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Cell Transplantation, Vol. 18, pp. 405–422, 2009 Printed in the USA. All rights reserved. Copyright  2009 Cognizant Comm. Corp.

Transplantation of Allogeneic and Xenogeneic Placenta-Derived Cells Reduces Bleomycin-Induced Lung Fibrosis Anna Cargnoni,* Lucia Gibelli,* Alessandra Tosini,* Patrizia Bonassi Signoroni,* Claudia Nassuato,* Davide Arienti,† Guerino Lombardi,† Alberto Albertini,‡ Georg S. Wengler,* and Ornella Parolini* *Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, 25124 Brescia, Italy †Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Reparto del Benessere animale, IZSLER, 25124 Brescia, Italy ‡Istituto di Tecnologie Biomediche, CNR, 20090 Segrate-Milano, Italy

Fetal membranes (amnion and chorion) have recently raised significant attention as potential sources of stem cells. We have recently demonstrated that cells derived from human term placenta show stem cell phenotype, high plasticity, and display low immunogenicity both in vitro and in vivo. Moreover, placenta-derived cells, after xenotransplantation, are able to engraft in solid organs including the lung. On these bases, we studied the effects of fetal membrane-derived cells on a mouse model of bleomycin-induced lung fibrosis. Fetal membrane-derived cells were infused 15 min after intratracheal bleomycin instillation. Different delivery routes were used: intraperitoneal or intratracheal for both xenogeneic and allogeneic cells, and intravenous for allogeneic cells. The effects of the transplanted cells on bleomycin-induced inflammatory and fibrotic processes were then scored and compared between transplanted and control animals at different time points. By PCR and immunohistochemistry analyses, we demonstrated the presence of transplanted cells 3, 7, 9, and 14 days after transplantation. Concomitantly, we observed a clear decrease in neutrophil infiltration and a significant reduction in the severity of bleomycin-induced lung fibrosis in mice treated with placenta-derived cells, irrespective of the source (allogeneic or xenogeneic) or delivery route. Our findings constitute further evidence in support of the hypothesis that placenta-derived cells could be useful for clinical application, and warrant further studies toward the use of these cells for the repair of tissue damage associated with inflammatory and fibrotic degeneration. Key words: Fetal membrane-derived cells; Placenta; Amnion; Cell transplantation; Lung fibrosis; Inflammation

INTRODUCTION

A number of studies performed in experimental models have shown that cell therapy may represent a promising approach for treatment of lung fibrotic diseases, although conflicting results have been reported by different groups to date (9). On the one hand, a number of authors have demonstrated that bone marrow-derived cells (12,23) and stem cells isolated from cord blood (40) are able to engraft in mouse lungs and differentiate, in vivo, into alveolar epithelial cells, and result in repair of various forms of lung damage (17,33,44). On the other hand, some studies have suggested that both unfractionated bone marrow cells or purified hematopoietic stem cells are not able to reconstitute lung epithelium (4,20) but can instead act as a potential source of fibroblasts, which play an active role in the lung fibrotic process (9,16).

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in lung parenchyma, leading to progressive remodeling of pulmonary tissue and loss of normal architecture and, eventually, to organ failure. Fibrosis is a typical component of many interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF), which is the most frequent in humans, as well as fibrotic diseases caused by tuberculosis, connective tissue disorders, or exposure to chemical agents, inorganic fibers, radiation, or pathogens (35,43). Despite extensive research aimed at understanding the molecular mechanisms involved in fibrogenesis, no therapy has yet been able to effectively reverse or stop progression of the fibrotic process.

Received September 19, 2008; final acceptance December 9, 2008. Online prepub date: April 6, 2009. Address correspondence to Ornella Parolini, Ph.D., Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Via Bissolati 57, I-25124 Brescia, Italy. Tel: 390302455754; Fax: 390302455704; E-mail: [email protected] or [email protected]

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In addition, transplantation of stromal bone marrow cells (21) and stem cells isolated from amniotic fluid (3) have been shown to result in engraftment of cells with morphology and phenotype of epithelial lung lineages in recipient animals with injured lung parenchyma. More recently, transplantation of allogeneic mesenchymal stem cells has resulted in reduction of fibrosis in the lungs of mice treated with bleomycin (30), with this effect most likely to be due to blocking of IL-1 and TNFα production by macrophages (29). Finally, transplantation of syngeneic alveolar type II epithelial cells (36) into bleomycin-treated rodents has been shown to result in a marked reduction in lung inflammation and fibrosis. Recently, we have demonstrated that amniotic and chorionic cells from human term placenta show a stem cell phenotype and functional potential (32). In particular, human amniotic epithelial cells (hAEC) express embryonic cell markers and are capable of differentiation toward tissues of all three germ layers (19,28,41). Meanwhile, human amniotic mesenchymal stromal cells (hAMSC), like mesenchymal stromal cells of other origins, have the capacity to differentiate toward cells of the mesodermal lineages (38). In addition, these cells display low immunogenicity both in vitro (25) and in vivo (1) and are able, after xenotransplantation, to engraft in solid organs including the lung (1). The potential application of fetal membrane-derived cells as a therapeutic tool for disorders characterized by inflammation and fibrosis is supported by previous studies, which report that when applied in the treatment of skin and ocular disorders (5,22,27,39), amniotic membrane results in downregulation of fibrotic and inflammatory responses [reviewed in (8)]. Here, we set out to evaluate the effects of fetal membrane-derived cell transplantation in bleomycin-treated mice, which represent a widely accepted model of lung interstitial fibrosis (2,7,11). We observed that intratracheal and intraperitoneal transplantation of either allogeneic or xenogeneic fetal membrane-derived cells can result in a reduction in bleomycin-induced lung fibrosis. These findings provide evidence that fetal membranederived cells may prove useful for cell therapy of fibrotic diseases in the future. MATERIALS AND METHODS Animals and Experimental Groups Experiments were carried out in a total of 197, 8– 9-week-old female C57BL/6 mice (Charles River, Italy), which have previously been reported to be bleomycin sensitive (10,15). Placentas from female BALB/c (Charles River, Italy) and BCF1 green fluorescent protein (GFP)expressing mice (kindly provided by Dr. Alessandro Vercelli, Department of Anatomy, Pharmacology and

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Forensic Medicine, University of Turin) were used to obtain allogeneic fetal membrane-derived cells. All animal experiments were carried out in accordance with current Italian and European regulations and laws on the Use and Care of Animals for research (DL. 116/27 January 1992). Each C57BL/6 mouse was weighed before any type of treatment was performed, and also before euthanasia. C57BL/6 mice were randomly divided into four main treatment groups: Saline control group—mice intratracheally instilled with saline, euthanized after 14 days; Cell control group—mice receiving allogeneic or xenogeneic cell transplants and euthanized 3, 7, 9, and 14 days after cell transplantation; Bleomycin-treated group— mice intratracheally instilled with bleomycin and euthanized after 3, 7, 9, and 14 days; Bleomycin + cell-treated group—mice intratracheally instilled with bleomycin and transplanted with allogeneic or xenogeneic cells and euthanized after 3, 7, 9, and 14 days. Induction of Lung Injury With Bleomycin C57BL/6 mice were anesthetized by intraperitoneal injection of xylazine (2 mg/kg) and ketamine (100 mg/ kg). The trachea was exposed and 50 µl of bleomycin (4 U/kg) were slowly instilled into the tracheal lumen (31). Control mice received the same volume of sterile saline. Intratracheal instillation was performed during a deep inspiration after compression of the thorax, in order to aid distribution of bleomycin to distal airspaces. Human Fetal Membrane-Derived Cell Preparation Human term placentas were obtained with maternal consent according to the guidelines of the Ethical Committee of the Catholic Hospital (CEIOC), and were immediately processed as previously described (1). Briefly, the amnion and chorion were manually separated and washed in phosphate-buffered saline (Sigma-Aldrich, St Louis, MO, USA) containing 100 U/ml penicillin and 100 µg/ml streptomycin (Lonza, Basel, Switzerland). Amnion fragments (⬃2 × 2 cm) were enzymatically digested with 2.4 U/ml dispase (Becton Dickinson, NJ, USA) for 9 min at 37°C, followed by a second digestion with 0.75 mg/ml collagenase (Roche, Mannheim, Germany) and 20 µg/ml DNAse (Roche, Mannheim, Germany) for 150 min at 37°C. Chorion fragments were subjected to two 9-min dispase digestions, separated by one 9-min wash step in RPMI-1640 (Lonza, Basel, Switzerland) containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FBS (SigmaAldrich, USA). The chorionic stromal layer was then separated from the trophoblastic layer and digested with collagenase and DNAse for 120 min at 37°C. After gentle centrifugation (150 × g for 3 min), the supernatant containing amniotic and chorionic mesenchymal stromal

PLACENTA-DERIVED CELLS REDUCE LUNG FIBROSIS

cells (hAMSCs and hCMSC) was filtered through a 100µm cell strainer (Becton Dickinson, NJ, USA). The collagenase-undigested amnion fragments were further incubated with 0.25% trypsin for 2 min at 37°C (SigmaAldrich, USA) to isolate amniotic epithelial cells (hAECs). The fetal membrane-derived cellular fractions (hAMSCs, hCMSCs, and hAECs) were cryopreserved separately in 10% DMSO supplemented with 90% FBS until use in cell transplantation procedures. Murine Fetal Membrane-Derived Cell Preparation Placentas were harvested from pregnant BALB/c and GFP+ BCF1 mice, at gestation day 16–18, and immediately processed, without separation of the amniotic and chorionic membranes, through sequential enzymatic digestions similar to those applied for the isolation of cells from human placenta. Briefly, placentas were digested at 37°C with 2.4 U/ml dispase for 5 min, followed by incubation with 0.75 mg/ml collagenase and 20 µg/ml DNAse for 90 min at 37°C. After a gentle centrifugation step (150 × g for 3 min) and filtration through a 100-µm cell strainer, a mix of amniotic and chorionic cells was obtained. The collagenase-undigested fragments were then further incubated with 0.25% trypsin for 2 min at 37°C in order to detach fetal membrane-derived epithelial cells. Each murine placenta yielded around 1 × 106 cells. Transplantation of Fetal Membrane-Derived Cells Xenogeneic fetal membrane cells derived from human term placenta were transplanted as a mixture composed of approximately 50% mesenchymal cells (hAMSCs + hCMSCs) and 50% epithelial cells (hAECs). Allogeneic fetal membrane cells derived from mouse placenta were used as a total fraction due to the impossibility of separating the amniotic and chorionic membranes in mice. Cryopreserved human and murine cells were thawed and their vitality was checked by trypan blue exclusion immediately before transplantation. In all cases, at least 85% of the transplanted cells were viable. Cell transplantation was performed both in control mice (Cell control group) and in mice that had been treated with bleomycin 15 min before cell administration (Bleomycin + Cell-treated group). Transplantation was performed under anesthesia via three different routes, namely intrajugular and intraperitoneal (systemic deliveries), or intratracheal (local delivery). Mice received either 4 × 106 cells for intraperitoneal transplantation, or 1 × 106 cells for intrajugular or intratracheal transplantation. Cells were resuspended in serum and phenol red-free DMEM and injected in a final

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volume of 200 µl for intraperitoneal delivery, or 100 µl for intrajugular or intratracheal delivery. Sample Collection After euthanasia, lungs were removed, sectioned, and formalin fixed for microscopy analysis, or frozen at −80°C for PCR analysis. Histological Evaluation of Lung Injury Formalin-fixed (10% neutral formalin, Bio-Optica, Milano, Italy) lung tissues were embedded in paraffin. Serial sections 4 µm in thickness were cut and stained with hematoxylin and eosin and Masson’s trichrome to identify inflammatory cells, fibroblasts, and collagen deposition. Two to four sections for each lung sample and for each stain were analyzed. Histological grading of inflammation and fibrosis was performed by a pathologist using a blinded semiquantitative scoring system adapted from Hagood et al. (14). Specifically, inflammation severity was evaluated based on the presence of macrophages, neutrophils, and lymphocytes and of edema. Subjective scores from 0 to 3 were given to reflect absent, mild, moderate, or extensive cell infiltration. Similarly, scores from 0 to 3 were assigned for edema intensity, which indicates the presence of luminal proteinaceous material. The sum of the scores reflecting cell infiltration and edema intensity was used to define the “severity of the inflammation.” The “extent of inflammation” was indicated as the percentage of the examined area (two to four entire sections for each sample) that was affected by inflammatory processes. Examined sections were assigned to four percentage groups: 0–25%, 25–50%, 50–75%, or >75%. The fibrotic process was evaluated based on the presence of fibroblasts and collagen deposition. Subjective scores from 0 to 3 were given to reflect absence, mild, moderate, or extensive cell infiltration. Similarly, scores from 0 to 3 were assigned for collagen deposition evaluated by Masson’s staining. By adding the scores relative to fibroblast infiltration and collagen deposition, we determined the “severity of fibrosis.” The “extent of fibrosis” was represented as the percentage of the examined area, which was affected by the fibrotic process as reported above for the assessment of the inflammation process. Detection of Allogeneic and Xenogeneic Fetal Membrane-Derived Cells in Host Lungs PCR Analysis. Total DNA (50–100 ng) was extracted from lung tissue using a Bio robot EZ1 (Qiagen, Hilden, Germany) and the EZ1 Tissue Kit (Qiagen) according to the manufacturer’s instructions, and was then amplified in 50-µl reactions containing dNTPs (200 µmol) and GoTaq DNA polymerase reagents (Promega,

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Madison, WI, USA) and the following primers (25 pmol) specific for the human sequence of the cytochrome B mitochondrial gene: 5′-CCCATACATTGG GACAGACC-3′ (forward) and 5′-GACGGATCGGA GAATTGTGT-3′ (reverse) (37); GFP sequence was detected using a primer pair that we designed: 5′-AC GACGGCAACTACAAGACC-3′ (forward) and 5′-GT CCTCCTTGAAGTCGATGC-3′ (reverse). All PCR reactions included an initial step at 95°C for 10 min. This was followed by 40 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 1 min) for DNA amplification of human mitochondrial cytochrome B, or 45 cycles (94°C for 1 min, 58°C for 30 s, 72°C for 1 min) for GFP. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining, followed by Southern blotting. For Southern blot analysis, PCR products were transferred to nylon membranes (Hybond N+, Amersham Biosciences, Little Chalfont, UK) and hybridized to specific probes labeled with horseradish peroxidase enzyme (ECL Gold, Amersham Biosciences). Chemiluminescent signals were detected with a Gel Doc 2000 system (Bio-Rad, Hercules, CA). Immunohistochemistry. Immunohistochemical studies were performed on formalin-fixed and paraffinembedded tissue sections using the Vector M.O.M. Immunodetection kit (Vector, Burlingame, CA). To detect the presence of human cells, samples were immunostained with a monoclonal antibody specific for cytokeratin 19 (CK 19, Ab-4) (clone BA17, LabVision, Fremont, CA) diluted 1:50. Allogeneic GFP+ cells were detected using a monoclonal anti-GFP antibody (clone GFP01, LabVision, Fremont, CA) diluted 1:300. Before staining, sections 4 µm thick were deparaffinized and unmasked with WCAP citrate buffer pH 6 (Bio-Optica, Milan, Italy). The endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution. The primary antibody was then applied for 1 h at room temperature. Novared (Vector) was used as the chromogen, and hematoxylin for counterstaining. Photographs were taken with an Olympus BX41 microscope using 40× and 20× lenses using an Olympus SP-500UZ Camera. Statistical Analysis Data are expressed as means ± SEM. Group means comparison was performed by two-way ANOVA with treatment and time as factors. Raw p-values were adjusted by Holm-Bonferroni’s procedure for multiple comparisons (24). Values of p < 0.05 were considered significant. Analyses were performed using R software 2.7.0 (R Development Core Team, 2005).

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RESULTS Effect of Different Treatments on Body Weight Mice used in this study had a baseline body weight of 18.7 ± 0.1 g. After 14 days, the weight of control mice increased to 21.0 ± 0.2 g (n = 11). Animals transplanted with allogeneic and xenogeneic fetal membrane-derived cells, without bleomycin instillation, showed similar body weight increases to control mice, while the weight of bleomycin-treated mice decreased significantly to 17.5 ± 0.8 g (n = 19) (p < 0.05). Bleomycin-treated mice that received cell transplants showed a less dramatic decline in body weight and in some cases an increase in body weight was observed, as reported in Table 1. Bleomycin-Induced Lung Injury As expected (30), intratracheal instillation of bleomycin resulted in marked lung injury. Progression of lung inflammation and fibrosis are shown in Figures 1A and B, respectively, through representative photomicrographs of lung histopathological analyses performed at different time points (3, 7, and 14 days) after bleomycin treatment. Intratracheal instillation of bleomycin resulted in progressive and marked infiltration of inflammatory cells in lung interstitial and intra-alveolar spaces (Fig. 1A, a1– a3), which was associated with a progressive and extensive thickening of the interalveolar septa (Fig. 1A, a1–a2). Inflammatory cell infiltration, together with fibroblast accumulation and marked collagen deposition in the interstitial spaces, as revealed by Masson’s staining (Fig. 1B, b1–b3), led to almost complete obliteration of the alveolar spaces (Figs. 1A and B, a3 and b3). In order to quantify the effect of bleomycin treatment and the efficacy of placenta-derived cell transplantation, we scored severity and extent of inflammation and fibrosis in at least four different lung samples obtained from each analyzed animal. The severity of inflammation was calculated by summing the scores representing the level of infiltration by each inflammatory cell population. Figure 1A shows the correspondence between cell infiltration and assigned score (panels M1–M3 for macrophages; panels N1–N3 for neutrophils; panels L1–L3 for lymphocytes). The correspondence between fibroblast infiltration and collagen deposition (parameters used to define the severity of lung fibrosis) and the attributed scores are shown in Figure 1B (panels F1–F3 and C1–C3, respectively). The severity of inflammation and fibrosis that were monitored over the course of lung injury progression and expressed as score units are reported in Figure 2 on the left y-axis (black bars). The extent of both inflammation and fibrosis, expressed as the percentage of the lung


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Table 1. Changes in Mouse Body Weight With Respect to Different Experimental Time Points and Treatment(s)

Treatment Saline Bleomycin Allogeneic cells Allogeneic cells Xenogeneic cells Xenogeneic cells Bleomycin + allogeneic cells Bleomycin + allogeneic cells Bleomycin + xenogeneic cells Bleomycin + xenogeneic cells

Cell Delivery Route — — IP IT IP IT IP IT IP IT

Body Weight Changes (g) After Treatment 3 Days n.d. −1.0 ± 0.95 0.53 ± 0.31 0.13 ± 0.13 1.02 ± 0.53 −0.15 ± 0.23 −0.98 ± 0.27 −1.38 ± 0.46 2.15 ± 0.30 −0.25 ± 0.70

7 Days

(3) (3) (3) (4) (2) (6) (3) (4) (2)

n.d. −0.37 ± 0.82 1.62 ± 0.16 0.46 ± 0.44 n.d. 0.07 ± 0.22 −1.82 ± 1.42 −3.28 ± 0.85 n.d. −2.55 ± 1.72

(7) (3) (4) (4) (5) (4) (3)

9 Days

14 Days

n.d. −1.1 ± 0.75 (24) 2.01 ± 0.40 (3) 1.73 ± 0.16 (3) n.d. n.d. −0.60 ± 0.94 (10) −1.53 ± 1.01 (11) −1.7 ± 0.98 (8) −0.13 ± 1.07 (6)

1.77 ± 0.31 (11) −0.62 ± 0.87 (19)* 2.80 ± 0.30 (3) 1.66 ± 0.53 (3) 2.34 ± 0.48 (4) −0.62 ± 0.95 (3) −0.13 ± 0.97 (8) 0.82 ± 0.24 (7) 2.11 ± 1.21 (8) −0.40 ± 0.93 (7)

IP, intraperitoneal; IT, intratracheal; n.d., not determined. *p < 0.05 versus saline group.

area presenting with signs of these two processes with respect to the total area examined, are shown on the right y-axis (gray bars) (Fig. 2). Intratracheal saline injection produced only slight inflammation (Fig. 2), likely due to local trauma, while no signs of fibrosis were observed (Fig. 2). Bleomycintreated animals showed early development of inflammation, which at day 3 involved about 25% of the total lung area examined. The inflammatory process continued to progress with time in the lungs of these animals, reaching its maximum levels in terms of severity (5.35 ± 0.39 score units) and extent (50–75% of lung areas) by day 9 after bleomycin instillation (Fig. 2). Fibrosis was detected at day 7 after bleomycin treatment (Fig. 2), and progressively increased over the study period. By day 14, the severity of fibrosis had reached its maximum levels with an involvement of 50–75% of examined lung areas (Fig. 2). Allogeneic Transplantation of Fetal Membrane-Derived Cells Bleomycin-treated C57BL/6 mice were transplanted via the intrajugular route with cells isolated from the allogeneic fetal membranes (amnion and chorion) of BALB/c placentas obtained at gestation day 16–18. At day 9, mice that had been treated with both bleomycin and allogeneic cells showed a reduction, although not significant, in the severity of lung inflammation (4.14 ± 0.55 score units) with respect to bleomycintreated mice (5.35 ± 0.39 score units). The extent of inflammation between these two groups did not differ significantly and, furthermore, no differences were observed in severity and extent of fibrosis (data not shown). At day 14, bleomycin-treated mice that received cell trans-

plants again showed a nonsignificant difference in lung inflammation compared to mice treated with bleomycin alone (4.50 ± 1.17 vs 5.76 ± 0.47 score units, respectively) (Fig. 3A). Interestingly, however, significant reductions in both severity (2.08 ± 0.71 vs. 4.11 ± 0.39 score units; p < 0.05) and extent (18.75 ± 7.00% vs. 46.05 ± 5.75% lung area involved; p < 0.05) of lung fibrosis were observed in samples from bleomycin-treated mice that received cell transplants (Fig. 3B). To study the effect of another allogeneic cell source and different transplantation routes, we transplanted bleomycin-treated C57BL/6 mice with allogeneic BCF1, GFP+ murine fetal membrane-derived cells. Cell delivery in this case was performed by intraperitoneal and intratracheal injections. Irrespective of the delivery route, allogeneic BCF1 cell transplantation did not change lung inflammation and fibrosis at days 3 and 7 after bleomycin instillation (data not shown). At days 9 and 14, mice treated with bleomycin and allogeneic cells (administered either intraperitoneally or intratracheally) showed similar levels of inflammation to those observed in bleomycin-instilled mice that did not receive cell transplantation (Fig. 4A and B). However, similarly to what was observed in animals transplanted with BALB/c fetal membrane-derived cells, transplantation of BCF1 placenta cells caused a reduction in bleomycin-induced lung fibrosis at day 14, both after intraperitoneal or intratracheal cell delivery (from 4.11 ± 0.39 to 2.69 ± 0.34 and to 1.50 ± 0.49 score units, respectively; p < 0.05 and p < 0.01) (Fig. 4D). After intratracheal cell delivery, a significant decrease in the extent of fibrosis was also observed (from 46.05 ± 5.75% to 17.75 ± 5.25% lung area involved; p < 0.05) (Fig. 4D).

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Figure 1. Bleomycin-induced lung injury in C57BL/6 mice. (A) Representative images showing progression of inflammation in lungs of mice at different time points (a1–a3) after bleomycin treatment. Images demonstrating different levels (score 1–3) of cell infiltration are shown for macrophages (M1–M3), neutrophils (N1–N3), and lymphocytes (L1–L3). Histological sections of lungs were stained with hematoxylin/eosin. (B) Representative images showing progression of fibrosis in lungs of mice treated with bleomycin at different time points (b1–b3) after exposure. Images demonstrating different levels (score 1–3) of fibroblast infiltration (F1–F3) and of collagen deposition (C1–C3) are shown. Histological sections of lungs were stained with Masson’s trichrome to show collagen deposition (green staining).


Figure 1. Continued

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Figure 2. Bleomycin-induced lung inflammation and fibrosis were evaluated at different time points after bleomycin treatment and expressed as score units (see Materials and Methods for the detailed description of the scoring system). The severity of inflammation and fibrosis are reported in black bars on the left y-axis; the extents of these processes are represented by the gray bars on the right y-axis. Numbers of mice included in each group are indicated (n).

Allogeneic transplantation into bleomycin-untreated mice caused an inflammatory response that was still present 9 days after both intraperitoneal and intratracheal cell delivery (Fig. 4A). This inflammation was no longer observed at day 14 after intraperitoneal allotransplantation, while it persisted, albeit with lower severity and extent, in intratracheally cell transplanted animals (Fig. 4B). No signs of lung fibrosis were observed in any animals treated with cells alone (Fig. 4C and D). Transplantation of Xenogeneic Fetal Membrane Cells Derived From Human Term Placenta The same transplantation settings used for allogeneic BCF1 cell transplantation were also applied using xeno-

geneic fetal membrane cells derived from human term placenta. Three and 7 days after intraperitoneal or intratracheal transplantation with human fetal membrane-derived cells, lungs from bleomycin-treated mice showed inflammation and fibrosis similar to mice that had received no cell treatment (data not shown). At days 9 and 14 we continued to observe nonsignificant changes in the severity of bleomycin-induced lung inflammation in cell transplanted mice compared to nontransplanted mice (Fig. 5B). Interestingly, fetal membrane-derived cell xenotransplantation did not result in significant changes in either severity or extent of bleomycin-induced fibrosis at day 9 (Fig. 5C); however, by


day 14, severity of bleomycin-induced fibrosis was significantly reduced in xenotransplanted mice (from 4.11 ± 0.39 in nontransplanted mice to 2.38 ± 0.40 and 2.50 ± 0.31 score units after intraperitoneal and intratracheal cell injections, respectively; p < 0.01 and p < 0.05) (Fig. 5D). Representative examples of the reduction in collagen deposition, which was observed at day 14 after intraperitoneal xenogeneic placenta cell transplantation, are shown in Figure 6A. Xenogeneic cell transplantation did not cause any

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significant changes in the extent of bleomycin-induced fibrosis regardless of the days after treatment or the route of transplantation (Figs. 5C and D). As also observed for allotransplantation, xenotransplantation of cells into bleomycin-untreated mice caused an inflammatory response that was still present at day 14 after both intraperitoneal and intratracheal cell delivery (Figs. 5A and B). As seen after allogeneic transplantation, xenogeneic transplantation alone did not induce lung fibrosis (Figs. 5C and D).

Figure 3. Effect of transplantation of allogeneic BALB/c fetal membrane-derived cells on bleomycin-induced lung inflammation and fibrosis. Transplantation was performed via the intrajugular route 15 min after bleomycin instillation in C57BL/6 mice lungs. Inflammation and fibrosis scores were assigned as described in Materials and Methods. The left y-axis shows values for injury severity; the right y-axes show values for injury extent. All data shown were obtained 14 days after treatment. Treatment groups are indicated as follows: “Bleo”: bleomycin-treated mice, “Bleo+cells”: bleomycin-treated mice transplanted with allogeneic cells. Numbers of mice included in each group are indicated (n). *p < 0.05 versus bleomycin treatment group.

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Figure 4. Effect of transplantation of allogeneic BCF1 fetal membrane-derived cells on bleomycin-induced lung inflammation and fibrosis. Transplantation was performed via intraperitoneal (IP) or intratracheal (IT) injection 15 min after bleomycin instillation in C57BL/6 mouse lungs. Inflammation and fibrosis scores were assigned as described in Materials and Methods. Injury severity values are shown on the left y-axis, while injury extent values are shown on the right y-axis. Data shown are from mice sacrificed at days 9 and 14 after treatment(s). Treatment groups are indicated as: “Bleo”: bleomycin-treated mice, “Bleo+cells”: bleomycintreated mice transplanted with allogeneic cells, “Cells”: mice receiving only allogeneic cell transplantation. Numbers of mice included in each group are indicated (n). *p < 0.05; **p < 0.01 vs. bleomycin-treatment group.


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Figure 5. Effect of transplantation of xenogeneic human placenta-derived cells on bleomycin-induced lung inflammation and fibrosis. Transplantation was performed through intraperitoneal (IP) and intratracheal (IT) injections 15 min after bleomycin instillation in C57BL/6 mouse lungs. Inflammation and fibrosis scores were assigned as described in Materials and Methods. Inflammation and fibrosis severity values are shown on the left y-axis, while extent values for these parameters are shown on the right y-axis. The data shown were from mice sacrificed 9 and 14 days after treatment. Treatment groups are indicated as: “Bleo”: bleomycin-treated mice, “Bleo+cells”: bleomycin-treated mice transplanted with xenogeneic cells, “Cells”: mice receiving only xenogeneic cell transplantation. Numbers of mice included in each group are indicated (n). *p < 0.05; **p < 0.01 versus bleomycin treatment group.

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Figure 6. Reductions in collagen deposition and neutrophil infiltration after xenogeneic fetal membrane-derived cell transplantation. Representative images of lungs from bleomycin-treated mice 14 days after intraperitoneal injection (xeno-IP) of xenogeneic fetal membrane cells. Control samples of bleomycin-treated not transplanted mice are also shown. Histologic sections were stained with Masson’s trichrome to show collagen deposition (A) and with hematoxylin/eosin to allow neutrophil detection (B).

Effect of Fetal Membrane-Derived Cell Transplantation on the Presence of Specific Inflammatory Cell Types The inflammation score gives a comprehensive indication as to the severity of inflammation. In attempting to understand whether the different treatments could affect the relative involvement of each cell type (i.e., macrophages, neutrophils, and lymphocytes) in the inflammatory process, we evaluated the level of infiltration of each of these cell types in all lung sections examined for each treatment group and time point. By microscopy analysis, we counted the lung sections that showed no infiltration (level 0), low infiltration (level 1), moderate infiltration (level 2), or marked infiltration (level 3) for each cell type, and expressed the numbers of sections showing these varying levels of infiltration as a percentage of the total number of sections examined for each treatment group and time point. Table 2 summarizes the

percentage of analyzed sections that showed these different degrees of infiltration for each cell type, with the total number of sections examined reported. Saline-treated mice showed a very slight inflammatory response, with low levels of macrophages and neutrophils observed in 13.6% and 18.2% of examined sections, respectively, and absence of lymphocytes. In the lungs of bleomycin-treated mice, macrophages and lymphocytes could be detected as early as 3 days after bleomycin instillation. At this time point, macrophages were present at low or moderate levels in all examined sections, while lymphocytes were observed in only 16.7% of the sections examined, and only at low levels. Neutrophil infiltration began later, and was detected at day 7 after bleomycin instillation. Infiltration of macrophages and lymphocytes increased progressively and massively, as demonstrated by an increase in the number of lung sections from bleomycin-treated mice, which showed level 3 infiltration over the course


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Table 2. Infiltration of Lung Parenchyma by Individual Cell Inflammatory Types After Different Treatments

Treatment

Cell Delivery Route

Days After Treat- Mice ment (n)

Macrophages Infiltration Degree 0 (%)

Saline

—

14

11

Bleomycin

—

3 7 9 14

3 7 24 19

0 0 0 3.3

Allogeneic cells

IP

3 7 9 14

3 3 3 3

100.0 100.0 100.0 100.0

Allogeneic cells

IT

3 7 9 14

3 4 2 3

Xenogeneic cells

IP

3 7 9 14

4 0 0 4

Xenogeneic cells

IT

3 7 9 14

2 4 0 3

75.0 37.5 n.d. 66.7

Bleomycin + allogeneic cells

IP

3 7 9 14

6 5 10 8

Bleomycin + allogeneic cells

IT

3 7 9 14

Bleomycin + xenogeneic cells

IP

Bleomycin + xenogeneic cells

IT

Neutrophils Infiltration Degree

No. 1 2 3 Sec(%) (%) (%) tions

86.4 13.6 83.3 31.0 33.3 20.0 0 0 0 0

66.7 33.3 83.4 8.3 50.0 50.0 94.4 5.6

0

0

16.7 0 62.1 6.9 43.8 22.9 66.7 10.0

0 (%)

1 (%)

22

81.8

18.2

6 29 48 30

100.0 62.1 47.9 60.0

No. 2 3 Sec(%) (%) tions

0 (%)

1 (%)

22

100.0

0

0 0 0 17.2 10.3 10.4 27.1 14.6 10.4 10.0 30.0 0

6 29 48 30

83.3 17.2 6.3 10.1

0 0 0 0

0 0 0 0

0 0 0 0

18 18 18 18

100.0 100.0 100.0 100.0

0 8.3 0 0

0 0 0 0

6 24 4 18

0 100.0 100.0 0 25.0 75.0 100.0 0

58.3 41.7 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 100.0 0 0 0

Lymphocytes Infiltration Degree

0

0

No. 2 3 Sec(%) (%) tions 0

0

22

16.7 0 0 34.5 48.3 0 50.0 37.5 6.2 53.3 23.3 13.3

6 29 48 30

0 0 0 0

18 18 18 18

0 0 0 0

6 24 4 18

0 0 0 0

0 0 0 0

18 18 18 18

77.8 100.0 83.3 88.9

22.2 0 16.7 11.1

0 0 0 0

0 0 0 0

0 0 0 0

6 24 4 18

100.0 54.1 75.0 61.1

0 0 29.2 16.7 0 0 38.9 0

24 n.d. n.d. 8

75.0 25.0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 87.5 12.5 0 0

24 33.3 50.0 16.7 0 24 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 8 100.0 0 0 0 8

25.0 0 0 62.5 0 0 n.d. n.d. n.d. 33.3 0 0

4 8 n.d. 18

0 100.0 0 0 37.5 62.5 0 0 n.d. n.d. n.d. n.d. 72.2 27.8 0 0

4 100.0 0 8 100.0 0 n.d. n.d. n.d. 18 100.0 0

0 0 4 0 0 8 n.d. n.d. n.d. 0 0 18

50.0 50.0 35.0 6.3

50.0 0 0 27.3 9.1 13.6 10.0 35.0 20.0 18.8 31.2 43.7

12 22 20 16

66.7 72.7 65.0 93.8

33.3 0 0 0 9.1 18.2 15.0 10.0 10.0 0 6.2 0

12 22 20 16

0 22.7 20.0 0

58.3 59.1 70.0 75.0

41.7 18.2 10.0 25.0

3 4 11 7

33.3 0 9.1 36.7

66.7 0 0 12.5 87.5 0 45.5 40.9 4.5 13.3 30.0 20.0

6 8 22 30

50.0 62.5 63.7 100.0

50.0 12.5 13.6 0

0 0 0 25.0 9.1 13.6 0 0

6 8 22 30

16.7 0 0 20.0

83.3 0 0 0 62.5 37.5 70.0 20.0 10.0 50.0 23.3 6.7

3 7 9 14

4 0 8 8

25.0 n.d. 6.3 6.3

75.0 0 0 8 100.0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 25.0 62.5 6.2 16 75.0 18.8 6.2 0 18.7 53.1 21.9 32 96.9 3.1 0 0

8 n.d. 16 32

75.0 25.0 0 0 8 n.d. n.d. n.d. n.d. n.d. 0 100.0 0 0 16 0 53.1 46.9 0 32

3 7 9 14

2 3 6 7

75.0 0 16.6 6.7

25.0 0 0 16.7 66.6 16.7 50.0 16.7 16.7 26.7 33.3 33.3

4 6 12 30

4 6 12 30

0 100.0 0 0 50.0 50.0 75.0 25.0 0 63.3 10.0 20.0

0 0 0 6.7

100.0 0 8.3 3.3

0 0 66.7 33.3 75.0 16.7 40.0 50.0

0 0 0 0

0 0 0 6.7

12 22 20 16 6 8 22 30

4 6 12 30

Infiltration degree: 0 = no infiltration, 1 = low infiltration, 2 = moderate infiltration, 3 = massive infiltration; No. sections: total number of examined sections; IP, intraperitoneal; IT, intratracheal; n.d., not determined.

418

CARGNONI ET AL.

Figure 7. Detection of allogeneic and xenogeneic fetal membrane-derived cells in host lungs. (A) Representative PCR analysis of DNA from lung samples from bleomycin-treated mice transplanted with allogeneic (Allo) fetal membrane-derived cells via intratracheal (IT) and intraperitoneal (IP) routes, or with xenogeneic (Xeno) human fetal membrane-derived cells via intratracheal (IT) and intraperitoneal (IP) routes, are shown. Lung samples were collected at different time points after transplantation, as indicated. For PCR H2O was used as negative control. (B) Representative images of lungs from bleomycin-treated mice transplanted with either allogeneic fetal membranederived cells (b1–b4) or with xenogeneic (human) fetal membrane-derived cells (b5–b8) are shown. Lung samples collected at days 3 and 7 after intratracheal allogeneic transplantation (b1 and b2), and at days 3 and 14 after intraperitoneal allogeneic transplantation (b3 and b4) show immunohistochemical positivity for GFP, as indicated by arrows. Xenogeneic positivity revealed by anti-human CK19 is shown at days 3 and 14 after intratracheal (b5–b6) and intraperitoneal (b7–b8) transplantation.

of this study. On the contrary, at day 14 neutrophils were absent in 60% of the sections analyzed, or were present at low or moderate levels (Table 2). Allogeneic intratracheal cell transplantation and xenogeneic intraperitoneal and intratracheal cell transplantation all resulted in mild lung infiltration (level 1) by all three cell types, with sporadic presence of macrophages and lymphocytes at moderate levels also observed at days 3 and 7. After intraperitoneal and intratracheal cell transplantation of either allogeneic or xenogeneic cells, we detected lower proportions of infiltrating macrophages in lung sections from bleomycin-instilled mice compared to mice treated with bleomycin only, and this difference was particularly evident at early time points. Lymphocyte infiltration did not show notable differences between bleomycin-instilled, cell-treated mice compared to mice treated only with bleomycin. Interestingly, at day 14, with the exception of sections from intratracheally xenotransplanted mice, all other conditions of cell source (xeno- or allogeneic) and delivery route (intraperitoneal and intratracheal) showed an evident reduction in neutrophil infiltration when compared to bleomycin-treated mice. Indeed, neutrophil infiltration was detected in 40% of the examined sections from bleomycin-treated mice at day 14, with 10% showing level 1 infiltration and 30% showing level 2 infiltration,

while neutrophils were absent in bleomycin-treated mice that had been intratracheally transplanted with allogeneic cells. Only a low proportion of sections were found to be positive for neutrophils in mice that had been intraperitoneally transplanted with allogeneic (6.2%, at level 2) and xenogeneic cells (3.1%, at level 1). Representative photomicrographs demonstrating reduced lung infiltration by neutrophils at day 14 after intraperitoneal xenogeneic placenta cell transplantation are shown in Figure 6B. Assessment of Allogeneic and Xenogeneic Fetal Membrane-Derived Cells in Host Lungs As reported in Figure 7A, PCR analysis was performed to investigate the presence of allogeneic (BCF1 GFP+) and xenogeneic (human) DNA in the lungs of transplanted mice. GFP or human DNA was detectable until day 14 in the lungs of mice that had received intratracheal or intraperitoneal cell transplants (Fig. 7A). The stronger intensity of the human signal versus the GFP signal is very likely due to the fact that the system used to detect human DNA is based on PCR amplification of a mitochondrial DNA sequence, which is present at higher copy number with respect to the genomic GFP DNA sequence (37). Representative examples of immunohistochemical analyses that confirm the presence of allogeneic GFP-


Figure 7. Continued

419

420

positive cells at 3 and 7 days after intratracheal injection, and at 3 and 14 days after intraperitoneal injection are shown in Figure 7B. The presence of human cells in mouse lungs, as shown by positive staining for antihuman CK 19, was observed at days 3 and 14 after xenogeneic intratracheal and intraperitoneal transplantation (Fig. 7B). DISCUSSION In this study we have demonstrated that both allogeneic and xenogeneic transplantation of fetal membranederived cells can reduce bleomycin-induced lung fibrosis in C57BL/6 mice. Even though cell therapy-based treatments for lung fibrosis have shown conflicting results to date regarding the ability of bone marrow-derived cells to differentiate into pneumocytes (9), decreased lung fibrosis has been observed after transplantation of bone marrow-derived mesenchymal stromal cells (29,30,33,45) and alveolar type II epithelial cells (36). We have previously demonstrated that hAEC, hAMSC, and hCMSC obtained from fetal membranes of human term placenta contain a variety of stem cells that display high plasticity and low immunogenicity in vitro (25,32). We have also shown that, after intraperitoneal transplantation, these cells can engraft into different organs including bone marrow, thymus, brain, and lung (1). In addition, amniotic cells are capable of inhibiting T-cell allogeneic proliferation responses in vitro both in a direct contact or transwell setting, thereby suggesting that these cells produce soluble factor(s) that could act as immunomodulatory agent(s) (25). These data are in line with the hypothesis that cells derived from the amniotic membrane have immunomodulatory properties, which has long been supported by the fact that amniotic membrane has been used as an anti-inflammatory agent in ophthalmology applications (8) and also explains the effect of these cells on tissue regeneration and reduction of scar formation in animal models of spinal cord (34) and myocardial injury (42). Based on the positive results observed from the application of bone marrow-derived mesenchymal stromal cells in lung fibrosis (30), as well as the immunomodulatory/anti-inflammatory characteristics that have been reported for placenta-derived cells (32), we set out to test the effects of transplanting amniochorionic membrane cells into a mouse model of bleomycin-induced lung fibrosis. We designed both xenogeneic and allogeneic transplantation models in order to avoid potential limitations in cell interactions, which may have been caused by interspecies differences. In previous studies, either local (intratracheal) (13,36) or systemic (intrajugular) (26,29,30) routes of cell delivery have been used. Given that the large size of human placenta-derived cells frequently resulted in occlusion of

CARGNONI ET AL.

the jugular vein upon injection, we used intraperitoneal transplantation as an alternative systemic delivery route for these cells, also based on our previous results showing that amniochorionic cells are able to migrate to the lung, as well as other organs, after intraperitoneal xenotransplantation (1). Our results show that, regardless of the route of inoculation and the cell source (allogeneic or xenogeneic), transplantation of fetal membrane-derived cells resulted in a significant antifibrotic effect in bleomycin-treated animals. Interestingly, preliminary results from additional studies that we conducted by applying intrapulmonary cell injection also yielded similar results (data not shown). Based on previous reports showing that maximum lung fibrosis and eventual resolution is observed by day 14 in bleomycin lung-injured mice, a 14-day transplantation model was also used in this study. Over this time, we observed persistence of transplanted cells by PCR amplification of specific allogeneic or xenogeneic donor DNA sequences, as well as by immunohistochemistry. In a previous study, we have demonstrated engraftment of human placenta-derived cells in various organs, including lung, at day 90 after xenogeneic intraperitoneal transplantation (1). However, microchimerism was detectable only by PCR in those studies, suggesting that out of the cells present at day 14 after injection, only a relatively small percentage will survive and engraft long term. Therefore, although questions remain as to whether the beneficial effects on lung fibrosis observed in this study are due to cell engraftment or to transient paracrine actions of the injected cells, the fact we have been able to consistently observe a reduction in lung fibrosis by administering both allogeneic or xenogeneic cells via different routes provides convincing proof of the principle that placenta-derived cells do indeed confer beneficial effects in bleomycin-induced lung injury. Even though the severity of inflammation did not show an overall reduction over the time course of this study with the different types of cells used, a marked reduction in neutrophil infiltration was observed both after xeno- and allotransplantation. The mechanism by which placenta-derived cells might affect infiltration by neutrophils remains to be elucidated. However, our observations are in accordance with the finding that transplantation of bone marrow MSC into the injured lung of mice results in a dramatic decrease in neutrophil levels in the bronchoalveolar lavage (BAL) samples (29). A reduction in neutrophil levels after syngeneic MSC transplantation has also been observed in the BAL of mice in which lung injury was induced by lipopolysaccharide (26). Therefore, the reduction in the levels of neutrophils that we observed may be partly responsible for the con-


comitant reduction in fibrotic damage. It is also worth noting that the presence of neutrophils is associated with a poor prognosis in IPF (18), while neutrophil elastase has been associated with the pathology of several lung diseases, whereas mice lacking neutrophil elastase are resistant to bleomycin-induced pulmonary fibrosis (6). Because the reduction in bleomycin-induced lung fibrosis caused by MSC has been suggested to result from interference with important inflammatory cytokines such as IL-1α and TNF-α (29), it is tempting to speculate that these cells may produce soluble factors that act through paracrine mechanisms to induce anti-inflammatory effects. Support for the hypothesis that amniotic cells may also act in a similar manner comes from the observation that amniotic membrane exerts anti-inflammatory effects when used in different clinical applications (8), even though the precise mechanism of action of amniotic cells in these settings remains to be defined. To this regard, it is worth noting that yet unidentified soluble factors released by amniotic cells have been shown to be responsible for the inhibition of allogeneic T-cell proliferation (25). Together with the evidences presented above, the results of this study lend support to the possibility that soluble factors produced by placenta-derived cells may also act on neutrophil recruitment, although further investigation would be required in order to fully validate this hypothesis. In conclusion, we have demonstrated that transplantation of fetal membrane-derived cells from allogeneic and human xenogeneic placenta can reduce the fibrotic process in bleomycin-injured lungs. Even though open questions remain regarding the mechanisms by which these cells reduce the inflammatory and fibrotic processes, this is, to our knowledge, the first study describing the antifibrotic effect of placenta-derived cell types in a lung injury animal model. Our results open the way for further investigations aimed at extending the potential use of the placenta to include cell therapy for both acute and chronic lung fibrotic diseases. ACKNOWLEDGMENTS: The authors thank the physicians and midwives of the Department of Obstetrics and Gynaecology of Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy. The authors are indebted to Dr. Fabio Candotti for critically reviewing the manuscript and to Dr. Marco Evangelista for help in editing the manuscript. This study was supported by a grant from Fondazione Cariplo (Bando 2004) and from MIUR (Bando FISS 2006). A European patent application has been filed with the application No. PCT2008-004845.

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