Gastrointestinal stem cells. II. Intestinal stem cells

Current views of the identity, distribution, and regulation of small intestinal epithelial stem cells and their immediate progeny are discussed. Recent works implicating Wnt signaling in stem and progenitor proliferation, the involvement of Notch signaling in epithelial lineage specification, and the role of hedgehog and bone morphogenetic protein families in crypt formation are integrated. We had the good fortune that many of these papers came in pairs from independent groups. We attempt to identify points of agreement, reinterpret each in the context of the other, and indicate directions for continued progress.     

Gastrointestinal stem cells. I. Pancreatic stem cells

The transplantation of islets isolated from donor pancreas has renewed the interest in cell therapy for the treatment of diabetes. In addition, the capacity that stem cells have to differentiate into a wide variety of cell types makes their use ideal to generate beta-cells for transplantation therapies. Several studies have reported the generation of insulin-secreting cells from embryonic and adult stem cells that normalized blood glucose values when transplanted into diabetic animal models. Finally, although much work remains to be done, there is sufficient evidence to warrant continued efforts on stem cell research to cure diabetes.     

Guidepost cells.

Guidepost cells, as classically defined in the grasshopper embryo have only rarely been found in other systems. If the concept of guidepost cells is expanded, recognizing that any special role of specific cells in axon guidance is a function of the entire landscape in which axons are growing, and that growth cone--guidepost interactions may share mechanisms with many other cell--cell interactions, then numerous examples are found in both the peripheral and central nervous systems of many species.     

Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Avian antigen-binding cells. II. Enrichment of antigen-binding B and T cells.

Antigen-binding B and T cells from chicken spleens were selected on plates of antigen-derived gelatin. Hapten-specific B cells from DNP-immune normal chicken spleens were selected on DNP-gelatin. As much as 165-fold functional enrichment of precursors of anti-DNP antibody-producing cells, as measured in an adoptive transfer system, was achieved. However, the enrichment of DNP-binding cells assessed by rosette formation and autoradiography was no more than 25-fold. HGG-specific T cells from bursectomized agammaglobulinemic chickens immunized with deaggregated HGG were selected on HGG-gelatin. In the fraction adherent to HGG-gelatin, at least a 20-fold enrichment of suppressors of the antibody response to TNP-HGG, as measured by adoptive transfer, was accomplished. In contrast, no more than 6-fold enrichment of HGG-binding cells was detected by autoradiography. Antigen-specific depletion and enrichment of suppressor T cells and of HGG-binding cells occurred in parallel, suggesting that suppressor cells can bind soluble antigen and can be isolated on antigen coupled to a solid support.     

Interaction of arterial cells. I. Endothelial cells alter cholesterol metabolism in co-cultured smooth muscle cells

Results of previous in vivo experiments indicated that the presence of arterial endothelium modifies cholesteryl ester (CE) metabolism and the retention of low density lipoproteins (LDL) in injured arteries. We describe herein the effects of bovine arterial endothelial cells (ENDO) on the CE cycle, fluid phase endocytosis, and cell proliferation in co-cultured bovine arterial smooth muscle cells (SMC). Following several days of cultivation on confluent SMC, ENDO were removed from SMC by treatment of the co-cultures with 1.0% collagenase (type II). Removal of only ENDO from the co-culture dishes was confirmed by immunofluorescent staining for Factor VIII antigen, hemotoxylin-eosin staining, and biochemical analyses. We observed that ENDO grown to 75% confluency on confluent SMC induced: 1) a reduction of CE hydrolysis as a result of decreased lysosomal CE hydrolytic activity in SMC as compared to SMC cultured alone; and 2) an increase in the rate of incorporation of labeled oleate into CE as a result of increased acyl CoA:cholesterol O-acyltransferase activity in SMC as compared to SMC cultured alone. Neither endothelial cell-derived culture media (ECDM) nor fibroblasts modulated CE metabolism in co-cultured SMC. Additional experiments showed that the presence of endothelial cells or ECDM decreased the proliferation of co-cultured SMC by 50%, but enhanced the endocytotic rate of labeled sucrose into SMC threefold. Results of experiments described herein demonstrate that, in addition to providing a thrombo-resistant surface and regulating permeability, endothelial cells may also serve to modulate cholesteryl ester metabolism in smooth muscle cells derived from the arterial wall.     

Development of mast cells in vitro. II. Biologic function of cultured mast cells.

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 mug/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.     

Macrophage-like suppressor cells in rats. I. Inhibition of natural macrophage-like suppressor cells by red blood cells

When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal.After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro, Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro. Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later.Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.     

Specific, transient suppression of the immune response by HGG tolerant spleen cells. II. Effector cells and target cells.

In a previous report, it was shown that spleen cells from mice made tolerant to human gamma-globulin (HGG)5 could specifically inhibit the immune response of normal spleen cells after adoptive transfer to lethally irradiated recipients. However, that report also showed that the suppressive activity was only transiently associated with tolerant spleen cell populations. It was concluded from those experiments that while suppressive activity could be demonstrated in tolerant spleen cells under certain conditions, such activity was not obligatory for the maintainance of the tolerant state. The experiments presented here were performed to determine the nature of the effector cell(s) and the target cell(s) involved in this system of suppression of the immune response. Treatment of cells from tolerant animals with anti-thymocyte serum and complement to remove thymus-derived (T) cells completely abrogated suppresive activity. Removal of adherent cells from tolerant spleen cells by passage over glass wool columns resulted in partial loss of the suppression. The inhibitory activity of the suppressor cells was resistant to 900 R irradiation regardless of whether the tolerant spleen cells were irradiated before or after adoptive transfer. The cellular target(s) for the supprssor cells was examined by using lipopolysaccharide (LPS) as an alternative source of helper activity for the response to HGG. LPS, injected at the time of the initial antigenic challenge of mice that had been reconstituted with tolerant and normal spleen cells, prevented the expression of suppression against bone marrow-derived (B) cells. However, when LPS was presented only at the time of secondary antigenic challenge, it was unable to overcome suppression of the immune response of reconstituted recipients. Thus, LPS could produce a state where the B cells were resistant to suppression, but LPS could not rescue the responsiveness of B cells once the cells in the reconstituted recipient had been suppressed. In addition, the immune response to both the hapten dinitrophenol (DNP) and the carrier (HGG) were suppressed when recipients of tolerant and normal spleen cells were challenged with DNP6HGG. This indicates that T helper cells are also a target for suppression. The results presented in this paper are discussed in relation to a possible mechanism of suppression which proposes that suppressive activity represents the induction of tolerance in immunologically competent cells by HCG which is closely associated with the tolerant spleen cells.     

Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells

The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. The length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state, however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach approximately equal to 50% were incapable of either increasing their RNA levels to that of proliferating G1 cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Since clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.