Derivation of Distal Lung Epithelial Progenitors from Murine Embryonic Stem Cell - 《stemcells》干细胞学杂志干细胞之家



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作者：Helen J. Rippon, Julia M. Polak, Mingde Qin, Anne E. Bishop作者单位：Tissue Engineering and Regenerative Medicine Centre, Faculty of Medicine, Imperial College London, Chelsea and Westminster Campus, London, United Kingdom ) y# K+ i7 C! N* V/ q
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      【摘要】& Q( H# p: ~, u# }/ C* P
   Embryonic stem cells (ESCs) are a potential source for the cell-based therapy of a wide variety of lung diseases for which the only current treatment is transplantation. However, distal lung epithelium, like many other endodermally derived somatic cell lineages, is proving difficult to obtain from both murine and human ESCs. We have previously obtained alveolar epithelium from ESCs, although final cell yield remained extremely low. Here, we present an optimized three-step protocol for the derivation of distal lung epithelial cells from murine ESCs. This protocol incorporates (a) treatment of early differentiating embryoid bodies with activin A to enhance the specification of the endodermal germ layer, followed by (b) adherent culture in serum-free medium and (c) the final application of a commercial, lung-specific medium. As well as enhancing the specification of distal lung epithelium, this protocol was found to yield cells with a phenotype most closely resembling that of lung-committed progenitor cells present in the foregut endoderm and the early lung buds during embryonic development. This is in contrast to our previous differentiation method, which drives differentiation through to mature type II alveolar epithelial cells. The derivation of a committed lung progenitor cell type from ESCs is particularly significant for regenerative medicine because the therapeutic implantation of progenitor cells has several clear advantages over the transplantation of mature, terminally differentiated somatic cells. " t9 _+ S0 Y) `) X2 i
      【关键词】 Embryonic stem cell Lung epithelium Progenitor cell; Y- R3 h: e, A5 g' d* s
   INTRODUCTION

Currently, end-stage pulmonary disease can be cured only by transplantation, yet a scarcity of donor lungs means there is an acute need for clinical alternatives. Regenerative medicine and tissue engineering are relatively new, multidisciplinary fields of research aimed at producing healthy cells and tissue ex vivo for implantation and the augmentation of tissue function in vivo. Tissue engineering for the treatment of lung disease will inevitably require the production of alveolar tissue, the gas-exchange unit of the lung. The alveolar epithelium contains two cell types, the type I and type II pneumocytes, which comprise 95% and 5% of the area of the alveolar lining, respectively . If an abundant, renewable source of alveolar type II cells were to be created in vitro, this could form the basis for tissue engineering of functional gas-exchange units for transplantation.6 }6 X: r( ]" t/ h1 u
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Type II cells can be isolated from primary lung tissue of various mammals; however, their yield is low and they frequently lose their phenotype and fail to proliferate in the culture environment . In this study, definitive endoderm differentiation was also improved by the replacement of fetal bovine serum (FBS) with KnockOut Serum Replacement (KOSR; Invitrogen, Paisley, U.K., http://www.invitrogen.com), a defined medium supplement designed to directly replace FBS in ESC culture medium.

We have combined this approach with our own lung differentiation protocol to develop a multistep strategy for the optimized derivation of distal lung epithelium from ESCs. Unexpectedly, the epithelial cells that were derived by this method had a gene expression profile resembling that of lung-committed progenitors found in the foregut endoderm, rather than mature alveolar epithelium. During murine embryonic development, expression of the SPC gene is first activated in lung epithelial progenitors within the foregut endoderm, eventually becoming restricted to the type II pneumocyte during branching morphogenesis and epithelial differentiation . This is the first report of a lung epithelial progenitor cell being derived from ESCs. As well as being a significant advance in lung tissue engineering, this work may also provide a new model to study epithelial cell fate specification in the maturing murine lung.
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MATERIALS AND METHODS% G/ X( ^. b9 G/ K
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Maintenance of Murine ESCs- ~! b/ `8 m8 s$ q2 p2 [5 O, j

The murine ESC line E14-Tg2a (a kind gift of Prof. A. Smith, University of Edinburgh, Edinburgh, Scotland, U.K.) was routinely cultured on gelatin-coated tissue culture plates in high-glucose Dulbecco¡¯s modified Eagle¡¯s medium supplemented with 10% batch tested FBS, 2 mM L-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, Dorset, U.K., http://www.sigmaaldrich.com), and 1,000 U/ml leukemia inhibitory factor (Chemicon, Temecula, CA, http://www.chemicon.com). Cultures were maintained in a humidified atmosphere of 5% CO2/95% air at 37¡ãC.9 T# T1 D) r0 U3 y# {

Generation of the SPC-EGFP Cell Line
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A 4.8-kb murine SPC promoter/EGFP construct was transfected into undifferentiated ESCs and murine lung epithelial (MLE-12; positive control; American Type Culture Collection, Manassas, VA, http://www.atcc.org) and murine embryonic fibroblast (MEF; negative control) cells using Lipofectamine 2000 according to the manufacturer¡¯s instructions (Invitrogen). The transfected ESCs were selected in ESC culture medium containing 300 ng/ml geneticin (Invitrogen) for 2 weeks. To test transfection efficiency, a constitutively active GFP construct (pTracer-GFP; Invitrogen) was also transfected into all three cell types using same protocol.& O+ J- w& G" p- S2 o) y

Differentiation of ESCs

Embryoid bodies (EBs) were formed from ESCs by limited trypsin digestion of ESC colonies 24 hours post-passage and suspension culture of the cell clusters in non-tissue culture-treated Petri dishes. Generally, one T25 flask of ESCs was sufficient to seed one 90-mm Petri dish. EBs were cultured in suspension for 10 days and then placed intact into gelatinized six-well plates (two six-well plates were generated per 90-mm dish of EBs) for further differentiation in adherent culture. EBs were allowed to outgrow for 15¨C25 days, giving a total differentiation culture time of 25¨C35 days. Throughout this manuscript, "day 1" refers to the first day of EB suspension culture. For directed differentiation of ESCs, suspension and adherent EBs were treated with several media regimes in a three-step protocol. These steps and the nomenclature used for them in the rest of this manuscript are summarized in Figure 1.! U) s3 G  Z# I, z* K
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Figure 1. Three-step differentiation protocols for the derivation of distal lung epithelium from mouse embryonic stem cells. Embryoid bodies were cultured in suspension for 10 days under three alternative media treatment schedules (step 1). Embryoid bodies were then returned to adherent culture for 10 days (step 2) before the addition of one of two specialized culture media for 4 or 14 days (step 3). Abbreviations: AA, activin A; DMEM, Dulbecco¡¯s modified Eagle¡¯s medium (high-glucose); FBS, batch-tested fetal bovine serum; KOSR, KnockOut Serum Replacement; SAGM, small airway growth medium.9 X6 }, C- v- p
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For cell-counting experiments, EBs were initially formed in 24-well low-adherence tissue culture plates (Corning Incorporated/Life Sciences, Acton, MA, http://www.corning.com), then transferred into tissue culture-treated 24-well plates (Corning Incorporated/Life Sciences) on day 11 to adhere. Cell counts were performed by dissociating EBs into a single-cell suspension with a mixture of 0.05% trypsin/0.53 M EDTA/2% chicken serum in phosphate-buffered saline. Cells were counted on a hemocytometer using trypan blue dye exclusion to identify viable cells.
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Flow Cytometry
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Differentiated SPC-EGFP ESCs were dissociated into a single-cell suspension as described above, fixed with 4% paraformaldehyde, and quantified for EGFP fluorescence on a FACScaliber flow cytometer (BD Biosciences, Oxford, U.K., http://www.bdbiosciences.com). Untransfected E14-Tg2a cells differentiated in parallel with the SPC-EGFP cell line were used to control for background fluorescence. Listmode data were analyzed using WinMDI 2.8 software (http://www.cyto.purdue.edu/flowcyt/software/Winmdi.htm).
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Quantitative Reverse Transcription-Polymerase Chain Reaction: z4 c. X# \1 r1 S
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RNA was extracted from differentiated ESCs using TRIzol reagent (Invitrogen) and quantified by spectrophotometry. RNA was DNaseI-treated using RQ1 RNase-free DNaseI (Promega, Madison, WI, http://www.promega.com), and then 3 µg of RNA was reverse-transcribed into cDNA using the Thermoscript reverse transcription-polymerase chain reaction (RT-PCR) system (Invitrogen) and a random hexamer primer. Three percent of the RT products were used as a template for each real-time PCR analysis.
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Real-time PCR was performed using a GeneAmp SDS 5700 thermal cycler and the SYBR Green PCR Core Reagents Kit (both Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). PCR reaction mixtures contained cDNA template, primers, 1x SYBR Green PCR buffer, 3 mM MgCl2, 200 nM each of dATP, dCTP, dGTP, and 400 nM dUTP, 0.75 U of AmpliTaq Gold, and 0.25 U of AmpErase UNG in a final volume of 25 µl. Each PCR reaction was performed in triplicate to control for pipetting errors, and positive controls and no-template negative controls were also included in each PCR run. Primers were designed using Primer-Express software (Applied Biosystems), and primer concentrations were optimized for each primer pair according to the PCR kit protocol. Primer sequences, reaction concentrations, and amplicon length are shown in Table 1. Thermal cycling parameters were 50¡ãC, 2 minutes; 95¡ãC, 10 minutes; followed by 40 cycles of 95¡ãC, 15 seconds; and 60¡ãC, 1 minute. After the PCR reaction, baseline fluorescence was measured between cycle number 5 and two cycles prior to the initiation of PCR product amplification, to adjust each sample for background fluorescence. Threshold cycle (CT) values were then obtained by manually setting a threshold of fluorescence such that it intersected the logarithmic phase of every amplification plot within each PCR run. Dissociation curves were also generated at the end of each PCR reaction in order to confirm the presence of a single specific reaction product in each positive sample.+ F/ s- H2 U/ V7 A  s
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Table 1. Quantitative reverse transcription-polymerase chain reaction primers

Relative quantification of gene expression by the comparative CT method was chosen as the most appropriate method of analysis. Two housekeeping genes with very different cellular functions were originally chosen for data normalization, ß-actin and glycer-aldehyde-3-phosphate dehydrogenase (GAPDH); however, ß-actin was found to be significantly downregulated in media containing KOSR (data not shown). Therefore, results were normalized to GAPDH only. For the  CT calculation to be valid, the amplification efficiency of the GAPDH PCR product must be approximately equal to that of each test gene product. This was confirmed by examining whether CT varied with 1:2 serial dilution of the positive control cDNA template (data not shown). Results derived from differentiated ESC were then analyzed by the comparative CT method according to the equation:

Statistical Analysis8 Y0 f: w7 n. T: ?' E4 q) f* S0 b
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Quantitative RT-PCR results were analyzed by one-way ANOVA (analysis of variance) with Tukey post-test. This stringent statistical test was chosen because of the variability between replicates in some of the qRT-PCR data.
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RESULTS
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Activin A Treatment of EBs Increases the Expression of Early Distal Lung Epithelial Markers
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The early distal lung epithelial marker SPC was upregulated approximately 20-fold over that of control cultures by the replacement of FBS in the ES differentiation medium with KOSR (Fig. 2). A further three- to fourfold increase over KOSR-only control cultures was seen when early differentiating EBs were treated with 100 ng/ml activin A. This expression pattern appeared to be paralleled by the marker TTF-1, a second early distal lung epithelial marker. Unfortunately, the extremely low-level expression of this transcription factor meant that it was difficult to accurately quantify, and this created large standard deviations between replicates. Therefore, differences in expression level between media treatments did not quite reach statistical significance for this marker gene.
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Figure 2. Activin A treatment of embryoid bodies enhances the expression of distal lung epithelial markers. Quantitative reverse transcription-polymerase chain reaction analysis of 21-day differentiated embryonic stem cells shows that activin A treatment of embryoid bodies (Fig. 1) induces the expression of the early lung markers TTF-1 and SPC. Adherent cultures did not undergo the final (step 3) media treatment in this experiment. Results for each gene are normalized to the level of expression in the FBS-only control (relative expression level set at 1). Bars represent the mean of six experiments, and error bars are one standard deviation from the mean. Note that the y-axis is on a logarithmic scale. Abbreviations: AA, activin A; FBS, batch-tested fetal bovine serum; KOSR, KnockOut Serum Replacement.' v1 x2 k; a3 K( @  c
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In contrast, the expression of three markers of mature distal lung epithelium (SPA, SPB, and CC10) exhibited no response to either serum removal or activin A treatment. As a control, the expression of SPD was also analyzed. This gene is abundantly expressed in mature distal lung epithelium but is also widely expressed in other mucosal epithelia as part of host defense systems and is therefore not lung-specific. Again, SPD expression showed no change regardless of media treatment regime.

Activin A Treatment Enhances Endoderm Formation and Accelerates Early Lung Epithelial Differentiation6 k" P( j- Y3 k9 f# p+ M7 ^6 S

To further investigate the mechanism underlying the upregulation of early but not late distal lung epithelial markers by activin A, we performed an experiment to study the temporal expression of genes marking mesendoderm, endoderm, and early and late lung epithelial developmental stages. Brachyury T expression during the early phase (days 3¨C5) of EB differentiation suggested that activin A enhanced mesendodermal differentiation by two- to threefold over untreated EBs (Fig. 3). The expression of endodermal markers Foxa2 and Sox17 began to be detected between days 7 and 10 of differentiation. In activin A cultures, the expression of both markers peaked at day 10 and declined, following a typical pattern of temporal developmental expression. In control cultures, Sox17 and Foxa2 expression peaked at much later time points. Consistent with this, the expression of TTF-1 and SPC was also induced at significantly earlier time points in activin A-treated cultures. However, both the mature distal lung epithelial markers SPB (Fig. 3) and CC10 (data not shown) showed only a basal level of gene expression and were not induced at any time point or under any medium treatment examined.% U1 v: E; |$ u. _0 H# ?
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Figure 3. Developmental staging of differentiating embryonic stem cells (ESCs). Quantitative reverse transcription-polymerase chain reaction analysis of developmental marker gene expression during ESC differentiation demonstrates that activin A treatment of EBs enhances the formation of mesendoderm and endoderm, as shown by Brachyury, Sox17, and Foxa2 expression; x-axes show differentiation time in days, and y-axes show expression levels of each marker gene relative to expression in the adult mouse lung positive control. Adherent cultures did not undergo the final (step 3) media treatment in this experiment. Points represent the mean of three experiments, and error bars are one standard deviation from the mean. Abbreviations: AA, activin A; EB, embryoid body; FBS, batch-tested fetal bovine serum; KOSR, KnockOut Serum Replacement.
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Lung Differentiation Is Further Enhanced by Exposure of Late Differentiating Activin A-Treated EBs to Specialized Growth Media

To enrich cultures specifically for lung epithelium, activin A treatment of EBs was combined with an application of small airway basal medium (SABM) or SAGM during the terminal phase of differentiation. Consistent with our previously reported findings , a final treatment of cells differentiated in FBS-containing medium with SAGM induced SPC expression (Fig. 4). This occurred concomitant with an upregulation of the primitive distal lung marker TTF-1 and the mature distal lung markers SPA, CC10, and SPB. Unexpectedly, activin A-treated cultures did not respond similarly to SAGM. By far the most significant changes in gene expression were observed in response to SABM, the basal medium upon which SAGM is based. In activin A-treated EBs, SPC expression and TTF-1 expression were both upregulated fourfold in SABM over cultures remaining in the original differentiation medium and approximately 100-fold over cells differentiated following our previously published protocol (FBS SAGM). Intriguingly, SPA, SPB, and CC10, markers of mature distal lung differentiation, were not upregulated in parallel, in contrast to the observations made with the FBS SAGM regime.
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Figure 4. Distal lung marker gene expression in embryonic stem cells (ESCs) after three-step differentiation. Quantitative reverse transcription-polymerase chain reaction analysis of differentiated ESCs following the full three-stage differentiation protocol. Results show that the activin A SABM treatment protocol is optimal for lung differentiation, evidenced by the marked induction of SPC and TTF-1 expression. Results for each gene are normalized to the level of expression in the FBS only control (= 1). Media treatment during the EB stage (days 0¨C10) is shown on the x-axis, whereas column color indicates the final differentiation medium (days 21¨C25). Bars represent the mean of six experiments, and error bars are one standard deviation from the mean. Note that the y-axis is on a logarithmic scale. Abbreviations: AA, activin A; EB, embryoid body; FBS, batch-tested fetal bovine serum; KOSR, KnockOut Serum Replacement; n/s, not significant; SABM, small airway basal medium; SAGM, small airway growth medium.
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The Frequency of SPC-Expressing Cells Is Increased in Activin A SABM Cultures8 b* U9 ~% H3 t( v2 }" g# ~
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To determine whether the upregulation of SPC gene expression in activin A SABM cultures was a consequence of an increase in the number of SPC-expressing cells, a murine ESC line containing a stably integrated SPC promoter/EGFP reporter construct was employed. In transient transfections with the SPC-EGFP construct, no EGFP expression was detected in undifferentiated ESCs or MEFs whereas all cell types transfected with a constitutively active EGFP reporter construct exhibited EGFP fluorescence (data not shown). However, SPC-EGFP was strongly expressed in transfected MLE-12 cells (data not shown), illustrating the tissue-specificity of the SPC-EGFP construct.0 t* Y# g/ V, X) s2 u

The stable SPC-EGFP ESC line was subjected to either the "new" activin A SABM differentiation protocol or the "old" FBS SAGM differentiation protocol. EGFP fluorescence was visible in activin A SABM cultures from day 17, whereas no fluorescence was detectable in FBS SAGM cultures until day 21, consistent with the results from time course analysis of endogenous gene expression. At the final day-25 time point, EGFP-expressing cells were visible as clusters in activin A SABM cultures (Fig. 5A). At least one cluster was present in the majority of adhered EBs, and approximately 10% of EBs contained two or more foci. In contrast, EGFP-expressing cells were seen only as infrequent and isolated cells in FBS SAGM cultures (Fig. 5A). Under this differentiation protocol, the majority of adhered EBs did not contain any EGFP-positive cells.

Figure 5. Visualization of SPC-expressing cells in living cultures using an SPC promoter/EGFP reporter cell line. (A): A mouse embryonic stem cell (ESC) line carrying an integrated SPC promoter/EGFP reporter cassette was differentiated according to the optimal protocol for SPC expression established in this work (activin A SABM) or our previously published protocol (FBS SAGM) . ESCs committed to a distal lung lineage were detected by fluorescence microscopy for EGFP expression (EGFP). Activin A SABM cultures exhibited sizable clusters of EGFP-expressing cells within most adhered embryoid bodies (EBs) (shown at x4 magnification; phase-contrast images are also shown to indicate overall cell density). In contrast, only individual, isolated EGFP-positive cells were observed very infrequently in FBS SAGM cultures under high-power magnification (x20). (B): The proportion of EGFP-expressing cells in activin A SABM cultures was quantified by flow cytometry. Dying cells and cell debris were excluded from analysis by tightly gating the viable cell population to minimize background fluorescence (i). A threshold was set such that approximately 1% of negative control cultures scored as positive for green fluorescence (ii). This fluorescence threshold was then used to quantify the percentage of EGFP-expressing cells in activin A SABM-treated cultures (iii and iv). The number of EGFP-positive cells in FBS SAGM cultures was not detectable above autofluorescence (data not shown). Abbreviations: EGFP, enhanced green fluorescent protein; FBS, batch-tested fetal bovine serum; SABM, small airway basal medium; SAGM, small airway growth medium.& W: H# f/ e) R  A: B8 W5 n' K5 H; ]

To determine the overall frequency of lung-committed cells, day-25 cultures were dissociated into single cells for flow cytometry. Unfortunately, flow cytometric analysis was hindered by cell suspensions exhibiting a high degree of autofluorescence from apoptotic cells and debris, apparently due to the combination of serum-free medium (SABM/SAGM) and the vigorous dissociation procedure. However, by tightly gating the viable cell population (Fig. 5B, i), some quantification was possible, although we acknowledge that this is may represent only the higher end of the EGFP-expression spectrum. Untransfected E14-Tg2a cells were differentiated in parallel to SPC-EGFP cells and used to control for background fluorescence. A threshold was set such that an average of 1% of the negative control samples was scored positive for green fluorescence (Fig. 5B, ii), ensuring that the maximum number of EGFP-expressing cells would be counted. Using this method of quantification, EGFP-expressing cells represented approximately 3.04% ¡À 1.26% of activin A SABM cultures (average of six experiments corrected for 1% background fluorescence; Fig. 5B, iii, 5B, iv). EGFP fluorescence in FBS SAGM cultures was not detectable above background.

Effects of Differentiation Protocol on Culture Expansion

The observation of sizable EGFP-expressing cell clusters in activin A SABM cultures suggested that the new differentiation protocol stimulated the proliferation of lung epithelial precursors. To determine whether this was an effect of the activin A SABM protocol on overall culture proliferation, viable cell counts were performed at regular intervals (Fig. 6). EBs formed in KOSR (with or without activin A treatment) contained much higher cell numbers in initial stages of EB differentiation than those formed in serum-containing medium, suggesting that the removal of serum from the medium generally allowed for greater cell proliferation. Activin A treatment of EBs reduced overall cell numbers slightly compared with the KOSR-only control, suggesting that the endoderm-enhancing effects of activin A are not a result of global cell proliferation. Cell numbers decreased after day 5 in both categories of KOSR-formed EBs (concomitant with the development of necrotic centers), whereas EBs in serum-containing medium continued to slowly proliferate. After plating in adherent culture at day 11, EBs cultured in all media variants expanded to fill the available space in the culture vessel, reaching approximately the same culture density by day 21. At day 21, the lung-selective medium was added, and as expected this initiated the death of a large proportion of the cultures. Cells cultured in KOSR-containing medium with or without early activin A treatment were selected in SABM, the optimal method for deriving SPC-expressing cells. Cells cultured in serum-containing medium were exposed to SAGM (SABM plus a commercial cocktail of growth factors) according to the "old" protocol. SABM selection had a much more profound effect on culture survival than did SAGM, with viable cell numbers decreasing to only 8¨C9 x 104 (less than double the input number of cells at the start of the experiment ). This indicates that the more minimal SABM medium is much more effective than SAGM at selecting out ESC-derived lung progenitors from a mixed differentiated ESC population.
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Figure 6. Viable cell counts during murine embryonic stem cell (mESC) differentiation. Viable cell numbers were counted at regular intervals during mESC differentiation under three media regimes: KOSR SABM (solid line), activin A SABM (hatched line), and FBS SAGM (dotted line). Points represent the mean of three experiments, and error bars are one standard deviation from the mean. Abbreviations: FBS, batch-tested fetal bovine serum; KOSR, KnockOut Serum Replacement; SABM, small airway basal medium; SAGM, small airway growth medium.
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ESC-Derived Lung Epithelial Progenitors Continue to Mature Slowly in Culture

ESCs differentiated under the activin A regime were exposed to SABM for 14 (rather than 4) days at stage 3 of the differentiation process to determine whether SPC-expressing progenitor cells could induce the expression of more mature lung markers during extended culture. Real-time RT-PCR results showed a statistically significant downregulation of TTF-1 expression concomitant with induction of the Clara cell-specific marker CC10 (Fig. 7), consistent with a further degree of maturation. However, induction of the late type II cell developmental markers SPA and SPB was not observed.

Figure 7. Embryonic stem cell (ESC)-derived progenitor cells mature slowly during prolonged culture. Prolonged culture in the final differentiation media promotes only a slight maturation of ESC-derived lung progenitors. Cells were cultured for 4 days (total 25 days) or 14 days (total 35 days) at stage 3 of the differentiation process and then harvested for quantitative reverse transcription-polymerase chain reaction analysis of marker gene expression. Media combinations are indicated on the x-axis. Note that the y-axis is on a logarithmic scale. Abbreviations: AA, activin A; FBS, batch-tested fetal bovine serum; n/s, not significant; SABM, small airway basal medium; SAGM, small airway growth medium.
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DISCUSSION

The results presented here describe a strategy to derive distal lung-committed epithelial progenitor cells from murine ESCs. First, differentiating cultures were enriched for the endodermal germ layer by exposure of EBs to high levels of activin A, an approach originally reported by Kubo et al.  that activin A alters the relative proportion of mesendoderm and neuroectoderm during the earliest stages of EB differentiation.$ v1 y1 g; n: v. p' r' C
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Recently, there have been several more reports of the up-regulation of definitive endoderm production by activin A in differentiating mESC, including two that avoid the formation of EBs during differentiation and thereby claim to achieve a purer cell population . Therefore, it appears that the action of activin A on hESC differentiation status may be highly context-dependent.; w* V  g5 A5 o% E6 d! {2 x
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Our laboratory has previously reported the derivation of mature type II pneumocytes from murine ESCs using a commercially available medium developed for the culture of distal lung epithelial cells . We have shown that under control conditions spontaneous type II pneumocyte differentiation cannot be detected in mESC populations. Even under permissive conditions, type II pneumocyte-like cells are present but are very infrequent and can be reliably detected only by highly sensitive techniques such as quantitative RT-PCR. We hypothesized that a more efficient differentiation protocol would involve multiple steps designed to target and enhance the development of each lung-progenitor cell type in turn. The combination of early activin A-mediated endoderm enhancement with a late step to enrich for lung epithelium using SABM significantly improved distal lung epithelial differentiation from mouse ESCs. Experiments with a murine ESC line carrying an SPC promoter/EGFP reporter construct confirmed that this was a result of an increase in the frequency of SPC-expressing cells, which appeared as clusters as opposed to isolated cells in the differentiated ESC population. Compared with the FBS SABM protocol, in the activin A SABM regime, the number of EGFP-positive foci as well as the number of EGFP-positive cells within each cluster were increased. Although flow cytometric analysis indicated that distal lung epithelial cells still represented only a small proportion of the overall cell population, this still represents a marked improvement over the earlier differentiation protocol.
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Interestingly, the cells derived under this differentiation protocol exhibited a gene expression profile that most closely resembles lung epithelial progenitor cells present in the foregut endoderm and early branching lung at approximately embryonic day 10 (E10) to E11. These cells express TTF-1 and SPC (detectable by RT-PCR at E8.5 and E9.5, respectively) but are yet to mature further into small airway and alveolar epithelial cells expressing CC10, SPA, and SPB . In comparison, cells differentiated under our previously reported protocol, in which cells were initially allowed to differentiate spontaneously in serum-containing medium prior to SAGM application, had a more mature distal lung epithelial phenotype and induced several late developmental lung markers. Together with the results of the cell-counting experiment (Fig. 6), these findings suggest that the activin A SABM differentiation strategy increases the number of lung-committed cells in two ways, first by stimulating proliferation of progenitors early in the differentiation process, then by SABM-mediated selection of lung epithelial cells, which causes widespread death of non-lung epithelial cell populations. In contrast, the FBS SAGM strategy yields a much smaller number of mature lung epithelial cells, suggesting a trade-off between proliferative activity and the acquisition of a mature differentiated phenotype.6 _# V& u% d4 S5 |

Murine ESC-derived lung progenitor epithelial cells continued to slowly mature during prolonged culture but did not induce the expression of SPB or SPA, indicating that an alternative environment will be required to drive progenitor cells derived by activin A SABM culture further along the developmental program within reasonable timescales. We are currently beginning to implant purified mESC-derived lung progenitors into animal lung injury models, which will allow us to assess differentiation capacity, cell function, and repair capability in the physiological environment.

For the purposes of tissue engineering, the derivation of progenitor cell types for transplantation has several clear advantages. First, the implantation of proliferative progenitor cells, rather than post-mitotic terminally differentiated cells, is likely to result in more efficient integration with host tissue. Furthermore, if progenitor cells can divide after implantation, this may minimize the number of cells which need to be transplanted. Second, early progenitor cells can usually form one or more mature somatic cell types, enabling single cells to mature into multifunctional tissue units. In this case, early distal lung epithelial progenitors in the foregut endoderm form type II pneumocytes, type I pneumocytes, and Clara cells during lung development in vivo. The stimulus for terminal differentiation will likely be provided by the host micro-environment itself, further promoting the functional integration and structural organization of ESC-derived progenitor cells. Consistent with this, we have previously shown that ESCs can respond to differentiation-promoting signals in coculture with other cell types .
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SUMMARY

We have developed a multistep differentiation strategy from which early distal lung epithelial progenitors can be derived from murine ESCs. These findings represent a significant step forward for lung tissue engineering and may also provide an in vitro model for the study of lung epithelial differentiation.

ACKNOWLEDGMENTS
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This work was funded by the Medical Research Council, Wellcome Trust, Rosetrees Trust, and Chelsea and Westminster Trust. We thank J. Whitsett for the kind donation of the SPC promoter/EGFP plasmid construct and Kian-Leong Lee and Richard Lubman for comments on the manuscript." O% Y. S% A$ \+ `0 o

DISCLOSURES3 h  F' Q2 ]" n% b& B) ?7 S( B$ z
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The authors indicate no potential conflicts of interest./ ?2 Z5 M7 V: g  ~) e8 Q% C' @% R$ a
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