Human airway stem cell development - Journal of Cell Science - The ...

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Journal of Cell Science 113, 767-778 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1125

Epithelial stem cell-mediated development of the human respiratory mucosa in SCID mice Aurélie Delplanque1, Christelle Coraux2, Rabindra Tirouvanziam1, Ibrahim Khazaal1, Edith Puchelle2, Peter Ambros3, Dominique Gaillard2 and Bruno Péault1,* 1INSERM U506, Hôpital Paul Brousse, Villejuif, France 2INSERM U514, IFR 53, Université de Reims, France 3Children’s Cancer Research Institute, Vienna, Austria

*Author for correspondence (e-mail: [email protected])

Accepted 8 December 1999; published on WWW 14 February 2000

SUMMARY We have developed an in vivo assay for progenitor cells of the human tracheobronchial epithelium relying on the transplantation of human prenatal respiratory tissues into severe combined immunodeficiency mice. Engrafted embryonic or fetal open tracheobronchial rudiments are rapidly closed at each end by a neoformed membrane that we named the operculum. After 2-4 weeks, differentiated human respiratory epithelium covers both the native airway matrix and the new operculum. Human epithelial cells dissociated from either emerging embryonic lung primordia or mature xenografts were seeded in host human airway grafts, of which native epithelium had been eliminated by several cycles of freezing and thawing. All grafts seeded with donor epithelial cells and implanted back into SCID mice recovered a surface mucociliary

epithelium expressing expected markers and secreting mucus. Spontaneous epithelium regrowth was never observed in control unseeded, denuded grafts. In some experiments, donor epithelial cells and host denuded airway were sex-mismatched and the donor origin of newly formed epithelial structures was confirmed by sex chromosome detection. After two rounds of seeding and reimplantation, a normal epithelium was observed to line the 3rd generation operculum. These observations substantiate a functional assay for human candidate airway epithelium stem cells.

INTRODUCTION

trachea depleted of its own epithelium, which was then engrafted into a nude mouse (Engelhardt et al., 1992, 1993). This model traced progenitor cells for submucosal glands (Engelhardt et al., 1995) and proved, by clonal analysis, the existence of several stem cell subsets in the tracheobronchial epithelium (Zepeda et al., 1995). We have previously described an in vivo model of human airway complete development relying on the transplantation of embryonic and fetal rudiments of the respiratory tree into xenotolerant severe combined immunodeficiency (SCID) mice (Péault et al., 1994). Proximal or distal, healthy or cystic fibrosis human airway primordia enlarge remarkably in the SCID mouse and fully differentiate into tracheobronchial or pulmonary structures. Regardless of their initial developmental stage, such xenografts reach similar end-stage differentiation and include either a pseudostratified, ciliated and secretory surface epithelium, submucosal glands and cartilage rings, or alveolar structures and interstitium (Péault et al., 1994). The human respiratory mucosa developed in the SCID mouse also displays regular functional traits: mucus accumulates in the engrafted airway (Baconnais-Minon et al., 1999), the lining epithelium of which acts as a selective barrier to ion transports (Tirouvanziam et al., 1998). SCID-hu air ducts

Human conducting airways are lined with a pseudostratified epithelium that includes three major cell types: ciliated, secretory and basal (Jeffery, 1998). The developed epithelium is constantly, albeit very slowly, renewed and can be efficiently repaired in injuring pathologies such as asthma and cystic fibrosis (CF; Ayers and Jeffery, 1988). This suggests that epithelial ‘stem’ cells exist within the respiratory mucosa, possibly among basal or secretory cells, but such progenitors have not been identified yet (Emura, 1997; Mason et al., 1997). Human airway epithelial cells can be propagated in vitro from dispersed cells or explant outgrowth but do not retain features of differentiated cells for long, nor do immature epithelial cells differentiate reliably and stably in culture, probably because an appropriate balance between such key factors as extracellular matrix, growth factors and hormones has not been defined yet (Gruenert et al., 1995). It also appears to be important to culture airway epithelial cells at an air-liquid interface: only under a capillary layer of medium could ciliated cells differentiate in vitro (De Jong et al., 1994). A closer approximation of the human airway has been obtained by seeding dispersed human epithelial cells into a recipient rat

Key words: Human development, Airway epithelium, Progenitor, Xenograft, SCID mouse

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A. Delplanque and others

therefore represent faithful surrogate human tissues in which the physiopathology of cystic fibrosis can be studied in the absence of preexisting infection. Human epithelium neogenesis in SCID-hu airway grafts is dramatic. We earlier observed that both open ends of engrafted tracheal or bronchial segments became closed in the host by tissue membranes, the inner aspect of which supported the development of human mature respiratory epithelial cells. These structures also sustain repeated human epithelium development on iterative transplantation into at least 3 successive host mice. Using light and transmission electron microscopy and immunohistochemistry, we show here that normal cell migration, proliferation and differentiation events are involved in the neogenesis of human airway epithelium in SCID-hu mice. Based on these observations, we assumed that the human tracheobronchial epithelium could also be regenerated in this model from transplanted progenitor cells: to that end, recipient human airway grafts have been conditioned by a physical epithelioablative regimen, then colonized with populations of sex chromosome-marked donor differentiated airway epithelial cells. The same experimental scheme was repeated using donor cells prepared from the embryonic endodermal rudiment of the respiratory tree, which represents a homogenous population of epithelial stem cells. The full reconstitution of the airway inner lining observed in all these experiments suggests the transplantability of respiratory epithelium stem cells, and stresses the usefulness of the SCID-hu mouse as a functional in vivo assay for sorted candidate subsets of such progenitors.

MATERIALS AND METHODS Construction of SCID-hu mice Animals C.B.17 scid/scid (SCID) mice were bred in our own facility, in isolators supplied with sterile-filtered, temperature-controlled air. Cages, bedding and drinking water were autoclaved. Food was sterilized by X-ray irradiation. All experiments on SCID mice were performed under laminar flow hoods. Human tissues and graft processing Human embryonic and fetal tissues were collected following spontaneous or therapeutic abortions performed in compliance with the current French legislation and with approval of both national (CCNE) and institutional (COPE) ethics committees. Developmental stages were estimated from the duration of amenorrhea, and confirmed on anatomic criteria (O’Rahilly and Müller, 1987). Engrafted tissues ranged from 5 to 36 weeks post-conception. Known or suspected infections excluded the use of the tissues concerned. Tracheobronchial fragments were excised under a dissecting microscope in phosphatebuffered saline (PBS) containing penicillin and streptomycin. Only fresh, undamaged rudiments were used in order to optimize engraftment success. When implanted in the host, tracheobronchial segments were surrounded by cartilaginous rings and supporting mesenchyme with submucosal glands, and lined by epithelial cells. As previously described (Péault et al., 1994; Tirouvanziam et al., 1998), segments of human fetal trachea or stem bronchus were implanted subcutaneously in the flank of 6- to 8-week-old SCID mice anaesthetized by intraperitoneal injection of 0.4 ml Hypnomidate (Janssen-Cilag, France). To harvest xenografts, host mice were either sacrificed by cervical dislocation or anaesthetized in the case of iterative transplantations.

Fig. 1. 15-week human fetal trachea implanted subcutaneously for 17 weeks in the SCID mouse. The airway cylinder is now closed at each end by an ‘operculum’ (op) and richly vascularized (arrow) (×8.5). Endogenous airway epithelial development in xenografts Xenografts from 7 fetuses (one with spina bifida, five spontaneous abortions and one with unknown pathology, gestational age: 20.1±4.0 weeks (w); range 17-28; duration of engraftment: 23.1±16.9 w; range 13-60) were biopsied and the epithelium developed in the host was studied by immunohistochemistry. A portion of each engrafted structure was preserved before engraftment, frozen and analyzed as a control. To study epithelium neogenesis chronologically, tracheas from 3 fetuses (one with spina bifida, one spontaneous abortion and one with renal malformation, gestational age: 20.0±1.0 w; range 19-21) were dissected into 5 or 6 segments which were engrafted and then serially harvested after 4, 7, 11, 14, 28 and 56 days. After biopsy, xenografts were cut longitudinally into halves. One half was fixed for light and transmission electron microscopy, and the other one was analyzed by immunohistochemistry. To characterize the epithelial cells lining the membrane (or operculum) developed at each end of the engrafted airway cylinder, 15 mature opercula were dissected from individual xenografts (5 spontaneous and 10 medically-induced abortions, gestational age: 19.4±4.2 w; range 14-30; duration of engraftment: 15.8±13.9 w; range 3-60). Iterative transplantations Closed xenografts from 4 fetuses (one with renal malformation, one with spina bifida and two with unknown pathologies, gestational age: 18.1±3.0 w; range 15.5-22; duration of engraftment: 14.2±7.8 w; range 8-25) were first biopsied and cut transversally into halves. One Fig. 2. Semi- and ultrathin sections of a 21-week fetal trachea implanted from 4 to 14 days in the SCID mouse. (A) At day 4, each end of the graft is closed by an operculum formed by mesenchymal cells (mc) lined internally with flattened human epithelial cells (he) migrating from the native mucosa (nm). Mouse inflammatory cells (mi) are present in the center of the operculum, where closure is not yet complete (×80). (B) Detail of A. Central part of the operculum. A loose network of mesenchymal cells is lined by migrating epithelial cells (×500). (C) At day 14, the operculum completely closes the engrafted tracheal tube. Fibroblastic tissue (f) has thickened. The epithelium (ep) is now pseudostratified (×80). (D) Detail of C. Basal (b), columnar ciliated (cc) and secretory (s) cells are present within the epithelium lining the operculum (×500). (E) At day 4, epithelial cells migrating on the operculum are monolayered, flattened and undifferentiated (×4000). (F) Detail of A. Intermeshed, flattened human epithelial cells display short microvilli on their luminal aspect. Arrows show frequent invaginations of lateral plasma membranes (×20,000). (G) Ciliated cells lining the operculum at day 14 (×10,000). (H) Same section as in E. Basal cells (b) of the mature epithelium are attached to a basement membrane (arrow) (×3000).

Human airway stem cell development

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portion, including one operculum, was kept as a control and studied immunohistochemically while the other half bearing the other operculum and preserving one cartilaginous ring, in order to prevent crushing in the host, was reengrafted into a secondary SCID mouse host after the external mesenchyme had been stained with carbon. After 5, 10 or 20 weeks of secondary engraftment, reimplanted grafts with a newly developed operculum were harvested again and either cut transversally into halves, one half being reimplanted again in a third host and the other one kept as a control, or entirely processed for immunohistochemistry.

2% NCS, sections were first exposed to the primary antibody overnight at 4°C. Biotinylated rabbit anti-mouse IgG antibody (Dako, France), then a streptavidin-biotin complex (Dako) were added, and the reaction was visualized using aminoethylcarbazole (Sigma). Sections were briefly counterstained with Gill’s hematoxylin and mounted in aqueous medium (Aqueous Mounting Media, Biogenex, USA). Alternatively, fluorescein isothiocyanate (FITC)-coupled goat anti-mouse IgG (Immunotech) was used, in which case slides were mounted in PBS-glycerol-agar (Citifluor, France). Observations and photographs were made on a Nikon epifluorescence photomicroscope.

Epithelium reconstitution Donor cells Five well-differentiated human airway xenografts (one with renal malformation, one with Down’s syndrome and three with unknown pathologies, gestational age: 21.3±8.6 w; range 16.5-22; duration of engraftment: 18.2±10.3 w; range 8-29) were harvested, filled intraluminally with 1% pronase in RPMI medium and left at 4°C overnight. Epithelial cells were then flushed out with RPMI medium supplemented with 10% newborn calf serum (NCS) to produce a single-cell suspension. Cell viability, assessed by trypan blue exclusion, was higher than 90%. A portion of each dissociated graft was preserved, frozen and controlled by immunohistochemistry. Human airway rudiments from 6 embryos (voluntary abortions, gestational age: 6.7±1.08 w; range 5-8.5) were carefully dissected and incubated 5 minutes on ice with pancreatin 1× (Sigma, France). Epithelium was further separated from mesenchyme in PBS, 0.1% EDTA, under a microscope, with sterile microsurgery instruments. Depending on the gestational age of the anlage, dissected epithelial tubes were either cut longitudinally into halves (5-7 weeks of development) or gently pipetted into a viable single-cell suspension (7-8 weeks of development).

Electron microscopy Tissues were fixed in 2.5% glutaraldehyde (Serva, Germany) in 0.1 M cacodylate buffer and postfixed in 1% osmium tetroxide. Dehydrated material was embedded in Araldite. Semi-thin sections were stained with toluidine blue and examined under light microscopy. Ultrathin sections were mounted on copper grids and stained with uranyl acetate and lead citrate for examination under a JEOL 1200 EX transmission electron microscope.

Host graft conditioning and transplantation Host xenografts (one with spina bifida, two with renal malformation, two with Down’s syndrome and five with unknown pathologies, gestational age: 20.2±4.2 w; range 16-30; duration of engraftment: 15.9±10.9 w; range 5-41) were deprived of their own native epithelium by 3 rounds of freezing and thawing in NCS, 10% dimethylsulfoxide (DMSO). Early intact epithelial rudiments or cell suspensions (4×104 cells) or 1×105 differentiated epithelial cells were then infused intraluminally into each conditioned host graft. Control grafts were denuded but did not receive donor epithelial cells. After 24 hours in vitro, structures were implanted back into SCID mice. In two experiments, donor mature epithelial cells and host denuded grafts were sex-mismatched (male and female, respectively). Reconstituted and control grafts were harvested after 2 to 16 weeks and processed for immunohistochemistry and fluorescent in situ hybridization. Histology and immunohistochemistry Tissues were rapidly dissected, immersed overnight in PBS 30% sucrose or fixed first in 3.7% formaldehyde (Merck, Germany) in PBS for two hours, then embedded in Cryo-M-Bed medium (Bright, UK), frozen in liquid nitrogen and stored at –20°C. 5-7 µm serial frozen sections were cut and, in the case of previously unfixed tissues, immersed in methanol at –20°C for 10 minutes. Slides were dried for 1 hour at room temperature and stored at –20°C until use. Mouse monoclonal antibodies to (i) cytokeratins 7, 13 and 14 were, respectively, purchased from Dako (OV-TL 12/30; IgG1), Sigma (KS1A3; IgG1) and a gift from Dr E. Lane (London, UK) (LL-002; IgG3); (ii) Mib-1 was purchased from Immunotech, France (Ki-67; IgG) and (iii) integrin subunits were gifts from Dr F. Watt (London, UK) (anti α2: HAS-6; IgG. anti α3: VM-2; IgG. anti α6: MP4F10; IgG. anti β1: p5D2; IgG) or purchased from Immunotech (anti α5: SAM1; IgG2b) or Gibco (anti β4: 3E1; IgG1). After rehydration and blocking of non-specific binding sites with

Fluorescent in situ hybridization 6 µm frozen sections of paraformaldehyde-fixed tissues were hybridized with labeled DNA probes specific for the centromeric regions of human sex chromosomes X and Y. These DNA probes were labeled with either biotin or digoxigenin. Sections were first enzymatically digested with pepsin (Sigma) for 2 to 15 minutes at 37°C to improve probe penetration. Slides were further washed in two changes of PBS for 5 minutes each, dehydrated in an ascending ethanol series and air-dried. Digoxigenin-labeled Y chromosome probe or biotin-labeled X chromosome probe diluted in hybridization buffer were applied to slides using coverslip chambers. Sections were denatured at 80°C for 10 minutes and incubated in a humidified chamber overnight at 37°C. After hybridization, slides were washed in two changes of 2× SSC at room temperature, 15 minutes in 50% formamide in 2× SSC, at 42°C, followed by two washes in 2× SSC, each for 5 minutes at 42°C and finally washed in 0.1% Tween-20 in 4× SSC for 5 minutes at room temperature. After blocking unspecific binding sites with 2% BSA in PBS for 15 minutes at 37°C probes were detected by incubating sections with mouse antibiotin and sheep anti-digoxigenin antibodies (Boehringer Mannheim), followed by rabbit anti-mouse-TRITC and rabbit anti-sheep-FITC antibodies (Dako) for 15 minutes at 37°C in a dark humidified chamber. Unbound antibodies were removed through two washes in 0.1% Tween-20 in 4× SSC for 7 minutes at 42°C. Slides were fixed with 4% paraformaldehyde in PBS and washed twice in PBS for 5 minutes each. Slides were mounted in small volumes of DAPI/antifade medium (Vectashield). Sections were analyzed using the ×63 or ×100 oil lenses of an Axioplan-2 microscope (Zeiss). Image documentation was performed with the ISIS software from Metasystems (Altlussheim).

RESULTS Endogenous development of human respiratory epithelium in SCID-hu airway grafts: the ‘operculum’ We observed that both open ends of human fetal tracheobronchial cylinders implanted subcutaneously into SCID mice were rapidly closed by thin membranes (Fig. 1). Each such occluding structure, that we named operculum, was seen on tissue sections to support respiratory epithelium development on its internal luminal side. As soon as day 4 post-implantation, extremities of the engrafted airway cylinder were already nearly closed by


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Fig. 3. Epithelial cell marker expression in a 20-week trachea graft maintained 4 and 14 days in the SCID mouse. (A-F) At day 4, flattened migrating epithelial cells and some exfoliated cells in the lumen express cytokeratin 7 (A). All human epithelial cells migrating on the operculum express CK13 (B) and CK14 (C). The β1 integrin subunit is also expressed by migrating epithelial cells, and more intensely on basal ones (arrow) (D). The α6 integrin is expressed on the basal aspect of all migrating epithelial cells (E). The β4 chain is solely present on the basal side of epithelial cells, in contact with the mesenchyme (F) (×220). (G-L) At day 14 post-engraftment, CK7 is in apical and, to a lesser extent, basal cells of the developed pseudostratified epithelium (G). Most epithelial cells express CK13 (H) while CK14 is restricted to basal and to a few intermediate cells (I). β1 integrin expression is maintained on all epithelial cells and higher on their basal side (J) α6 (K) and β4 (L) integrin chains are clearly restricted to the basal membrane of basal cells. Submucosal glands are also present in the mesenchyme, with myoepithelial cells expressing α6 (×220).

fibrous membranes (Fig. 2A). At the periphery, several layers of mouse mesenchymal cells were lined with a monolayer of flattened human epithelial cells that appeared to emigrate from the native mucosa. Then, numerous mouse fibroblasts migrated centripetally to eventually completely obturate the lumen. This structure was still loose, unorganized and was transiently invaded by mouse inflammatory cells (Fig. 2B). Native

epithelium along cartilaginous rings was only partially preserved, showing areas of exfoliation (Fig. 2A). At day 7, opercula lined with a continuous layer of human epithelium on their luminal aspect completely closed the xenograft. Mesenchymal cells contacting the epithelium were arranged parallel to the operculum surface, just beneath the newly synthetized lamina propria. At the periphery, epithelial cells

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Fig. 4. Epithelium development in a human 19-week fetal trachea implanted 15 weeks in the SCID mouse. (A) Mature surface epithelium (ep) and glands (g) within the submucosa. cr: cartilaginous ring. HPS staining (×85). (B,C,D) Cytokeratin 7 is present in apical cells (B), while CK 13 is only found in basal cells (C) and α6 in the basal membranes of basal cells (D) (×220).

were piled up in two or three layers, while in the center a single layer of flattened epithelial cells laid on thick mesenchymal tissue. All epithelial cells appeared undifferentiated at that stage. At day 11, several layers of cuboidal and columnar epithelial cells were present in the middle of the structure, while at the periphery epithelial cells were even more columnar, a few being ciliated (not shown). At day 14, the epithelium was pseudostratified, constituted by basal, ciliated and even some secretory columnar cells (Fig. 2D). Beneath epithelial cells laid an increased number of fibroblastic cell layers. Capillaries with turgescent endothelium were identified (Fig. 2C). At day 28, the newly developed epithelium was mature, entirely pseudostratified and included all cell types encountered in the adult human airway mucosa. Submucosal glands were numerous at the junction between the native mucosa and operculum (Fig. 2E). At 4 days post-engraftment, electron microscopy confirmed the undifferentiated state of epithelial cells migrating on the operculum membrane, which were flattened, intermeshed and covered with short microvilli swelling out into the lumen. Epithelial cells were rich in glycogen particles, anchored in the forming basement membrane through a large contact surface (Fig. 2E) and joined by numerous junctional complexes and invaginations of the plasma membrane (Fig. 2F). By 14 days post-engraftment, basal bodies could be observed in a few ciliated cells, perpendicular to the apical surface. Cilia

displayed normal axonemas and microvilli were numerous (Fig. 2G). Occasional columnar cells showed electron-dense cytoplasm and included secretory granules (not shown). Basal cells had a dark cytoplasm with a large central nucleus and laid on a thin and continuous basement membrane (Fig. 2H). Cytokeratins (CK) and integrin subunits were used to discriminate epithelial cell subpopulations. Cell division was assessed with Ki-67, which marks human cells out of the G0 phase. At day 4, the single-layered flattened epithelial cells covering the operculum strongly expressed CK7, CK13 and CK14 (Fig. 3A,B,C). At the periphery, all cells in the pluristratified epithelium expressed CK13 while CK7 and CK14 were restricted to upper and lower layers, respectively (not shown). Punctate expression of the β4 integrin subunit was seen along the basal membrane of some flattened cells (Fig. 3F), while the α6 subunit was present in all epithelial cells (Fig. 3E). The α3 and β1 chains were expressed in all epithelial cells, but more strongly on the side contacting the basement membrane (Fig. 3D). The α5 integrin was not expressed at that stage. At day 7, numerous epithelial cells expressed Ki-67, indicating intense proliferation. At later stages of epithelium development, CK7 (Fig. 3G) and CK14 (Fig. 3I) were expressed by their specific cell types, respectively apical ciliated and secretory cells for the former one and basal cells for the latter. At day 14, CK13 was still present in numerous differentiated cells (Fig. 3H). The α6 (Fig. 3K) and β4 (Fig. 3L) integrin subunits were


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Table 1. Epithelial regeneration in denuded airway grafts seeded with epithelial cells from differentiated fetal xenografts or embryonic lung rudiments (lower part) ‘Host’ graft WG: weeks of gestation W/S: weeks in the SCID mouse

‘Donor’ airway epithelial cells

Duration of engraftment (weeks)

Epithelial regrowth

30WG + 12W/S 30WG + 12W/S

None 16.5WG + 11W/S

4 4

None +

20WG + 7W/S 20WG + 7W/S

None 22WG + 8W/S

5 5

None +

16WG + 9W/S 乆 16WG + 9W/S 乆

None 16WG + 25W/S 么

3 3

None +

20WG + 10W/S 乆 20WG + 10W/S 乆

None 16WG + 29W/S 么

5 5

None +

16WG + 25W/S 16WG + 25W/S

None 5.5WG

12 12

None +

21WG + 5W/S 21WG + 5W/S

None 7WG

16 16

None +

23WG + 12W/S 23WG + 12W/S

None 7.5WG

15 15

None +

21WG + 5W/S 21WG + 5W/S

None 5WG

14 14

None +

16WG + 41W/S 16WG + 41W/S

None 8WG

14 14

None +

Each experiment included, as a control, a conditioned host graft that received no epithelial cells prior to retransplantation.

restricted to basal cells in the newly formed epithelium while α3 and β1 were maintained in all epithelial cells (Fig. 3J). Terminally differentiated grafts were entirely lined internally with pseudostratified, ciliated and columnar epithelium and numerous glands were present in the submucosa (Fig. 4A). Cytokeratin 7 was expressed by columnar ciliated and secretory surface cells and by gland duct cells (Fig. 4B). Conversely, strong expression of CK13 (Fig. 4C) and CK14 (not shown) was restricted to basal cells in the surface epithelium and to myoepithelial cells in the glands. α2 and β1 integrin chains were expressed on all epithelial cells, albeit at a higher level on basal ones. α3 was on all cells in the surface epithelium, ducts and glandular acini, with also higher expression on their basal aspect. The most restricted distribution was detected for the α6 (Fig. 4D) and β4 subunits, only present on the basal membrane of basal cells. In conclusion, native and newly developed human respiratory epithelium in SCID mice exhibited a regular pattern of expression of molecular markers, similar to that described in control airways. Closed airway grafts were harvested and cut transversally into halves which were reimplanted into secondary SCID mouse hosts. Each reimplanted half-cylinder supported on its open end the development of a parallel new operculum, lined with human mature, mucus-secreting epithelium expressing typical markers (Fig. 5A-C). So far, such iterative transplantations have been repeated 3 times from one original tissue, and always induced the formation of a new operculum supporting epithelium development. Regeneration of respiratory epithelium from engrafted donor progenitor cells We next explored the possibility that appropriately conditioned human tracheobronchial grafts in SCID mice could support the

expansion and differentiation of extrinsic epithelial cells, and hence provide a functional assay in vivo for human candidate airway epithelial stem cells. To that end, host grafts explanted from SCID mice and with one closed end preserved were frozen and thawn three times sequentially, which resulted in complete disappearance of the surface pseudostratified epithelium. Donor epithelial cells were then isolated as follows. Differentiated tracheobronchial grafts were harvested from mice and treated intraluminally with pronase, resulting in complete dissociation of the surface epithelium which was then recovered as a single-cell suspension. Donor epithelial cells were then injected into the denuded lumen of host grafts, which the day after were retransplanted into SCID mice. Control host grafts inoculated with medium alone and engrafted for 4 to 16 additional weeks never showed spontaneous epithelial regrowth nor did they support new operculum development, although endogenous cartilage, mesenchyme and submucosal glands were preserved (Table 1). In contrast, grafts seeded with airway epithelial cells were closed in 4 weeks by a new operculum. Both native airway walls and opercula in these grafts were then covered by typical human airway surface epithelium (Fig. 6A,B; Table 1). As early as at 2 weeks postengraftment, the original operculum was already covered by several layers of epithelial cells. All apical cells expressed CK7 (Fig. 6C), while CK13 was detected in the whole epithelium (Fig. 6D). Integrin subunits α3 and β1 were similarly expressed by all epithelial cells, albeit more strongly on basal ones (Fig. 6E) while α6 and β4 integrins were restricted to the basal cell membranes contacting the basal lamina (Fig. 6F). The newly formed operculum was bordered then with two layers of flattened epithelial cells, expressing CK7 (Fig. 6G) and CK13 (Fig. 6H). Integrin chains were expressed like in the original parallel operculum (Fig. 6I-J). After 4 weeks of engraftment,

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the epithelium along the whole inner side of the reconstituted was observed after 12 to 16 weeks of residency in the host graft was pseudostratified and contained basal, secretory and mouse, a relatively long period of engraftment that was chosen ciliated cells expressing regular differentiation markers (not arbitrarily to guarantee optimal development. shown). Glands also emerged from the epithelium covering the original operculum (not shown) and mucus was secreted into the lumen. The presence of numerous epithelial gland buds, DISCUSSION besides more developed glands, suggested the generation of new glands from the forming epithelium. It is far less likely Developmental studies rely principally on experiments that preexisting glands resisting the denuding process regrew, performed in vivo as only very limited tissue ontogeny can be since no epithelial development was ever observed in the recapitulated in culture. This severe limitation to investigations absence of seeded donor epithelial cells. In some experiments on human development has been to some extent surmounted female host grafts were seeded with male-derived donor by devising human xenochimeras. Most popular are epithelial cells and sex chromosomes were detected in the immunodeficient mice, notably of the SCID and SCID/NOD reconstituted airway by fluorescent in situ hybridization (Fig. strains, as hosts for human normal and malignant 7). As expected, no human chromosomes were detected in the hematopoiesis. The observation that human blood-forming mesenchymal operculum, confirming its mouse origin. In tissues implanted into SCID mice sustain an essentially normal contrast, operculum-lining epithelial cells were all human and most were derived from the donor, as they contained a Y chromosome (Fig. 7). On iterative transplantation each newly formed operculum bordered with donorderived epithelium supported the development of a third-generation operculum which was also lined internally with normal mucus-secreting human epithelium. After 4 weeks, both opercula were lined by a normal pseudostratified epithelium (not shown). These observations suggested that the bare mesenchyme and developing operculum in conditioned host grafts had been seeded by progenitor cells present in the injected respiratory epithelium cell suspensions. In order to assay in the same conditions a cell population much enriched in epithelial progenitors, we next used as a source of donor cells the endodermal layer of the embryonic lung bud, from which all epithelial cells in the developed respiratory tree are derived. Due to its very small size (1 to 2 mm), the endodermal tube dissociated from the 5- to 7-week rudiment was cut longitudinally and each half was deposited intact into the lumen of a conditioned host graft. From 7 to 8 weeks of development, the endodermal pouch was amenable to physical dissociation by gentle pipetting and cell suspensions therefrom were regularly instilled intraluminally into recipient grafts. Similar to what had been observed on transplantation of mature airway epithelium cell suspensions, denuded tracheobronchial explants seeded with embryonic endodermal cells Fig. 5. Iterative transplantations of the fetal airway. (A) One of the operculum-closed supported development and end-stage extremities dissected from a 15.5-week trachea engrafted 9 weeks in a SCID mouse was differentiation of a continuous, tagged with carbon (arrow) and reimplanted 5 more weeks in a secondary mouse host. An pseudostratified and mucus-secreting other operculum developed and closed the reimplanted structure (arrowhead). Both original respiratory epithelium expressing the and newly formed opercula are lined with human pseudostratified epithelium, which expected structural and adhesion proteins secretes mucus (m) in the closed lumen. Gill’s hematoxylin staining (×40). (B,C) Columnar (Fig. 8A-F). This complete reconstitution cells in the original (B) and newly formed (C) opercula express CK7 (×260).


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Fig. 6. Epithelium regeneration from extrinsic donor airway epithelial cells. (A) A graft seeded with donor airway epithelial cells has closed after 4 weeks of reengraftment in the SCID host. A newly developed parallel operculum (N-op) covers the original seeded operculum (O-op) and the whole inner surface is lined with airway epithelium (arrows). Gill’s hematoxylin staining (×40). (B) Detail of B. Basal, ciliated and secretory cells are present within the reconstituted human normal pseudostratified epithelium. Gill’s hematoxylin staining (×100). (C-F) At 15 days of engraftment, all apical epithelial cells in the original seeded operculum express the cytokeratin 7 while basal cells are CK7-negative (arrow) (C). All human epithelial cells lining the operculum express CK13 (D). Expression of the α6 and β4 (E) integrin subunits is restricted to the basal membrane, while the α3 and β1 (F) integrin chains are both expressed by all human epithelial cells, albeit at higher levels on basal cells. β1 is also expressed in smooth muscle (arrowhead) (×220). (G-J) At 15 days of engraftment in the newly developed operculum, bilayers of human epithelial cells express CK13 (G) and CK7 (H). In this bistratified epithelium, expression of integrin chains is similar to that described in the original parallel operculum (×220).

pattern of development (McCune et al., 1988; Baum et al., 1992; Kyoizumi et al., 1992; Legrand et al., 1997) has stimulated attempts to model in these mice the formation of other human tissues such as skin (Levy et al., 1998) and respiratory structures. In the latter case, portions of human fetal airways reach endstage histologic differentiation, irrespective of their initial stage of development, a few weeks after implantation in SCID mice (Péault et al., 1994). Xenografts, which retain cylindrical shape, are then closed at both ends with mouse tissue expansions or ‘opercula’, filled with mucus and lined on their whole inner aspect with a pseudostratified, ciliated and secretory surface epithelium that, like its natural human counterpart, drives selective ion fluxes (Tirouvanziam et al., 1998). The initial focus of the present study was the neogenesis

Fig. 7. After 4 weeks of development in the host, the female (red spots) host graft reconstituted with male-derived (green spots) epithelial cells is lined with a chimeric pseudostratified epithelium.

of human respiratory epithelium in SCID-hu airway grafts, notably on the inner side of the operculum. The operculum emerges, in the first days post-engraftment, as epithelial cells migrate from the native human fetal epithelium on mesenchyme condensed at the extremities of the tracheal walls. We were convinced that epithelial neogenesis was actually a consequence of cell migration from the native epithelium, and not merely readherence of exfoliated cells since (i) free cells encountered in the lumen of the grafts were mostly mouse inflammatory cells, (ii) rare epithelial exfoliated cells were of differentiated type and died shortly and (iii), when airway with damaged epithelium was engrafted, significant exfoliation ensued but no operculum ever formed. Finally flattened cells on the developing operculum exhibit the typical shape of migrating cells (Fig. 2A,B) and are similar to the socalled poorly differentiated (PD) cells (Shimizu et al., 1994), suggested to represent a pivotal stage in the regeneration process. These migrating cells were described in vivo in the regenerating rat (Shimizu et al., 1994; Horiba and Fukuda, 1994), hamster (Keenan et al., 1983) and human (Pilewski et al., 1997; Dupuit et al., 1999) respiratory epithelium, as well as in an in vitro model of human airway epithelium wound repair (Zham et al., 1991, 1997). In these models, like on the operculum, epithelial cells spread, migrate until they form tight junctions, then proliferate and differentiate to restore a novel epithelial structure (reviewed by Puchelle et al., 1997).

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Fig. 8. Airway epithelium reconstitution mediated by embryonic progenitors. (A) After 12 weeks of engraftment in the mouse, a host graft seeded with lung bud endoderm supports the development of a normal mucociliary epithelium. A newly developed operculum closes the structure, the whole inner surface of which is lined by epithelial cells. Gill’s hematoxylin staining (×40). (B) Detail of A. The epithelium derived from endodermal progenitor cells is pseudostratified, and includes basal, ciliated and secretory cells. (C-F) Apical cells express CK7 (C) while CK13 (D), the adhesion molecule CD44 (E) and integrin subunit α6 (F) are present in basal cells (arrow) (×220).

Migrating cells in the repairing respiratory epithelium overexpress the α5β1 integrin in vitro (Herard et al., 1996) like in vivo (Pilewski et al., 1997), while this fibronectin receptor was not found on the flattened cells migrating on the operculum. Furthermore, operculum PD cells express both basal cell-specific cytokeratins 13 and 14 but also the columnar

cell marker CK7. Expression of the latter at this early step also distinguishes operculum development from other regeneration models, in which this ciliated cell marker is detected much later (Shimizu et al., 1992; Randell et al., 1993). Yet, it is likely that all differentiated cells eventually lining the operculum derive from these migrating PD cells. The β4 integrin subunit is

Human airway stem cell development expressed, however, as soon as migration begins, while it appears later in normal development (Virtanen et al., 1996; Coraux et al., 1998). Migrating PD cells may originate from basal cells present in the engrafted epithelium, of which they express the CK13, CK14 and α6β4 markers. The numerous submucosal glands gathered at the mucosa/operculum junction can also suggest the participation of glandular cells in epithelium emergence. The next stages of epithelium development on the operculum mimic both repair and fetal maturation. During the first days post-engraftment, until the lumen is nearly closed at day 7, and lined with at least two layers of epithelial cells, migrating cells proliferate very slowly as is the case during repair (Puchelle et al., 1997). At day 14 ciliated cells differentiate first and segregation of CK7 and CK14 in apical and basal cells occurs gradually. Epithelial differentiation pursues essentially like in normal intrauterine development (Gaillard et al., 1989), irrespective of the age of the implanted trachea. At difference with normal ontogeny (Jeffery et al., 1992), though, no transient hypersecretory surface epithelium develops on the operculum. At day 28, glandular buds are present within differentiated epithelium, invaginating in the mesenchyme and later on forming submucosal glands (Fig. 3). In conclusion at this point, human airway grafts in SCID mice support the essentially normal development of novel layers of pseudostratified epithelium expressing the molecular markers encountered in normal tissues (Broers et al., 1989; Virtanen et al., 1996; Coraux et al., 1998). Among other potential uses of that model of human respiratory epithelium ontogeny, we examined in priority whether SCID-hu airway grafts could also support epithelium development from extrinsic progenitor cells. Repeated freezing and thawing appropriately conditioned host grafts, since no residual surface epithelial cells were left, confirming previous observations (Terzaghi et al., 1978; Hook et al., 1987), nor did epithelium recover endogenously on transplantation into SCID mice. As a proof of concept, donor-derived total dissociated airway epithelial cells seeded into such conditioned grafts fully reconstituted in 4 weeks, in 4 out of 4 experiments performed, surface epithelium and secretory glands. Therefore, neither physical conditioning of the host organs nor enzyme dissociation of donor cells compromised their ability to interact and resume normal epithelial development. Although unseeded, control grafts never recovered a surface epithelium we confirmed that the regenerating respiratory mucosa in these experiments is donor-derived by using a chimerism marker (see Fig. 8). It must be noticed, however, that in some instances such sex-mismatched combinations of host airway transplants and donor epithelial cells revealed only partial contribution of the latter to ciliated border renewal. Some endogenous progenitor cells thus survive the freezing/thawing conditioning regimen used. These may be too rare, though, to ensure epithelial recovery autonomously but may later reveal their developmental potential if the appropriate environment of a regenerating epithelium is provided. While we show unambiguously that human respiratory epithelium can be fully restored by allogeneic tracheobronchial epithelial cells, these experiments give no clue as to the identity of the repopulating subset, which can include true progenitors or/and de-differentiated cells. One cannot even exclude that all

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dissociated donor epithelial cells merely readhered orderly to host bare mesenchyme, although chronologic analysis suggested that a regular sequence of epithelium development was taking place in that setting. To ascertain that the method described permits stem cell-mediated epithelium restoration, repopulation experiments were performed again using donor cells intrinsically devoid of differentiated elements. We assumed that the endodermal rudiment of the respiratory tree harvested on embryos during the second month of gestation fulfilled that criterion, representing a pure progenitor cell population for the respiratory epithelium. Complete epithelial repletion observed in denuded airway grafts seeded with 5-8week lung endoderm argues that the experimental setting we describe guarantees a full ontogenetic sequence of epithelium development. Yet, it is not known whether such early embryonic anlagen already comprised a hierarchy ranging from stem cells to diversely committed progenitors that may have unequally participated in epithelium renewal in the grafts. It is now well established that immunodeficient mice are amenable to functionally testing human hematopoietic stem cells, including the rarest, most primitive candidate subsets thereof (Baum et al., 1992; Bhatia et al., 1997). The present study suggests the feasability of assaying human stem cells for other lineages in related xenochimeras. A current target is the progenitor cell(s) for renewal and repair of the differentiated respiratory epithelium, candidate subsets of which being presently identified and selected by immunohistochemistry and flow cytometry. Low numbers of such sorted cells will be available for functional assessment in SCID-hu airway assays, the sensitivity of which in terms of quantity of input cells is presently unknown. We estimated that about 104 cells constituted the youngest embryonic endodermal rudiments that readily ensured full reconstitution of airway graft epithelium. Cell numbers in the same order of magnitude were used by Engelhardt et al. (1992) to restore a differentiated human epithelium, in 6 weeks, in the rat trachea system. While we did not try so far to estimate the quantitative limits of the assay it is likely that significantly lower progenitor cell numbers can be read out in these conditions since all seeded host grafts were readily reconstituted. Of special interest in the perspective of developing a stem cell assay was our observation that iterative airway transplantations into secondary and tertiary hosts consistently ended up in full respiratory epithelium development on the newly formed opercula, including in the case of grafts repopulated with donor-derived epithelial progenitors. This is clearly the first assay system in which the long-term, possibly unlimited potency of such progenitors can be tested. Demonstrating in that setting the sustained expression of a marker or therapeutic gene transduced into epithelium-building cells would be of outstanding significance in a clinical perspective. Besides professional, tissue-specific stem cells, recent experiments suggest that progenitors endowed with unexpectedly broad developmental capacities exist in mouse differentiated organs, even at adult stages (Ferrari et al., 1998; Bjornson et al., 1999; Petersen et al., 1999), that may reflect either the underestimated plasticity of lineage-committed progenitors or the persistence during the whole life of totipotent stem cells. Xenochimeras, and notably SCID-hu mice are presently the only dynamic assays in which the existence of such cells in human tissues can be explored.

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We are grateful to C. Carrière and Prof E. Aubeny for procurement of human embryos and to Drs M. Catala, F. Dahmane, A.-L. Delezoide, F. Menez, F. Narcy and C. Nessman for providing fetal tissues. We also acknowledge the expert technical assistance of MarieFrance Hallais with electron microscopy and Françoise Viala for the preparation of figures. Supported in part by EC network n°BIO-CT 95-0284.

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