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Am J Physiol Lung Cell Mol Physiol 292: L1241-L1247, 2007. First published January 12, 2007; doi:10.1152/ajplung.00440.2006
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A murine respiratory-inducing niche displays variable efficiency across human and mouse embryonic stem cell species

Mark Denham ,1,* Brock J. Conley,1,* Fredrik Olsson,2 Lerna Gulluyan,1 Timothy J. Cole,2 and Richard Mollard1,2

1Centre for Reproduction and Development, Monash Institute of Medical Research, and 2Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia

Submitted 8 November 2006 ; accepted in final form 29 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differentiating hESCs into respiratory lineages may benefit respiratory therapeutic programs. We previously demonstrated that 24% of all mouse embryonic stem cell (mESC) derivatives cocultured with embryonic day 11.5 (E11.5) mouse lung rudiments display immunoreactivity to the pneumonocyte II specific marker surfactant-associated protein C (Sftpc). Here we further investigate the effects of this inductive niche in terms of its competence to induce hESC derivative SFTPC immunoreactivity and the expression of other markers of terminal lung secretory units. When hESCs were cocultured as single cells, clumps of ~10 cells or embryoid bodies (EBs), hESC derivatives formed pan-keratin-positive epithelial tubules at high frequency (>30% of all hESC derivatives). However, human-specific SFTPC immunoreactivity associated with tubule formation only at low frequency (<0.1% of all hESC derivatives). Human-specific SFTPD and secretoglobin family 1A member 1 (SCGB1A1, also known as CC10) transcripts were detected by PCR after prolonged culture. Expression of other terminal lung secretory unit markers (TITF1, SFTPA, and SFTPB) was not detected at any time point analyzed. On the other hand, hESC derivatives cultured as plated EBs in media previously demonstrated to induce Sftpc expression in isolated mouse fetal tracheal epithelium expressed all terminal lung secretory unit markers examined. mESCs and hESCs thus display fundamental differences in their response to the E11.5 mouse lung inductive niche, and these data provide an important step in the delineation of signaling mechanisms capable of efficiently inducing hESC differentiation into terminal secretory units of the lung.

coculture; respiratory; surfactant; Clara cell; adipocyte; development

MOUSE EMBRYONIC STEM CELLS (mESCs) are in vitro cultivated, theoretically immortal cells derived from the inner cell mass of developing blastocysts (20, 38). Tetraploid complementation studies demonstrated that mESCs are capable of giving rise to functionally relevant representatives of all cells of the adult mouse (45). More recently, the isolation of embryonic stem-like cells from human blastocysts (hESCs) has been reported (49, 59). Their pluripotentiality has been suggested due to the ability of clonal populations to form tissue representatives of all three embryonic germ layers in teratomas following injection under the testicular capsule of NOD/SCID mice (3). It is envisaged that the discovery of methods facilitating the controlled differentiation of hESCs into specific and desired cell derivatives will greatly benefit the development of therapies aimed at treating a variety of diseases and injuries. Such derivatives may have applications in, for example, direct cellular replacement therapies or screening platforms for high-throughput drug discovery programs [for reviews, see Denham et al. (17) and Conley et al. (13)]. Progress towards realizing these goals has been made by many groups, and a number of methods directing differentiation of ESCs into a specific and desired lineage have been documented. These methods include, but are not limited to, coculture with an inducing tissue source, the addition to culture of fate-specifying cytokines, lineage selection using cell type-specific markers, and overexpression of fate-specifying genes (9, 16, 18, 25, 28, 32, 36, 43).

In both the mouse and human, differentiation of the terminal lung secretory units is characterized by the expression of several genes, including: surfactant-associated protein A (Sftpa), Sftpb, Sftpc, Sftpd, and secretoglobin family 1A member 1 (Scgb1a1; also known as uteroglobulin and Clara cell 10). Sftpa and Sftpd play defense roles against pathogens and allergens, Sftpb and Sftpc are active in reducing surface tension at the air-liquid interface, and Scgb1a1 mediates cellular responses to inflammation and allergy (8, 21, 31). In the mouse and human, Sftpc is considered a lung-specific marker demarcating type II pneumonocytes and respiratory progenitors (21, 29, 47). Sftpa, Sftpb, Sftpd, and Scgb1a1, on the other hand, although representing major secretory components of the mouse and human lung, are also synthesized in a number of extrapulmonary sites (for review, see Refs. 6, 21, and 31). The combined presence of these gene products, however, can be used to describe the phenotype of the dominant secretory cell types of the terminal lung units, i.e., alveolar type II and Clara cells (39).

Development of the respiratory system during organogenesis depends on reciprocal morphogenetic interactions between the relatively undetermined epithelium and adjacent and instructive mesenchyme (1, 7, 34, 42). In the absence of the mesenchyme, the epithelium does not bud or branch, and the cells transform from a columnar to a squamous phenotype and lose orientation with each other and die. The mesenchymal signals inducing patterning and differentiation of the associated epithelium are often conserved across heterotypic tissue sources and species. For example, isolated fetal mouse lung bud mesenchyme induces ectopic budding, branching, and Sftpc expression in denuded mouse tracheal epithelium (1, 55, 56). Similarly, fetal mouse lung mesenchyme induces a lung-like branching pattern when grafted to mouse salivary epithelium and the normally nonbranching chick air sac epithelium (22, 33, 34).

Recent work by our group has demonstrated that mESCs can be directed toward a respiratory cell-like phenotype with high efficiency (greater than 24% of all mESC derivatives) in vitro by coculture with dissociated embryonic day 11.5 (E11.5) mouse lung explants (18). Systems that similarly drive differentiation of hESCs into respiratory phenotypes in an efficient manner are yet to be described. We demonstrate here that the E11.5 mouse lung inductive niche is supportive of hESC differentiation into epithelial tubules at high frequency, yet SFTPC immunoreactivity associates with these tubules only at a very low frequency. To demonstrate that hESCs used in this system are capable of displaying a terminal lung unit phenotype, we also show that SFTPA, SFTPB, SFTPC, SFTPD, SCGB1A1, and TITF1 transcripts can be detected in hESC derivatives following plating of embryoid bodies (EBs) in a cocktail of defined factors previously shown to induce Sftpc expression in isolated fetal mouse tracheal epithelium (57). These data thus demonstrate that embryonic stem cells display species-specific differences in response to the E11.5 mouse lung inductive niche and aid in the identification of molecular signaling mechanisms relevant to the differentiation of hESCs into respiratory-specific lineages.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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MESC, HESC, and EB culture. All protocols were approved by the appropriate and independent institutional review committee. The HES-2 hESC line was previously developed in conjunction with the Department of Obstetrics and Gynecology at the National University of Singapore (49). HES-2 cells are registered as eligible for funding with the National Institutes of Health and have previously been shown to form teratomas when injected under the testis capsule of severe combined immune-deficient mice (http://stemcells.nih.gov/research/registry/). HES-2 culture and differentiation as EBs were performed as described previously (14, 15). Briefly, hESCs were cultured on mitomycin C- (Sigma) treated mouse embryonic fibroblasts (MEFs) in media consisting of high-glucose DMEM (Invitrogen/GIBCO), 20% FBS (Invitrogen/GIBCO), 1% nonessential amino acids, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 1x insulin-transferrin-selenium supplement (Invitrogen/GIBCO), and 0.1 mM beta-mercaptoethanol (Sigma) at 37°C-5% CO2. Colonies were mechanically dissected with finely pulled glass micropipettes (1-mm outer diameter; Clark Electromagnetic Industries) every 7 days and either transferred to a freshly prepared MEF layer or placed in suspension in bacteriological Petri dishes (Sarstedt) for EB formation.

Histology. Histological analysis was performed on hematoxylin and eosin-stained 3-µm sections of 7-day-old paraffin-embedded human EBs according to standard procedures (12).

Generation of HESC/fetal mouse lung aggregates. Mouse fetuses from F1 (C57B/6J WEHI female x CBA/CaH WEHI male) natural matings were dissected at E11.5 into PBS, and fetal lungs were excised. Approximately 24 lungs were transferred to a 1-ml Eppendorf tube containing 10 mg/ml DNase I (Roche) in 200 µl of PBS for 10 min at 37°C before mechanical dissociation. Cells were subsequently diluted with 800 µl of hESC medium, centrifuged at 600 g for 5 min, and resuspended in fresh high-glucose DMEM supplemented with 10% FBS (JRH Biosciences), 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomyocin (Invitrogen/GIBCO) at 37°C/5% CO2. hESCs were combined with the fetal lung cell suspension for both 14 and 21 days at a ratio of 1:20 in one of three ways: 1) hESCs were dissected into fragments containing ~10 cells as described for passaging and added to the lung cell suspension, 2) hESCs were dissected into fragments, and then 18–20 fragments were dissociated into a single cell suspension by incubation in 1 ml of cell dissociation solution (Sigma) for 10 min at 37°C, and 3) hESCs were cultured to form EBs for 4 days, and two cultivated EBs were mixed with the fetal lung cell suspension in 2 µl of BGJb medium (BGJb Fitton-Jackson modified; Invitrogen/GIBCO), cultured in BGJb medium containing 5% FBS, 180 mg/ml ascorbic acid (Sigma), 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomyocin, and placed on Millipore filters (Invitrogen/GIBCO) at 37°C/5% CO2. EBs cultured for 4 days were selected for the induction experiments because this is the first day a cohesive EB structure is formed, yet also a time when the hESC derivatives continue to display a relatively primitive phenotype (14) (Conley BJ, unpublished observations).

The presence of hESC derivative TITF1, SFTPA, SFTPB, SFTPC, SFTPD, and SCGB1A1 transcripts was assessed by semiquantitative PCR using primers to human specific sequences of the respective mRNA species (for primer sequences, see Table 1). PCR reactions were run to saturation with respect to beta-actin expression and then analyzed for either the presence or absence of markers of the terminal lung secretory system (41, 53).


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  		Table 1. Mouse and human PCR primer sequences

 
Double immunohistochemistry using primary antibodies from two different species. Fetal lung/stem cell derivative aggregates were fixed in 4% paraformaldehyde for 30 min at 4°C and then washed briefly in PBS. Aggregates were embedded in Tissue-Tek OCT compound (Miles Laboratories), cut at 8 µm on a cryostat, and sections were placed on superfrost slides according to previously described methods (42). Sections were blocked overnight at 4°C in blocking solution (150 mM NaCl, 50 mM Tris, pH 6.8, 0.5% Nonidet P-40, and 5 mg/ml BSA).

Rabbit anti-Sftpc (1:300, Research Diagnostic) and mouse anti-human nuclear (1:100, Chemicon) antibodies diluted in blocking solution were applied to sections for 1 h at room temperature (RT). Following 3x 3-min washes in PBS, biotinylated rabbit anti-mouse IgG1 (1:300, Zymed Laboratories) and goat anti-rabbit IgG Alexa Fluor 568 (2 µg/ml, Molecular Probes) secondary antibodies were applied for 30 min at RT. Streptavidin Alexa Fluor 488 was bound to the biotinylated secondary antibody according to the manufacturer's instructions (Molecular Probes). Sections were counterstained for 30 min with Hoechst 33342 (5 µg/ml). Slides were mounted in PBS for viewing under an immunofluorescent microscope (Leica DMR), and images were captured using the Leica MPS60 photo system.

Double immunohistochemistry using primary antibodies from the same species. Mouse anti-human nuclear antibody was preconjugated with anti-mouse Alexa Fluor 488 Fab fragments for 5 min at RT using the Zenon mouse IgG1 Alexa Fluor 488 labeling kit. Cryosections were prepared as above and incubated with mouse anti-pan-keratin or mouse anti-thyroid transcription factor 1 (Titf1; also known as Nkx2.1, T/EBP, Ttf-1; Refs. 40 and 64) primary antibodies diluted 1:300 in blocking solution at RT for 1 h, washed three times in PBS, and incubated at RT for 30 min with goat anti-mouse Alexa Fluor 546 fluorescent secondary antibody. Sections were then incubated with preconjugated mouse anti-human nuclear antibody for 1 h at RT, washed three times in PBS at RT, counterstained, and photographed as above.

Semiquantitative PCR. Total RNA was isolated from cell pellets using the RNeasy Mini Spin Kit (Qiagen) as per the manufacturer's instructions. RNA was quantified using a spectrophotometer, and 3 µg were transcribed using Superscript II (Invitrogen/GIBCO) as per the manufacturer's instructions. Negative controls were treated in the same fashion, but no Superscript II was added to the reaction mix. PCR reactions consisted of 2 µl 10x PCR buffer (Amersham), 0.4 µl 2 mM dNTP mix (Promega), 0.8 µl forward primer (final concentration 100 ng/ml), 0.8 µl reverse primer (final concentration 100 ng/ml), 2 µl cDNA, and 0.1 µl Taq polymerase (5 U/µl; Amersham). Primer sequences are listed in Table 1. Reverse transcription negative controls were similarly subjected to PCR to assay for the presence of contaminating genomic DNA in each cDNA preparation. PCR reactions were performed using a GeneAMP PCR System 9700 (Perkin Elmer). A typical PCR reaction consisted of a single 5-min denaturation at 95°C, followed by 32 cycles of 1-min steps at 95°C, 56°C, and 72°C. PCR products were assessed by DNA gel electrophoresis.

Culture and plating of EBs in complex growth medium. Human and mouse EBs were cultured for 5 days in hESC medium (as described above for EB culture) and then plated on 0.1% gelatin-coated dishes for a further 8 days in HESC medium supplemented with either 1) 100 ng/ml nerve growth factor (NGF; PeproTech), 2) 25 ng/ml hepatocyte growth factor (HGF; PeproTech), or 3) BFGM culture medium, which is a combination of 10 µg/ml insulin (Sigma), 1 µg/ml cholera toxin (Sigma), 1 µg/ml EGF (Research Diagnostics), 25 ng/ml HGF, 10 ng/ml fibroblast growth factor 7 (FGF7; PeproTech), and 25 ng/ml FGF1 (PeproTech) (57).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Induction of mESC derivative Sftpc immunoreactivity is most efficient when combining dissociated E11.5 lung anlage with dissociated mESCs at a cell-cell ratio of 20:1 (18). Ratios of 4:1 and 40:1 are inferior as is coculture with E12.5 or E13.5 lung anlagen. To determine whether the dissociated E11.5 mouse lung environmental niche can similarly induce hESC derivative SFTPC immunoreactivity, cocultures with hESCs at a cell-cell ratio of 20:1 were performed. Aggregates were prepared according to three different strategies: 1) hESCs dissociated into single cells, 2) hESCs dissected into clumps of ~10 cells, and 3) hESCs differentiated as EBs (see Fig. 1, A and B).


Figure 1
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  		Fig. 1. Embryoid body (EB) production from HES-2 human embryonic stemlike cells (hESCs). A: HES-2 hESCs passaged by mechanical dissection on mouse embryonic fibroblasts (MEFs). B: hESCs dissected into small fragments and cultured for 7 days in suspension form spherical structures resembling EBs. C: low-power magnification of a representative EB serial sectioned and stained with hematoxylin and eosin displays a polarized phenotype with adipocytes (a) and neural rosettes (r) contained between epiblast (e) and visceral endodermal-like (ve) layers. D: high-power magnification demonstrates unilocular adipocytes (a) displaying eccentric nuclei and a thin cytoplasm. The presence of mitotic nuclei (a') is suggestive of proliferation. Epiblast-like cells (e) display apico-basal polarization and apical protrusions and appear to ingress by downward migration. The visceral endoderm-like cells (ve) appear as a defined basally located epithelium containing numerous vacuoles and low nuclear-to-cytoplasmic ratios. Neural rosettes (r) display characteristic wedge-shaped cells arranged radially around a central cavity.

 
The ability of hESCs to form EBs in culture was first assessed by morphological analysis of day 7 cultivated hESCs grown in suspension as spontaneously differentiating spheres. Hematoxylin and eosin staining of semithin serial paraffin sections revealed polar structures containing a pit-like depression in a peripheral epithelial-like layer, a central area displaying adipocyte-like cells (mesoderm) and neural rosettes (ectoderm), and an underlying visceral endodermal-like layer (Fig. 1, C and D). Although not fully characterized, the epithelial-like cells forming the pits were columnar, polarized, and displayed plump apical cytoplasmic protrusions, low nuclear-cytoplasmic ratios, and prominent nucleoli. The gross morphology and organization of these epithelial-like cells were reminiscent of the pits described following removal of the rhesus monkey embryonic stem cell line R366.4 from fibroblast feeders (5). Adipocytes displayed characteristic eccentric and flattened nuclei, a thin cellular cytoplasm, and a single and large clear unilocular vesicle (10). The presence of nuclei in mitosis suggested adipocytes were capable of clonal expansion. Visceral endoderm-like cells displayed a flattened morphology with numerous vacuoles and high nuclear-cytoplasmic ratios (14). Neural rosettes displayed radial wedge-shaped/pseudostratified cells surrounding a well-defined cavity (27). hESCs used in this study, therefore, display trademark features of EB formation.

During coculture with hECSs as single cells, clumps, or EBs, dissociated E11.5 lung explants readily underwent organotypic regeneration (Fig. 2A). Double immunofluorescence to pan-keratin-specific and anti-human-specific antibodies demonstrated that hESC derivatives incorporated as both tall columnar and cuboidal/squamous epithelial-like glandular structures at high frequency after 6, 8, 10, 14, and 21 days of coculture (Fig. 2B). The average percentage of pan-keratin-immunopositive hESC derivatives in all three treatment groups across all time periods assessed was 33.4%. No significant difference was observed between different treatment groups (n > 3 aggregates/group, n > 300 cells/aggregate; P = 1) or treatment times (n > 4 aggregates/time point, n > 300 cells/aggregate; P = 1). To determine whether hESC derivative epithelial-like cells within these glandular structures displayed characteristics of respiratory differentiation, hESC derivatives were tested for double immunoreactivity to both the human-specific and Sftpc-specific antibodies. On only one occasion, a cluster of hESC derivatives displaying immunoreactivity to the anti-Sftpc-specific antibody was observed (Fig. 2B). The incidence of such cells appeared to be more indicative of spontaneous rather than induced differentiation and was too low to afford meaningful statistical analysis (<0.1% of greater than 7,000 hESC derivatives scored).


Figure 2
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  		Fig. 2. Aggregate coculture of dissociated embryonic day 11.5 (E11.5) mouse lung with hESCs and EBs. A: gross morphology of hESCs cocultured for 6 days with dissociated E11.5 mouse lung. Lung organotypic regeneration is not inhibited following coculture with hESCs as either dissociated single cells, colony fragments containing ~10 cells, or partially differentiated day 4 EBs. In all cases, a large number of glandular ducts contained within the supportive mesenchyme appear. B: double immunohistochemistry for hESCs (green) and either pan-keratin (red) or Sftpc (red) demonstrates these glandular ducts contain hESC derivatives differentiated as epithelial-like cells. Ducts contain pan-keratin-positive tall columnar hESC epithelia (t) or less-organized cuboidal and squamous-like epithelia (c). Hoechst counterstain (blue) reveals both human and mouse nuclei within the aggregates. Ducts also display Sftpc immunoreactivity as do the occasional hESC derivative associated with these ducts (*). PCR using human specific primers to SFTPD and SCGB1A1 transcripts demonstrate detectable levels after 21 days of coculture. No transcripts were detectable after 14 days of coculture. Human, control human adult lung; mouse, control adult mouse lung; –RT, reverse transcriptase control.

 
Immunoreactivity to the TITF1-specific antibody was readily detectable in sections of control human respiratory tissue, yet no hESC derivative TITF1 immunoreactivity was detected at either 6 or 21 days of coculture (data not shown). Although SFTPA and SFTPB transcripts were not detectable at any time point examined, the predicted SFTPD and SCGB1A1 amplicons were detected at 21 days, but not at 14 days, of coculture (Fig. 2B and data not shown). These data thus demonstrate that hESC derivatives readily form pan-keratin-immunopositive glandular structures during E11.5 mouse lung coculture. However, unlike mESC derivatives, hESCs possess a limited capacity to respond to the E11.5 mouse lung niche with expression of the type II pneumonocyte phenotype.

To determine whether HES-2 hESCs could be induced to display all characteristics of the respiratory terminal secretory unit phenotype, HES-2 EBs were dissociated, plated in BFGM, and analyzed by semiquantitative PCR for the presence of SFTPA-, SFTPB-, SFTPC-, SFTPD-, SCGB1A1-, and TITF1-specific transcripts. SFTPA and SCGB1A1 transcripts were detected in control and all treatment groups (Fig. 3). SFTPB transcripts were detected following the addition of HGF or BFGM, whereas the incidence of TITF1 transcript detection increased following the addition of NGF, HGF, or BFGM. SFTPC and SFTPD transcripts were most readily detectable following treatment with BFGM. Thus, as previously reported, NGF and HGF appeared to increase the incidence of endoderm marker expression in hESC derivatives cultured as plated EBs (53). Furthermore, culture in BFGM facilitated the detection of all makers of the terminal secretory phenotype tested. Therefore, although HES-2 hESCs show a limited capacity to respond to the E11.5 mouse lung inductive niche with expression of the terminal lung phenotype, they possess the potential to display key markers of the respiratory airways following culture as EBs in the presence of a complex growth medium.


Figure 3
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  		Fig. 3. Semiquantitative PCR for respiratory transcripts within plated EBs. After 5 days in suspension culture, EBs were plated and cultured for a further 8 days either without supplement (control) or in nerve growth factor (NGF), hepatocyte growth factor (HGF), or the BFGM cocktail. SFTPA and SCGB1A1 transcripts were detected in control and all treatment groups, TITF1 transcripts were more readily detectable in treatment groups, SFTPB transcripts were more readily detectable in HGF treatment groups, SFTPD transcripts were more readily detectable following culture in BFGM, and SFTPC transcripts were detectable only following culture in BFGM. SFTPA and SCGB1A1 transcripts were detected in control and all treatment groups, yet SFTPA was detected in only 3 of 4 treatments in both HGF and BFGM. The sum incidence of all respiratory transcripts analyzed was highest following culture in BFGM.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
These data demonstrate that the dissociated E11.5 mouse lung is permissive to the organization of HES-2 hESCs and EB derivatives into pan-keratin-immunopositive epithelial-like glandular tubules at high frequency. Derivatives displayed SCGB1A1 and SFTPD expression, however, no SFTPC or TITF1 immunoreactivity and no SFTPA or SFTPB expression were detected. The addition of BFGM to HES-2 hESCs grown as EBs and then plated on gelatin, on the other hand, enabled detection of all terminal respiratory secretory unit transcripts examined, including: SFTPA, SFTPB, SFTPC, SFTPD, SCGB1A1, and TITF1. These data are consistent with previous reports demonstrating that hESC derivatives cultured as EBs and then plated in the presence of HGF or NGF express markers of endoderm and that E13 mouse tracheal epithelial cells cultured in BFGM express Sftpc and Scgb1a1 transcripts (53, 57). Derivatives of the HES-2 hESC line therefore display a competence to express a terminal respiratory secretory unit-like phenotype, yet this competence is realized only to a limited extent in the presence of the same E11.5 mouse lung morphogenetic signals that efficiently induce respiratory-like differentiation of mESCs (18). Further studies aimed at dissecting precisely the degree of expression of each transcript following culture with each additive would involve the use of quantitative PCR techniques.

Morphogenetic signals guiding organogenesis during embryogenesis have proven efficient inducers of both mESC and hESC differentiation into specific tissue derivatives in a number of systems. These signals have also often acted efficiently across species boundaries. For example, the mouse P19 embryonal carcinoma cell-derived visceral endoderm-like END-2 cell line induces both mESC and hESC differentiation into beating cardiomyocytes (43, 44). Matrix components of the human amniotic membrane induce neural differentiation of both mESCs and hESCs (61). Mouse S17 bone marrow stromal and C166 yolk sac endothelial cell lines induce differentiation of hESCs into a range of hematopoietic lineages (25). Furthermore, mouse PA6 and MS5 bone marrow stromal cell lines induce differentiation of mESCs, hESCs, and monkey ESCs into dopaminergic neurons (4, 26, 48). It may, therefore, appear unexpected that dissociated E11.5 mouse lung efficiently induces mESC derivatives to display an Sftpc-immunopositive respiratory-like phenotype, yet displays only a limited SFTPC inductive effect on hESC derivative differentiation. However, mESCs and hESCs are known to display a broad range of differences in respect to cell surface markers, transcriptional regulation of self-renewal, and passaging requirements (for a review, see Ref. 17). Together these factors may explain alternative interpretations of external environmental cues due to either the absence or presence of permissive and/or inductive signaling pathways.

Signaling pathways instigating and maintaining the distal respiratory phenotype during mouse lung organogenesis have been described. At the two to five somite stage, cardiac mesoderm-derived FGF induces respiratory epithelial Titf1 expression, thus inducing distal lung morphogenesis and the expression of respiratory epithelial Sftpa and Sfptc (54, 64). Differentiation and maintenance of the Sftpc-immunopositive phenotype within the developing and adult mouse lung is dependent on expression of Titf1, and similarly, TITF1 expression is associated with normal respiratory function in humans (19, 24, 40, 64). It must be noted that Titf1 expression is restricted to the developing mouse and human lung, thyroid, and neural populations, and can thus be used only as an indicative (yet not exclusive) marker of distal respiratory differentiation (30, 35, 60). Titf1 also regulates transcription of other respiratory markers, having been demonstrated to regulate Scgb1a1 and Sftpb expression in HeLa and H441 adenocarcinoma cells and foregut endoderm (37, 54, 63, 65). The phenotype of the terminal respiratory secretory system is then maintained by reciprocal and local epithelial-mesenchymal interactions regulated through Titf1 and FGF signaling pathways (11, 46, 58, 62, 64).

In the human lung, TITF1 expression similarly overlaps with that of SFTPA and SFTPC, and TITF1 haploinsufficiency is compatible with reduced surfactant protein, atelactasis, and respiratory distress (19, 23, 24). The absence of significant SFTPC immunoreactivity and SFTPA expression in the hESC derivatives following coculture may thus be explained at least in part by the failure of E11.5 mouse lung to induce TITF1 expression in the hESC derivative epithelial layer. In agreement, hESC derivatives cultured as plated EBs in the presence of BFGM demonstrated both TITF1 and SFTPC expression, whereas control cultures demonstrated variable TITF1 expression and no detectable SFTPC expression. Furthermore, BFGM contains the Titf1 inducers FGF1 and FGF7. Addition of FGFs to culture may thus represent a crucial factor for respiratory-like differentiation of hESCs.

mESCs and hESCs cultured in media that maintains the alveolar type II phenotype have also been reported to differentiate into derivatives displaying alveolar type II characteristics, yet at low frequency (2, 50, 51). For example, both Ali et al. (2) and Samadikuchaksaraei et al. (51) demonstrated Sftpc expression by RT-PCR and Sftpc-immunopositive cells, whereas Rippon et al. (50) demonstrated Sftpc expression by RT-PCR and SP-C/EGFP transgenics. Yields in these systems remain low (<3% of all stem cell derivatives), and evidence of alveolar type II differentiation has been based on the detection of Sftpc immunoreactivity or expression and/or the presence of lamellar bodies. It must be noted, however, that although Sftpc is predominantly lung specific, some thyroid precursors may also express Sftpc early during development (47). Furthermore, in addition to alveolar type II cells, human nasal and stomach mucosa, tongue papillae, oral epithelium, and gastrointestinal tract similarly contain lamellar bodies (52). Thus although markers used in this study and by others are indicative of a respiratory phenotype, they are not exclusive, and before real claims for respiratory differentiation can be made, more explicit data are required. For example, appropriate functional studies demonstrating that the ESC-derived alveolar type II cells can give rise to alveolar type I cells and a demonstration that lamellar bodies are found within alveolar type II cells using dual Sftpc immunogold/electron microscopic techniques would go much further to demonstrating the existence of truly differentiated alveolar type II cell derivatives.

Our previous work utilizing dissociated fetal lung as an inductive source successfully induced Sftpc immunoreactivity in 24% of all mESC derivatives (18). A similar system utilizing isolated fetal lung mesenchyme to instruct mESC differentiation into respiratory derivatives has also been reported (61a). The same dissociated whole fetal lung system as described here resulted in a less than 0.1% induction of hESC cells and was associated with an absence of detectable TITF1 expression. Whether hESCs in this system were induced to differentiate into mesenchymal-like cells, other cells of the respiratory lineage, or nonrespiratory lineage cell types, or whether the incidence of hESC derivative SFTPC immunoreactivity would be increased by coculture with fetal human tissue remains to be determined. Ethical difficulties surrounding the acquisition of fetal human material for experimentation, in addition to the possibility of sourced material harboring genetic mutations or anti-glucocorticoid receptor antagonists, renders the use of such systems difficult. However, FGF signaling and the ability to drive TITF1 expression appear to be associated with key determinants of the SFTPC differentiation process. These data thus reiterate innate differences in the molecular biology of embryonic stem cell species and provide further clues to the development of in vitro culture conditions that may efficiently induce respiratory differentiation of hESCs.


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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was funded by the Strategic Monash University Research Fund, the Australian Research Council, ES Cell International, the U.S. Cystic Fibrosis Foundation, and a Program Grant (application number 384100) from the National Health and Medical Research Council of Australia.


   ACKNOWLEDGMENTS
 
We acknowledge Dr. Martin Pera for helpful discussions and support and Dr. Henry Sathananthan for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Mollard, Dept. of Biochemistry and Molecular Biology, Monash Univ., Clayton 3800, Australia (e-mail: mollard{at}med.monash.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* M. Denham and B. J. Conley contributed equally to this work. Back


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
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