Inductive properties of fibroblastic cell cultures derived from rat intestinal mucosa on epithelial differentiation

The present study represents a first attempt to elucidate the regulatory properties displayed by the non-epithelial portion of the intestinal mucosa, growing as fibroblasts in monolayer cultures. Thus, we compared the inductive action of 6-day suckling rat duodenal fibroblasts with that displayed by chick embryonic intestinal mesenchyme on the heterotypic cytodifferentiation of 5 1/2-day chick embryonic gizzard endoderm. The latter, isolated by 0.03% collagenase, was surrounded by intestinal intramucosal fibroblastic cell sheets. As control experiments, fibroblastic cells derived from the intestinal muscle or from 20-day fetal rat skin and lung were used. Every type of association was grafted into the coelomic cavity of 3-day chick embryos for 11 to 12 days, a system providing their vascularization and growth. The results clearly demonstrate that the mucosal fibroblastic cells of rat intestine were as potent as embryonic intestinal mesenchyme in inducing brush-border enzymes like sucrase and maltase, in conformity with an induced intestinal morphology. In contrast, the control fibroblastic cells were completely ineffective.     

Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes

The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of $\text{5-}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones.     

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.     

CHRONOLOGICAL CHANGES IN THE INDUCTIVE ABILITY OF THE MESENCHYME OF THE DIGESTIVE ORGANS IN AVIAN EMBRYOS

Dissociation and reassociation experiments were carried out to study the inductive ability of mesenchyme of the oesophagus, gizzard and intestine of the chicken embryo, using 3-day-old quail embryonic allantoic endoderm as an effector tissue. The mesenchyme of the oesophagus and gizzard possesses inductive ability until the Ilth day of incubation. Thereafter, it no longer has inductive influence upon the allantoic endoderm. The intestinal mesenchyme was favourable to differentiation of allantoic endoderm into intestinal epithelium even on the I5th day of incubation. In all types of recombination tested, goblet cells differentiated among allantoic endodermal cells.     

DIFFERENTIATION OF THE DIGESTIVE TRACT EPITHELIUM UNDER THE INFLUENCE OF THE HETEROLOGOUS MESENCHYME OF THE DIGESTIVE TRACT IN THE BIRD EMBRYOS

The endoderm of the oesophagus, proventriculus, gizzard or small intestine of the 5-day-old chick or quail embryo was cultivated in combination with homologous or heterologous mesenchyme on a WxxxOLFFyyy and HxxxAFFHNyyy medium for 7 to 21 days or on the chorio-allantoic membrane (CAM) for 8 days. With homologous mesenchyme the epithelium always differentiated homotypically. In association with heterologous mesenchyme, the differentiation of the epithelium was both homotypical and heterotypical depending on the region of the digestive tract. The oesophagus and small intestine differentiate mainly homotypically both in culture and on CAM, but the gizzard and proventriculus show heterotypic differentiation particularly on CAM. Thus, the endoderm of the digestive tract of the 5-day-old chick or quail embryo, though rather "determined", still reacts to the heterologous stimuli of the mesenchyme to some degree.     

Effects of human fetal gastroenteric mesenchymal cells on some developmental aspects of animal gut endoderm

Human intestinal and gastric mesenchymal cells were associated with chick and rat intestinal endoderm in order to test their species-specific capacity on epithelial differentiation. Primary cell cultures were established from human intestinal and gastric mesenchyme. Animal intestinal endoderms were associated with both cell types, grafted in ovo and allowed to develop for 12 days. The morphologic and enzymatic differentiation of the recombinants demonstrated two types of inductive properties exerted by human fetal intestinal and gastric mesenchymal cells, respectively. Firstly, human intestinal mesenchymal cells triggered intrinsic developmental capacities in chick and rat endoderm, i.e. enhanced structural brush-border maturation in both species and precocious sucrase induction in rat endoderm. Secondly, human gastric mesenchymal cells provoked the partial conversion of chick intestinal endoderm into gastric structures. Such properties were not found in homologous animal mesenchymes.     

Intestinal mesenchyme provokes differentiation of intestinal endocrine cells in gizzard endoderm

The gizzard (muscular stomach) of chicks is deficient in endocrine cells at hatching. It has previously been shown that proventricular types and proportions of endocrine cells can be induced in gizzard endoderm under the influence of proventricular (glandular stomach) mesenchyme. In order to test its capacity to form nongastric endocrine cell types, gizzard endoderm of 3.75- to 5-day chick embryos was combined with mesenchyme from the small intestine of 3.5- to 4-day quail embryos. The combinations were grown as chorio-allantoic grafts until they attained an incubation age comparable to that of hatching chicks. Controls comprised reassociated endoderm and mesenchyme of chick gizzard and of quail intestine. In the experimental grafts, morphogenesis was predominantly intestinal but some grafts showed gizzard-like features, particularly if the endoderm had been provided by older donors. All intestinal endocrine cell types, including those also found in the normal proventriculus (serotonin-, glucagon-, pancreatic polypeptide-, neurotensin- and somatostatin-immunoreactive cells) differentiated in experimental grafts, some even where morphogenesis was gizzard-like. Hence progenitors of not only gastric, but also intestinal, endocrine cells are indeed present in gizzard endoderm. The possibility that gizzard mesenchyme is inhibitory to endocrine cell differentiation is mooted. Motilin- and secretin-immunoreactive cells, which are characteristic of the intestine but not of the proventriculus of chicks at hatching, were respectively sparse or absent when the endoderm was derived from older donors. Thus the ability of gizzard endoderm to differentiate into nongastric endocrine cell types declines before its capacity to form gastric types. The unexpected appearance of gastrin-releasing peptide (GRP)-immunoreactive cells, a proventricular type not found in normal chick intestine, suggests that the intestinal mesenchyme, at least in this instance, was exercising a permissive role.     

Differentiation of lens and pigment cells in cultures of neural retinal cells of early chick embryos

Dissociated cells of neural retinas of 3.5-day-old chick embryos (stages 20–21) were cultured as a monolayer in order to examine their differentiation in vitro. These cells started to grow actively soon after inoculation and formed a confluent sheet within which neuroblast-like cells with long cytoplasmic processes were differentiated by 8 days. At about 16 days the differentiation of both lentoid bodies and foci of pigment cells was observed, while neuronal structure disappeared. The numbers of lentoid bodies and foci of pigmented cells continued to increase up to 30 days, when primary cultures were terminated. The increase in δ-crystallin content, as measured by quantitative immunoelectrophoresis assay using rabbit antiserum against δ-crystallin, was consistent with the increase in the number of lentoid bodies in cultures. The amount of α-crystallin per culture, estimated by the same technique as above, reached a maximum at 16 days and decreased slightly during further culture. The differentiation of both lentoid bodies and pigment cells was observed also in cultures of the second generation. The results demonstrate that cells of the undifferentiated neuroepithelium of 3.5-day-old embryonic retinas can achieve at least three differentiations, neuronal, lens, and pigment cells, in vitro. We discuss several differences between the present results and the previous ones from in vitro cultures of 8- to 9-day-old embryonic neural retinas.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

Two distinct regulatory steps in cartilage differentiation

The effect of developmental stage on chondrogenic capacity in high-density cell cultures of chick embryonic wing bud mesenchyme is examined. Mesenchyme from stage 19 embryos forms aggregates of closely associated cells which do not form cartilage matrix, nor contain significant levels of type II collagen that are detectable by immunofluorescence, unless they are treated with dibutyryl cyclic AMP. Mesenchyme from stage 24 embryonic wing buds in high-density cell cultures will spontaneously form cartilage, as defined by electron microscopy and immunofluorescence with antibody to type II collagen. Cultures prepared from stage 26 wings form numerous aggregates which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells morphologically. However, because these cells show considerable intracellular immunofluorescence for type II collagen, they are actually unexpressed cartilage cells. Several treatments, including exposure to dibutyryl cyclic AMP, ascorbic acid and an atmosphere of 5% oxygen, or mixture with small numbers of stage 24 wing mesenchyme cells, stimulate expression, as determined by the accumulation of an Alcian blue-staining matrix and an ultrastructurally recognizable cartilage matrix. Since the addition of similar numbers of differentiated cartilage cells does not stimulate expression of stage 26 cells, it is proposed that initial cartilage expression is dependent on a mesenchyme-specific influence which might be removed by cell dissociation. These studies demonstrate that there are at least two distinct transitions in cartilage differentiation: one involves the conversion of mesenchyme to unexpressed chondrocytes and the second involves mesenchyme-dependent expression of chondrogenic differentiation.     

Proteomic Profiling of Enteroid Cultures Skewed toward Development of Specific Epithelial Lineages

Recently, 3D small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small‐molecule drug treatment can skew organoid epithelial cell differentiation toward particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. This study utilises label‐free quantitative proteomics to determine whether skewing murine enteroid cultures toward the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Here, proteomics data confirms that skewed enteroids recapitulate important features of the in vivo gut environment, demonstrating that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, comparison of mass spectrometry data with histology data contained within the Human Protein Atlas identifies putative novel markers for goblet and Paneth cells.     

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10⁶ cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [³H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.     

Immunocytochemical studies of chicken somatotrophs and somatotroph granules before and after hatching

Immunocytochemical methods were used to gain information about the embryonic development of chicken somatotrophs before and after hatching. To localize growth hormone, anterior pituitary sections were incubated with growth-hormone antibody, and then an indirect peroxidase method was used for light microscopy and an immunogold method for electron microscopy. The earliest evidence of embryonic somatotrophs was seen at 12 days. At this stage somatotrophs were sparse (0.2% of parenchymal cells) and their granules were pleomorphic with elongated ovoid and lozenge shapes predominating. Few of the immunogold-labeled somatotroph granules of the embryo were spherical until 15 days after fertilization. At 18 days, most of the granules were spherical (their shape in the adult chicken). During the six days between the 15-day-old embryo and the 1-day-old chick, the number of gold particles per granule section approximately doubled suggesting an increase in growth hormone content of the granules. This rise was the result of increases in the size of the granule sections and in the concentration of gold particles in the sections. During the embryonic period of 12–20 days, somatotrophs were not more than 3.6% of the anterior pituitary cell population. During the following two days, between the 20-day-old embryo and the 1-day-old chick, the percentage of somatotrophs in the pituitary parenchymal cell population rose rapidly from 3.6% to 20.7% and then increased slowly to 24.6% during the period of 1–5 days after hatching. Both the sharp percentage rise in somatotrophs (20-day-old embryo to 1-day-old chick) and the rise in growth hormone content of the granules (15-day-old embryo to 1-day-old chick) suggested by gold-particle counts occur close to the time of hatching. These morphological changes may reflect an increased synthesis of growth hormone that is responsible for the rise in plasma growth-hormone concentration that begins about the same time and is especially abrupt two days later (1–3 days after hatching).     

Failure of insulin cells to develop in cultured embryonic chick pancreas: A model system for the detection of factors supporting insulin cell differentiation

Summary Little being known about factors necessary for insulin cell differentiation, we tested the chance observation that these cells were virtually absent from collagen gel cultures of embryonic avian pancreas in which the other pancreatic endocrine cells were numerous. Five-day dorsal buds stripped of their enveloping mesenchyme were embedded in gel and overlaid by a defined medium containing serum, then cultured for 7 days. Immunocytochemical evaluation showed a very low proportion of insulin cells. Substitution of the gel by a polyamino acid coating slightly increased the proportion. In an attempt to test for ability of insulin cell formation to recover, we transferred explants first cultured in collagen gel to polyamino-acid-coated dishes for a further 7 days. No improvement resulted. In controls grown for 14 days on a polyamino acid coating, insulin cells disappeared completely. We conclude that collagen gel does not support survival and differentiation of chick embryonic insulin cells and that the medium used is lacking in some essential factor(s). Determination of their identity should prove possible by exploitation of this model.     

Characterization of a bone-specific alkaline phosphatase in chick limb mesenchymal cell cultures

Previous morphometric and biochemical studies suggested that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. Others have shown that osteoblast expression is marked by an increase in bone-specific alkaline phosphatase activity. Our results indicate that chick limb mesenchymal cells develop alkaline phosphatase activity that is identical to that of the chick embryonic bone-specific isoenzyme. The alkaline phosphatase isozymes were partially purified from samples of chick intestine, liver, stage 38 embryonic limbs, and cultures of stage 24 limb mesenchymal cells. These tissues were separately extracted with butanol, acetone precipitated, redissolved, and passed over a DEAE-Sephacel ion-exchange column and ion-filtration column (Sephadex A-25). From the data obtained during this purification scheme, we conclude that the alkaline phosphatase from stage 38 limbs (bones) and Day 4 cultures are identical, and this activity is different from the enzyme purified from intestine and liver. The cell culture isozyme has an apparent K_m, heat lability, response to specific inhibitors, electrophoretic mobility, and molecular weight similar to those of bone-specific alkaline phosphatase. These observations support the view that osteoblastic progenitor cells are present in the stage 24 limb mesenchyme and that under specific culture conditions, bone development can be uniquely observed in vitro.     

Enzymatic response to glucocorticoids of the chick intestinal endoderm associated with various mesenchymal cell types

The aim of the present study was to test the morphological and functional maturation of recombinants composed of chick intestinal endoderms associated to different mesenchymal supports and their enzymatic response to glucocorticoids. For this purpose 5.5-day chick embryonic intestinal endoderm has been associated to 14-day fetal rat gut mesenchyme, to rat intestinal fibroblasts (6-day neonatal rat intramucosal fibroblasts) or to rat control fibroblasts, originating from 20-day fetal rat skin and lung and from 6-day neonatal rat intestinal muscle. The recombinants were grown as intracoelomic grafts either for 12 days or for 10 days plus 2 days in organ culture in the presence of dexamethasone. The data show that heterospecific recombinants achieve subnormal morphogenesis and enzymatic maturation. The organ culture experiments further reveal that sucrase activity is insensitive to dexamethasone in all types of recombinants whereas, alkaline phosphatase is highly stimulated over the levels present in the intestine developed in situ whatever the stromal support, except when this support is provided by rat gut mesenchyme. These results support the view that in the intestine the hormonal response is mediated by epithelial-mesenchymal interactions.     

Development of sucrase in the chick small intestine

Development of sucrase in the chick small intestine was studied biochemically and immunologically using antiserum prepared against purified chick intestinal sucrase. Sucrase activity was first detectable at 10 days of incubation and increased with age. After a transient drop at 20 days, the activity rapidly increased to the adult level. Immunodiffusion and polyacrylamide gel electrophoretic studies suggested that the sucrase of the embryonic and hatched chick intestines was identical except for a difference in the content of sialic acids. In immunofluorescence and immunoelectron microscopy, sucrase was found to appear on the luminal surface of epithelial cells at 8-10 days of incubation, soon after the start of morphological differentiation from an undifferentiated thick epithelium to a thin simple epithelium.     

Embryonic neurons as in vitro inducers of differentiation of nephrogenic mesenchyme

Nephrogenic mesenchyme differentiates into epithelium as a result of morphogenetic tissue interactions. In vivo, the ureter bud is thought to induce tubular differentiation of the mesenchyme. In vitro recombination experiments have shown that various embryonic tissues can act as inducers when put in close proximity to nephrogenic mesenchyme. Induction also occurs across a porous filter. In the present study we show that only a few embryonic tissues are potent inducers in transfilter cultures in which mesenchyme and inducing tissue are separated by a membrane filter. Of the tissues tested, only embryonic spinal cord and brain were effective, whereas the ureter bud did not induce. All tissues tested sent processes through the filter. Weak inducing capacity of embryonic tissues is thus not due to a failure of the cells to make contact with the mesenchyme. To analyze which cell type within the embryonic brain possesses inducing capacity, neurons were selectively removed from primary cultures of chick tectal cells by antibody and complement-mediated cell lysis. These cultures, consisting of glial and undifferentiated cells, were then recombined with nephrogenic mesenchyme. They proved to be ineffective in inducing tubulogenesis, whereas cell populations containing neurons retained their inducing capacity. In transfilter cultures, ingrowth of neuronal processes deep into the mesenchyme, as assayed by anti-neurofilament staining, occurred within the first 24 hr of culture. Thus, it is not the time needed for processes to grow through the filter, but the time needed to grow into the mesenchyme that corresponds to the minimal induction time. These studies suggest that embryonic neurons are the most effective inducers of nephrogenic mesenchyme in vitro. Differentiation may be triggered by neuronal processes that establish cell contacts deep within the mesenchyme. Neurons might be important for nephrogenesis in vivo as well, although we can present no direct evidence to support this idea, since we failed to detect neurons at early stages of kidney development when the first tubules are induced.     

Endoderm- and mesenchyme-dependent commitment of the differentiated epithelial cell types in the developing intestine of rat

During organogenesis, the intestinal tract progressively acquires a functional regionalization along the antero-posterior axis. Positional information needed for enterocytes has been studied, but the mechanisms that control Paneth and endocrine cell differentiation are poorly understood. We have used a model of endoderm/mesenchyme cross-associations to evaluate the respective roles of endoderm and mesenchyme in the cytodifferentiation of these epithelial cells. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and colon were developed as xenografts in nude mice. Our results show that endoderm from the presumptive proximal jejunum when associated with colonic mesenchyme generate small intestinal enterocytes. Interestingly, no lysozyme-producing cells were generated. On the other hand, associations comprising colon endoderm and jejunal mesenchyme showed heterodifferentiation with typical small intestinal morphology with sucrase-isomaltase expression and Paneth cell differentiation. Heterotopic associations developed enteroendocrine cell patterns according to the normal fate of the endodermal moiety. As enteroendocrine cell commitment seems to occur before the other intestinal cell types, we cannot exclude a role of instructive signals from the mesenchyme on endocrine cell differentiation earlier in the development. These results identified a complex pattern of cell commitment, dependent of the differentiation type of the epithelial cell, on the regional origin of the endoderm and the associated mesenchyme.