(Fig. 1) presents a chart flow showing the design of the experiments and the specific outcomes measured. Fetal rat lung type II cells or fibroblasts were co-cultured with adult rat BMSCs using Transwell^® inserts. In this way cells were able to communicate without direct cell contact. This co-culture system, as shown in Fig. (1), was set up in several ways. Each cell type was grown on plastic in one group of experiments and on the inserts in a second group of experiments. After 48h we studied: 1) proliferation (by thymidine incorporation) in the type II cells, the fibroblasts and the BMSCs cultured on plastic; 2) surfactant synthesis (choline incorporation into disaturated phosphatidylcholine (DSPC)) in each cell cultured on plastic; and 3) cell surface markers using flow cytometry of the BMSCs cultured on plastic. The specific procedures for these studies are described in the following paragraphs.

Fig. (1). Schematic of study flow design. Co-cultures were prepared with either fetal rat lung fibroblasts or type II cells cultured on the bottom of 6-well plates with BMSC cultured on the Transwell®insert, or BMSC cultured on the bottom of the plates with either fetal rat lung fibroblasts or type II cells cultured on the Transwell® insert. All cell studies were performed on the cells cultured on the plate bottoms after 48 hours of co-culture. Cell proliferation was measured by thymidine incorporation. Differentiated type II cell-like function was studied by choline incorporation into DSPC. Cell surface markers that help define BMSC were studied by flow cytometry in the BMSC only.

Fig. (2). Paracrine effects on cell growth: Paracrine effects of BMSC (bar 1+2) on the proliferation of fetal rat d21 type II cells and fibroblasts and of type II cells (3. grey bar) and fibroblasts (4. light grey bar) on proliferation of BMSC. Proliferation was measured by thymidine incorporation. Results are expressed as percent changes of their specific untreated controls and presented as Mean ± SEM (n=8, *p<0.05).

Fig. (3). Paracrine effects of BMSC on type II cell surfactant synthesis: The paracrine effects of BMSC (black bars) on fetal rat d21 type II cell surfactant DSPC synthesis was measured by choline incorporation into DSPC. Results are expressed as percent changes of their specific untreated controls and presented as Mean ± SEM (n=7, *p<0.05).

Fig. (4). Paracrine effects of BMSC and fetal lung fibroblasts on Sftpb and Sftpc mRNA expression in fetal type II cells: Fetal rat E21 type II cells were grown in co-culture with fetal lung fibroblasts (light grey bar) or BMSC (black bar) and the paracrine effects on sftpb mRNA (A) and sftpc mRNA (B) expression evaluated. Results are displayed as the DDCt and presented as Mean ± SEM (n=4, *p<0.035). A negative DDCT value represents an increase in expression.

Fig. (5). Paracrine effects of fetal lung fibroblasts and type II cells on BMSC surface marker expression: The effect on BMSC marker expression after co-culture with fetal lung fibroblasts (FCM) and type II cells (TCM) for 48 h was analyzed by fluorescence activated cell sorting (FACS) and compared to non-exposed BMSC. Representative fluorescence intensity histograms with specific antibodies for membrane antigens (green area) and a negative control (grey area) are shown. Experiments were performed in quadruplicate.

The animal research protocol was approved by the institutional IACUC. Time-dated pregnant Sprague-Dawley rats were sacrificed on E21 of gestation by CO₂ inhalation as previously published [22]. Briefly, the fetal lungs were removed, washed in Hank’s buffered saline solution (HBSS), minced with a razor blade, dissociated in HBSS containing 0.25% trypsin and 20µg/ml DNAse and incubated at 37° C for 12 minutes. The reaction was stopped by DMEM containing 10% charcoal stripped fetal bovine serum (FBS^-). The cells were filtered through sterile Nitex filters and centrifuged for 10 minutes at 650 x g at 4° C. The pellet was resuspended in DMEM containing 10% FBS^-, the cells were plated in culture dishes, and incubated for 45 minutes at 37° C (21%O₂/5%CO₂) to allow the differential adherence of lung fibroblasts. Dishes with adherent fibroblasts were washed with DMEM, then DMEM containing 10% FBS^- was added and the cells were incubated at 37°C overnight. The supernatant containing non-adherent type II cells was removed and centrifuged for 5 minutes, the pellet was resuspended in DMEM containing 10% FBS^- and the cells were plated into culture dishes and incubated for 60 minutes at 37° C for a second differential adherence. The supernatants were again collected and centrifuged. The pellet was resuspended in DMEM containing 20% FBS^- and the cells were plated at a concentration of 0.7 x 10⁶/ml into 24- or 6-well tissue culture plates or cell culture inserts overnight. Non-adherent cells were washed away with DMEM.

Bone marrow derived mesenchymal stem cells (BMSC) were kindly provided by Dr. Bernard Thébaud (University of Alberta, Alberta, Edmonton Canada). The BMSC were harvested from adult Sprague-Dawley rats by excising the femur and tibia and flushing the marrow with DMEM as previously published [12]. The isolated marrow was dissociated with a 21-gauge needle and plated into a tissue culture flask. After overnight adherence, the media was aspirated, adherent cells were rinsed with PBS, and the media was replenished. Adherent cells were allowed to grow to ~75% confluence, then trypsinized and reseeded at a density of 10⁵ cells/cm². This procedure was performed for 2 passages. Cells were then shipped to Tufts Medical Center, where they were immediately placed in culture and expanded for further use. Experiments were done with BMSC that had undergone a total of 4 – 6 passages.

BMSC were plated into 24- or 6-well tissue culture plates or onto cell culture inserts with DMEM + 10% FBS^-. Type II cells plated in the wells were exposed to BMSC- or fibroblast-conditioned media (CM) by placing inserts plated with BMSC or fibroblasts into the wells with type II cells, allowing the cells to grow in the same medium (DMEM with 10% FBS^-) without direct contact between the type II cells and either the BMSC or the fibroblasts. Fibroblasts plated in the wells were exposed to BMSC conditioned media in the same manner. BMSC plated in the wells were exposed similarly to type II cell or fibroblast conditioned media. All cells were harvested after 48 hours of co-culture.

Type II cells and fibroblasts exposed to BMSC CM and BMSC exposed to type II cell or fibroblast CM were serum starved for 2 hours and exposed to [³H] thymidine (0.5 µCi/ml) for an additional 24 hours in serum-free DMEM. Cells were washed three times with ice-cold PBS and detached with trypsin. Total DNA was measured in duplicate aliquots and used as an internal standard. The cells were transferred into scintillation fluid and incorporated [³H] thymidine was counted in a beta scintillation counter as previously published [23]. The results from exposed cells were expressed as percent of sample-specific non-exposed control cells.

Type II cells were co-cultured with BMSC, and BMSC were co-cultured with type II cells for 24 hours and exposed to [³H] choline (0.5 µCi/ml) for an additional 24 hours. Cells were washed three times with ice cold PBS, lysed in trypsin, and sonicated. Total protein amount per sample was measured in duplicate aliquots and used as an internal standard. Chloroform/methanol extraction of lipids was performed as previously described [23]. Disaturated phosphatidylcholine (DSPC) was isolated from the extracted lipids by osmium tetroxide and separated by thin-layer chromatography on silica gel H chromatography sheets. The resulting spots were scraped into scintillation fluid, counted in a beta scintillation counter, expressed as disintegrations per min per µg protein, and the results expressed as percent of sample specific controls.

RNA was harvested on day 3 of culture using a Qiagen RNeasy MicroKit. The first strand complementary DNA was synthesized using the Super Script^TM III Reverse Transcriptase Kit with oligonucleotide dTs (0.5µg/µl). The cDNA levels from the surfactant proteins B and C (sftpb, sftpc), and actin gene transcripts were measured by real-time PCR. One microgram of the cDNA product was used for amplification in a 20µl reaction volume containing 10µl SYBR Green PCR Master Mix, 7µl DEPC-H₂O and 1µl forward and reverse primers [24]. The amplification protocol consisted of an initial denaturation and enzyme activation at 95°C for 10 minutes, followed by a DNA amplification with 40 cycles each consisting of 30 seconds at 95°C, an attachment of primers for 1 minute at 55°C, and the extension at 72°C for 30 seconds, and finally 1 cycle at 72°C for 10 min for final elongation. The relative expression level of the sftp genes was calculated by calculating the delta (D)Ct value representing the difference in the Ct values of the target and the reference gene. From this, the DDCt value was calculated as the difference between the DCt of co-cultured cells and their non-exposed controls. The DDCt value, which is a negative number when the treatment condition is stimulated compared to the control condition, is a standard representation of comparative real time PCR results. The value [–(DDCT)] is the power to which 2 is raised to calculate fold changes in mRNA levels between treatment and control conditions. The DDCt therefore is geometrically proportional to the change in levels of sftp mRNA [25].

On day 3 of culture cells were harvested, centrifuged, resuspended in 5% normal horse serum, and incubated with the primary antibody for 0.5 hours at 4°C. The cells were washed with PBS and probed with the appropriate secondary antibody. After an incubation time of 30 minutes at 4°C, cells were washed extensively with PBS, transferred into ice-cold PBS containing 0.5% BSA, and kept in BD Falcon tubes on ice until read in the Beckman-Counter MoFlo high speed sorter.

The effects of BMSC CM exposure on proliferation and surfactant synthesis were expressed as percentages of their specific non-treated controls. All treatment values are presented as mean ± SEM of experiment-specific controls unless otherwise stated. The results were evaluated for statistical significance using a two-tailed t-test or a Mann-Whitney U test and corrected for multiple comparisons when appropriate.

Timed pregnant Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY); plastic tissue culture dishes, 6- and 24-well culture plates, and 6- and 24 –Transwell^® (0.4 µm pore sized) cell culture inserts were obtained from Becton Dickinson Labware (Franklin Lakes, NJ). [³H] choline (specific activity 70.3 Ci/mmol) [³H] thymidine (specific activity 20.0 Ci/mmol), Dulbecco’s modified eagle’s medium (DMEM), dipalmitoylphosphatidylcholine (DSPC) standard and osmium tetroxide were from Sigma Aldrich (St. Louis, MO). Charcoal-stripped fetal bovine serum was from Hyclone (Logan, UT); silica gel-coated PE sheets came from Analtech (Newark, CE). Antibodies were obtained as follows: mouse monoclonal IgG anti CD54 antibody and goat polyclonal IgG anti CD105 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal IgG anti CD90 antibody and mouse monoclonal IgG anti CD45 antibody were from Cedarlane (Burlington, NC); mouse monoclonal IgG anti CD73 antibody was from BD Bioscience Pharmingen (Franklin Lakes, NJ); monoclonal anti-ß Actin was from Sigma (St. Louis, MO); Alexa Fluor secondary antibody 488 was from Invitrogen (Eugene, OR). PCR primers for actin, sftpb and sftpc were obtained from the Tufts Medical Center Core Facility (Boston, MA). RNeasy Micro Kit was from Qiagen (Valencia, CA), SuperScript^TM III Reverse Transcriptase Kit was from Invitrogen (Carlsbad, CA), and SYBR Green PCR Master Mix was from Stratagene (La Jolla, CA).

Purity of type II cells was confirmed by staining [26] and was more than 92% as we have previously published [27]. BMSC CM exposure significantly decreased type II cell proliferation to 72.5 ± 7.1% (n=8, p=0.009) compared to non-exposed control type II cells (100 ± 7.6%, n=8) (Fig. 2). Fetal lung fibroblast proliferation decreased to 83.7 ± 8.7% (n=8, p=0.16) when exposed to BMSC CM compared to non-exposed fibroblasts (100 ± 6.2%, n=8) (Fig. 2). Proliferation of BMSC was significantly reduced to 72.2 ± 5.8% (mean ± SE, n=8, p<0.001) when co-cultured with type II cells, and to 76.2 ± 3.2% (mean ± SE, n=8, p<0.001) when exposed to fibroblasts, compared to BMSC grown in monoculture (100 ± 3.9%) (Fig. 2).

As a marker of surfactant phospholipid synthesis we measured the amount of incorporated [³H] choline into DSPC in type II cells with and without exposure to BMSC CM. Surfactant phospholipid synthesis was significantly increased to 84.0± 8.3 disintegrations/min/μg protein (121.1 ± 9.6%, mean ± SE, n= 7, p=0.046) in fetal primary type II cells when co-cultured with BMSC compared to type II cells cultured without BMSC exposure (67.92 ± 6.4 disintegrations/min/μg protein, 100 ± 4.4%, n=7) (Fig. 3). This increase after BMSC CM exposure is similar to, albeit less than, the stimulatory effect of fibroblast CM or NRG1-β on type II cell DSPC as reported in the literature [23,24,28], which we have studied previously. Exposure of BMSC to either fetal lung fibroblast or type II cell CM did not induce the BMSC to incorporate choline into DSPC (data not shown), confirming that they have not been induced to transform into lung type II cells.

We then studied the effect of BMSC CM on sftpb and sftpc mRNA in type II cells and compared it to the known stimulatory effect of fetal lung fibroblast CM on type II cell surfactant protein mRNA expression. Co-culture with BMSC significantly increased sftpb mRNA expression in type II cells: DCt of sftpb/actin was 7.6 ± 0.5 cycles for type II cells exposed to BMSC CM compared to 9.6 ± 0.3 cycles for non exposed control type II cells. The DDCt between treatment and control was -2.1 ± 0.5 (n=6, p=0.002). The increase in sftpb mRNA expression was similar to the stimulatory effect of fibroblast CM: the DCt for sftpb/actin was 7.7 ± 0.5 cycles, the DDCt between treatment and control was -1.9 ± 0.4 (n=5, p=0.003) (Fig. 4A). The stimulatory effects of both conditioned media were in alignment with our previous published effect of NRG on Sftpb mRNA, though somewhat more potent [28,29].

However, BMSC CM stimulation of sftpc mRNA expression (DDCt -2.4 ± 0.8; n=5, p=0.065) was significantly (p=0.035) more pronounced than that of fibroblast CM (DDCt -0.5 ± 0.4 n=6, p=0.33). The DCt value of sftpc/actin was 10.0 ± 0.3 cycles for type II cells alone, 9.3 ± 0.5 for type II cells exposed to fibroblast CM and 7.9 ± 1.0 for type II cells exposed to BMSC CM. (Fig. 4B).

BMSC used for all experiments are able to differentiate into adipogenic, osteogenic and chondrogenic tissue and express CD90, CD73, CD105, CD54, CD44, but do not CD31 and CD45 [12]. These characteristics comply with the position statement for minimal criteria to define MSC [30]. We followed the expression patterns of CD90, CD73, CD105, CD54, and CD45 as markers of multipotent mesenchymal stem cell characteristics, before and after the exposure to lung cell CM. BMSC stained positive for CD90, CD105, CD54, and CD73 (data not shown), with no change after fibroblast CM (n=4) or type II cell CM exposure (n=4), (Fig. 5). The BMSC stained negative for CD45 in all three conditions (data not shown).