Impact of bone marrow-derived mesenchymal stem cells on remodeling the lung injury induced by lipopolysaccharides in mice

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1 Reserch Article For reprint orders, plese contct Impct of one mrrow-derived mesenchyml stem cells on remodeling the lung injury induced y lipopolyscchrides in mice Aim: This study evluted the potentil of one mrrow derived mesenchyml stem cells (MSCs) to regulte cytokines nd remodel the lung induced y lipopolyscchride (LPS; O-ntigen). Mterils & methods: A group of mice (n = 21) ws inoculted intrperitonelly with one dose 0.1 ml contining mg LPS/mouse, nd nother treted intrvenously with one dose of leling one mrrow derived MSCs t cell/mouse 4 h fter LPS injection. All nimls were scrificed on the 1st, 7th nd 14th dys post-injection. Results: MSCs incresed the level of IL-10 with suppression of TNF-α, decrese of collgen fiers nd renewl of lveolr type I cells, together with lung tissue remodeling. Conclusion: MSCs were shown to modulte inflmmtory cytokines (TNF-α nd IL-10) nd to differentite into lveolr type I cells, which prevented firosis in lung tissue from LPS-treted mice. Ly strct. This study sought to confirm the remodeling effect of mesenchyml stem cells (MSCs) on lungs injury resulting from lipopolyscchride infection, s lipopolyscchride hs n importnt role in cute lung injury pthogenesis. MSCs decresed the level of TNF-α nd incresed the level of ntinflmmtory cells (IL-10), leding to prevention of firosis with renewl of lveolr type I cells. This reserch ids our understnding of the utility of MSCs in chronic lung injury tretment. Mouchir M Mohi El-Din*,1, Lil A Rshed 2, Mohi A Mhmoud Hridy 1, Atef Mohmed Khlil 1 & Mohmed A Mohmed Aldry 1 1 Pthology & Clinicl Pthology Deprtment, Fculty of Veterinry Medicine, South Vlley University, Qen, Egypt 2 Biochemistry & Moleculr Biology Deprtment, Medicine Fculty, Ciro University, Ciro, Egypt *Author for correspondence: Tel.: mesho_pth@yhoo.com First drft sumitted: 16 My 2016; Accepted for puliction: 8 Novemer 2016; Pulished online: 17 Jnury 2017 Keywords: cytokines flow cytometry fluorescent technique histopthology immunohistochemistry lipolyscchride lungs mesenchyml stem cells mice Lipopolyscchrides (LPSs) from Grmnegtive cteri re one of the most potent innte immune-ctivting stimuli. LPSinducile genes cn regulte the production of cytokines y humn monocytes nd mcrophges [1]. However, LPS lso plys n importnt role in the pthogenesis of cute lung injury in humns nd nimls [2]. The inflmmtion induced y cute lung injury cuses disruption of the lung endothelil nd epithelil rriers nd remins significnt source of moridity nd mortlity [3]. Stem cells my e defined s cells tht re clonogenic, self-renewing nd cple of differentiting into multiple cell lineges [4,5]. Recent findings suggest tht exogenous stem cells derived from emryonic nd dult tissues cn e used for the repir nd regenertion of injured or disesed orgns, including the lungs [6]. Bone mrrow (BM) stem cells cn e moilized to migrte to the injured orgn to mintin physiologic hemostsis [7]. These stem cells re derived from dult BM nd re le to differentite into wide vriety of nonhemtopoietic cells; they lso produce numer of growth fctors (cytokines) tht re importnt for tissue repir nd remodeling [8,9]. Cell-sed therpies using dult stem cells hve emerged s tretment for certin lung diseses, such s emphysem, prt of /fso Mohi El-Din Future Sci. OA (2017) FSO162 eissn

2 Reserch Article Mohi El-Din, Rshed, Mhmoud Hridy et l. pulmonry firosis, pulmonry hypertension nd cute respirtory distress syndrome [10]. The im of this work ws to determine the modultory effects of BM-derived mesenchyml stem cells (BM-MSCs) on inflmmtory rections nd their ility to remodel the lungs, mking BM-MSCs potentil therpy for lung injury. The effects of BM-MSCs were detected using ELISA, rel-time PCR, immunohistochemistry nd histopthologicl techniques. Mterils & methods Experimentl nimls Eighty-four dult mle BALB/c lino mice (6 weeks old in ge) weighing g were purchsed from the Animl House of Misr University Sciences nd Technology nd mintined in specific pthogen-free environment. The study protocol ws pproved y the Animl Ethics Committee t South Vlley University, Qen, Egypt. All nimls were cclimtized in plstic cges (seven nimls per cge) inside well-ventilted room for 1 week prior to the experiment. The nimls were mintined under stndrd conditions (23 ± 3 C temperture, 60 70% reltive humidity nd 12 h light/drk cycle), fed diet of stndrd commercil pellets nd given wter d liitum. LPSs (O-ntigen) from Escherichi coli The LPS product (serotype O127:B8, product numer L 3880, stored t 2 8 C, Sigm-Aldrich, MO, USA) ws extrcted from E. coli (source strin ATCC 12740). This LPS serotype hs een used to study septic shock [11], to induce NOS ctivtion in murine mcrophges [12] nd to induce PAF synthesis in rt glomerulr mesngil cells [13]. LPSs re mde up of hydrophoic lipid (lipid A, which is responsile for the toxic properties of the molecule), hydrophilic core polyscchride chin nd hydrophilic O-ntigen polyscchride side chin. Isoltion & chrcteriztion of BM-MSCs Twenty-one dult mle lino mice (6 weeks old) underwent BM hrvesting y flushing the tii nd femur with Dulecco s modified Egle medium (GIBCO/BRL, ThermoFisher Scientific, Pisley, UK). The nucleted cells were isolted with density grdient (Ficoll/Pque [Amershm Bioscience, NJ, USA]) nd then resuspended in complete culture medium supplemented with 1% penicillin-streptomycin nd 10% fetl ovine serum (GIBCO/BRL). The cells were incuted in 50-cm culture flsk (Flcon, Ciro, Egypt) t 37 C in 5% humidified CO 2 incutor for dys s the primry culture or upon formtion of lrge colonies (80 90% confluent), trypsinized t dy 14 with 0.25% trypsin in 1 ml methylenediminetetrcetic cid (GIBCO/BRL) for 5 min t 37 C [14] nd then counted with hemocytometer. BM-derived MSCs were chrcterized y their dhesiveness nd fusiform shpe nd identified y stining with surfce mrkers CD29, CD90 nd CD105 for MSCs nd CD45 for hemtopoietic cells using flow cytometry [15]. Leling of stem cells with PKH26 dye MSCs were hrvested when the numer of suitle vile cells ( )/mice hd een otined nd were leled ccording to Sigm protocol using PKH26 red fluorescent kit (Sigm-Aldrich, MO, USA) [16,17]. The cells were centrifuged nd wshed twice in serumfree medi then pelleted nd suspended in dye solution nd injected intrvenously into til vein of the mice. The lung tissue ws exmined on the 1st, 7th nd 14th dys post injection to detect nd trce the cells using fluorescence microscopy. Experimentl design Sixty-three dult mle lino mice were divided into three groups (gp; n = 21). Group 1 (control gp): The mice were injected intrperitonelly with one 0.1 ml/mouse dose of phosphte uffer sline (PBS); Group 2 (LPS-infected group): The mice were injected intrperitonelly with one dose of LPS (serotype 0127:B8) t 0.1 ml PBS contining mg LPS/mouse) [18,19]; Group 3 (MSC-treted, LPS-infected group): The mice received n intrvenous injection of one dose of leled BM-derived MSCs t dose of cell/ mouse dissolved in PBS 4 h postinocultion long with LPS t dose of mg/mouse). The nimls were exmined dily, nd clinicl signs nd mortlity nd moridity rtes were recorded. The mice were euthnized with xylzine (40 mg/kg) nd ketmine (400 mg/kg) [20], lood smples were collected from the medil eye cnthus nd roncholveolr lvges (BALs) were collected from the lungs of scrificed mice y intrtrchel injection with 1 ml/mouse of norml sline. The superntnts of the BAL plsm were used for the estimted TNF-α nd IL-10 cytokines evlution crried out y ELISA. Two smples from the lungs were collected from ll scrificed nimls t 1st, 7th nd 14th dys post injection (DPI): one smple ws used for histopthologicl nd imunohistochemistry nlysis nd the other ws kept frozen t -20 C to determine myeloperoxidse expression y rel-time PCR (RT-PCR) /fso Future Sci. OA (2017) FSO162

3 Impct of BM-MSCs on remodeling the lung injury induced y LPS in mice Reserch Article Estimtion of TNF-α & IL-10 levels in plsm & BAL y ELISA The quntittive evlution of the TNF-α nd IL-10 levels in the plsm nd BAL were crried out using mouse ELISA kits tht were purchsed from Boster Biologicl Technology Co. (CA, USA). The test smples nd cytokine stndrds were dded to 96-well pltes coted with coting ntiody, nd the pltes were then incuted t 37 C for 90 min. After incution t 37 C for 30 min, the pltes were developed with tetrmethyl enzidine t 37 C for min. The rection ws stopped y the ddition of 100 μl of stop solution. The sornce ws mesured using n ELISA reder t 450 nm. The concentrtions of TNF-α nd IL-10 were clculted ccording to the stndrd curve using ech of the recominnt cytokines in the ELISA kits [21]. Detection of myeloperoxidse gene expression y RT-PCR The RNA ws extrcted from the lung tissue homogente using EZ-10 Spin Column Blood Mini-Preps Kit (Bio Bsic Inc., ON, Cnd). The extrcted RNA ws then reverse trnscried into complementry DNA (cdna) using Reverse Aid First Strnd cdna Synthesis Kit (Thermo Scientific, Vilnius, Lithuni). cdna ws generted from 10 μl of the totl RNA tht hs een extrcted with 0.5 μl of Oligo (dt) 18 primer nd 0.5 μl of moloney murine leukemi virus reverse trnscriptse enzyme (50 U/μl) for 60 min t 42 C in progrmmed therml cycler (HYBAID, MA, USA). The reltive undnce of mrna species ws ssessed with n ABI prism 7500-sequence detector system (Applied Biosystems, CA, USA). PCR primers were designed using Gene Runner softwre (Hsting Softwre, Inc., NY, USA) from RNA sequences from Gene Bnk myeloperoxidse (MPO) forwrd (5 -ACCTACCCCAGTACCGATCC-3 ) nd reverse (5 -AACTCTCCAGCTGGCAAAAA-3 ) primers nd P-ctin: forwrd (5 -TCT GGC ACC ACA CCT TCT ACA ATG-3 ) nd reverse (5 -AGC ACA GCC TGG ATA GCA ACG-3 ). All primer sets hd clculted nneling temperture of 60 C. Quntittive RT-PCR ws performed in duplicte in 25 μl rection volume consisting of 12.5 μl 2x SiriHot Mster Mix (BIORON GmH, Ludwigshfen, Germny), 1 μl of ech primer, 5 μl of cdna nd 5.5 μl RNAse-free wter using the Applied Biosystem Step One Plus TM RT-PCR system therml cycling lock. The mplifiction conditions were 2 min t 50 C nd 40 cycles of denturtion t 95 C for 15 s nd nneling t 60 C/extension t 72 C for 1 min. Dt from the rel-time ssys were clculted using the v17 Sequence Detection Softwre from PE Biosystems (CA, USA). The reltive expression of MPO ws clculted using the comprtive Ct method. All vlues were normlized to the P-ctin gene [22]. Sttisticl nlysis Sttisticl nlysis ws induced using one-wy nlysis of vrince. It ws done to compre the control nd ll other treted groups nd ws followed y post hoc nly sis (Dunnett s test) using the Sttisticl Pckge for the Socil Sciences, version 17 [23]. The dt were presented s the men ± stndrd devition. The difference ws considered sttisticlly significnt when p < Fluorescent microscope exmintion Five microns of prffin-emedded, unstined lung sections were prepred nd deprfinized then exmined using fluorescent microscopy to ensure homing of leled-msc cells in the lung tissues, which hd n excittion of 551 nm nd n emission of 567 nm. Immunohistochemistry method (CD105 immunostining) The immunohistochemistry method used DkoCytomtion s Envision system nd polyclonl primry rit CD105 ntiody (Bioryt, Cmridge, Englnd). Five-micron thick seril sections were prepred nd plced on positively chrged slides. Antigen retrievl ws performed using progrmmed PT-Link contining Envision FLEX trget retrievl solution, High ph (50 ), for 20 min. Afterwrd, the sections were treted with 100 μl of primry CD105 ntiody (1:200) except for the negtive control slides (100 μl of ntiody diluents were pplied without ny primry ntiody) t 4 C overnight. After wshing in the Envision FLEX Wsh Buffer (Bioscience, Eremodegem, Belgium), nd treted with the Envision FLEX/HRP Buffer (Bioscience) for 30 min. The sections were stined with Myer s hemtoxylin for 3 5 min [24]. Control stining ws performed, nd no positive stining ws found on the control slides. Histopthologicl exmintion Lung specimens from ll groups were fixed, in 10% neutrl uffer formlin fter intrtrchel infltion of the lung with 10% neutrl uffer formlin, then dehydrted in lcohol nd prepred in prffin sections. Five-micron sections were prepred nd stined using Hrris hemtoxylin nd eosin nd Msson s trichrome stin [25] for the histopthologicl exmintion. Results Identifiction of MSCs in mice The microscopic exmintion for flushing nd determining the centrifugtion BM yield just efore the incution showed ptches of ggregted cells. How /fso

4 Reserch Article Mohi El-Din, Rshed, Mhmoud Hridy et l. Proinflmmtory (TNF- α) & nti-inflmmtory (IL10) levels in the plsm & BAL Quntittive ELISA nlysis of TNF-α in oth (plsm & BAL) TNF-α level in Plsm As shown in Figure 4, the men level of plsm TNF-α ws significntly incresed in gp 2 t 1st nd 7th DPI (p < 0.001) when compred with the control group. Similrly, the men level of plsm TNF-α ws significntly incresed in gp 3 on the 1st DPI (p < 0.001) nd the 7th DPI (p < 0.05) when compred with the control group. In contrst, it ws significntly decresed in gp 3 on the 1st nd 7th DPI (p < 0.001) when compred with gp 2. Menwhile, on the 14th DPI, gp 2 (p = 0.650) nd gp 3 (p = 0.435) were significntly decresed nd pproximtely reched the prmeters of gp 1. Figure 1. Bone mrrow mesenchyml stem cells. Ptches of ggregted cells (rrow) efore incution (A). Elongted, fusiform, nd spindle cells t the 3rd dy post injection (DPI), (rrow) (B). Reltively homogenous cell cultures ofmesenchyml stem cells resemle the firolst morphology on the 7th DPI (C). Reltively homogenous cell cultures on the 14th DPI (x200). ever, fter 3 dys of incution, elongted, fusiform nd spindle-shped cells were extensively proliferted nd dhered to the wll of the flsk. A reltively homogenous cell culture of BM-MSCs with wht resemled firolst morphology ppered fter 7 dys of incution. The cell popultion reched 70 80% confluence on the 14th dy of incution with firolstoid morphology (Figure 1). The identifiction nd purifiction of MSCs y flow cytometric nlysis sed on cell surfce mrker expression detected tht MSCs were uniformly negtive for CD45 nd positive for CD29, CD90 nd CD105 (Figure 2). TNF-α level in BAL As shown in Figure 5, the men level of BAL TNF-α ws significntly incresed (p < 0.001) in gp 2 on the 1st nd 7th DPI when compred with the control group. Moreover, the men BAL TNF-α level in gp 3 hd significntly decresed (p < 0.001) on the 1st nd 7th DPI when compred with gp 2, ut no significnce ppered on the 1st nd 7th DPI (p = nd p = 0.437, respectively) when compred with the control group. In ddition, BAL TNF-α ws nonsignificntly chnged in gp 2 (p = 0.303) nd gp 3 (p = 0.832) on the 14th DPI when compred with the control group. Quntittive ELISA nlysis of IL-10 in plsm & BAL IL-10 level in plsm As shown in Figure 6, the men level of plsm IL-10 ws nonsignificntly chnged in gp 2 on the 1st, 7th nd 14th DPI (p = 0.102, p = nd p = 0.132, respectively) when compred with the control group. Moreover, the men level of plsm IL-10 ws significntly incresed (p < 0.001) in gp 3 on the 1st, 7th nd 14th DPI when compred with the control group nd gp 2. The men level of plsm IL-10 in gp 3 on the 14th DPI ws significntly incresed (p < 0.001) when compred with tht of the 1st nd 7th DPI in the sme group. Fluorescent microscope results Fluorescence microscopy for the slides prepred from lung sections fter deprffiniztion with unstined sections nd no tretment showed no uto-fluorescence for oth gp 1 nd gp 2. Red fluorescent spots (MSCs leled with PKH26 fluorescent dye) were displyed in gp 3, confirming the movement of these cells into the lung tissues on the 1st, 7th nd 14th DPI with the most diffuse concentrtion on the 7th DPI (Figure 3). CD CD90 CD CD Figure 2. Flow cytometric chrcteriztion nlysis of one mrrow mesenchyml stem cells. The cells were uniformly negtive for CD45 (A) nd positive for CD29 (B), CD90 (C), nd CD105 ntiodies (D) /fso Future Sci. OA (2017) FSO162

5 Impct of BM-MSCs on remodeling the lung injury induced y LPS in mice Reserch Article chnge ws oserved on the 7th DPI (p = 0.241) or the 14th DPI (p = 0.147) in gp 3 compred with gp 1. Figure 3. Photomicrogrph of fluorescent microscopy for the lung sections of mice. No uto-fluorescence ws oserved in the unstined (gp 1) nd no treted group (gp 2) (A). In the treted group (gp 3), with PKH26 leled-mscs on the 1st dy post injection (DPI), red fluorescent spots (rrows) (B). Diffuse of red fluorescent spots (rrow) on the 7th DPI (C). Vrile sized red fluorescent spots show homing of the cells in the lung tissue (rrows) on the 14th DPI (x200) (D). IL-10 level in BAL As shown in Figure 7, the men level of BAL IL-10 ws significntly decresed (p < 0.001) in gp 2 on the 1st, 7th nd 14th DPI when compred with the control group. In contrst, gp 3 s men level of BAL IL-10 hd significntly incresed on the 1st, 7th nd 14th DPI (p < 0.005, p < nd p < 0.001, respectively) when compred with gp 2. While the men level of BAL IL-10 in gp 3 ws significntly decresed on the 1st DPI (p < 0.001) when compred with the control group, no significnt Quntittive RT-PCR for myeloperoxidse gene expression in lung tissue As shown in Figure 8, the men MPO gene expression in the lung ws significntly incresed on the 1st, 7th nd 14th DPI (p < 0.002, p < nd p < 0.001, respectively) in gp 2 when compred with the control group. At the sme time, the men MPO gene expression in gp 3 ws nonsignificntly chnged t 1st nd 7th DPI (p = nd p = 0.057, respectively) in comprison with the control group, while slightly significnt increse showed t 14th DPI (p < 0.05) when compred with the control group. Moreover, the men MPO gene expression in gp 3 ws significntly decresed in gp 3 on the 1st, 7th nd 14th DPI (p < 0.05, p < nd p < 0.001, respectively, when compred with gp 2. Menwhile, no significnt difference ws identified in the men MPO gene expression in gp 3 for the 7th nd 14th DPI (p = nd p = 0.170) when compred with the sme group for the 1st DPI. Immunohistochemistry results The microscopic ppernce of lung tissues stined y CD105 ntiody for BM-MSCs showed no rection (rown grnules) in the pulmonry tissues in the noninfected mice (gp 1) on the 1st, 7th nd 14th DPI (without or with ppliction of primry ntiody) (Figure 9A C). The sme ppernce with no positive rection for peroxidse ctivity ws detected in the infected pulmonry tissues in LPS-infected mice (gp 2) Plsm TNF-α level gp.1 gp.2 gp.3 Concentrtion (pg/ml) c c 0 1d 7d 14d Time p.i. of LPS Figure 4. Histogrm of the men vlues (± stndrd error of the men) of the TNF- level in the mice plsm (gps 1, 2, nd 3) on the 1st, 7th, nd 14th dys post injection. Different letters indicte significnt chnge when p < LPS: Lipopolyscchride; p.i: Post-inocultion /fso

6 Reserch Article Mohi El-Din, Rshed, Mhmoud Hridy et l BAL TNF-α level gp.1 gp.2 gp.3 Concentrtion (pg/ml) d 7d 14d Time p.i. of LPS Figure 5. Histogrm of the men vlues (± stndrd error of the men) of the TNF- level in the BAL of mice (gps 1, 2, nd 3) on the 1st, 7th, nd 14th dys post injection. Different letters indictes significnt chnge when (p < 0.05). BAL: Broncholveolr lvge; LPS: Lipopolyscchride; p.i: Post-inocultion. on the 1st, 7th nd 14th DPI, respectively (Figure 9D F). However, the lungs in gp 3, which ws treted with BM-derived MSCs on the 1st DPI, displyed positive grnulr rown rection inside the lumen of perilveolr lood vessels nd surrounded the lveolr tissue (Figure 9G). All cses of LPS infected mice treted with BM-derived MSCs on the 7th DPI noticed tht MSCs formed into spindle-shped cells nd replced most of the lveolr epithelil cells nd lrge rown cells (ctivted mcrophges with positive grnulr rown rection in their cytoplsm in perironchiolrs nd mong the septl cells; Figure 9H). The sme group on the 14th DPI detected rownish colortion (mcrophges) nd rown color rection. MSCs covered most of the pulmonry tissues nd surrounded the ronchioles inside the perironchil lood vessels (Figure 9I). Pthologicl results With respect to the recorded clinicl signs, the mortlity rte ws 0% in ll experimentl groups. The mor gp.1 gp.2 gp.3 Plsm IL-10 level Concentrtion (pg/ml) d 7d 14d Time p.i. of LPS Figure 6. Histogrm of the men vlues (± stndrd error of the men) of the IL-10 level in the plsm of mice (gps 1, 2, nd 3) on the 1st, 7th, nd 14th dys post injection. Different letters indicte significnt chnge when p < LPS: Lipopolyscchride; p.i: Post-inocultion /fso Future Sci. OA (2017) FSO162

7 Impct of BM-MSCs on remodeling the lung injury induced y LPS in mice Reserch Article BAL IL-10 level gp.1 gp.2 gp.3 Concentrtion (pg/ml) c d 7d 14d Time p.i. of LPS Figure 7. Histogrm of the men vlues (± stndrd error of the men) of the IL-10 level in BAL of mice (gps 1, 2, nd 3) on the 1st, 7th, nd 14th dys post injection. Different letters indicte significnt chnge when p < BAL: Broncholveolr lvge; LPS: Lipopolyscchride; p.i: Post-inocultion. idity rte reched up to 100% in gp 2 (LPS-induced lung injury) in comprison to 0% in gp 1 (control) nd vrile moridity rtes in gp 3 (MSC-treted), which differed ccording to the time of tretment (90% on the 1st DPI, 40% on the 7th DPI nd <10% on the 14th DPI). Moridity lwys included loss of ppetite nd rpid respirtory rte. The microscopic ppernce of lung tissues stined with hemtoxylin nd eosin in noninfected mice (gp1) on the 1st, 7th nd 14th DPI showed norml structure of pulmonry tissues (lveoli, ronchi nd ronchioles) (Figure 10A C). The lungs of LPS-infected mice (gp 2) on the 1st DPI showed focl res of lveolitis with thickening in the sept nd prolifertion in the epithelil lining of the ronchioles. Edemtous lungs found mnifested y vesicle densities in type I epithelil cells of the lveolr sept (Figure 10D). The sme group displyed severe interstitil pneumoni with emphysem nd hemorrhge, esides congestion in ll lood vessels on the 7th DPI. Alveolitis ws mnifested y thickening of the interlveolr sept with ggregtions of inflmmtory cells (minly neutrophils nd firolsts), esides vsculitis gp.1 gp.2 gp.3 MPO gene expression c d 7d 14d Time p.i. of LPS Figure 8. Histogrm of the men vlues of MPO gene expression in the lungs of mice (gps 1, 2, nd 3) on the 1st, 7th, nd 14th dys post injection. Different letters indicte significnt chnge when p < LPS: Lipopolyscchride; p.i: Post-inocultion /fso

8 Reserch Article Mohi El-Din, Rshed, Mhmoud Hridy et l. Figure 9. Lung tissue stined y immunohistochemistry. No rection (rown grnules) in the lung tissues (gp 1) (A, C & D) (rs = 200 μm), with no positive rection for peroxidse ctivity in tissues from lipopolyscchridetreted mice (gp 2) (B, E & F) (rs = 100 μm). Mesenchyml stem cells were confirmed from positive grnulr rown rection (rrow) inside the lumen of the lveolr lood vessels, formed s spindle-shpe cells (mesenchyml stem cells), nd replced the lveolr epithelil cells nd lrge rown cells (ctivted mcrophges with positive grnulr rown rection in their cytoplsm; MQ), which covered the perironchiolr tissues on the 1st, 7th, nd 14th dys post injection (G, H & I) (All rs = 50 μm). were seen (Figure 10E). However, LPS-infected mice on the 14th DPI displyed inflmmtory edem in the lung mnifested y vesicle densities in type I epithelil cells of the lveolr sept, with ggregtion of inflmmtory cells minly neutrophils mong the lveoli (Figure 10F). The common lesions tht ppered in these cses were nrrowing in the lumen of the ronchioles, congestion in perironchil lood vessels nd inflmmtory cells nd firolsts cells infiltrted mong the pulmonry tissue. MSC-treted LPS infected mice (gp 3) on the 1st DPI, displyed signs of lung recovery where the min inflmmtory cells, prticulrly mcrophges nd few neutrophils, were scttered mong the wll of the ronchioles nd the lveolr cells (Figure 10G). MSCs (gp 3) restored lveolr epithelil type I (ATI) cells to their norml structure, on the 7th DPI, where numerous spindle cells light in color filled the lveolr tissues, nd the lungs ppered norml in the lveolr nd ronchiolr structures in some cses (Figure 10H). In contrst, focl res of mononucler cells were detected in the pulmonry tissue, with few emphysemtous res. Multi ple ggregtions of inflmmtory cells, pr /fso Future Sci. OA (2017) FSO162 ticulrly mcrophges nd neutrophils, re commonly seen in the lung tissues of some nimls. Other cses showed greter degree of progression s indicted y the decline in inflmmtory cells t the lveolr sept nd nerly norml structure in the lungs tissues. On the 14th DPI, the lungs in gp 3 displyed prolifertion of the perironchil lymphoid tissues with nerly norml lveolr nd ronchiolr structures (Figure 10). The microscopic ppernce of the lung tissues stined y Msson s trichrome in the noninfected mice (gp 1) on the 1st, 7th nd 14th DPI indicted tht few collgen fiers were stined lue in color in the perironchil nd perivsculr tissues in norml lungs (Figure 11A C). The injured lungs in gp 2 on the 1st DPI hd only few collgen fiers stined lue in color in the perivsculr nd perironchil tissues (Figure 11D). However, the sme group on the 7th DPI demonstrted tht undles of collgen fier hd replced the lveolr cells (Figure 11E). On the 14th DPI, the lungs in ll cses in gp 2 showed tht collgen fiers stined lue hd replced most of the injured lveolr cells (Figure 11F). The MSC-treted LPS-infected

9 Impct of BM-MSCs on remodeling the lung injury induced y LPS in mice mice (gp 3) displyed few perivsculr collgen fiers in the lungs on the 1st DPI (Figure 11G). On the 7th DPI, the injured lungs of the sme group displyed normlly ppering lveolr tissues with few collgen fiers, perilveolr lood vessels nd perironchil tissues (Figure 10H). The sme lesions were displyed in the lungs in gp 3 on the 14th DPI, where few collgen fiers dherent to the ronchil nd p erironchil tissue were oserved in some mice (Figure 11I). Discussion BM-MSCs plyed n integrl role in the heling of LPSinduced lung injury, which ws mnifested y edem, lveolitis with deposits of collgen mtrix, leuko cyte recruitment nd ronchiolitis (gp3). Previous studies hve shown tht some of these roles detected tht BMderived MSCs convert the systemic endotoxin response from proinflmmtory one to n nti-inflmmtory milieu y suppressing the genertion of proinflmmtory meditors without hmpering the genertion of ntiinflmmtory meditors [19]. Our results showed tht for the LPS infected group (gp 2) on the 1st nd 7th DPI, there re significntly incresed plsm nd BAL levels of TNF-α s well s MPO gene expression levels. However, in comprison with the control group on the 1st, Reserch Article 7th nd 14th DPI, the plsm level of IL-10 showed no significnt chnge in the LPS-treted gp (2), while there ws significnt increse in the MSC-treted gp (3). This result confirmed the immuno-modultory effect of MSCs tht lters cytokine secretion nd elevtes the nti-inflmmtory response y incresing the IL-4 secretion nd n inhiited the production of TNF-α [26,27]. Additionlly, MSCs suppressed endotoxin- induced lung inflmmtion in cute lung injury y decresing neutrophilic infiltrtion nd edem. In ddition, MSCs inhiit inflmmtory cells nd suppress the genertion of proinflmmtory meditors (such s TNF-α), while mintining locl BAL level of IL-10, which contriutes to lung injury repir [19]. MSCs significntly decresed collgen deposition, which cn e ttriuted to the ction of MSCs in locking the production of TNF-α nd IL-1 tht medite firotic lung injury [28]. MSCs secrete fctors to upregulte the secretion of IL-10 vi peripherl lood mononucler cells, tolerogenic mcrophges [29] nd tolerogenic DCs [30 32]. BM-derived MSC hve the ility to engrft nd differentite into specific nd distinct lung cell phenotypes, ssocited with the suppression of inflmmtion, reduction in the totl neutrophils counts, decrese in inflmmtory cytokines (TNF-α) nd triggering the Figure 10. Lung tissue stined with hemtoxylin nd eosin. Norml lung structure (gp 1) (A, B & C) (rs = 200 μm). Lipopolyscchride-induced edemtous lungs mnifested y vesicle densities in type I epithelil cells of the lveolr sept (gp 2) on the 1st nd 14th dys post injection (DPI) (D & F). In ddition, interstitil pneumoni (rrowhed), firosis (thin rrow), nd vsculitis (thick rrow) ws seen on the 7th DPI (E) (rs = 100 μm). Mesenchyml stem cells restored lveolr epithelil type I cells to their norml structure, with mild inflmmtory rection noticed in gp 3 on the 1st nd 7th DPI (G & H) (rs = 50 μm), on the 14th DPI (I) (r = 200 μm) /fso

10 Reserch Article Mohi El-Din, Rshed, Mhmoud Hridy et l. Figure 11. Lung tissue stined y Msson s trichrome. Norml structure with no collgen fier seen in mouse lung tissue (gp 1) (A & B) (rs = 100 μm), (C) (r = 200 μm). Lipopolyscchride-induced firosis round the perironchiolr nd perivsculr tissues (rrowhed) in gp 2 on the 1st nd 7th dys post injection (D & E) (rs = 100 μm), incresed on the 14th dy post injection (F) (rs = 50 μm). Mesenchyml stem cells limit l ipo poly scchride-induced firosis in mouse lung tissue (rrowhed; gp 3) (G) (r = 200 μm), (H & I) (rs = 100 μm). production of reprtive growth f ctors, to protect the lungs from injury nd firosis [33,34]. To confirm the ction of MSCs inside lung tissue, we looked for the presence of MSCs in lung tissue y using fluorescent detection which clrifies tht the PKH26leled MSCs in gp 3 were ttrcted to this tissue fter the 1st DPI nd remined in the lung until the 14th DPI. These results sustntite the ility of cells to migrte nd set up long-term engrftment t the site of injury fter the intrvenous injection of MSCs [35]. In contrst, PKH26-leled MSCs were detected in the lood nd lungs t 2 6 h fter the injection [36]. Furthermore, MSCs homed in on dmged tissues y moving from the loodstrem to inflmmtory sites vi the utiliztion of dhesion molecules, such s selectins nd integrins, nd chemokines nd their receptors [37]. Moreover, MSCs express lrge rnge of receptor tyrosine kinse growth fctors, such s PDGF or IGF-1, to home in on MSCs [38]. By immunohistochemistry stining using CD105 ntiody ( surfce mrker for MSCs), our studies trcked the progress of MSCs inside lung tissues to discover other modultory roles for MSCs in lung tissue heling. The CD105 ntiody is used to screen nd determine tissue-specific incorportion of donor-derived /fso Future Sci. OA (2017) FSO162 cells in recipient nimls [39]. Our work indicted tht spindle-shped cells (with cytoplsmic rown grnules confirming the presence of MSCs) were in the intrvsculr spce nd dispersed mong septl cells in tissues from MSC-treted mice (gp 3), prticulrly on the 1st DPI, nd in distriuted pttern on the 14th DPI. MSC homing ws first loclized inside the lumen of the lveolr lood vessels on the 1st DPI nd then MSCs migrted nd covered ll dmged lung tissues on the 7th nd 14th DPI. Trnsplnted MSCs interct with the lood vessel wll during extrvstion. MSCs exhiited rolling nd firm dhesion onto endothelil cells (ECs), which incresed when ECs were prestimulted with TNF-α nd inding ws vi P-selectin in vivo [40]. Alveolr cpillry ECs ply role in promoting lveolr regenertion [41]. A previous study showed tht dult stem cells cn undergo oth self-renewl nd differentition in multiple line ges, nd from these properties, it ws suggested tht BM-derived stem cells could repir dmged tissues y differentiting intoepithelil cells in disprte sites [34]. In vitro differentition studies hve demonstrted the potentil of MSCs to differentite into lveolr nd irwy epithelil cells [42,43]. Histopthology confirmed the ction of MSC in the pulmonry tissue of gp 3 mice in response to LPS-

11 Impct of BM-MSCs on remodeling the lung injury induced y LPS in mice Reserch Article induced lung injury, which cused mny chnges, including lveolr edem mnifested y vesicle densities in type I epithelil cells of the lveolr sept on the 1st, 7th nd 14th DPI. At the 1st DPI, focl res of lveolitis were evident from diffuse neutrophil infiltrtion nd few mcrophge cells ssocited with undles of collgen fiers (confirmed y Msson s trichrome stin). At the 7th nd 14th DPI, there ws progression of interstitil pneumoni. The improvement of MSCs to repir the lveolr nd ronchiolr tissues ws mnifested y reduced inflmmtion nd limited collgen depositions on the 7th nd 14th DPI. These positive effects were ttriuted to the fct tht MSCs secrete lrge quntities of ioctive fctors, which suppress the locl immune system, inhiit firosis nd poptosis, esides enhncing ngiogenesis nd the differentition of tissue intrinsic progenitor cells [44]. In ddition, one study demonstrted tht murine MSCs re home to the lungs in response to injury, dopt n epithelil-like phenotype, nd reduce inflmmtion nd collgen deposition in the lung tissue of mice chllenged with leomycin [45]. The reduction in hemorrhge nd edem tht ws oserved in MSC-treted gp 3 cn e ttriuted to the preservtion of endothelil nd epithelil tissue integrity tht is medited y MSCs, which is essentil for the mintennce of dequte homeostsis in oth the pulmonry nd systemic circultions [46]. When MSCs come into contct with injured tissues, they relese solule fctors tht re cple of modulting cell prolifertion [47]. In our work, the endotoxin (LPS) induced chnges in lung tissue nd the engrftment of MSCs inside lung tissues nd dherence to ECs nd lveolr tissues is ttriuted to the MSCs ility to help in the initition of lveolr epithelil type II (ATII) cell ctivtion. ATII cells hve een considered to e similr to stem cells in dult lungs, nd they ehve s progenitor cells y proliferting nd differentiting into type I cells following injury, during lveolr homeostsis nd during repir [48 51]. Developmentl studies hve confirmed tht the progenitor cell properties of ATII cells re controlled y the cell mtrix nd cell cell interctions relted to their prticulr environments efore or fter injury [52 55]. It is not cler if these puttive regenertive ATII cells, which pper postinjury, re derived from the expnsion of existing stem cell pools locted in n undefined niche or re derived from quiescent, terminlly differentited ATII cells. Therefore, it would e interesting to identify which signls or fctors induce the formtion of progenitor-like ATII cell sugroups following injury [56]. BM-derived cells re cple of forming lung lveolr epithelium, nd it ws demonstrted tht cultured BM cells could ct s type I pneumocyte precursors [57]. In contrst, our findings suggest tht ATI cells re the most ffected y LPS (endotoxin) leding to chnges in the extrcellulr mtrix. A solution to LPSinduced lung tissue injury ws evident y lung tissue improvement in the mjority of mice treted with MSCs derived from BM (gp 3). We conclude tht MSCs my hve progenitor cell properties such s ATII cells or tht they my e le to serve s ATII tht cn convert to ATI cells in order to remodel lung tissue. Conclusion MSCs could regulte inflmmtory cytokines through the ccumultion of mcrophges tht stimulted the nti-inflmmtory ctivity of IL-10 nd suppressed TNF-α y reduction of neutrophils in lung inflmmtion. In ddition, MSCs proly ct s progenitors for remodeling lveolr tissues nd prevent the firosis, s well s, ATII cells tht cn convert to ATI cells. Future perspective Our work reported on the remodeling effect of MSCs on cute lung injury. In the future, this study my llow us to look for the effect of MSCs on tret chronic lungs injury nd will progress to how it cn lysis the firosis in lungs y using MSCs in gene therpy. Acknowledgements The uthors thnk MB El-Begwy, Professor of Pthology, Fculty of Veterinry Medicine, Beni Suef University, Egypt for gret ssistnce in immunohistochemistry stining. Authors contriutions All uthors contriuted in the prcticl prts, the sttisticl nlysis, interpreted the results nd prepred the mnuscript ut MM Mohi El-Din supervised the work, prepred nd gve input to ll res of this mnuscript. All uthors hve red nd pproved the finl mnuscript. Finncil & competing interests disclosure The uthors received finncil id from South Vlley University, Qen, Egypt. The uthors hve no other relevnt ffilitions or finncil involvement with ny orgniztion or entity with finncil interest in or finncil conflict with the suject mtter or mterils discussed in the mnuscript prt from those disclosed. No writing ssistnce ws utilized in the production of this mnuscript. Ethicl conduct of reserch The protocols of this study ws pproved y the Animl Ethics Committee t South Vlley University, Qen, Egypt. Open ccess This work is licensed under the Cretive Commons Attriution 4.0 License. To view copy of this license, visit /fso

12 Reserch Article Mohi El-Din, Rshed, Mhmoud Hridy et l. Executive summry Lipopolyscchride (LPS) lso plys n importnt role in the pthogenesis of cute lung injury, resulting in significnt moridity nd mortlity in oth humns nd nimls. Regenertion of lung tissue y one mrrow-derived mesenchymls We evluted the modultory effect of one mrrow-derived mesenchymls (BM-MSCs) on inflmmtory cytokines, including TNF-α nd IL 10, nd their differentition into lveolr type I (ATI) cells through their direct contct with injured murine lung tissue. MSCs in culture were chrcterized y CD29, CD90 nd CD105 surfce mrkers of mouse MSCs using flow cytometry. Experimentl design on mice Sixty-three mice were divided into three groups (n = 31). The first group ws the control group. The second group ws inoculted with one 0.1-ml intrperitonel dose of mg LPS/mouse. The third group ws treted with intrvenous injection with one dose of leling (BM-MSCs) t ( cell/mouse), 4 h post-inocultion with (0.1 ml LPS/mouse). All mice were euthnized on the 1st, 7th nd 14th dys post injection. Blood, roncholveolr lvges (BALs) smples nd lung tissues were collected to mesure levels of IL-10, TNF-α nd myeloperoxidse with rel-time PCR, histopthologicl nd immunohistochemistry nlysis, respectively. Alterntive effects of cytokines in the presence of BM-MSCs The levels of proinflmmtory cytokines (TNF-α) were significntly decresed in BAL nd plsm, wheres the levels of nti-inflmmtory cytokines (IL-10) were significntly higher in BAL nd plsm in MSC-treted LPS-infected group (gp 3). This finding confirmed the immuno-modultory effect of MSCs on LPS-induced lung injury. Histopthologicl & immunohistochemistry results from of the LPS-induced lung injury & MSCs treted lung injury The lungs in the LPS-infected group (gp 2) exhiited lveolitis, interstitil edem nd interstitil pneumonitis, in ddition to vesicle densities in ATI cells from ll scrificed nimls on the 1st, 7th nd 14th DPI. The lungs in the MSC-treted LPS-infected lung, displyed decrese in pulmonry edem nd decrese in collgen fiers with renewl in ATI cells leding to fst recovery. Conclusion MSCs showed n ility to oth modulte inflmmtory cytokines (TNF-α nd IL-10) nd to differentite into cells, which led to the prevention of lung firosis in mice. References Ppers of specil note hve een highlighted s: of interest; of considerle interest. 1 Rossol M, Heine H, Meusch U et l. LPS-induced cytokine production in humn monocytes nd mcrophges. Crit. Rev. Immunol. 31(5), (2011). 2 Frevert CW, Wrner AE. Respirtory distress resulting from cute lung injury in the veterinry ptient. J. Vet. Intern. Med. 6(3), (1992). 3 Johnson ER, Mtthy MA. Acute lung injury: epidemiology, pthogenesis, nd tretment. J. Aerosol. Med. Pulm. Drug Deliv. 23(4), (2010). 4 Weissmn IL. Stem cells: units of development, units of regenertion, nd units in evolution. Cell 100(1), (2000). 5 Weissmn IL. Trnslting stem nd progenitor cell iology to the clinic: rriers nd opportunities. Science 287(5457), (2000). 6 Loeinger MR, Jnes SM. Stem cells for lung disese. Chest 132(1), (2007). 7 Korling M, Estrov Z. Adult stem cells for tissue repir: new therpeutic concept? N. Engl. J. Med. 349(6), (2003). 8 D Agostino B, Sullo N, Sinisclco D, De Angelis A, Rossi F. Mesenchyml stem cell therpy for the tretment of chronic ostructive pulmonry disese. Expert Opin. Biol. Ther. 10(5), (2010). 9 Suelinvong V, Weiss DJ. Cell therpy pproches for lung diseses: current sttus. Curr. Opin. Phrmcol. 9(3), (2009). 10 Suelinvong V, Weiss DJ. Stem cells nd cell therpy pproches in lung iology nd diseses. Trnsl. Res. 156(3), (2010). 11 Xu H, Gonzlo JA, St Pierre Y et l. Leukocytosis nd resistnce to septic shock in intercellulr dhesion molecule 1-deficient mice. J. Exp. Med. 180(1), (1994). 12 Stuehr DJ, Mrlett MA. Mmmlin nitrte iosynthesis: mouse mcrophges produce nitrite nd nitrte in response to Escherichi coli lipopolyscchride. Proc. Ntl Acd. Sci. USA 82(22), (1985). 13 Wng J, Kester M, Dunn MJ. The effects of endotoxin on pltelet-ctivting fctor synthesis in cultured rt glomerulr mesngil cells. Biochim. Biophys. Act 969(3), (1988). 14 Alhdlq A, Mo JJ. Mesenchyml stem cells: isoltion nd therpeutics. Stem Cell Dev. 13(4), (2004). 15 Sung JH, Yng HM, Prk JB et l. Isoltion nd chrcteriztion of mouse mesenchyml stem cells. Trnsplnt. Proc. 40(8), (2008). 16 Muirhed KA, Trio JDJ, Wllce PK. Cell Lines: Models of Disese. Cell Trcking with Lipophilic Memrne Dyes. Biowire Spring, (2011). 17 Trio JD Jr, Muirhed KA, Pn D, Munson ME, Wllce PK. Trcking immune cell prolifertion nd cytotoxic potentil using flow cytometry. Methods Mol. Biol. 699, (2011) /fso Future Sci. OA (2017) FSO162

13 Impct of BM-MSCs on remodeling the lung injury induced y LPS in mice Reserch Article 18 Rojs M, Woods CR, Mor AL, Xu J, Brighm KL. Endotoxin-induced lung injury in mice: structurl, functionl, nd iochemicl responses. Am. J. Physiol. Lung Cell Mol. Physiol. 288(2), L333 L341 (2005). 19 Xu J, Woods CR, Mor AL et l. Prevention of endotoxininduced systemic response y one mrrow-derived mesenchyml stem cells in mice.. Am. J. Physiol. Lung Cell. Mol. Physiol. 293(1), L131 L141 (2007). 20 Lery S, Underwood W, Anthony R et l. AVMA Guidelines for the Euthnsi of Animls: 2013 Edition. Americn Veterinry Medicl Assocition, Schumurg, IL, USA (2013) Sun H, Zhng J, Chen F, Chen X, Zhou Z, Wng H. Activtion of RAW264.7 mcrophges y the polyscchride from the roots of Actinidi erinth nd its moleculr mechnisms. Crohydr. Polym. 121, (2015). 22 El-Denshry ES, Rshed LA, Elhussiny M. Mesenchyml stroml cells versus etmethsone cn dmpen disese ctivity in the collgen rthritis mouse model. Clin. Exp. Med. 14(3), (2014). 23 Borenstein M, Rothstein H, Cohen J. Smple Power Sttistics 1.0.SPSS, Inc Elsevier Inc Chicgo, IL, USA (1997). 24 Tylor CR, Rudeck L. Immunohistochemicl Stining Methods Eduction Guide (6th Edition). Dko Denmrk, Glostrup, Denmrk (2013). 25 Drury RB, Wllington EA. Crleton s Histologicl Technique (5th Edition). Oxford University Press, New York, NY, USA (1980). Demonstrtes the immune-medited role of mesenchyml stem cells (MSCs) in regulting the cytokine secretions. 26 Aggrwl S, Pittenger MF. Humn mesenchyml stem cells modulte llogeneic immune cell responses. Blood 105(4), (2005). 27 Nut AJ, Fie WE. Immunomodultory properties of mesenchyml stroml cells. Blood 110(10), (2007). Demonstrtes the immune-medited role of MSCs in remodeling the lung injury through decresing of the inflmmtory response nd regulting cytokine secretions. 28 Ortiz LA, Dutreil M, Fttmn C et l. Interleukin 1 receptor ntgonist medites the ntiinflmmtory nd ntifirotic effect of mesenchyml stem cells during lung injury. Proc. Ntl Acd. Sci. USA 104(26), (2007). 29 Eggenhofer E, Luk F, Dhlke MH, Hoogduijn MJ. The life nd fte of mesenchyml stem cells. Front. Immunol. 5, 148 (2014). 30 Bssi EJ, Ait CA, Cmr NO. Immune regultory properties of multipotent mesenchyml stroml cells: where do we stnd? World J. Stem Cells 3(1), 1 8 (2011). 31 Ivnov-Todorov E, Bochev I, Dimitrov R et l. Conditioned medium from dipose tissue-derived mesenchyml stem cells induces CD4 + FOXP3 + cells nd increses IL-10 secretion. J. Biomed. Biotechnol. 2012, (2012). 32 Ivnov-Todorov E, Bochev I, Mourdjev M et l. Adipose tissue-derived mesenchyml stem cells re more potent suppressors of dendritic cells differentition compred with one mrrow-derived mesenchyml stem cells. Immunol. Lett. 126(1 2), (2009). 33 Rojs M, Xu J, Woods CR et l. Bone mrrow-derived mesenchyml stem cells in repir of the injured lung. Am. J. Respir. Cell Mol. Biol. 33(2), (2005). 34 Sge EK, Loeinger MR, Polk J, Jnes SM. The role of one mrrow-derived stem cells in lung regenertion nd repir. In: StemBook. Lis Girrd, Ken Chien, Fion Wtt (Eds). Hrvrd Stem Cell Institute, Cmridge, MA, USA (2008). Demonstrtes the homing of MSCs in lung. 35 Shke JG, Gruer PJ, Bumgrtner WA et l. Mesenchyml stem cell implnttion in swine myocrdil infrct model: engrftment nd functionl effects. Ann. Thorc. Surg. 73(6), ; discussion 1926 (2002). 36 Schfer R, Dominici M, Muller I et l. Progress in chrcteriztion, preprtion nd clinicl pplictions of non-hemtopoietic stem cells, Septemer 2006, Tuingen, Germny. Cytotherpy 9(4), (2007). 37 Fox JM, Chmerlin G, Ashton BA, Middleton J. Recent dvnces into the understnding of mesenchyml stem cell trfficking. Brit. J. Hemtol. 137(6), (2007). 38 Ponte AL, Mris E, Glly N et l. The in vitro migrtion cpcity of humn one mrrow mesenchyml stem cells: comprison of chemokine nd growth fctor chemotctic ctivities. Stem Cells 25(7), (2007). 39 Anjos-Afonso F, Sipti EK, Bonnet D. In vivo contriution of murine mesenchyml stem cells into multiple cell-types under miniml dmge conditions. J. Cell Sci. 117(23), (2004). The mechnism y which MSCs remodeled the lung dopting n epithelil-like phenotype. 40 Ruster B, Gottig S, Ludwig RJ et l. Mesenchyml stem cells disply coordinted rolling nd dhesion ehvior on endothelil cells. Blood 108(12), (2006). 41 Ding BS, Noln DJ, Guo P et l. Endothelil-derived ngiocrine signls induce nd sustin regenertive lung lveolriztion. Cell 147(3), (2011). 42 Wng GS, Bunnell BA, Pinter RG et l. Adult stem cells from one mrrow strom differentite into irwy epithelil cells: potentil therpy for cystic firosis. Proc. Ntl Acd. Sci. USA 102(1), (2005). 43 Potpov I, Plotnikov A, Lu ZJ et l. Humn mesenchyml stem cells s gene delivery system to crete crdic pcemkers. Circ. Res. 94(7), (2004). 44 Cpln AI. Why re MSCs therpeutic? New dt: new insight. J. Pthol. 217(2), (2009). 45 Ortiz LA, Gmelli F, Mcride C et l. Mesenchyml stem cell engrftment in lung is enhnced in response to leomycin exposure nd meliortes its firotic effects. Proc. Ntl Acd. Sci. USA 100(14), (2003). 46 Orfnos SE, Mvrommti I, Korovesi I, Roussos C. Pulmonry endothelium in cute lung injury: from sic science to the criticlly ill. Intensive Cre Med. 30(9), (2004). 47 Lee RH, Oh JY, Choi H, Bzhnov N. Therpeutic fctors secreted y mesenchyml stroml cells nd tissue repir. J. Cell Biochem. 112(11), (2011) /fso

14 Reserch Article Mohi El-Din, Rshed, Mhmoud Hridy et l. 48 Brkusks CE, Cronce MJ, Rckley CR et l. Type 2 lveolr cells re stem cells in dult lung. J. Clin. Invest. 123(7), (2013). 49 Desi TJ, Brownfield DG, Krsnow MA. Alveolr progenitor nd stem cells in lung development, renewl nd cncer. Nture 507(7491), (2014). 50 Evns MJ, Crl LJ, Stephens RJ, Freemn G. Renewl of lveolr epithelium in the rt following exposure to NO2. Am. J. Pthol. 70(2), (1973). 51 Rock JR, Brkusks CE, Cronce MJ et l. Multiple stroml popultions contriute to pulmonry firosis without evidence for epithelil to mesenchyml trnsition. Proc. Ntl Acd. Sci. USA 108(52), E1475 E1483 (2011). 52 Leiner KA, Newmn D, Li CM, Wlsh E, Khosl J, Snnes PL. Heprin nd firolst growth fctors ffect surfctnt protein gene expression in type II cells. Am. J. Respir. Cell Mol. Biol. 35(5), (2006). 53 Olsen CO, Iskson BE, Seedorf GJ, Lumn RL, Boitno S. Extrcellulr mtrix-driven lveolr epithelil cell differentition in vitro. Exp. Lung Res. 31(5), (2005). 54 Rice WR, Conkright JJ, N CL, Ikegmi M, Shnnon JM, Wever TE. Mintennce of the mouse type II cell phenotype in vitro. Am. J. Physiol. Lung Cell Mol. Physiol. 283(2), L256 L264 (2002). 55 Demio L, Tseng W, Blverde Z et l. Chrcteriztion of mouse lveolr epithelil cell monolyers. Am. J. Physiol. Lung Cell Mol. Physiol. 296(6), L1051 L1058 (2009). 56 Liu Y, Type II cells s progenitors in lveolr repir. In: Stem Cell Biology nd Regenertive Medicine. Firth A, Yun JXJ (Eds). Humn Press, (2015). 57 Kruse D, Grdner R, Hwng S, Collector MI, Shrkis SJ, Theise ND. Multi-orgn, multilinege engrftment y single one mrrow-derived stem cell fter trnsplnttion in mice. Blood 96(11), (2000) /fso Future Sci. OA (2017) FSO162