Human peripheral blood cells enriched for B lymphocytes were stimulated to focal proliferation in semisolid cultures with lymphocyte-conditioned media, Protein A (Prot A), lipopolysaccharide (LPS) and 2-mercaptoethanol (2-ME). After 6-8 days of incubation, two morphologically distinct colony types were observed. Type I colonies were diffusely proliferating aggregates within the agar layer, whereas another subset of B-cell-colony-forming cells (CFU-BL) formed round compact type II colonies which appeared to leave the agar layer and continued to proliferate in the liquid overlayer of our culture system. They reached maximum proliferation 2 days earlier than type I colonies. Cells derived from both colony types were positively identified as B lymphocytes by monoclonal antibodies using immunoperoxidase staining. In addition to this distinct growth pattern, both colony types exhibited different proliferative responses which were dependent on the kind of conditioned media used and the mitogen concentration. In secondary cultures both type I and type II colony-derived cells showed recloning capacity. However, after replating, both colony types gave rise to round compact type II colonies. These results demonstrate that there exist at least two subpopulations of colony-forming B lymphocytes, possibly one more primitive than the other, which can be distinguished by in vitro growth characteristics.

The micro-agar-culture technique for cloning early and late erythropoietic progenitor cells (BFU-E and CFU-E) was further modified and miniaturized in order to study the optimal growth conditions with a minimal consumption of erythropoietin (EP). Using microtiter plates, the total incubation volume was lowered from 0.5 to 0.1 ml and thus reduced the necessary amount of EP and cells by a factor of 5. The method consists of a 50 microliter agar layer, in which the mononuclear cells are suspended, and a 50 microliter liquid overlayer containing bovine serum albumin (BSA), transferrin (TF), and EP. After a seven- or 14-day incubation, the whole agar layers were fixed, transferred to microscopic slides, dried, stained using the Pappenheim method, and permanently preserved. The influences of FCS, BSA, and TF in the presence of EP were studied on the proliferation of human bone marrow CFU-E and BFU-E. The variation of FCS concentration showed an optimum at 10%. The addition of BSA in the presence of optimal concentrations of EP markedly increased the number of BFU-E, but not CFU-E. Furthermore, the threshold concentration of EP required for the initial burst formation could be reduced by half in the presence of BSA. By the addition of TF, a further increase in the number of BFU-E was obtained.

The recently described micro-agar culture system for cloning erythropoietic progenitor cells was used to study the optimum conditions for the growth of CFU-M. In this system human mononuclear cells from normal human bone marrow were suspended in agar and incubated for 12 days. Various concentrations of phytohaemagglutinin lymphocyte conditioned medium (PHA-LCM) and prostaglandin E (PGE) were added to the liquid overlayer in the presence of 2-mercaptoethanol (2-ME) for the stimulation of CFU-M. Human AB serum was used instead of fetal calf serum (FCS) in all experiments. A sigmoidal dose-response curve, with a plateau at a concentration of 5 to 10%, was obtained by the addition of different concentrations of PHA-LCM in the presence of 10(-6)PG-E. Under optimal conditions (5% PHA-LCM, PGE 10(-6)M) a linear relation was obtained between the number of seeded cells and the megakaryocytic colonies formed. For routine morphological analysis the whole agar layer was stained using the Pappenheim method. For further characterization of CFU-M, an immunofluorescence test with rabbit antihuman factor VIII related antigen was performed on the whole agar layer.

Human bone marrow cells were grown in a micro-agar culture system in the presence of human placenta (HPCM) and giant cell tumor conditioned media (GCT). The effects of HPCM and GCT conditioned media on linearity, growth dynamics, and morphological composition of colonies were studied after 7 and 14 days of incubation. Under the described conditions the dose-response curves for HPCM and GCT were different: on day 7 maximal stimulation was obtained with 2.5% HPCM and 20% GCT; on day 14 a maximal response was reached with 1.25% HPCM and 5% GCT. Both stimuli produced maximal growth after 7 days of incubation, followed by a rapid decrease in the number of formed colonies up to day 14. The morphological study of aggregates showed that after 7 days of incubation 80% pure granulocyte and 20% mixed granulocyte-macrophage colonies were found in the presence of both stimuli. However, on day 14 the incidence of granulocyte-macrophage colonies increased to 60%, whereas the percentage of pure granulocyte colonies decreased to 20%. The frequency of eosinophil colonies was relatively low (median 15%) with both stimuli. The described system can be applied successfully for studies of myelopoiesis in vitro. Both sources of colony stimulating activitites (CSA) employed had no significant difference in their ability to stimulate myelopoiesis.

A recently developed micro agar culture system has been optimized for the in vitro growth of human B-lymphocytes. Enriched B-lymphocytes from the peripheral blood of normal individuals were suspended in an agar layer, above which a liquid overlayer containing 20% PHA-LCM, 2-ME and Prot A or LPS as stimulants was added. Two morphologically distinguishable colony types were observed using this culture technique: diffusely proliferating colonies (Type I) were found within the agar layer, and round, compact colonies (Type II) appeared to leave the agar layer and continue growth in the liquid overlayer. For both colony types a linear relationship was demonstrated between the number of seeded cells and the number of formed colonies. The appearance of the two colony types in vitro was not concurrent, and they exhibited differing sensitivity to mitogen concentration and to the type of serum used as additive to the culture medium. The implications of clonal in vitro cultivation of lymphocytes, both normal and pathological, are discussed.

A simple and reproducible micro-agar culture technique for cloning human CFU-M is described. Human bone marrow mononuclear cells were suspended in agar and incubated for 12 days. Stimulation was provided by the direct addition of phytohemagglutinin-P (PHA-P), erythropoietin (Epo) and 2-mercaptoethanol (2-ME) to the liquid overlayer. A shift from BFU-E and CFU-C proliferation to CFU-M and CFU-TL was observed with increasing PHA concentrations. Under optimal conditions (PHA 50 micrograms, Epo 1.2 IU, 2-ME 2 x 10(-4) M, 1% purified BSA, 0.04% human transferrin, saturated with Fe C13) a linear relationship between colonies formed and plated cell number were observed. For the routine morphological analysis, the whole agar layers were stained using the Pappenheim method. For further characterization of CFU-M, cytochemical stainings and immunofluorescence tests with rabbit-antihuman factor VIII-related antigen were performed on the whole agar layers.

Axon and terminal degeneration were studied in the cat dorsolateral pontine nucleus (DLPN) after lesion of the inferior colliculus. In separate experiments the acoustic responses of 111 units of the lateral pontine nuclei were studied in cats anesthetized with chloralose-urethane. Lesions of all three nuclei of the inferior colliculus (central, pericentral and external) lead to a very similar pattern of terminal degeneration in a discrete region of ipsilateral DLPN. This is suggestive of a highly convergent projection in which topography may be blurred. Most units responded to binaural stimulation, and the most common binaural response consisted of excitatory inputs from each ear which were facilitated at some binaural intensity levels and occluded at others. Discharge rates changed as a result of alterations in the number of spikes evoked at the onset of the stimulus, and sustained discharges were rarely encountered. Units were broadly and irregularly tuned; binaural inhibition was very uncommon. Unit response characteristics suggested that, while the projection from the inferior colliculus was highly convergent, only a subclass of inferior colliculus neurons may be involved. However, the acoustic properties of lateral pontine units were strikingly similar to those of the cerebellar vermis, a region to which DLPN is known to project.

Auditory responses of single units in the pontine nuclei of the anaesthetized cat were studied electrophysiologically. Many units in the pontine nuclei, and in particular the dorsolateral pontine nucleus were strongly driven by the onset of auditory tonal stimuli in a broadly tuned manner. Most units were excited by stimulation of each ear and demonstrated binaural facilitation. Also, many pontine units responded to visual stimuli and some were bimodally active. These response properties are similar to those of units recorded in the cerebellar vermis which probably receives auditory and visual input from the pontine nuclei.

The responses of 146 cerebellar neurons to tone stimuli were studied in 29 cats anesthetized with chloralose-urethan and in 7 decerebrate preparations. Units were classified as onset or sustained firing. Onset spikes occurred on stimulation of either ear and showed binaural facilitation, while sustained discharges were frequently only excited by monaural stimulation. The latent periods of sustained discharges appeared to be shorter than those of onset responses, and sustained discharges were also more sharply tuned than the onset units. Evidence was presented suggesting that onset responses reflected input from the inferior colliculus and sustained responses, the cochlear nucleus. The sterotyped facilitatory behavior of onset units suggested that a maximal discharge might occur if sounds were of equal intensity at each ear; 26 neurons were examined with variable interaural time or intensity differences and 10 of these exhibited maximal firing when the interaural time and intensity difference was zero--i.e., if the sound was located directly in front of the head.