Studies on the role of serum in long-term bone marrow cell cultures.

The effect of fetal calf serum (FCS) concentration on hemopoiesis in long-term bone marrow cell cultures (supplemented with 10(-6) M hydrocortisone) was investigated. The duration of hemopoiesis was directly related to the concentration of serum in the cultures. At each concentration, marked differences were seen between different batches of FCS.

Erythropoietin-dependent and erythropoietin-independent enhancement of colony formation by immature erythroid progenitors (BFUe).

The erythropoietin (epo)-dependent burst forming activities (BFA) of foetal calf serum (FCS), mouse bone marrow cells (BFA-cell) and lectin-stimulated mouse spleen conditioned medium (BFA-mscm) were investigated. Burst enhancement by BFA-mscm was independent of FCS concentration above 5% FCS. The activity of BFA-cell was directly proportional to FCS concentration.

Haemopoietic colony formation (BFU-E, GM-CFC) during the development of pure red cell hypoplasia induced in the cat by feline leukaemia virus.

The GM-CFC assay for granulocyte-macrophage progenitors and the BFU-E and CFU-E assay for early and late erythroid progenitors from cat bone marrow were characterized. GM-CFC gave 59 +/- 4 to 118 +/- 6 colonies per 10(5) bone marrow cells using colony stimulating factors (CSF) from cat, mouse or human sources. The CFU-E and BFU-E assays gave 114 +/- 7 and 58 +/- 7 colonies respectively with optimum doses of erythropoietin.

Quantification of prothymocytes in long-term bone marrow cultures.

Prothymocytes were present in long-term marrow cell cultures for 17 weeks, although at very low levels. The prothymocytes from the cultures appeared normal in their thymus repopulation ability since the growth curves for thymocytes derived from cultured cells, measured in thymuses of irradiated mice, were parallel to those for thymocytes derived from fresh bone marrow prothymocytes. The buoyant density distribution for prothymocytes from the cultures appeared normal as well, with a modal density of 1,069 g/cm3.

Corneal epithelial cell cultures on stromal carriers.

Exposure of denuded rabbit corneal stromal carriers for 24 hr at 37 degrees C to suspensions of rabbit corneal epithelial cells grown in tissue culture resulted in the establishment of a cell layer on the carriers. The cell layers persisted for at least 1 week of incubation and were one to three cells thick. They consisted of healthy-appearing cells with normal intracellular organelles and intercellular desmosomal connections.

The regulation of hemopoiesis in long-term bone marrow cultures. III. The role of burst forming activity.

The addition of burst forming activity (BFA) from mouse spleen conditioned medium (MSCM) to serum-free long-term cultures induced the formation of day 4 BFUE demonstrating that MSCM has an erythropoietin-independent activity on erythroid development. Conditioned medium (CM) from long-term mouse marrow cell cultures was inhibitory to burst formation at concentrations as low as 0.01% when bone marrow cells were used as the external source of BFA, whereas significant levels of inhibition were apparent only at 100-fold greater concentrations of CM in cultures containing MSCM.

Insect contamination of a cornea stored in M-K medium.

We report the unusual case of contamination with an insect of corneal donor tissue stored in M-K medium. Some possible explanations of this occurrence are proposed, and suggestions are made to help prevent its recurrence.

Evidence for cellular cooperativity in hemoglobin synthesis by erythroid bursts.

We have studied hemoglobin synthesis associated with erythroid bursts under various conditions. The slope of the curve relating log cell number to log hemoglobin synthesis is consistently greater than the slope of the curve relating log cell number to log bursts, suggesting that cellular interactions may be involved in maximal hemoglobin synthesis by the cells comprising the bursts. This suggestion if strengthened by experiments which show that gentle disruption of burst structure is accompanied by a decline in hemoglobin synthesis.

An ocular perfusion system.

An enclosed system is described utilizing an osmotically driven pump to continuously deliver a solution to a variety of intraocular locations. The behavior of this system is illustrated by using it to measure the fluorescein turnover rate in the anterior chamber.

Role of purified erythropoietin in the amplification of the erythroid compartment.

A single dose of Myleran induced a prolonged depletion of multi-potential haemopoietic cells and of early erythroid precursors to levels between 2 and 6 of control. In these mice, a priming dose of highly purified erythropoietin (Epo) or of any of 3 Epo preparations from different sources, and having different specific activities, increased the magnitude of the response to a test dose of Epo by erythropoietin responsive cells (ERC) in the hypertransfused mouse assay. As significant feeding into the ERC compartment from the most immature cells is unlikely because of the depletion induced by the Myleran, it may be concluded that highly purified Epo, not contaminated with other biologically active molecules, has a dual effect on the ERC: it causes increased amplification besides inducing differentiation into cells with the capacity to synthesize haemoglobin.

Erythropoietin-stimulated erythropoiesis in long-term bone marrow culture.

The proliferation of multipotential haematopoietic stem cells (CFU-S) is possible in some long-term bone marrow cultures. Granulocyte and macrophage progenitor (CFU-C) and megakaryocyte precursor cells (CFU-M) are present in these cultures and undergo full development into mature cells. In contrast, while immature erythroid progenitors ('early' BFU-E) are maintained in long-term culture, none of the more differentiated progeny (CFU-E) have been detected, and no morphologically recognisable erythroid cells have been observed.

The relationship of hemoglobin synthesis to erythroid colony and burst formation.

We have demonstrated that the cyclohexanone method for the extraction of hematin can be used to measure hemoglobin synthesis induced by erythropoietin (epo) in mouse bone marrow cells cultured in medium containing methyl cellulose. The time course of hemoglobin synthesis by mouse marrow cells showed two effects due to epo: an increase in hemoglobin synthesis at day 2, which corresponded to the formation of small erythroid colonies resulting from the CFU-E (colony-forming unit, erythroid), and a very large increase in hemoglobin synthesis, which was maximal at days 7-8 and corresponded to the formation of large erythroid colonies (bursts) resulting from the BFU-E (burst-forming unit, erythroid). The epo dose-response curves for CFU-E colony counts and day-2 hemoglobin synthesis were similar, and the cell-number-response curves for these two paramaters were parallel.

Leukocytes and experimental corneal vascularization.

The growth of blood vessels toward corneal burns was compared in normal rabbits and those depleted of their white cells by exposure to X-irradiation. By the fourth day following injury, new vessels were present in all control and leukopenic animals. Histologic examination failed to demonstrate infiltrating cells in the leukopenic animals.

An assay for erythropoietin in vitro at the milliunit level.

A method is described for the assay of erythropoietin using primary cultures of adult rat bone marrow cells. Either total labeled iron uptake by the cells or hematin synthesis from labeled iron may be used as the measure of erythropoietin action. The method is useful in the range 0.