Lung organoid culture

An organoid culture system for lung cells is described in which morphogenesis of lung histotypic structures and differentiation of both pneumocytes type II and mesenchyme occur. The principle of this technique is the culture of mouse fetal lung cells at high density on a membrane filter at the medium/air interface. In the course of cultivation, cell sorting-out, epithelial cell aggregation, formation of an alveolar-like lumen in the organoids and formation of a basal lamina occur. Epithelial differentiation culminates in the production of lamellar bodies, and the mesenchyme develops into mature connective tissue. Morphogenesis and differentiation depend on the stage of fetal development from which the lung cells were derived but appear independent of the formation of a basal lamina. Various drugs have been tested for their effects on morphogenesis and differentiation in this lung organoid culture: some of them inhibit differentiation or damage the mesenchyme, others stimulate surfactant production. Due to the quite complex morphogenetic and cellular events occurring in lung organoid culture, it may be an applicable tool for alternative in vitro screening methods.     

Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.     

Lamellar body formation precedes pulmonary surfactant apoprotein expression during embryonic mouse lung development in vivo and in vitro

Abstract. The purpose of this investigation was to determine whether lamellar inclusion body (LB) formation and surfactant apoprotein (SP-35) production are directly coordinated by temporal and positional information during development. In the present study we report a comparison between embryonic B10.A mouse lung morphogenesis and cytodifferentiation in vivo with that observed during organ culture in serumless medium. Precursor LB were first detected at embryonic day 12 (E12d), and progressively larger numbers and forms were produced during subsequent differentiation of respiratory alveolar duct epithelium. SP-35 was first detected during the canalicular period (E16.5d). Lung cultures (E12 d) showed pseudoglandular and canalicular periods of morphogenesis, and both ciliated epithelial and type II cell differentiation. Nonciliated cells produced increasing numbers of lamellar inclusion bodies throughout the culture period. SP-35 was detected at 9 days in vitro (d.i.v.). These observations indicate (i) precursor LB formation precedes SP-35 expression and is not dependent on apoprotein synthesis; (ii) E12d lung development in vitro using serumless medium proceeds at a rate equivalent to 0.5 days in vivo through 11 d.i.v.; and (iii) morphogenesis and differentiation occur in the absence of exogenous hormones and growth factors. The cell-cell interactions that play a role in morphogenesis and cell differentiation appear to be intrinsic to the developmental program for embryonic lung development and are likely to be mediated by autocrine and/or paracrine factors.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.     

Embryonic mouse lung morphogenesis and type II cytodifferentiation in serumless, chemically defined medium using prolonged in vitro cultures

The timing, position and mechanism(s) for determining type II cytodifferentiation during mammalian lung development are not known. To approach this problem, we have cultured Theiler stage 16 embryonic B10.A strain mouse lung primordia (12-days gestation, E12) in serumless, chemically defined medium in the presence or absence of dexamethasone (DEX) for periods up to 27 days in vitro. Morphogenesis and cytodifferentiation were evaluated by light and transmission electron microscopy and immunochemical techniques. Pulmonary surfactant-associated apoproteins (PSAP) were initially expressed by type II cells at 16.5-day gestation in vivo. DEX-supplementation to the culture medium resulted in the accelerated expression of PSAP; the apoprotein isoforms (A1, A2, and A3) produced in vitro were comparable to those synthesized during fetal and postnatal in vivo development by high resolution, two-dimensional gel electrophoresis coupled with immunoblot staining. Cultures without DEX produced PSAP A2 and A3 isoforms, but did not produce A1 (26-31 kDa, pI 5.2-5.3). DEX-treated cultures produced more lamellar bodies within type II cells than non-treated controls. The results demonstrate that long-term cultures of embryonic lung primordia express morphogenesis, cytodifferentiation and the synthesis and secretion of PSAP in the absence of exogenous hormones or growth factors. The data set further supports the hypothesis that morphogenesis and type II cytodifferentiation are regulated by autocrine and paracrine factors intrinsic to the embryonic lung developmental program and independent of exogenous hormone controls.     

Lectin Histochemistry of Normal Human Lung

This study aimed to identify and specify the glycotypes of cell populations in normal human lung including types I and II pneumocytes, alveolar macrophages and mast cells, and also in the larger tissue structures of lung, including blood vessels and bronchi/bronchioles, using lectin- and immuno-histochemistry on paraffin-embedded tissue from 11 normal cases. The alveolar macrophages were anti-CD68 positive whereas the cells lining the alveolar walls were positive for cytokeratins. The alveolar macrophages in normal lung tissues showed a broad spectrum of staining for different subsets of N-linked saccharides, N-acetylgalactosamine, N-acetylglucosamine, terminal beta-D-galactose and sialyl groups. This study showed that some lectins could be used as specific markers for some cell types i.e. Galanthus nivalis and Narcissus pseudonarcissus lectins for macrophages, Psophocarpus tetragonolobus lectin-II for capillary endothelium, Dolichos biflorus agglutinin for bronchial epithelial cells, Lycopersicon esculentum, Phytolacca americana or Triticum vulgaris (succinylated) for type I pneumocytes and Hippeastrum hybrid or Maclura pomifera lectins for type II pneumocytes. Patchy staining of type I pneumocytes by peanut agglutinin indicated the possibility of two distinct populations of these cells or a pattern of differentiation that is unapparent morphologically.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.     

Comparison of glycoconjugates at the surface of developing type II pneumocytes and Clara cells

Summary The apical surface coat of type II pneumocytes and Clara cells in pre- and post-natal rat lung was examined with lectin histochemical methods. Lectins fromHelix pomatia (HPA), peanut (PNA) andMaclura pomifera (MPA) were conjugated with horseradish peroxidase and used to stain paraffin sections of fixed lung with or without certain pre-treatments. HPA and MPA were observed to react with almost all type II pneumocytes at postnatal day 1. Type II pneumocytes that stained with a sialidase—PNA sequence increased from a few positive cells at postnatal day 5 to many in the adult. It has been reported that the surface coat of type II pneumocytes closely resembles that of Clara cells in its staining with histochemical methods employing cationic dyes or lectins including MPA and PNA. However, staining with HPA, especially after periodic acid oxidation, revealed many type II pneumocytes with strong reactivity but showed only a few Clara cells that were faintly positive. HPA also stained alveolar macrophages. The HPA affinity of macrophages, however, was labile to oxidation with periodic acid or galactose oxidase unlike that of type II pneumocytes. This difference suggests that HPA recognizes more than one type of sugar structure.To whom all correspondence and reprint requests should be addressed.     

Human dendritic cell lysosome-associated membrane protein expressed in lung type II pneumocytes

Human dendritic cell LAMP (hDC-LAMP) is a unique member of the lysosome-associated membrane protein (LAMP) family with a tissue distribution initially described as restricted to major histocompatibility class II (MHC II) compartments of activated DC before the translocation of MHC II to the cell surface [Immunity 9 (1998) 325]. In this report, we show that hDC-LAMP is also expressed by lung type II pneumocytes, another cell type with constitutive expression of MHC II. A recombinant hDC-LAMP protein and a monospecific anti-hDC-LAMP polyclonal antibody were prepared. The antibody reacted specifically with hDC-LAMP sequences of hDC-LAMP protein expressed in transfected cells and with a 54 kDa protein of normal human lung tissue with properties corresponding to those of transgene expressed hDC-LAMP. Immunohistochemical analysis of hDC-LAMP in human lung showed its presence in alveolar type II epithelial cells (type II pneumocytes) as well as in cells in the interfollicular area of bronchus-associated lymph nodes, where interdigitating DCs are concentrated, and with lesser staining of alveolar macrophages. The native protein contained approximately 16% carbohydrates, most of which are sialyl N-linked oligosaccharides, with an acidic isoelectric point (pI 4.8). The restricted localization of this protein to lung type II pneumocytes and DCs is in contrast to hLAMP-1, which was present in many cell types of the lung and lymph node. Type II pneumocytes are known to express MHC II and the abundant expression of hDC-LAMP in these cells as well as in DCs suggests its possible relationship to specific MHC II related function(s) of DC and type II pneumocytes.     

Immunocytochemical distribution of E-cadherin in normal and injured lung tissue of the rat

Affinity purified rabbit anti-mouse E-cadherin antibodies, reacting with diverse rat epithelia, were used to characterize epithelial changes in a radiation-induced fibrosis model of rat lung by immunoblotting techniques, immunoperoxidase and immunofluorescence microscopy. Immunostaining of normal rat lung tissues revealed a predominant staining of type II pneumocytes. Immunoelectron microscopy confirmed the immunohistochemical data of normal lung tissue obtained at the light microscopic level. In severely injured rat lung, we found enhanced immunoreactivity for E-cadherin at the surface of type I alveolar epithelial cells. The results suggest that E-cadherin is an adhesion molecule that is modulated after pathological alteration of the alveolar epithelium and that the antiserum may be useful for the characterization of normal and diseased rat epithelia.     

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.     

Epithelial-mesenchymal interactions control basement membrane production and differentiation in cultured and transplanted mouse keratinocytes

Summary The effects of mesenchyme and substratum on epidermal differentiation and formation of a basement membrane (BM) were analyzed in vitro and in vivo. Primary epidermal cell cultures (PEC) from neonatal mice were grown: (1) on plastic culture dishes; (2) on lifted collagen gels, either alone or (3) in recombination with mesenchyme; (4) after reimplantation in vivo either directly on mesenchyme or (5) on collagen interposed between keratinocytes and mesenchyme. Differentiation of the epithelium and formation of a BM were examined by electron microscopy, and expression of BM constituents (type IV collagen, laminin, fibronectin, bullous pemphigoid antigen, and heparan sulfate proteoglycan) by indirect immunofluorescence. PEC on plastic or on collagen gels showed poor differentiation, a structured BM was not visible, and the expression and deposition of BM constituents was incomplete. Upon reimplantation in vivo, differentiation was normalized, expression of BM components complete and a structured BM reformed. This effect does not depend on immediate contact of epidermal cells with mesenchyme. When PEC on collagen gel were similarly associated with dermal mesenchyme in vitro, epidermal differentiation and expression of BM components were almost normalized, but a structured BM was absent. These findings demonstrate that formation of the BM in epidermis is a function of keratinocytes and, like differentiation is subject to mesenchymal control. A structural BM is not a prerequisite but rather an additional criterion of normal epidermal differentiation. Send offprint requests to: Norbert E. Fusenig, M.D., Division of Differentiation and Carcinogenesis in vitro, Institute of Biochemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-6900 Heidelberg, Federal Republic of GermanyPart of the work was performed when I.C. Mackenzie was guest scientist at the DKFZSupported by the Deutsche Forschungsgemeinschaft (Fu 91/2)     

Depletion of alveolar glycogen corresponds with immunohistochemical development of CD208 antigen expression in perinatal lamb lung.

CD208 DC lysosomal-associated protein is a marker of activated human dendritic cells; however, recently it was described as a marker of adult type II pneumocytes in many species including humans and sheep. Our hypothesis was that CD208 is developmentally regulated in lung pneumocytes. Lamb lungs at varying stages of development were stained immunohistochemically for CD208 and with Nile red (a fluorescent stain for lamellar bodies of type II cells) along with pulmonary markers of maturation (glycogen stores and surfactant protein A [SP-A] expression) or proliferation (Ki-67). CD208 staining and Nile red were localized to rare pneumocytes in young fetal lambs (day 115), increasing in frequency and stain intensity with age. Periodic acid-Schiff staining of glycogen granules was most prominent in the young lambs (day 115) with reduced staining through advancing lung development. SP-A was detected in pulmonary epithelia and staining in alveoli increased through gestation with decreased staining at 2 weeks of age. Intranuclear Ki-67 staining decreased through late gestation but was increased in 2-week-old lambs. Ontogeny of CD208 staining and depletion of glycogen were correlated (p<0.0001) and consistent with the premise that CD208 is localized to developing lamellar bodies. The findings suggest that CD208 antigen expression may serve as a marker for pneumocyte maturation in the developing fetal lung.     

T17b murine embryonal endothelial progenitor cells can be induced towards both proliferation and differentiation in a fibrin matrix

Endothelial progenitor cells (EPC) may enhance blood vessel formation in a variety of clinical settings such as ischaemia and tumour angiogenesis as well as in tissue-engineered matrices. In the present study, we cultured a murine endothelial progenitor cell line, T17b, in vitro in cell culture as well as in an FDA-approved fibrin matrix and investigated cell proliferation, differentiation and secretion patterns of the angiogenic growth factor VEGF under hypoxia and differentiation. We show that T17b EPC remain viable for at least 8 days in the fibrin matrix where they proliferate and form clusters including lumen-like structures. Proliferation in fibrin clots overlayed with basal medium (BM) was confirmed morphologically and immunohistochemically by positive Ki67 staining, indicating mitotic activity. Significant cell proliferation and Ki-67 expression were absent when cells were incubated with dibutyryl-cAMP and retinoic acid (RA). Incubation with dibutyryl-cAMP and RA stimulated the expression of the EPC differentiation markers von Willebrand Factor (vWF) and VEGF receptor 2 (VEGFR-2), indicating successful differentiation in the fibrin clot. EPC differentiation induced by dibutyryl-cAMP and RA was confirmed in 2-D chamber slide cultures by positive vWF immunostaining, which was absent in BM controls. EPC chamber slides also displayed positive vWF staining when exposed to hypoxia under BM conditions, indicating EPC activation and differentiation could also be induced by hypoxia. Taken together, T17b EPC secrete increased levels of VEGF when submitted to either hypoxia or differentiation and can be differentiated into mature endothelial cells not only in cell and matrigel cultures but also in a fibrin matrix that is FDA approved for clinical application.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.