate that IV artesunate is effective and well-tolerated in European travellers lacking immunity. However, increased attention needs to be paid to the possible development of haemolytic anaemia 2-3 weeks after start of treatment. Treatment of IV artesunate should be limited to the period that IV treatment is required and should be followed by a full oral course of an appropriate anti-malarial drug.     

Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone

Dexamethasone induces morphological differentiation in murine neuroblastoma cells in culture. The percentage of differentiated cells depends on the concentration of dexamethasone in the medium and duration of treatment. After drug removal (with or without replating), many cells maintain their differentiated phenotype. Dexamethasone-treated cells have larger soma and contain more protein. Dexamethasone also produces a concentration-dependent inhibition of population growth. Growth inhibition is complete by 2 days following treatment with dexamethasone 50 micrograms/ml. Complete growth inhibition is achieved prior to the complete expression of morphological differentiation. Androstenedione, testosterone, and 17-beta-estradiol--all steroids without glucocorticoid activity--have no differentiating effect, although they inhibit growth or cause cell death at higher concentrations.     

Effect of retinoic acid on the differentiation and growth of murine mastocyte precursors

In cultures of normal mouse hematopoietic cells containing Interleukin-3 develop cells with many features of mast cells. These cells seem heterogeneous with respect to morphological and biochemical examination. Nevertheless, most of the cells show many granules and a low ability to self-renew. In the present report we describe the development of a blastic cell population, termed mastoblasts, when normal mouse hematopoietic cells are exposed continuously to retinoic acid (RA: 10(-6) to 10(-5) M/l). Using H*3-thymidine incorporation, cell cycle measurement and protein content by flow cytometry, transmission and scanning electron microscopy, we show that these cells seem to be of mast cell lineage but with a high self-renewing capability. So, RA is able to inhibit mast cell differentiation and to provide us a "mastoblastic" population which could be used as a model to study mast cell differentiation.     

Simplified process for preparation of schizophyllan solutions for biomaterial applications

Schizophyllan is a biopolymer commercially produced for pharmaceutical and cosmetics uses. However, schizophyllan also has potential biomaterial applications. Schizophyllan is conventionally produced from glucose and recovered by diafiltration and ultrafiltration to produce a highly purified product. Here we demonstrate a simplified process for preparation of schizophyllan solutions for biomaterial applications. Schizophyllan was produced in 1.5-L bioreactors from distiller’s dried grains with solubles (DDGS), an abundant coproduct of dry grind fuel ethanol production. Downstream processing eliminated filtration and concentration steps, providing solutions containing 4.2 ± 0.3 g schizophyllan/L. Solutions contained high-molecular-weight schizophyllan and exhibited viscosity properties similar to those of commercial schizophyllan. Schizophyllan solutions showed promise as a component of biolubricants in friction and wear tests and by dynamic surface and interfacial tension measurements.