Anne-Laure Leblond

Patrice Naud

Virginie F Forest

Clothilde Gourden

Christine Sagan

Bénédicte Romefort

Eva Mathieu

Bruno Delorme

Christine Collin

Jean-Christophe Pagès

Jean-Christophe Pag

Luc Senseb

Bruno Pitard

Patricia Lemarchand
email: <patricia.lemarchand@univ-nantes.fr

Developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered

Keywords: Analysis of Variance, Animals, Bronchoalveolar Lavage, DNA Primers, genetics, Embryonic Stem Cells, transplantation, Enzyme-Linked Immunosorbent Assay, Galactosides, Genetic Engineering, methods, Genetic Vectors, Indoles, Male, Mesenchymal Stem Cell Transplantation, methods, Mice, Respiratory Tract Diseases, therapy, Tissue Therapy, methods, Trachea, cytology, Transfection, beta-Galactosidase

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In most of these studies, systemic administration of SCs required total-body irradiation of the recipient to promote SC bone marrow engraftment, a difficult condition to apply in the clinical setting, especially in patients suffering from airway disease associated with chronic infections such as cystic fibrosis. Alternatively, considering the advantages of the intratracheal route to target the airway and the respiratory epithelium, more recent studies reported the intratracheal administration of adult SCs in reagent-injured lungs (Gupta et al., ; ). Using fluorescent techniques SC engraftment was enhanced with the intratracheal route as compared to the 2007 ). The intratracheal route was also used for the administration of differentiated cells and showed both positive (Serrano-Mollar 2007 et al., The aims of our study were to evaluate the survival and the engraftment of different types of exogenous SCs and differentiated cells after intratracheal injection. We used a murine model presenting acute epithelial airway injury without total-body irradiation. This model was developed for gene therapy experiments on cystic fibrosis and consisted in intranasal injection of polidocanol detergent (Parsons et al., ). Quantification of cell survival rates and cell location within lungs were performed using ELISA, biochemical assay and PCR. 1998

Materials and methods

Animal model and intratracheal administration

Male, 8-to 10-week old Swiss mice were obtained from Janvier Laboratories (Le Genest St Isle, France). Animal experiments were performed under the approval of the published by the U.S. National Institutes of Guide for The Care and Use of Laboratory Animals Health (NIH Publication No. 85-23, revised 1996). Mice were anesthetized with an intraperitoneal injection of xylazine (15mg/kg) and ketamine (70mg/kg), and placed supine. Briefly, intratracheal administration was performed using a 22-gauge catheter placed within an intubation canula (Harvard Apparatus) that was removed immediately before injection. Airway injury was induced after anaesthesia by intratracheal administration of 25 l of 2 polidocanol (PDOC: polyoxyethylene 9-lauryl ether, Sigma, ( ; intratracheally injected 24hr after PDOC administration using the same protocol. Mice were sacrificed by an intraperitoneal lethal injection of nesdonal 24hr or 7 days after intratracheal administration.

Bronchoalveolar lavages

Bronchoalveolar lavages (BALs) were performed 24hr after CAT-transfected cell administration in healthy or injured airway. Mice were anesthetized and thoracic cavity was opened by careful dissection. The trachea was exposed, and a small transverse incision performed just below the level of the larynx. BAL was then performed using one dose of 1 ml of PBS, ensuring that both lungs inflated during the lavage process and that there was no leakage of lavage fluid from the trachea. BAL fluid was centrifuged at 400 g for 5 min.

The supernatant was removed and stored at 80 C until ELISA. Cell pellet was suspended in lysis buffer and stored at 80 C until ELISA. Undifferentiated murine mesenchymal stem cells (BMC9s, ( )) were cultured as previously described [START_REF] Dennis | Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse[END_REF] in alpha MEM medium with nucleosides (Invitrogen) supplemented with 10 FCS (Invitrogen), 2mM Chateauvieux 2007 et al., % L-Glutamine (Invitrogen), penicillin/streptomycin (100U/ml and 100 g/ml respectively, Invitrogen). Total bone marrow was obtained from wild type adult male Swiss mice and male Rosa26 lacZ-mice (background C57Bl/6J x 129S2, kindly provided by Dr M.F. Gardahaut, Nantes, France) by flushing femurs and tibias with culture medium. Cells were plated at a density of 500 000 cells/cm in medium composed of alpha MEM with nucleosides supplemented with 10 FCS, 2 mM L-Glutamine, 2 % penicillin/streptomycin (100U/ml and 100 g/ml respectively) and 2ng/ml of human FGF2 (AbCys). The culture medium was changed at μ day 3 to remove non-adherent cells and was subsequently replaced weekly. The cells were grown for 2 3 weeks until confluence.

-Adherent cells were then detached by 0.5 trypsin-EDTA and plated at a density of 10 000 cells/cm . Subsequent passages and seeding of % 2 the cells were performed at a density of 5 000 cells/cm . From passage 8, MSC cultures were characterized by FACS analysis (FACS 2 Calibur Instrument, BD Biosciences, CellQuestPro Software) after incubation with anti-CD45-PE, anti-CD90-PE, anti-CD29-CyChrome, anti-Sca1-PE, anti CD106-CyChrome (BD Biosciences) (

). [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF] In vitro cell transfection with CAT or GFP reporter genes and evaluation of gene transfer system efficacy ESCs, BMC9s and differentiated cells were transfected with pCIK-CAT (4.7 Kb) and pEGFP-C1 (4.7 Kb) plasmids encoding for Chloramphenicol AcetylTransferase (CAT) protein and GFP, using synthetic vectors just prior to intratracheal administration (Pitard et al., ). DNA plasmids were complexed with ICAfectin 441 according to the manufacturer s instruction (In-Cell-Art, Nantes, France). 2001

® '
After 2hr, transfection complexes were removed by changing growth medium and cells were lysed for dosages or kept in culture for 24hr, 7 days, or injected intratracheally as indicated above.

MSCs from Swiss mice were transfected with pCIK-CAT and pEGFP-C1 plasmids using nucleofection (Nucleofector Solution, Amaxa Biosystem) just prior to intratracheal administration ( ). After 2hr, growth medium was changed and cells were Aluigi 2006 et al., lysed for dosages or kept in culture for 24hr, 7 days, or injected intratracheally as indicated above. The percentage of GFP-expressing cells was evaluated 24hr after GFP nucleofection using FACS analysis.

The efficacy of gene transfer systems was evaluated using cytometry for GFP-expressing cells and ELISA for CAT-expressing cells.

The percentage of GFP-expressing cells was evaluated 24hr after GFP transfection using FACS analysis (FACS Calibur Instrument, BD Biosciences, CellQuestPro Software). CAT protein quantity per stem cell at 24hr was then calculated using data: CAT protein in vitro quantity in cell lysis buffer was divided by the cell number and by the percentage of GFP-expressing cells (obtained by FACS analysis).

Finally, this value was used to determine the minimum number of CAT-expressing stem cells required to be detected by ELISA or in vitro considering that CAT ELISA detection threshold was 50pg CAT protein (see below). in vivo

In vitro stem cell transduction with nls-lacZ reporter gene

BMC9s were transduced with an amphotropic recombinant murine retrovirus vector containing the nls-LacZ reporter gene (LacZ gene encoding galactosidase ( gal) coupled to a nuclear localization signal (nls), kindly provided by Dr. N. Ferry, Nantes, France). This

β β
retrovirus vector was obtained from the human producer cell line TELCeB6AF7 derived from the Te671 cell line as described in (Aubert ). 24hr recombinant retroviral supernatant was harvested from the confluent producer cell line and filtered through a 0.45 m 2002 et al., μ membrane. The titer of the gal supernatant, determined by endpoint dilution using Te671 target cells, was 2 10 transducing β × 8 particles/ml. After BMC9 transduction, retrovirus vector was removed by changing growth medium and cells were lysed for dosages or kept in culture or injected intratracheally as indicated above.

To evaluate the percentage of gal-transduced BMC9s, cells were fixed in 4 paraformaldehyde, washed with PBS and incubated in β % 5mM K4Fe(CN)6, 5mM K3Fe3(CN)6 and 2mM MgCl2 in PBS containing 0.5mg/ml X-Gal ( 5-bromo-4-chloro-3-indolyl-

Reporter gene assay and estimation of survival rate

CAT-transfected cells, BAL cells and BAL fluid, or whole frozen trachea-lungs were homogenized in reporter lysis buffer (Roche Diagnostics) supplemented with protease inhibitors (Roche Diagnostics). After centrifugation at 10 000 rpm for 5min, CAT quantity was measured in supernatant with a VICTOR multilabel counter (PerkinElmer), using a CAT enzyme-linked immunosorbent assay (ELISA)

To estimate cell survival rate 24hr after intratracheal administration, we first calculated CAT protein quantity per CAT-expressing cell, using data: CAT quantity in cell lysis buffer was divided by the cell number and by the percentage of GFP-expressing cells in vitro (obtained by FACS analysis). Finally, the total CAT protein quantity per animal was divided by this value, in order to in vivo in vitro estimate the number of CAT-expressing cells per animal and subsequently the cell survival rate.

BMC9s and Rosa26 MSCs or whole frozen murine trachea-lungs were homogenized in 1ml reporter lysis buffer (Roche Diagnostics) supplemented with a protease inhibitor cocktail (Roche Diagnostics). After centrifugation at 10 000 rpm for 5min, gal activity was β determined by enzymatic fluorimetric assay using 4MUG (4-methylumbelliferyl--D-galactoside, Sigma) as a fluorescent substrate. Each nuclear fast red (Sigma).

Detection of nls-lacZ reporter gene

Total DNA from whole murine trachea-lungs was isolated for PCR analysis. Tissues were digested overnight with lysis buffer containing proteinase K (10mg/ml, Sigma). Total DNA was extracted with phenol/chloroform/isoamyl alcohol (Sigma) method and then precipitated in ethanol. Total DNA from 10 transduced MSCs was also extracted for PCR experiments. PCR were performed using 6 primers that specifically bind the nls sequence and the lacZ gene encoded in the nls-lacZ lentivirus vector: 5 -GTA ACA ACT CCG CCC ′ CAT T -3 and 5 -GAC AGT ATC GGC CTC AGG AA -3 .

′ ′ ′

Statistical analysis

Data are expressed as means S.E.M. Statistical significance was evaluated using the analysis of variance (ANOVA) method to ± compare control and treatment groups of 3 or more. Tukey s test was subsequently used for pair-wise comparisons. A non parametric ' Mann and Whitney test was performed to compare two groups. Statistical significance was set at p<0.05.

Results

Transient epithelial airway injury induced by polidocanol intratracheal administration

Intratracheal administration of 2 PDOC induced macroscopic lung injury at 24hr with haemorrhage ( ). The major ). PDOC effect did not alter significantly animal survival ( ). Figs. 1f,i Table S1 in supplementary data

ESC and BMC9 survival in murine airway with acute epithelial airway injury

In a first set of experiments, embryonic stem cells (ESCs) and adult mesenchymal stem cells (BMC9s) from well-characterized cell lines were intratracheally injected. ESCs and BMC9s were injected 24hr after PDOC administration, when epithelial airway injury induced by PDOC was acute ( ). Stem cell survival was evaluated 24hr after stem cell administration. with plasmids encoding CAT or GFP reporter genes 2hr prior to intratracheal injection. CAT-transfected stem cells in vitro were then intratracheally injected, at a time where they did not yet express the foreign genes (see below). We hypothesized that only surviving stem cells would express CAT protein in trachea-lungs 24hr after intratracheal cell injection. in vivo Two hours after the beginning of cell incubation with both plasmids and synthetic vectors, transfection complexes were removed and reporter gene expression was evaluated. Neither CAT protein nor cell fluorescence was detected in stem cells using ELISA (

) and Fig. 2b microscopy (not shown), demonstrating that stem cells did not express CAT nor GFP protein at the time of injection. in vivo

In vitro reporter gene expression was also evaluated 24hr and 7 days after incubation with plasmids and synthetic vectors. As evaluated by FACS analysis, 19.4 0.2 ESCs (n 6) and 37.0 4.4 BMC9s (n 6) expressed GFP at 24hr ( ). However, CAT and GFP

± % = ± % =
in vitro Fig. 2a were not detected at 7 days after transfection ( ), suggesting that transgene expression was transient. Using CAT ELISA quantitative Fig. 2b results and GFP-expressing cell rates, we calculated that CAT-expressing ESCs and CAT-expressing BMC9s contained at 24hr 0.03 ± 0.01pgCAT/cell (n 10) and 0.08 0.02pgCAT/cell (n 6), respectively. Cell values were further used to evaluate the number of survival rate was significantly less with BMC9s (0.43 0.12 , n 17, Tukey s test, p 0.02). CAT cell quantities were also used to

± % = ' =
calculate the cell detection threshold. A minimum of 10 1.2 10 ESCs and 4.3 1.1 10 BMC9s could be detected in one animal in vivo

± × 3 ± × 3
, corresponding to 0.4 1 injected cells. These results suggest that even if the percentage of cells expressing reporter gene in vivo -% in vitro was low, this was still a sensitive method for detection of a small cell number. in vivo

Influence of differentiation state and airway environment on ESC and BMC9 survival

To determine whether cell survival ability in murine injured airway was specific to stem cells, differentiated cells from various origins (Hep3B, mOS-J and COS-7 cells) were transfected with CAT plasmids in conditions and results similar to that of ESCs and in vitro BMC9s (not shown). CAT-transfected differentiated cells were then intratracheally injected 2hr after the beginning of incubation with CAT plasmid. No CAT protein was detected in any animal injected with PDOC and differentiated cells at 24hr ( ), suggesting that Fig. 2c only stem cells survived after intratracheal injection in injured airway. CAT-transfected ESCs and BMC9s were injected in control healthy animals. No CAT protein was detected in any animal with healthy airway 24hr after stem cell injection ( ), suggesting that stem cell Fig. 2c survival was favoured by airway injury.

Cell survival in the epithelial lining fluid

To evaluate whether cells expressing CAT reporter protein were present within the epithelial lining fluid or after phagocytosis by alveolar macrophages, bronchoalveolar lavages (BALs) were performed at 24hr in conditions when phagocytosis was expected in vivo maximal, e.g. when CAT ELISA results were negative: mesenchymal stem cells (BMC9s) in healthy airway and murine differentiated cells (mOS-J) in injured airway. As further controls, BALs were also performed in control and PDOC animals and in animals intratracheally injected with PDOC and BMC9s. No CAT protein was detected in any BAL fluid nor BAL cell sample from any animal ( ), suggesting that if stem cells or differentiated cells were phagocytosed by alveolar macrophages, such phagocytosis did not alter Fig. 2d CAT protein measurement nor cell survival detection. Importantly, in animals intratracheally injected with PDOC and BMC9s, high levels of CAT protein were measured in trachea and lung homogenates whereas no CAT protein was detected in BAL fluid nor BAL cell samples, suggesting that CAT-expressing cells were mostly present within the lungs and did not survive in the epithelial lining fluid (Fig.

). 2d

BMC9 location at 24hr and 7 days

Next, we investigated BMC9 location at 7 days when the airway epithelium was known to be spontaneously regenerating ( ). In contrast to previous experiments, gal-transduced BMC9s expressed nlslacZ gene Fig. 3c β at the time of intratracheal injections. Therefore, as a further control, we injected lysates of gal-transduced BMC9s, and no significant β increase in gal activity was observed as compared to endogenous gal activity (Tukey s test, p 0.98). In animals intratracheally injected

β β ' =
with PDOC and gal-transduced BMC9s, 24hr after injection gal activity significantly increased as compared to that of control animals β β

(Tukey s test, p 0.003) and to animals injected with PDOC and lysate of gal-transduced BMC9s (Tukey s test, p 0.004), confirming

' = β ' =
results with CAT gene transfer. However, 7 days after stem cell injection gal activity was similar to that of control animals (Tukey s test, β ' p 0.98) and animals injected with PDOC and lysate of gal-transduced BMC9s (Tukey s test, p 0.94), suggesting that the number of

= β ' =
surviving BMC9s in the airway epithelium decreased significantly between 24hr and 7 days.

To locate BMC9s after intratracheal injection into injured airway, we performed X-Gal staining of trachea-lungs at 24hr and 7 in toto days (

). No blue staining was observed in trachea-lungs ( ) and histological sections ( ) from any control animal ( Fig. 4 Fig. 4a Figs. 4b-d , n 3), animal injected with PDOC ( , n 3), or animal intratracheally injected with gal-transduced BMC9s only ( , Fig. ) and injured bronchi or bronchioles ( ), but not in lung parenchyma ( ). Fig. 4k Figs. 4l,m Fig. 4l At 7 days, blue spots were observed in 7/7 animals, in the lumen of bronchioles ( ). No blue cell was observed in trachea or in Figs. 4n-p pulmonary parenchyma (not shown). Clusters of blue cells were sometimes located in polyp-like structures, located in bronchioles and large bronchi. Polyp-like structures were not observed in any animal receiving PDOC only. Further quantitative and statistical analyses demonstrated that the development of polyp-like structures at 7 days was not due to stem cell administration but rather to a second intratracheal administration (PBS or BMC9) 24hr after PDOC injection (

). Histological analyses at high Table S2 in supplementary data magnification showed that gal-expressing cells did not have a respiratory phenotype, with no cilia at 24hr or 7 days (not shown).

β

MSC survival in murine airway with acute epithelial airway injury

In a second set of experiments, to avoid cell survival linked to cell line transformation, we repeated previous experiments with in vivo the same experimental design, using murine MSCs from primary cultures. MSC cultures were characterised according to (Dominici et -Fig. 5a ± % = -± 0.2 , n 6) and CD29 , Sca-1 ( , 96.9 1.3 and 98.1 0.7 respectively, n 4) and CD106 (96 0.8 , n 4).

% = + + Fig. 5a ± % ± % = + ± % =
MSCs were transiently transfected with plasmids encoding CAT or GFP reporter genes. In contrast to ESCs and BMC9s, MSC in vitro transfection with synthetic vectors was ineffective (not shown), therefore nucleofection was used to obtain significant gene transfer rate.

As evaluated by FACS analysis, 56.8 2.9 MSCs expressed GFP at 24hr ( , n 3), but CAT and GFP were not detected 7

± % in vitro Fig. 5c = days after nucleofection ( ), suggesting that foreign gene expression was again transient and that nucleofection could not be used for Fig. 5b 7 days experiments. CAT-transfected MSCs contained 0.35 0.02pg CAT/cell at 24hr (n 6). CAT-transfected MSCs were then in vivo

± =
intratracheally injected 2hr after the beginning of incubation with CAT plasmid. In control mice and mice injected with PDOC only, no CAT protein was detected at 24hr ( ). Importantly, CAT protein was detected in each animal injected with PDOC and MSCs ( Fig. 5d Fig.

, n 8, Tukey s test, p<0.001), suggesting that MSCs from primary cultures survived in injured airway and expressed CAT protein after 5d = '

intratracheal administration. MSC survival rate was 5.52 1.9 (n 8), a minimum of 710 45 CAT-expressing MSCs could be detected

± % = ±
in one animal , corresponding to 0.07 injected cells, confirming CAT ELISA sensitivity for experiments. in vivo % in vivo

MSC location at 24hr and 7 days

To investigate whether MSCs could be located into the airway epithelium 7 days after cell injection, we first used MSCs in primary cultures from gal transgenic (Rosa26) mice, thus expressing continuously cytoplasmic gal (

). Using the 4 MUG method ( injection in injured airway. However, no nls-lacZ gene was detected in animals at 7 days after gal-transduced MSC injection, suggesting β that no gal-transduced MSCs from primary culture survived at this time once the epithelium is regenerated ( , n 6).

β Fig. 6e =

Discussion

Using different and independent methods based on reporter gene transfer we demonstrated the short term survival of different types of exogenous stem cells, including embryonic and adult mesenchymal stem cells, after intratracheal injection in murine airway presenting acute epithelial airway injury and without total-body irradiation. In contrast to differentiated cells, 0.43 to 5.5 stem cells were capable of % surviving within the injured lungs at 24hr. Importantly no SCs survived in healthy airway or in the epithelial lining fluid. Biochemical staining showed that transduced MSCs were located in the lumen of conducting airway at 24hr and at 7 days. However, the amount in vivo of engrafted MSCs decreased dramatically with time. No primary MSCs were located within lungs at 7 days.

In our study, several gene transfer systems were used, according to our aims and to cell types. Using careful timing, non-viral vectors were useful to allow expression of the foreign gene only after intratracheal cell injection. Gene delivery with viral vectors was used for in studies at 7 days, but we had to use different viral vectors for transducing MSCs from cell lines and primary MSCs. Depending on the vivo gene transfer system and the cell type, gene transfer efficacy was variable . Studies with retrovirus vectors showed high efficacy in vitro and stable MSC transduction (up to 97 , (

)) whereas studies with lentivirus vectors showed variable efficacy between gal-expressing cells, even when gal activity in the whole lungs was not significantly different from baseline.

β

We investigated whether stem cells would be capable of locating specifically on the epithelial side of airway after intratracheal administration. BAL experiments showed that stem cells expressing reporter protein did not survive in the epithelial lining fluid. X-Gal staining showed that stem cells were localised into conducting airway . By injecting stem cells in healthy airway, we also in vivo confirmed the major role of injured environment on the engraftment ( ). Stem cells may not engraft into healthy MacPherson 2005 et al., airway because of mucociliary clearance mechanism that could impede access to airway epithelium, as described for gene transfer in vivo to the lungs (

). We speculated that mucociliary clearance would be disrupted in polidocanol (PDOC)-treated airway and that [START_REF] Weiss | Delivery of gene transfer vectors to lung: obstacles and the role of adjunct techniques for airway administration[END_REF] the freshly denuded basement membrane may also promote specifically stem cell adherence and engraftment in the airway epithelium ( ). In the first set of experiments, murine ESCs were intratracheally injected and a significant Another hypothesis to explain MSC negative results at 7 days is the spontaneous regeneration of the airway epithelium that may have hampered MSC incorporation into epithelial airway. At 24hr, e.g. when there is a desquamation of the epithelial surface and a denuded basement membrane, the PDOC murine model is a good model to evaluate stem cell survival and presence in injured airway but stem cell differentiation into airway epithelial cells is unlikely at this time. At 7 days after PDOC administration, the epithelium was spontaneously regenerated, demonstrating that PDOC did not alter endogenous lung capability to regenerate. Therefore, the PDOC model may not be an adequate model to evaluate stem cell capability in regenerating lung epithelium, since the endogenous repair mechanisms may impede MSC contribution to achieve structural lung regeneration. This limitation was described in (

) study in which the Wong 2007 et al., naphthalene-injured airway epithelium was still undergoing rapid cell turnover and regeneration and the number of administered stem cells also decreased with time. A more appropriate animal model to study stem cell contribution to structural lung regeneration would be a model with chronic epithelial airway injury and a recurrent loss of the epithelial surface, due to a lack or an exhaustion of endogenous progenitor cells. This hypothesis was evaluated in animal models with permanent retinal epithelial degeneration such as the RCS rat model ) may be more appropriate to evaluate MSC contribution to lung regeneration. 2008

Finally, an important limitation of our study was also the lack of phenotypic characterisation of surviving cells. This characterisation of stem cell-derived epithelial cells has been hampered by methodological problems, in the lungs as in many other organs such as the heart (

) or the brain ( ), and remains difficult and controversial. In this regard, additional studies using

Ackowledgements:

We thank Nicolas Ferry for providing the nls-lacZ retrovirus vector (Nantes, France) and Marie-France Gardahaut for providing Rosa26 mice (Nantes, France). We are grateful to Beatrice Delasalle for statistical analyses. This work was supported in part by a grant from Vaincre la " Mucoviscidose (Paris, France).

"

Footnotes:

No competing financial interests exist.

)

  techniques, which could explain these conflicting results.

  phosphate-buffered saline (PBS) through the 22-gauge catheter. Cells (10 cells/25 l) or PBS were MacPherson 2005 et al., 6 μ

  ). The culture medium was composed of Dulbecco minimum essential medium with 4.5g/l glucose (DMEM, Invitrogen) supplemented with 15 FCS (Invitrogen), 0.1 non essential amino acids (Invitrogen), 1mM sodium pyruvate (Invitrogen), 2mM % % L-Glutamine (Invitrogen), 10 M mercaptoethanol (Sigma), penicillin/streptomycin (100U/ml and 100 g/ml respectively, Invitrogen)

μ

  A human hepatocellular carcinoma cell line, transformed African green monkey kidney fibroblast cell line (COS-7, ATCC Number CRL-1651) and murine osteosarcoma cell line (mOS-J, ( )) were used for Joliat 2002 et al., control experiments. Cells were cultured in DMEM medium with 4.5 g/l glucose supplemented with 15 FCS, 2 mM L-Glutamine, % penicillin/streptomycin (100U/ml and 100 g/ml respectively). μ Primary culture of adult mesenchymal stem cells

  galactopyranoside dissolved in N,N-dimethylformamide at 20mg/ml before dilution into the reaction mixture, Sigma) for 2hr, at 37 C.] °Cellswere identified as positive for gal activity by blue nuclear staining after X-Gal reaction. The percentage of gal expressing cellsβ βin was obtained by counting the number of nuclear-blue cells in a total of 200 cells. vitro MSCs from Swiss mice were transduced with a lentivirus vector containing the nls-lacZ reporter gene. The vector was derived from pHR LacZ ( ) in which a nls from the SV40 T Ag was cloned in frame 5 to the LacZ cDNA. The lentivirus vector ' Infection), lentivirus vector was removed by changing growth medium and cells were lysed for dosages or kept in culture or injected intratracheally as indicated above. The percentage of gal-transduced MSCs was evaluated by X

β

  cell and trachea-lung samples were analyzed in duplicate. gal activity detection threshold was 230pg. Protein content was measured with β a BCA protein assay kit. Additional experiments were done with lysates of gal-transduced BMC9s or Rosa26 MSCs after three cycles of β freezing and thawing. Viability was ascertained by trypan blue exclusion. Detection of galactosidase activity β Tissues were fixed in 4 paraformaldehyde (at room temperature, 20min) and rinsed 3 times with PBS. Immediately after fixation, % trachea-lungs were stained by intratracheal infusion (though a 19-gauge needle, total volume 5ml) and immersion in 5mM K4Fe(CN)6, = 5mM K3Fe3(CN)6 and 2mM MgCl in PBS containing 0.5mg/ml X-Gal (as indicated above) for 6hr, at 30 C to avoid the detection of 2 °endogenous gal activity ( ). Tissues were again washed with PBS and immediately embedded in paraffin. Tissues were β Weiss 1997 et al., identified as positive for galactosidase activity by nuclear blue staining after X-Gal reaction. Sections (4 m thick) were stained with β μ

%

  Figs.1b,k,n histological findings at 24hr were acute injury of the murine airway epithelium with focal shedding areas observed in the epithelial surface, with only a remaining layer of basal cells or a total denudation of the basement membrane (). However, in some regions, the Figs.1e,h luminal layer of epithelial cells was just disrupted or sloughed whereas the basal cell layer and the basement membrane remained intact (not shown), as already described after intratracheal or intranasal administration ( and , Driscoll 2000 et al., Southam 2002 et al., respectively). Inflammation was present in pulmonary parenchyma ( , arrow heads). Trachea-lung sections at 7 days after PDOC Figs. 1k,n administration demonstrated significant improvement of haemorrhage ( ) and of airway injury with epithelial surface again Figs. 1c,l covered by cilia (). PDOC effect did not alter significantly animal survival ( ). Figs.1f,iTableS1in supplementary data

)

  Fig. 3a= β significantly increased just prior to injection as compared to endogenous gal activity in BMC9s ( , Mann and Whitney test, p β

  Fig.4b

  Fig. 4c n 5). No cell with nuclear blue staining was observed in any animal injected with PDOC and lysate of gal-transduced BMC9s ( , = β

Fig. 6b =

 6b Fig.6b

  -subunit of epithelial Na channel (ENaC ) and showing epithelial degeneration in newborn animals (

Figure 1

 1 Figure 1 Epithelial damage induced by intratracheal administration of polidocanol (PDOC). ( ) Trachea-lungs from ( ) control animal, ( ) animals a c -

Figure 2

 2 Figure 2 cell transfection with reporter genes and CAT gene expression into injured airway 24hr after intratracheal stem cell In vitro in vivo administration. To follow stem cell survival , murine embryonic stem cells (ESCs) or adult mesenchymal stem cells (BMC9s) were in vivo transfected with plasmids encoding CAT or GFP reporter genes just prior to intratracheal administration. ( ) Representative in vitro a histograms of FACS analysis of GFP-expressing ESCs or BMC9s 24hr after transfection. ( ) CAT protein quantification in ESC and in vitro b BMC9 lysates using ELISA, 2hr, 24hr or 7 days after incubation with plasmids and synthetic vectors. ( ) CAT protein quantification in whole c trachea-lung homogenates using ELISA, 24hr after ESC or BMC9 intratracheal injection. Evaluated groups included animals that were intratracheally injected with the followings: no injection (control), PDOC only (PDOC), ESCs or BMC9s only (ESC, BMC9), PDOC and ESCs or BMC9s 24hr later (PDOC ESC, BMC9), PDOC and differentiated cells 24hr later (PDOC Hep3B, mOS-J, COS-7). ( ) CAT + +

Figure 5

 5 Figure 5 cell transfection with reporter genes and CAT gene expression 24hr after intratracheal administration into injured airway of In vitro in vivo murine adult mesenchymal stem cells (MSCs) from primary cultures. ( ) MSC morphology and representative histograms of CD45, CD29 a and Sca-1 labeling, using FACS analysis just prior to intratracheal injection. ( ) To follow MSC survival , MSCs were transfected b d -

  These data confirmed that primary murine MSCs are more difficult to transduce than humanMSCs. 2005 Engraftment of exogenous stem cells into the lungs has been reported in several studies, but cell engraftment rate remains in vivo controversial (). Our results highlight the main advantage in using ELISA and biochemical methods to detect and Loebinger 2008 et al., quantify a small number of administered cells in the whole organ. Although lacZ gene is widely used to locate stem cells, some reports have shown that standard protocols for X-Gal staining can lead, especially in the lungs, both to false positive results as well as to failure to adequately detect gal expressing cells (). In our study, while gal activity significantly increased after intratracheal endogenous from exogenous gal activity. Using nls-LacZ gene, we were able to detect and locate nuclear

							% Sales	et al.,	2007
	murine MSCs (up to 50 , ( % Ricks	et al.,	2008	;	Santoni de Sio	et al.,	2008	;	Xu	et al.,	2008	)) and human MSCs (up to 93 , ( % Chan et al.,
	)). β							Weiss	et al.,	1997	β
	injection of MSCs from Rosa26 mice, surviving Rosa26 MSCs could not be located	in vivo	on histological slides, as already described in
	another report (	MacPherson	et al.,	2005	). The lacZ gene coupled with a nuclear localization signal had already been used in several
	studies from our group (	Lemarchand	et al.,	1994	;	Chapelier	et al.,	1996	) and others (	Mastrangeli	et al.,	1993	) and was shown to allow
	easy detection of β	β

  The negative results of PCR experiments on nls-lacZ gene at 7 days demonstrated that this was not the case. Allogeneic rejection 2005 seems unlikely to be responsible for the lack of survival of differentiated cells at 24hr since ESCs, although known to be targeted for

	Engelhardt	et al.,	1992	Nikolova	et al.,	2006
	survival cell rate was observed, although	in vitro	ESC transfection rate was low. This confirms the great potential of ESC pluripotency for
	regenerative medicine and in particular airway injury. Nevertheless, ESCs for cell-based therapy have met with ethical, moral and political
	challenges, and with inherent risks associated with immune rejection (	Chidgey	et al.,	2008	). In further experiments, we focused our
	attention on MSCs because they are already used in the clinical setting (	Uccelli	et al.,	2008	). Our ELISA data indicated that MSC survival
	rate varied according to immortalized or primary cultures, probably because of the different gene transfer systems we used. Survival rates
	(from 0.4 to 5.5 ) 24hr after intratracheal administration were higher than those described after intravenous administration (from 0.01 % %	%
	to 0.1 ) and in agreement with those usually described in stem cell therapy protocols, including cardiology ( %	Robey	et al.,	2008	) or
	diabetology (	Bottino	et al.,	2003	) protocols, and lung injury models (	Gupta	et al.,	2007	;	Wong	et al.,	2007	;	Wong	et al.,	2009	).
	We hypothesized that MSC incorporation in the lungs at 7 days could be masked by a down regulation of transgene expression into the
	lungs because of the common cytomegalovirus enhancer/promoter element in the virus vector constructs (	Alton	et al.,	1999	;	Pringle et al.,
	). rejection by the immune system (	Chidgey	et al.,	2008	), survived at 24hr. Nevertheless, allogeneic MSC rejection cannot be ruled out at 7
	days.

) and sometimes in conducting airway ( ; 2007 Xu 2007 et al., Loebinger 2008 et al., Sueblinvong 2008 et al., Pereira 1995 et al.,

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

kit according to the instructions of the supplier (Roche Diagnostics). Each sample was analyzed in duplicate. CAT detection threshold was

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF] Zufferey R ,Nagy D , Mandel RJ , Naldini L , Trono D . 1997 ; Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo . Nat Biotechnol . 15 : (9 ) 871 -5

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]

[START_REF] Humphries | Retinopathy induced in mice by targeted disruption of the rhodopsin gene[END_REF]