Human Amnion Epithelial Cell Transplantation ... - ATS Journals

Human Amnion Epithelial Cell Transplantation Abrogates Lung Fibrosis and Augments Repair Yuben Moodley1, Sivagami Ilancheran2, Chrishan Samuel3, Vijesh Vaghjiani4, Daniel Atienza1, Elizabeth D. Williams5, Graham Jenkin1, Euan Wallace2, Alan Trounson1*, and Ursula Manuelpillai2† 1 Monash Immunology and Stem Cell Laboratories, School of Biomedical Sciences, 2Centre for Women’s Health Research, Monash Institute of Medical Research (MIMR), 3Howard Florey Research Institute, 4Centre for Reproduction and Development, MIMR, and 5Centre for Cancer Research, MIMR, Monash University, Melbourne, Australia

Rationale: Chronic lung disease characterized by loss of lung tissue, inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. Objectives: To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell–like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Methods: Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. Measurements and Main Results: hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A–D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks post-transplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-a, IL-1 and -6, and profibrotic transforming growth factor-b in mouse lungs. In addition, lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors of matrix metalloproteinase-1 and 2. Conclusions: hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis. Keywords: amnion epithelial cells; fetal membranes; lung fibrosis; bleomycin

(Received in original form January 5, 2010; accepted in final form June 2, 2010) * Present address: California Institute for Regenerative Medicine, San Francisco, California †

Present address: Centre for Reproduction and Development, MIMR, Monash University, Melbourne, Australia Supported by NH&MRC project grant 491145 and a Monash University Collaborative Grant; NH&MRC Biomedical Career Development Award 519539 (E.D.W.); NHFA/NH&MRC RD Wright Fellowship (C.S.); and NH&MRC grant 436836 (U.M.). Correspondence and requests for reprints should be addressed to Yuben Moodley, M.D., Ph.D., F.R.A.C.P. School of Medicine and Pharmacology, University of Western Australia, Level 4, Medical Research Foundation Building, 50 Murray Street, Perth, Western Australia 6000, Australia. E-mail: yuben.moodley@uwa. edu.au This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 182. pp 643–651, 2010 Originally Published in Press as DOI: 10.1164/rccm.201001-0014OC on June 3, 2010 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Cellular therapies may become future therapeutic option for lung disease. Both murine and human stem cells have been shown to express some markers of lung phenotype, whereas predominantly murine stem cells reduce fibrosis in animal models of lung injury. What This Study Adds to the Field

We use human stem-like cells isolated from a plentiful tissue source to demonstrate that human amnion epithelial cells differentiate into lung epithelium in addition to reducing inflammation and abrogating fibrosis post–lung injury. Furthermore, this study demonstrates functionality of the differentiated stem cells and makes use of novel antibodies to isolate human cells from a mouse lung suspension.

Lung fibrosis is the central pathologic end point of many diseases characterized by progressive inflammation, cell death, and disordered repair. Such conditions as acute respiratory distress syndrome, with a mortality rate that can reach up to 40% and idiopathic pulmonary fibrosis represent a major burden to global health (1). Although immunosuppressive agents are frequently used in the therapeutic armamentarium, there are no effective pharmacologic agents that impact on the morbidity and mortality associated with acute respiratory distress syndrome and idiopathic pulmonary fibrosis. Studies have shown that allogeneic murine stem cell transplantation reduced inflammation and fibrosis after lung injury (2–4). However, investigations examining human stem cells in treating lung fibrosis are limited. Human bone marrow–derived stem cells, umbilical cord blood mesenchymal stem cells (uMSCs), and amniotic fluid stem cells (AFSCs) expressed some markers of alveolar epithelium after injection into mice, albeit at very low rates of engraftment in the lungs. However, there are no data on their ability effectively to treat disease models (5–7). Human placental stem cells (PSCs) and MSCs obtained from Wharton jelly of the umbilical cord have been shown to reduce inflammation and fibrosis in bleomycininjured mice (8, 9). The bleomycin-induced model of lung injury is a well-characterized model of initial inflammation and subsequent fibrosis. This model has an overlap with acute respiratory distress syndrome and idiopathic pulmonary fibrosis (10). Notably, however, PSCs and Wharton jelly MSCs did not differentiate into lung epithelium in bleomycin-treated mice. Recent studies have shown that human amnion epithelial cells (hAECs), an abundant cell type, isolated from amnion membranes of termdelivered placenta display features of embryonic and multipotent stem cells (11, 12). The amnion and chorion form part of the

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embryonically derived inner fetal membranes of the placenta with the maternally derived decidua comprising the outer layer. The amniotic epithelium arises from pluripotent embryonic epiblast cells about 8 days after fertilization in the human, whereas the chorion forms from the placenta several weeks later in gestation. Because the epiblast is also the origin of the three embryonic germ layers, it has been suggested that the amniotic epithelium could harbor stem cells even at term pregnancy (13). Although we and others have differentiated hAECs from term pregnancy into lineages derived from all three germ layers (11, 12), it is unknown whether they fulfill all the characteristics of stem cells. For example, clonal expansion is limited and stem cell properties of clonally expanded hAECs have not been determined. Furthermore, hAECs have morphologic features of differentiated epithelial cells unlike quiescent cells. hAECs are also known to have low immunogenicity and have been successfully transplanted into allogeneic recipients (14). Furthermore, hAECs and amnion membranes are widely used in ophthalmology and dermal applications for their antiinflammatory properties (14). Although these features suggest that hAECs may be attractive for potential cellular therapies, little is known about tissue-specific differentiation in vivo and if their antiinflammatory effects can be exploited after transplantation. We studied the capacity of hAECs to differentiate into alveolar epithelium and reduce inflammation and fibrosis in bleomycin-injured mice.

MATERIALS AND METHODS Human and Animal Research Ethics Committees of Monash Medical Centre and Monash University approved the study.

hAEC Isolation and Culture Amnion was obtained from normal term pregnancies delivered by caesarean section. hAECs were isolated, as described previously, and analyzed by flow cytometry for cytokeratins 7, 8/18 (12); batches greater than 99% purity as determined by the cytokeratins were used (12). For characterization, CD73, CD105, CD117 (c-kit), CD90, CD166, CD31, CD34, CD45, HLA-A-B-C, HLA-DP-DQ-DR, aquaporin-5, and caveolin were analyzed by fluorescence-activated cell sorter. Oct-4, Sox-2, SSEA-4, and GCTM2 were assessed by immunohistochemistry (12). Nkx2.1, occludin, and mucin mRNA expression was analyzed by reverse transcriptase–polymerase chain reaction (qRT-PCR) (n 5 6). To induce differentiation, primary hAECs were cultured in small airway growth medium (SAGM; Clonetics, San Diego, CA) or controls maintained in Dulbecco’s modified Eagle medium (DMEM)/F12 (Gibco, Grand Island, NY) plus 10% FCS (n 5 6) for 4 weeks and assessed for lamellar body formation (electron microscopy) and surfactant protein (SP) production (immunohistochemistry and ELISA).

hAEC Injection into Mice Bleomycin-sulfate (0.15 mg in 20 ml saline; Sigma-Aldrich, St. Louis, MO) was administered intranasally under weak anesthesia (isoflurane, Baxter Healthcare, Toongabbie, New South Wales, Australia) to induce pulmonary fibrosis in 8-week-old SCID mice. To determine the optimal cell dose, 0.5, 1, and 2 3 106 hAECs were injected into the tail vein 24 hours after bleomycin and culled 2 weeks later (n 5 4 mice per dose). Lungs analyzed for human DNA by PCR showed mice given 1 3 106 hAECs had the highest human DNA content; hence, this dose was subsequently used (15). hAECs were localized in murine lungs by immunohistochemistry for the human specific inner mitochondrial membrane protein (IMM; 1:100, Chemicon, Boronia, Victoria, Australia). Differentiation of hAECs in murine lungs was assessed by injecting hAECs obtained from pregnancies of male babies into female mice given bleomycin and culled 2 weeks later (n 5 8 mice). Lungs were digested in type I collagenase (3 mg/ml; Invitrogen) for 45 minutes and erythrocytes lysed. hAECs were recovered from lung cell suspensions by fluorescence-activated cell sorter using antihuman CD29 antibody. Cytospins were prepared from flow– sorted cells and analyzed for XY/

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XX chromosomes by fluorescence in situ hybridization (FISH) and SPs A–D by immunohistochemistry. To assess hAEC effects on inflammation and fibrosis, the control groups were as follows: healthy (intranasal saline, 20 ml); saline (tail vein, 100 ml); healthy mice 1 hAECs; bleomycin alone; bleomycin 1 fibroblasts; and bleomycin 1 cytotrophoblasts. Experimental groups comprised bleomycin 1 hAECs (24 hours postbleomycin) and bleomycin 1 hAECs (2 weeks postbleomycin) (n 5 8 mice per group). Animals were killed 2 and 4 weeks later. Inflammation and fibrosis was determined by the Ashcroft score on lung histology (16, 17). Cytokine mRNA expression in lungs was measured by RT-qPCR and protein by a cytometric bead array (BD Biosciences, San Jose, CA) or ELISAs (R&D Systems, Minneapolis, MN). Lung collagen content was measured by the hydroxyproline assay, matrix metalloproteinase (MMPs) by gelatin zymography, and tissue inhibitor of metalloproteinases (TIMPs) by RT-qPCR and reverse zymography (18, 19). METHODS are described in greater detail in the online supplement.

Statistics Data are expressed as the mean 6 SEM and analyzed by one-way analysis of variance and the Bonferroni post hoc test. P , 0.05 was considered to be significant. GraphPad Prism software (v5.03; La Jolla, CA) was used for data analysis.

RESULTS hAECs Produce Surfactant Proteins In Vitro

hAECs isolated from term amnion expressed some epithelial (cytokeratin-7, 8/18), pluripotent (Oct-4, Sox-2, SSEA-4, GCTM2), and MSC markers (CD73, CD117, CD166, CD90). HLA-A, -B, and -C was low, whereas HLA-DP, HLA-DQ, HLADR, endothelial (CD31, CD34), and hematopoietic (CD45) lineage markers were absent. The primary hAECs also expressed lung-associated markers Nkx2.1, mucin, occludin, aquaporin-5, and caveolin-1 (Table 1). Noting the expression of Nkx 2.1, an early lung lineage specification marker in primary hAECs, we cultured the cells in small airway growth medium (SAGM), a medium that induces differentiation of embryonic stem cells into type II pneumocytes (20). hAECs grown in SAGM produced SP-A (62 6 6% of cells); SP-B (73 6 8%); pro–SP-C (78 6 8%); and SP-D (48 6 6%). Cultures maintained in DMEM/F12 did not TABLE 1. CHARACTERIZATION OF PRIMARY HUMAN AMNION EPITHELIAL CELLS Marker Cytokeratin 7* Cytokeratin 8/18* Oct-4† Sox-2† SSEA-4† GCTM2† c-kit (CD117) * CD73* CD166* CD90* CD105* HLA-A, -B, -C* HLA-DP, -DQ, -DR* CD45* CD31* CD34* Nkx 2.1‡ Mucin‡ Occludin‡ Caveolin-1* Values are expressed as means 6 SEM. * Flow analyses. † Immunocytochemistry. ‡ Real-time polymerase chain reaction.

Percentage (n 5 4–7) 98.7 6 0.7 98.4 6 1.1 4.8 6 1.2 6.4 6 1.8 98.6 6 0.6 97.4 6 1.3 6.7 6 1.3 95.3 6 1.2 91.9 6 4.2 22.5 6 3.8 Not detected 2.73 6 0.3 Not detected Not detected Not detected Not detected Present Present Present 91.1 6 2.1

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Figure 1. Human amnion epithelial cells (hAECs) acquire an alveolar epithelial cell phenotype. (A) Primary hAECs cultured in small airway growth medium (SAGM) for 4 weeks produce prosurfactant protein (SP)-C compared with controls in DMEM/ F12. Similar results (not shown) were obtained for SP-A, SP-B, and SPD. hAECs grown in SAGM secreted SP-D when stimulated by dexamethasone treatment. (B) Transmission electron microscopy of hAECs grown in SAGM showed lamellar body– like organelles (arrows); these were absent in controls. (C) hAECs lining term amnion and isolated hAECs show strong immunostaining for human inner mitochondrial membrane (IMM) protein. (D) Lung tissue sections from bleomycin-injured mice and isotype controls (not shown) did not stain for IMM. Lung tissue sections from mice given bleomycin 1 hAECs, 4 weeks after cell injection, showed IMM1 cells in fibrotic and alveolar areas (brown staining, original magnification 350, 3100, and 3200). Scale bars 5 50 mm.

produce SPs (Figure 1A). We also examined if hAECs cultured in SAGM secreted SP-D, which would suggest functionality. After 4 weeks in SAGM, hAECs were stimulated with dexamethasone and SP-D measured in the supernatant by ELISA. SP-D was secreted by hAECs grown in SAGM alone, whereas dexamethasone stimulation increased SP-D by a further 30% (Figure 1A). Cells grown in DMEM/F12 did not secrete SP-D. Furthermore, ultrastructural examination revealed that hAECs grown in SAGM for 2–4 weeks had a distinct cell population with cytoplasmic organelles containing surfactant-associated lamellar bodies typical of type II pneumocytes. These features were absent in hAECs cultured in DMEM/F12 (Figure 1B). hAECs Localize to Injured Lung

hAECs injected into the tail vein 24 hours after bleomycininduced lung injury was shown to locate to the lung by different assays. Lung tissue retrieved 2 weeks post-hAEC injection and analyzed for human Alu repeat sequences by DNA-PCR (15) confirmed the presence of hAECs within the lung. Furthermore, hAECs were identified in the lung by immunostaining for human IMM. The specificity of the IMM antibody was demonstrated by immunostaining of human amniotic epithelium and hAECs but not mouse lung tissue (Figure 1C). IMM-positive hAECs were identified in fibrotic regions of injured lungs 4 weeks post–cell injection (10 6 0.7 hAECs per 100 lung cells) (Figure 1D). IMM-positive cells were also present in alveolar regions (6 6 0.4 hAECs per 100 lung cells) (Figure 1D). hAECs were absent in lung tissue from healthy mice injected with cells. There was no evidence of tumor formation in lung tissue examined at 4 weeks post-hAEC injection. hAECs Demonstrate a Type II Pneumocyte Phenotype In Vivo

To determine whether hAECs adopted characteristics of type II pneumocytes in mice, antihuman CD29 antibody was used to

recover human cells from mouse lung cell suspensions by flow cytometry. The specificity of the CD29 antibody was demonstrated by positively stained human amniotic epithelium and isolated hAECs, but not mouse lung tissue (Figure 2A). At 2 weeks post-hAEC injection, CD29-positive cells constituted 5.3 6 0.9% of the total lung suspension (Figure 2A). To verify that the CD29-positive cells were indeed human, we injected hAECs obtained from pregnancies with male babies into female SCID mice and analyzed the recovered cells by FISH for the XY chromosomes to identify human cells and XX chromosomes representing mouse cells. Greater than 98% of flow sorted cells were human by this criterion (Figure 2B). Notably, primary hAECs that were injected did not produce pro–SP-C, SP-A, SP-B, or SP-D (Figure 2C). The antibodies against the SPs showed specific cytoplasmic staining of human lung tissue, whereas SP-B and pro– SP-C also showed nonspecific nuclear staining of mouse lung cells (Figure 2C). The CD29-positive cells recovered from bleomycininjured lungs by fluorescence-activated cell sorter were positive for SP-A (65 6 5%), SP-B (72 6 7%), pro–SP-C (79 6 8%), and SP-D (70 6 8%) by immunohistochemistry (Figure 2D). hAECs Reduce Inflammation in the Injured Lung

Bleomycin instillation caused a significant inflammatory and fibrotic infiltration of the lung that was attenuated by hAEC injection (Figure 3A). Ashcroft scores of inflammation and fibrosis on histology showed that bleomycin-induced pneumonitis was significantly reduced at 2 and 4 weeks by hAEC administration (Figure 3B) (P , 0.03; Student t test). The inflammatory response to bleomycin was accompanied by increased mRNA expression of IL-1, IL-2, IL-6, IFN-g, tumor necrosis factor (TNF)-a, monocyte inflammatory protein, and transforming growth factor (TGF)-b compared with controls (P , 0.001 for all cytokines). The expression of these cytokines was significantly attenuated in mice receiving hAECs (P , 0.01

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Figure 2. Analysis of CD29-positive cells from mouse lung cell suspensions. (A) Human amnion epithelial cells (hAECs) lining term amnion and isolated hAECs show strong immunostaining for CD29 (diaminobenzidine-DAB staining). Haemotoxylin stained lung tissue sections from bleomycin-injured mice and isotype controls (not shown) did not stain for CD29. The CD291 population was easily identified and separated by flow cytometry 2 weeks after hAEC injection and comprised 5.3 6 0.9% of the total lung cell suspension. Magnification 100x. (B) hAECs from male placenta (XY) were injected into female (XX) SCID mice. Fluorescence in situ hybridization analysis for XY chromosomes (Poseidon RAB9B) confirmed that flow-sorted CD291 cells were human and that the CD292 population were murine (XX) cells. (C) Human lung alveolar epithelial cells stained positively for surfactant protein (SP)-A and pro–SP-C (DAB). Mouse lung cells showed no cross reactivity for SP-A. Nonspecific nuclear staining was seen for pro–SP-C. Primary hAECs that were injected lacked staining for surfactant proteins. Cells and tissue sections counterstained with haemotoxylin. (D) Immunohistochemistry showed that greater than 65% of flow-sorted CD291 cells recovered from murine lungs produced SP-A, SP-B, pro–SP-C, and SP-D (DAB) Magnification 100x (A-C and 50x in D). Magnification 100x (A-C and 50x in D). Scale bars 5 20 mM in cell spreads and 50 mM in tissue sections.

for all cytokines). IL-10 mRNA was significantly increased in mice given hAECs compared with bleomycin (P , 0.01) (Figure 4A). The expression of human specific cytokines in lungs of hAEC-injected mice was also investigated. hAECs expressed mRNA for IL-1, IL-10, TNF-a, TGF-b, migration inhibition factor, and IFN-g. We detected low levels of human specific cytokine mRNA for IL-1, IL-10, TNF-a, and TGF-b in lungs of mice culled two weeks after hAEC transplantation using realtime PCR. However, mRNA for migration inhibition factor and IFN-g were not detectable. Gel electrophoresis showing the PCR products generated is shown in Figure 4B. We also examined changes in cytokine protein levels in mouse lung tissue lysates; IL-1, IL-6, monocyte chemoattractant protein-1, and TGF-b were reduced significantly in mice treated with hAECs compared with animals given bleomycin alone (P , 0.05 for all cytokines) (Figure 4C). hAECs Reduce Fibrosis in the Injured Lung

We investigated whether hAECs reduced fibrosis by assessing collagen deposition using the hydroxyproline assay. Collagen was

elevated 4 weeks after bleomycin instillation (P , 0.05 versus healthy controls), but was significantly reduced in the cohort receiving hAECs (P , 0.05 versus bleomycin). Notably, injection of human lung fibroblast cells or placental cytotrophoblast cells into bleomycin-injured mice did not lower lung collagen content. Furthermore, hAECs did not influence collagen levels in lungs of healthy mice. The effect of hAECs on well-established injury was also investigated by administering the cells 2 weeks after bleomycin instillation. hAECs significantly reduced collagen in this cohort (P , 0.05 vs. bleomycin alone) (Figure 5A). hAEC Treatment Alters Protease Levels in the Injured Lung

Gelatin zymography demonstrated that active MMP-2 was significantly elevated in bleomycin-injured mice compared with healthy controls (P , 0.05), which is in keeping with previous studies (21). Notably, hAEC injection into bleomycin-exposed mice resulted in a significant increase in latent and active MMP2 compared with bleomycin alone or healthy controls (P , 0.05 for all comparisons). Active MMP-2 was not elevated in healthy controls treated with hAECs or bleomycin-injured mice given


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Figure 3. Human amnion epithelial cells (hAECs) reduce inflammation of the lung after bleomycin injury. (A) Bleomycin induced an inflammatory infiltrate at 2 weeks and an inflammatory and fibroblastic infiltrate of the lung at 4 weeks. The lung injury was attenuated by hAEC injection at both time-points. Scale bars 5 50 mM. (B) The Ashcroft score confirmed reduced inflammation and fibrosis after hAEC injection (*P , 0.03).

human fibroblasts (Figure 5B). Analyses of TIMP mRNA expression showed a down-regulation of TIMP-1, TIMP-3, and TIMP-4 in mice treated with hAECs (P , 0.05). Reverse zymography demonstrated that TIMP-1 and TIMP-2 proteins were up-regulated after bleomycin and significantly reduced by hAEC treatment (P , 0.05) (Figure 5C).

DISCUSSION This study described several novel findings that extend the field of stem cell therapy for lung disease. We have used a novel source of cells (hAECs) to treat lung injury. Furthermore, to our knowledge, this is the first study to describe human cells that not only differentiate into an alveolar epithelial phenotype but also rescue an injury model of lung fibrosis. Significantly, hAECs were not only positive for lung epithelial markers after both in vitro and in vivo differentiation assays, but also displayed functionality in the secretion of SP-D after stimulation with corticosteroids. Preliminary characterization studies of hAECs generated from our laboratory by Ilancheran and coworkers (12) revealed that the primary cells express a number of pluripotent and multipotent stem cell markers, are clonogenic, and express low levels of HLA Class IA and IIB antigens. Furthermore, hAECs differentiated into lineages derived from all three germ layers in vitro (12). Based on this study, we hypothesized that hAECs may be an effective modality of cellular therapy; we therefore explored their reparative and differentiation properties in a model of lung injury. As part of the focus on lung differentiation and repair, we found that hAECs express thyroid transcription factor or Nkx 2.1 mRNA, which is among the earliest lineage specification markers of the developing lung. Nkx. 2.1 is a transcription factor critical for branching lung morphogenesis and the formation of type II pneumocytes (22). Primary hAECs also expressed epithelial mRNAs occludin, mucin, caveolin-1, and aquaporin-5, but lacked the more lung-specific proSP-C. Upon noting the expression of Nkx 2.1 in primary hAECs, we cultured these cells in SAGM, which has been shown to induce differentiation of uMSC and embryonic stem cells into

type II pneumocytes (20, 23). SAGM contains hydrocortisone, human epidermal growth factor, and retinoic acid, which are factors required for lung development (24). Primary hAECs grown in SAGM produced SP-A, SP-B, pro–SP-C, and SP-D, whereas control cells cultured in DMEM/F12 did not express SPs. SP-A, SP-B, and SP-C are synthesized in the endoplasmic reticulum of type II pneumocytes and form lamellar bodies, whereas SP-D is synthesized through a different pathway (25). Collectively, the SPs contribute to lowering the surface tension between the air–liquid interfaces in the lung. SP-D and SP-A also play a part in the innate defense system of the lung (25). The production of all SPs is suggestive of hAEC differentiation to a phenotype of type II pneumocytes. Furthermore, ultrastructural examination revealed that cells grown in SAGM for 2 to 4 weeks contained a distinct cell population with cytoplasmic organelles containing lamellar bodies typical of type II pneumocytes. These features were absent in hAECs cultured in DMEM/ F12. We also examined whether hAECs cultured in SAGM secreted SP-D, which would suggest functionality. After 4 weeks of culture in SAGM, hAECs were stimulated with dexamethasone and SP-D measured in the supernatant. Corticosteroids are used to accelerate lung maturation in preterm infants by augmenting surfactant production and secretion (26). Notably, SP-D production was increased in hAECs after stimulation with corticosteroids, suggesting a phenotype that is functionally similar to type II pneumocytes. We noted other markers of type II pneumocytes including CD44v6 (27), CD208 (28), and gp600 (29). However, these markers share epitopes with other cell types and tissue and are not as specific as SPs for type II pneumocytes. Primary hAECs that lacked SP production were also injected into bleomycin-treated SCID mice and identified by IMM immunostaining in fibrotic and alveolar regions 4 weeks post– cell transplantation without evidence of tumor formation. Furthermore, the hAECs were recovered from lung suspensions by flow cytometry using CD29. FISH analysis of both human and mouse chromosomes verified that the flow–sorted CD29-positive cells were human. Notably, the flow–sorted cells produced all SPs demonstrating that hAECs were capable of differentiating into type II pneumocytes in vivo. Taken together, these data support

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Figure 4. Human amnion epithelial cells (hAEC) injection reduces cytokine levels in the bleomycin-injured lung. (A) Analysis of mouse lung mRNA transcripts by reverse transcriptase–polymerase chain reaction (qRT-PCR) showed an elevation in inflammatory and profibrotic cytokines 2 weeks after bleomycin injury compared with saline–injected controls (P , 0.001); however, this response was attenuated by hAEC injection (*P , 0.01 comparing bleomycin with bleomycin 1 hAECs). (B) Gel electrophoresis of PCR products for human-specific IL-1, IL-10, tumor necrosis factor (TNF)a, transforming growth factor (TGF)-b, migration inhibition factor (MIF), and IFN-g. The primers specific for human cytokines did not cross-react with mouse lung mRNA from animals given bleomycin alone. hAECs expressed mRNA for IL-1, IL-10, TNF-a, TGF-b, MIF, and IFN-g. We detected low levels of human-specific cytokine mRNA for IL-1, IL-10, TNF-a, and TGF-b in lungs of mice culled 2 weeks after hAEC transplantation using realtime PCR and on gel electrophoresis of PCR products. However, mRNA for MIF and IFN-g were not detectable. B 5 bleomycin alone; BH 5 bleomycin 1 hAECs; H 5 hAECs. (C) Analysis of lung tissue homogenates showed that the increase in IL-1, IL-6, MCP-1, and TGF-b protein after bleomycin was attenuated by hAEC injection (*P , 0.05).

the proposal that hAECs may be capable of longer-term alveolar restitution. Recently, human MSCs from gestational tissues including umbilical cord blood and Wharton jelly, AFSCs, and PSCs have been injected into injured mice (6–9). There are salient differences between these reports and our study. hAECs are epithelial in phenotype compared with the mesenchymal phenotype of umbilical cord–derived cells and AFSC (6, 9). The engraftment of hAECs is high compared with other studies; however, the type of cell, route of administration, the nature of the injury, and the use of SCID mice may all influence engraftment. The effect of each of these components is the subject of further investigation. Previous studies showed expression of cytokeratins by uMSCs and Clara Cell Protein (CC10) and SP-C by AFSCs after transplantation (6, 7). In contrast, hAECs produced all SPs by 2 weeks of administration and also rescued an injury model. Wharton jelly MSCs and PSCs have been shown to reduce bleomycin-induced lung injury in mice. However, in contrast to hAECs, there was no evidence of differentiation displayed by injected Wharton jelly MSCs and these cells were absent in the lung approximately 2 weeks after cell trans-

plantation (9). The PSCs injected by Cargnoni and coworkers (8) into bleomycin-injured mice consisted of a 1:1 mixture of amnion-chorion–derived MSCs and hAECs unlike the pure population of hAECs used in the present study. Although no evidence of functional differentiation into pneumocytes was reported, reduced neutrophil infiltration and collagen deposition was noted. However, it remains unclear if these effects are caused by either or both cell types that were transplanted. In addition, hAECs demonstrated a type II pneumocyte phenotype; this was not investigated in the PSC study. hAEC injection reduced the bleomycin-induced pneumonitis and increased expression of cytokines that have been shown to be central to the pathogenesis of lung injury, including monocyte chemoattractant protein-1, IL-1, IL-2, IL-6, TNF-a, IFN-g, and TGF-b (30–33). IL-1 is elevated during bleomycin lung injury and is usually secreted by monocytes and macrophages, promoting fibrosis by increasing fibroblast proliferation and collagen production (34). As such, the inhibition of IL-1 observed in our study supports an antifibrotic role for hAECs in the lung. In addition, IL-6 is significantly elevated during bleomycin lung injury and is postulated to synergize with TNF-a to perpetuate inflammation


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Figure 5. Human amnion epithelial cells (hAECs) reduce lung fibrosis after bleomycin injury and alter matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinases ( TIMPs). (A) hAEC injection resulted in a significant fall in collagen content compared with cellular controls placental trophoblasts and lung fibroblasts. hAECs injected into healthy mice did not induce a fibrotic response. hAECs also reduced well-developed fibrosis. *P , 0.05 compared with saline controls; 1 P , 0.05 compared with bleomycin alone. (B) Active MMP-2 increased significantly in mice receiving bleomycin and hAECs (*P , 0.05 versus bleomycin alone). (C) Compared with controls, TIMP mRNA expression is up-regulated in bleomycininjured mice (P , 0.001). However, hAEC treatment led to a reduction in TIMP-1, -3, and -4 mRNA expression (*P , 0.05). Reverse zymography of lung homogenates show that the increase in TIMP-1 and TIMP-2 after bleomycin instillation is reduced after hAEC injection (*P , 0.05).

and fibrosis (31). Therefore, the reduction in IL-6 after hAEC injection may serve to reduce further the development of fibrosis. TGF-b is a central cytokine in the pathogenesis of lung fibrosis (35). By Day 14 after bleomycin administration, TGF-b is increased in macrophages, epithelial cells, fibroblasts, and myofibroblasts (33). Khalil and coworkers (33) conclusively showed that the amelioration of bleomycin fibrosis is dependent on the inhibition of TGF-b. As such, the significant down-regulation of TGF-b also supports the antifibrotic role of hAECs. In addition, hAECs reduce fibrosis when injected after early (24 hours postbleomycin) and during established injury (2 weeks postbleomycin), suggesting that hAEC therapy may be useful in lung diseases with established fibrosis and tissue remodeling. The detection, albeit low levels, of human specific mRNA for IL-I, IL-10, TNF-a, and TGF-b suggests that hAECs engraft in the lung, remain viable, and are capable of synthesizing mRNA. However, despite the presence of human mRNA, there is a reduction in protein levels for IL-1, TNF-a, and TGF-b after hAEC injection. Furthermore, it is unlikely that the detected low levels of human IL-10 mRNA contributes significantly to the increase in IL-10 mRNA in the mouse lung. Therefore, we propose that the overall antiinflammatory effect of hAECs is to modify the response of the mouse lung to injury.

MMPs and their endogenous inhibitors (TIMPs) are primarily responsible for the degradation of extracellular matrix proteins, such as collagen. Consistent with our findings TIMP concentrations increase before collagen deposition in the bleomycin mouse model at 14–28 days after injury (36). Protein analysis of lung tissue lysates demonstrated an hAEC-induced increase in MMP2 and a reduction in TIMP-1 and TIMP-2 protein, which other studies suggest would augment lung repair (21). In contrast, Ortiz and colleagues (37) demonstrated down-regulation of MMP-2 when using murine bone marrow MSCs to reduce the fibrotic response to bleomycin injury. This difference may be caused by the source of cells and the mouse strains (C57/Bl6 versus SCID) used. The mechanisms delineating the salutary effects of stem cells on models of lung injury are largely unknown. However, studies in MSCs have found that several different molecules may play a part in augmenting repair. MSCs reduced lung fibrosis by inhibition of IL-1 receptor antagonist (IL-1Ra) after bleomycininduced lung injury (2). Furthermore, MSCs reduced endotoxininduced lung injury by decreasing inflammation and cytokines caused by the actions of angiopoeitin-1 (3, 38). In addition, hepatocyte growth factor reduced elastase-induced lung injury by mobilizing endothelial progenitor cells to the lung (39). In an

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elegant study, Lee and coworkers (40) found that keratinocyte growth factor was central to increasing alveolar fluid clearance and reducing capillary leak after injection of human MSCs in an ex vivo human lung model of LPS-induced lung injury. Although we have not identified a specific molecule by which hAECs mediated lung repair, we suggest these cells act through several mechanisms to reduce inflammation and fibrosis. The adoption of a lung phenotype in vivo by hAECs would assist in restituting alveolar epithelium, thereby reducing collagen deposition (41). In addition, the reduction in inflammation and cytokine expression may be important mechanisms that limit damage to the lung and subsequent scarring. Furthermore, the elevation in MMPs with concurrent inhibition of TIMPs would constitute a prodegradative environment for the breakdown of deposited collagen. To date, there are no safe and effective therapeutic agents to treat chronic fibrotic lung diseases. The use of hAECs for the treatment of lung disorders has distinct advantages because they are highly abundant, obtained noninvasively, and have been parentally administered into humans without adverse effects (14). In addition, there are no ethical controversies associated with the clinical use of these cells. Author Disclosure: Y.M. has a pending patent PCT/AU2008000753. S.I. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. V.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.D.W. received grant support from AstraZeneca for collaborative contact research and from the NHMRC (more than $100,001). G.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. U.M. has a temporary patent. Acknowledgment: The authors thank James Ngui for flow cytometry, Mark Malin and Jinhua Li for tail vein injections, Dhanya Sreedharan for DNA-PCR, Gregory Allen for FISH analysis, and A/Prof Phillip Bardin for providing human lung fibroblast cells.

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