Trypanosoma brucei: Differentiation of in Vitro-Grown Bloodstream Trypomastigotes into Procyclic Forms1

Trypanosoma brucei strain 366D trypomastigotes grown at 37°C in the presence of a human fibroblast cell line formed foci underneath the feeder cells whereas trypanosomes grown in the presence of a human epithelial cell line grew only in the culture supernatant. A culture system was developed to study the differentiation of bloodstream trypomastigotes grown in the epithelial cell system into procyclic trypomastigotes at 27°C. The morphological differentiation into the procyclic form was complete by 48 h. Cell division did not occur until 30–40 h after transfer to 27°C. Various characteristics of this system were examined, including the effect of the feeder layer, the type of medium, the presence of the metabolites cis-aconitate and citrate, the preadaptation period, and the trypanosome cell concentration. The respiration of the recently differentiated procyclic cells was less sensitive to inhibition by CN-than that of established procyclic forms, implying a delayed appearance of complete mitochondrial oxidative pathways. This trypanosome differentiation system has the advantage that the animal host is not needed and the entire process is carried out in in vitro culture.     

The effect of citrate/cis-aconitate on oxidative metabolism during transformation of Trypanosoma brucei

Monomorphic bloodstream forms of Trypanosoma brucei, grown in the mammal, are deficient in aconitase and 2-oxoglutarate dehydrogenase and they do not respire in the presence of the substrates citrate, cis-aconitate, succinate, proline or 2-oxoglutarate. When grown in vitro low levels of aconitase, succinate oxidase and proline oxidase are detected. Addition of citrate/cis-aconitate at 37 degrees C to bloodstream forms leads to the formation of aconitase and proline oxidase. Most cells undergo an 'abortive' transformation to non-dividing procyclic-like cells while some cells adapt to the presence of the citric acid cycle intermediates and continue to multiply as bloodstream forms. At 27 degrees C and in the presence of citrate/cis-aconitate bloodstream forms transform synchronously to dividing procyclic cells. Within 72 h the rate of respiration with proline, succinate and 2-oxoglutarate becomes similar to that in established procyclic cells while the rate of glucose oxidation decreases. The possible role of citric acid cycle intermediates in determining whether a trypanosome will retain the properties of a bloodstream trypomastigote or differentiate to a procyclic trypomastigote is discussed.     

Studies on the development of metacyclic Trypanosoma brucei sspp. cultivated at 27 degrees C with insect cell lines

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T.b. rhodesiense were grown at 27 degrees C in 25-cm2 flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 X 10(5) metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Trypanosoma rhodesiense: mitochondrial proteins of bloodstream and procyclic trypomastigotes

One- and two-dimensional gel electrophoresis of the solubilized mitochondrial proteins of bloodstream and procyclic trypomastigote Trypanosoma brucei rhodesiense and radiolabeling of proteins in the presence of cycloheximide were used to identify proteins synthesized in the trypanosome mitochondrion. The proteins which comprise the mitochondrion were found to be very similar in both bloodstream and procyclic trypomastigotes, but do differ in their level of synthesis. A protein putatively identified as subunit II of cytochrome oxidase (EC 1.9.3.1) was detected in mitochondria from both the procyclic and bloodstream organisms. The presence of this protein in bloodstream trypomastigotes and the overall similarity of protein content in the trypanosome mitochondria is noteworthy in view of the fact that bloodstream trypomastigotes have a repressed mitochondrion with no detectable tricarboxylic acid cycle or cytochrome electron transport chain.     

Differentiation of Trypanosoma brucei may be stage non-specific and does not require progression of cell cycle

Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei, we were able to knock down expression of each of the 11 non-ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock-down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei, in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn-depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild-type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37 degrees C to 26 degrees C in the presence of citrate and cis-aconitate.     

Infectivity of monomorphic and pleomorphic trypanosoma brucei stocks cultivated at 28 C with various tsetse fly tissues

Noninfective procyclic forms of Trypanosoma brucei stocks derived from the pleomorphic EVE 10 were cultivated at 28 C in Cunningham's liquid medium in the presence of head-salivary gland, alimentary tract, and abdominal body wall explants of Glossina morsitans morsitans. After 8 to 10 days of cultivation some of the procyclic forms transformed into metacyclic stages infective for mice. Infectivity persisted for varying periods up to 66 days, when the experiments were terminated. Only 10 explants of alimentary tract or abdominal body wall tissues were required in the flasks to render the culture infective for most of the mice inoculated. Similar trypanosome suspensions grown with 10 head-salivary gland explants produced an infection on only one occasion. Cultures of procyclic organisms derived from the monomorphic stock 427 grown inthe presence of all three types of tsetse fly explants produced only sporadic infections in mice. Metacyclic forms failed to develop in trypanosome populations of stock EVE 10 cultivated at 28 C in the liquid medium alone or supplemented with mouse embryo tissues.     

Reconstituted human gingival epithelium: Nonsubmerged in vitro model

Summary Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type I collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.     

Trypanosoma brucei brucei: In Vitro Production of Metacyclic Forms

ABSTRACT An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei . Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27° C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27° C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

A novel selection regime for differentiation defects demonstrates an essential role for the stumpy form in the life cycle of the African trypanosome

A novel selection scheme has been developed to isolate bloodstream forms of Trypanosoma brucei, which are defective in their ability to differentiate to the procyclic stage. Detailed characterization of one selected cell line (defective in differentiation clone 1 [DiD-1]) has demonstrated that these cells are indistinguishable from the wild-type population in terms of their morphology, cell cycle progression, and biochemical characteristics but are defective in their ability to initiate differentiation to the procyclic form. Although a small proportion of DiD-1 cells remain able to transform, deletion of the genes for glycophosphatidyl inositol-phospholipase C demonstrated that this enzyme was not responsible for this inefficient differentiation. However, the attenuated growth of the Delta-glycophosphatidyl inositol-phospholipase C DiD-1 cells in mice permitted the expression of stumpy characteristics in this previously monomorphic cell line, and concomitantly their ability to differentiate efficiently was restored. Our results indicate that monomorphic cells retain expression of a characteristic of the stumpy form essential for differentiation, and that this is reduced in the defective cells. This approach provides a new route to dissection of the cytological and molecular basis of life cycle progression in the African trypanosome.     

Identification and Partial Purification of a Stage-Specific 33 kDa Mitochondrial Protein as the Alternative Oxidase of the Trypanosoma brucei brucei Bloodstream Trypomastigotes

ABSTRACT. The glycerophosphate oxidase (GPO), the unique terminal oxidase of bloodstream trypanosome (TAO), appears to be functionally similar to the alternative oxidases of some plants and higher fungi. Immunoblotting of mitochondrial proteins of bloodstream trypomastigotes of Trypanosoma brucei brucei with monoclonal or polyclonal antibodies to Sauromatum guttatum (voodoo lily) and Symplocarpus foetidus (skunk cabbage) alternative oxidases respectively revealed two proteins of about 33 kDa (p33) and 68 kDa (p68). These proteins are not present in procyclic trypomastigotes. Electrophoresis under rigorous denaturing conditions indicated p68 to be the dimer of p33. Indirect immunofluorescent studies of bloodstream and procyclic trypomastigotes with monoclonal antibody to plant alternative oxidase also showed the localization of 33 kDa protein in the mitochondria of the bloodstream trypomastigotes. The functional TAO activity could be solubilized efficiently from the mitochondrial membrane of the bloodstream trypomastigotes by 1% NP-40 or 10 mM lauryl maltoside. When fractionated by Superose 12 gel filtration chromatography, p33 was co-purified with the TAO enzymatic activity. The apparent molecular size of the active enzyme complex was found to be 160 kDa. Gradual disappearance of the 33 kDa protein and the TAO enzymatic activity were well correlated during in vitro differentiation of the bloodstream to procyclic trypomastigotes. This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.     

IL-4 regulates differentiation and proliferation of human precursor B cells

The mechanism by which precursor and pre-B cells undergo differentiation is unclear; however, it is known that growth factors play an important role in this maturation process. The lymphokine, IL-4 has been shown to increase expression of class II Ag on B cells and induce B cell proliferation. In the murine system, IL-4 induced differentiation of precursor B cells into pre-B cells. In order to analyze growth factors on B cell development we have established an in vitro culture system for human bone marrow cells. We found that in the presence of IL-4, normal human precursor and pre-B cells can be induced to differentiate in the absence of cell proliferation with four days of culture. Furthermore, IL-4 depressed proliferation induced by supernatant from a T cell line. The differentiation was measured by an increase in both the number of cytoplasmic mu and surface IgM-positive cells. The effect of IL-4 on precursor and pre-B cell differentiation was detected as soon as 14 h of exposure to the lymphokine in the absence of an adherent feeder layer. These data suggest that IL-4 directly affects the differentiation process of normal human precursor and pre-B cells, and may antagonistically affect cell proliferation.     

Trypanosoma brucei: cis-aconitate and temperature reduction as triggers of synchronous transformation of bloodstream to procyclic trypomastigotes in vitro

Synchronous transformation of the monomorphic Trypanosoma brucei 427 variant clone MITat 1.4 (117) from bloodstream to procyclic trypomastigotes was studied in modified minimum essential medium plus 15% inactivated horse serum. Repression of variant surface glycoprotein synthesis, subsequent morphological transformation, and growth of procyclic cells was triggered by the simultaneous action of two signals: a reduction in temperature from 37 to 27 C and the addition of cis-aconitate. Repression of variant surface glycoprotein synthesis initiated by these two signals is reversible during the first hours, but becomes irreversible after about 1 day. Thereafter, cells are committed to differentiation at 27 C.     

Studies on the Development of Metacyclic Trypanosoma brucei sspp. Cultivated at 27°C with Insect Cell Lines1

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T. b. rhodesiense were grown at 27°C in 25-cm² flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 × 10⁵ metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

Trypanosoma brucei brucei: regulation of ornithine decarboxylase in procyclic forms and trypomastigotes

Ornithine decarboxylase (ODC) activity was measured in procyclic forms of Trypanosoma brucei brucei grown in semidefined medium. ODC activity rapidly increased in late log-phase cells which were resuspended in fresh medium. A biphasic induction curve similar to that observed in mammalian cells was observed over an 18-hr period. ODC activity increased 4.5- to 25-fold over control levels measured at zero time. Actinomycin D and cycloheximide inhibited induction by greater than 90%. Polyamines at a level not inhibitory to growth (10 microM) inhibited ODC induction, but only by 30-50%, late in the induction period. Putrescine inhibited the first peak of induction and suppressed activity at 14 hr by 75%. Polyamine analogs such as bis(ethyl)spermidine were not effective suppressors of ODC activity. The half-life of ODC in procyclic forms grown in the presence of cycloheximide was greater than 6 hr, while that of bloodstream trypomastigotes in mice treated with cycloheximide was 5 hr. A single dose of the ODC inhibitor DL-alpha-difluoromethylornithine given to infected rats or mice suppressed trypanosome ODC activity greater than 90% for more than 7 hr. These studies indicate that although trypanosome ODC increases rapidly under log growth conditions, it is less susceptible to fluctuation and external control than the enzyme from mammalian sources. The latter may be a factor in the clinical efficacy of ODC inhibitors.     

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols.     

Trypanosoma acomys (Wenyon, 1909): continuous culturing with a mouse fibroblast cell-line (A9)

The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37 degrees C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 x 10(4), 1.5 x 10(5) and 7.4 x 10(5) parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 x 10(6) parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.     

Culture of human main pancreatic duct epithelial cells

Summary Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated culture inserts suspended above a human fibroblast feeder layer. After primary culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-free medium continued to develop into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These cells were also positive for cytokeratin 19, 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry. Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-d-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passaged using a feeder layer system, with serum-free medium, and in organotypic cultures.     

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.