Human intestinal development in a severe-combined immunodeficient xenograft model

Differentiation (1995) 58:361-371

Differentiation

Onlogeny, Neoplsris and DinerentiationTherapy

0 Springer-Verlag 1995

Human intestinal development in a severe-combined immunodeficient xenograft model T.C. Savidge1.2,A.L. Morey-l, D.J.P. Ferguson-1, K.A. Fleming", A.N. Shmakovl, A.D. Phillips2 Laboratory of Intestinal Biology, Department of Cellular Physiology, The Babraham Institute, Cambridge CB2 4AT. UK Academic Department of Paediatric Gastroenterology,Queen Elizabeth Hospital for Children, Hackney, London E2 8PS, UK 3 Oxford University, Nuftield Department of Pathology and Bacteriology, John Radcliffe Hospital, Oxford OX3 9DU, UK

I 2

Accepted in revised form: 18 January 1995

Abstract. The present work describes a severe-combined immunodeficient murine xenograft model used to investigate human gastrointestinal ontogenesis. Specifically, the study has tested whether carefully selected regions of human fetal gut are able to undergo region-specific morphogenesis and epithelial cytodifferentiation when transplanted subcutaneously into immunodeficient mice. In addition, double-label in situ hybridisation techniques, utilising specific human and mouse DNA probes, have been adopted to characterise host and donor cell types and to investigate the potential developmental roles for non-epithelial cells in the regulation of epithelial differentiation pathways in vivo. Human fetal small and large bowel developed to form a characteristic mucosa 10 weeks after transplantation, which displayed Elear region-specific structural and functional gradients. The initial phase of xenograft epithelialisation closely resembled the stratified type of epithelium which is present during early fetal gastrointestinal development. Idiosyncratic epithelial differentiation pathways were recorded during xenograft regeneration, with an absence of Paneth cells and an abundance of enteroendocrine cells when compared with developed xenograft and paediatric intestine. Such differences may, therefore, be important in ensuring rapid and region-specific development in the absence of conventional luminal stimuli and hormonal changes that occur normally during pregnancy. In situ hybridisation demonstrated an exclusively human origin for the intestinal xenograft epithelium and muscularis mucosa and externa. Although the submucosa and lamina propria were comprised of a chimeric mixture, murine cells were rarely seen to contact with the epithelium, which interacted primarily with human myofibroblasts and human intraepithelial lymphocytes. It is proposed that a 'selection' process operates to maintain species-specific cellular interactions, and this mechanism may subsequently play an important role in regulating epithelial cell differentiation, orchestrated in part by juxtaposed non-epithelial cell types. Correspondence to: T.C. Savidge

Introduction The morphological and functional development of the gastrointestinal tract in vertebrates requires complex interactions between the mesenchymal cells originating from the splanchnopleuric mesoderm and the endodermally derived epithelium [ 151. Studies performed using a number of different experimental animal models have demonstrated that the tissue-specific morphogenesis, which is evident during early gastrointestinal development, results from a complex network of instructive and permissive information relayed between the mesenchyma1 components and the epithelium [ I I , 18, 20, 231. Epithelial gene expression and differentiation, which is unique to a particular tissue location, may be attributed to early instructive mechanisms. Permissive instructions, on the other hand, allow preprogrammed epithelial tissues, whose developmental fate has already been determined, to be maintained and stabilised by 'foreign' mesenchymal components [ 12, 13,471. In humans, studies of the mechanisms regulating intestinal ontogenesis have been less forthcoming, this being mainly due to the ethical considerations which are associated with clinical investigations. A number of recently established in vitro culture techniques e.g. organ culture, and primary or tumour intestinal cell lines, have now provided a partial solution to these limitations. There are, however, several problems associated with these techniques as they preclude long-term studies of differentiated and structurally intact intestine under normal microenvironmental conditions [24,41]. Syngeneic and xenogeneic transplantation of intestinal tissues into murine hosts has provided an attractive alternative method for investigating the physiological and genetic determinants which regulate intestinal development in vivo [3, 5, 8, 9, 10, 22, 37, 38, 43, 44.45, 46, 481. These studies demonstrate that donor fetal or postnatal intestine transplanted into ectopic sites in recipient host animals possesses an inherent ability to differentiate in a fashion which correlates temporally and in a sitespecific manner with non-transplanted fetal gut. It has

362

been proposed, therefore, that transplanted intestinal epithelial stem cells retain a 'positional address' [5, 381, as well as a long-term potential to proliferate [35]. Both of these features subsequently contribute to the regeneration of a morphologically and functionally distinct tissue. The present study investigates the developmental pattern of human fetal intestine transplanted into C.B- 17 severe-combined immunodeficient (scid) mice, a host strain which is able to accept and accommodate foreign donor tissues for extended periods [ 141. When intact segments of immature fetal gut are surgically implanted into subcutaneous tunnels on the back of scid mice, the xenografts develop and remain viable for several months. Characterisation of this model demonstrates that extensive xenograft vascularisation and development occurs within 10 weeks after transplantation, at which time the tissue closely resembles morphologically normal human intestine. This approach has, therefore, been used for long-term in vivo investigations of epithelial cell differentiation pathways during human gastrointestinal ontogenesis.

Methods Procurement and transplantation of human fetal intestinal tissues into scid mice. Human fetal intestine (12 samples; mean gestational age 13.5k0.5 weeks, range 10.7-15.9 weeks, as assessed by crown-rump length) was obtained from the MRC Tissue Bank, Royal Marsden Hospital, London, after therapeutic abortion. Procurement and procedures involving xenografting of human fetal tissues into C.B-17 scidkcid mice were performed with full approval from the Cambridge Local Ethics Committee and in accordance with the Home Office guidelines specified in the Polkinghorne Report (1989) [34]. Prior to transplantation, intestinal tissues were washed twice in ice-cold serum-free Dulbecco's Modified Eagle's Medium (Difco, East Molesey, UK). Mice were anaesthetised with 0.1 ml Hypnovel (Roche, Welwyn Garden City, UK), Hypnorm (Jansen, Oxford, UK) and pyrogen-free water, given intraperitoneally at a 1 : I :2 ratio, and a 2% halothane-air mixture delivered through a specially adapted face mask. Intact segments of fetal intestine (2-3 cm lengths) were transplanted subcutaneously on the back of 6-8 week old female and male scid mice, in a fashion described previously [22, 44,461. Fetal tissues were carefully designated into proximal and distal small intestine (subdivided into proximal and distal 50% lengths) and large bowel prior to transplantation, using the appendix as an additional marker for the ileocaecal junction. Scid mice were maintained in negative pressure isolators supplied with High Efficiency Particle Air (HEPA) filters and had access to sterilised food (Labsure, K&K Greef Ltd., Croyden, UK) and water ad libitum. Intestinal xenografts were harvested at set time-intervals (1, 2, 6, 8, 10, 12, 20 and 26 weeks) after transplantation and were compared to corresponding segments of fetal tissue collected during surgery. Xenograft tissues were also compared to histologically normal paediatric proximal small intestine (12 samples; median age 37 months) obtained from routine investigative procedures performed at the Queen Elizabeth Hospital for Children, London, UK. Analysis of small intestinal structure and epithelial cell proliferation. Tissues were fixed in 10% phosphate-buffered formal saline (pH 7.2) for 24 h and embedded in paraffin using routine procedures. Sections (5 pm thick) were stained with haematoxylin and eosin (H&E) or periodic acid Schiff's (PAS) stain. Villus height and crypt depth were measured using image analysis on a Magi-

scan 2A Analyser (Joyce-Loebl, Tyne and Wear, UK). Actively proliferating cells of human origin were identified using the MIBI monoclonal antibody [25] ( 1 :50 dilution; a generous gift from The Binding Site, Birmingham, UK). For ultrastructural studies of paediatric and xenograft intestine, tissues were fixed in 4% phosphate-buffered (pH 7.2) glutaraldehyde and processed for electron microscopy using conventional methods. Epithelial cell lineage determination. Quantitative counts of epithelial cell lineage representation in fetal, paediatric and xenograft intestine were performed on H&E - and PAS - stained paraffin sections to identify Paneth and goblet cells, respectively. Enteroendocrine cells were identified in paraffin sections using a monoclonal antibody to chromogranin ( 1 :200 dilution), a generous gift from Professor J.M. Polak, Hammersmith Hospital, London [7]. Goblet and enteroendocrine cell representation is expressed as a percentage of epithelial cells (a minimum of one thousand cells/sample were counted) and Paneth cells as the mean number of cells identified per 20 crypt sections. Epithelial cell lineages were also investigated by electron microscopy. Quantitative brush border enzyme cytochemistry. For cytochemical determination of brush border alkaline phosphatase (AP) activity, frozen sections (10 pm thick) of fetal, paediatric and xenograft intestine were incubated under initial rate conditions (2 min at 37" C) with naphthol-As-Bi-phosphate (Sigma, Poole, UK) as artificial substrate using Fast Blue B (Sigma) as the coupling agent [33]. Tissue sections were prefixed in formal calcium (pH 6.0), 1.1% CaCI, and 3.8% formaldehyde in deionised water for 10min at 4°C. prior to the enzyme reactions. All quantitative measurements of AP activity were performed on a Leitz MPV-3 microdensitometer (Milton Keynes, Bucks., UK), at a final magnification of x400. Sequential absorbance readings at 550 nm, using a scanning window size of 4x4 pm, were taken at 16-pm increments along the crypt-villus axis, starting from the crypt-villus junction and ending at the villus tip. Values from a minimum of five well-orientated villi were averaged to provide final profiles for enzyme activity. Preparation of probes f o r in situ hybridisation. Whole human and mouse DNA, obtained by standard phenol-chloroform extraction from peripheral blood leukocytes and spleen, respectively, were nick-translated with either biotin- I I-dUTP (Sigma) or digoxigenin-l I-dUTP (Boehringer Mannheim, Lewes. UK) to a mean fragment length of 200 bases. Probes were diluted in hybridisation mixture containing 50% deionised formamide, 10% dextran sulphate, 250 pg/ml sheared herring-sperm DNA and 2xSET buffer (300mM NaCI, 6 0 m M Tris, 4 m M EDTA; pH 7.0) at a final probe concentration of 2 pg/ml. Probe mixtures were denatured at 90" C for 15 min and then rapidly chilled to 4" C.

In situ hybridisation. Fetal and xenograft intestine was either fixed in 10% buffered formal saline (pH 7.4) for 24 h and paraffin-embedded, or fixed in 2% paraformaldehyde/O. I % glutaraldehyde in 0.1 M PBS for I h before dehydration and embedding in LR White resin. Double-label in situ hybridisation was performed essentially as previously described [3]. Probe mixture containing both digoxigenin-labelled probe to whole mouse DNA and biotinlabelled probe to whole human DNA was hybridised overnight to proteolytically digested paraffin sections. Proteolysis was performed with 0.05 mg/ml Protease VIIl (Sigma) in PBS at 37" C for 15 min. After post-hybridisation washes (3x5 min in TBS [50 mM Tris, 150 mM NaCI; pH 7.21 with 0.5% Triton at 37" C TBS at 65" C), endogenous peroxidase activand 3x5 min in 0 . 5 ~ ity was blocked by a 15-min incubation in 0.3% hydrogen peroxide in TBS. Blocking was performed using 15% skimmed milk powder in AW.5 buffer (0.1 M Tris, 0.1 M NaCI, 2 mM MgCI,; pH 7.5) with 0.5% Triton for 15 min at 37" C. Codetection of bound probes was achieved by simultaneous incubation with antidigoxigenin FA, fragments conjugated with peroxidase ( 1 :750; Boehringer Mannheim) and avidin conjugated with alkaline phos-

363 phatase ( I :250; Dako, High Wycombe, UK) in AP7.5Rriton buffer, with 2% BSA for 30 min at 37" C. After post-conjugation rinses (3x5 min in AP7.5/0.5% Triton, 3x5 min in AW.5) slides were rinsed in TBS and reacted with diaminobenzidine (DAB) substrate (6 mg DAB in 10 ml TBS with 100 pl 3% hydrogen peroxide) for brown visualisation of the human DNA signal. Slides were then rinsed for 3x5 minutes in AP7.5 and 3x3 minutes in AP9.0 (100 mM NaCI, 100 mM Tris, 100 mM MgCI,; pH 9.0) before immersion in NBT-BCIP substrate (40 pl 75 mg/ml NTB stock and 120 pI 50 mg/ml BClP stock in 40 ml AP9.0 buffer prewarmed to 37" C) for purple-black visualisation of the mouse DNA signal. The protocol was adapted for triple-labelling of different target antigens by performing standard APAAP immunolabelling with Fast Red substrate prior to the dual-probe in situ hybridisation. Antibodies to human common-leukocyte antigen (anti-human CD45; Dako) and intermediate filaments (anti-vimentin; Sigma) were employed. For ultrastructural in situ hybridisation of human and mouse probes, ultrathin sections on Formvar/carbon-coated nickel grids (200 mesh) were floated face-down on drops of millipore-filtered solutions on prafilm sheets[28]. Sections were denatured using 0.5 M NaOH at room temperature for 5 min, then washed for 5x2 min with distilled water and air dried. Sections were then placed on 15-pl drops of probe mixture at 42" C in a moist chamber for 4 h. Post-hybridisation washes consisted of 5x2 min in TBS/O.5% Triton at 42" C, then 5x2 min in 0.5xTBS at 42" C. Blocking was Performed with 1% BSA in TBS for 15 min at room temperature. An immunogold system was used to detect hybridised probes to human and mouse DNA, respectively. The protocol involved sequential 30-min incubations in: (i) sheep polyclonal antibody to digoxigenin (1 : 100 dilution; Boehringer Mannheim) or mouse anti-biotin ( I : 100 dilution; Dako); (ii) polyclonal rabbit antibodies to sheep or mouse (1:20 dilution; Dako), followed by (iii) goat anti-rabbit conjugated to either 10-nm (human probe) or 15-nm (mouse probe) colloidal gold (Biocell, Cardiff, UK) diluted 1:25. Grids were washed for 5x2 min on drops of TBS between steps and were finally stained with 2% aqueous uranyl acetate. Statistics. All statistical analyses were calculated using MINITAB statistical software (Minitab Inc., State College, PA 16801-2756, USA). The Welsh test was adopted to account for nonpooled variance present within each population analysed. Deviations from the null-hypothesis were additionally confirmed using the non-parametric Mann-Whitney U-test for ranks (*sem).

Results Morphological and proliferative changes during intestinal xenograft development

The human fetal small and large bowel used for transplantation displayed an immature mucosa which was characteristic of a mean gestational age of 13.5kO.5 weeks[6, 301. Villi were present throughout the intestine, including the large bowel. Primitive crypts of Lieberkuhn were identified as invaginations into the connective tissue lamina propria. This feature was particularly apparent in the proximal small intestine, corroborating previous reports describing the developmental process as progressing from proximal to distal sites during intestinal ontogenesis [6, 19, 211. The crypt invaginations often displayed a stratified type of epithelium, in contrast to the consistently columnar epithelium of the formed villi (Fig. lA, B and C). Both stratified and columnar types of epithelia were separated from a network of undifferented mesenchymal cells by a continuous

basement membrane. The whole mucosal mass was encompassed by a poorly developed muscularis externa (Fig. 1A). Use of the proliferative marker MIB-1, as well as identifying individual mitoses (M-phase cells) within tissue sections, demonstrated that epithelial cell proliferation occurred preferentially within the primitive crypt structures (Fig. 1C). However, a substantial number of dividing cells were also identified throughout the cryptvillus axis. This non-compartmentalised division process formed a characteristic feature in pre-transplanted tissues, and was especially apparent in younger and less well developed fetal gut. In the first week after transplantation the intestinal xenografts became visibly haemorrhagic and necrotic. The lumen filled with degenerating epithelial tissue which sloughed off the immature villi to leave denuded areas of mucosa showing signs of ischaemic tissue damage. In the second to fourth week following transplantation there was evidence of angiogenesis and focal epithelialisation in areas of necrosis. Initially, the epithelium formed a flattened stratified tissue which spread out to encompass large areas of regenerating mucosa (Fig. 1D and E). At this stage of development the epithelium was approximately two to four cells thick and closely resembled the stratified epithelium seen in early human fetal gut of approximately 7 to 10 weeks gestational age [6]. Similarly to pre-transplanted fetal gut, epithelial cell proliferation occurred throughout the mucosa and showed no evidence of compartmentalisation (Fig. IF). Small, superficial blood vessels formed a characteristic feature during this developmental stage. However, after approximately 4 to 8 weeks following transplantation this transformed into an extensive circumferential vascular array which supplied the serosa, muscularis propria, intermyenteric plexus, submucosa, lamina propria and epithelium (Fig. 1L). A characteristic mucosa formed approximately 10 weeks after transplantation and this pattern did not change substantially up to 6 months posttransplantation, which was the last time-point investigated (Fig. IG,H and J). Typically, small bowel xenografts possessed well developed villi and crypts, whereas in large bowel the villi were replaced by a characteristically flat mucosa. Developed xenograft tissue was, therefore, classified as possessing a histologically intact and differentiated mucosa when sampled at least 10 weeks after transplantation. Developing xenograft tissue was classified as possessing a histologically incomplete and undifferentiated mucosa, when sampled in the first 8 weeks after transplantation. In all cases the xenograft survival rate was greater than 9096, indicating the suitability of the scid mutation, as compared with nude mice, to maximise the use of available tissues [lo, 45, 461. Similar developmental profiles were recorded for both small and large bowel, although time-course studies demonstrated that proximal small intestine differentiated and matured more rapidly than distal small or large bowel. The resulting mucosal architecture was entirely dependent upon the region of fetal intestine chosen for transplantation. Typically, villi were tall and ridge-like in

364

Fig. 1A-L. Morphological and functional development of human fetal intestine xenografted subcutaneously into severe-combined immunodeficient (scid) mice. A, B and C Hematoxylin and eosin (H&E), periodic acid-Schiff (PAS-) and MIB- 1-stained pretransplanted fetal small intestine (gestational age of 14.2 weeks), respectively. Sections showing MIB- I positive (brown) nuclei were counter-stained with haematoxylin. D-F H&E, PAS and MIB-I immunoperoxidase stained sections of developing small intestinal xenograft tissue 6 weeks after transplantation, respectively. G I H&E, PAS and MIB-1 stained sections of developed small intesti-

nal xenograft mucosa 10 weeks after transplantation, respectively. J H&E-stained large intestinal xenograft mucosa 12 weeks after transplantation. K Profile for brush border alkaline phosphatase activity in a single villus from proximal small intestinal xenograft mucosa (arrows indicate crypt-villus junction). L Pattern of vascularisation in developed intestinal xenografts arrows, blood vessels; arrowhead, mitotic figure within crypt of Lieberkuhn). Scale bars represent 200 pm in D, G and J; 150 pm in A and K; 50 pm in B, E and H; 25 pm in C, F, I and L

365

'r 5

O t -1

V-243&8 C-40f2

V-122t8 C-3Qt4

I**

4 --

T

\

V-80*7 C-325&15

FETAL INTEST"

PAEDUTRIC INTESTINE XENOORAFT INTESTINE

PSI

DSI

LI

Fig. 2. Cephalocaudal differences in intestinal villus height and crypt depth. Developed xenograft villus height (V) and crypt depth (0,expressed individually (pm) and as a villus-crypt (VX) ratio for proximal small intestine (PSI), distal small intestine (DSI) and large intestine ( L o , are compared statistically with corresponding segments of pretransplanted fetal intestine. Data for paediatric gut is restricted to measurements from proximal small intestine (**, fcO.001;*s.e.m.)

developed proximal small intestine (Fig. 1G and H) and were significantly shorter in distal sites approaching the ileo-caecal junction (Fig. 2). Transplanted fetal large bowel initially possessed villus-like structures and developed to form a characteristic flat mucosa, which was supplied by crypts 2.4-fold longer than those measured in small intestinal xenografts (Fig. 1J). Regional measurements of villus height and crypt depth are shown in Fig. 2 and are expressed as a vi1lus:crypt ratio, as an additional sensitive measure of these cephalocaudal structural differences. In contrast to fetal and developing xenograft mucosa, epithelial cell proliferation was confined exclusively to the intestinal crypts in developed xenografts (Fig. 1I), and closely resembled measurements made in histologically normal paediatric gut (manuscript in preparation). MIB- 1 staining was also abundant in the muscularis layers and lamina propria, demonstrating a proliferative capacity for human-derived cells within these sites.

and externa in xenografted tissues remained exclusively human, although murine cells rapidly populated the lamina propria and submucosa (Fig. 3B and C). The chimeric nature of the lamina propria was highly dependent upon the stage of intestinal development. In developing xenograft tissues, murine cells comprised 43.9+4.1% (n=4) of the mesenchymal composition, which was densely packed under the stratified epithelium (Figs. 1 and 3B). In situ electron microscopy demonstrated that the infiltrating mouse cells consisted primarily of fibroblasts and smooth muscle cells, as well as inflammatory cells, especially eosinophils. In developed intestinal xenografts there was a 2.3-fold reduction in the number of infiltrating murine cells within the lamina propria ( I 8.7+1.1%; Pc0.001 (Mann Whitney U-test); n=4). At this stage, the mesenchyme appeared less densely packed when compared with developing xenograft intestine, a feature described previously during gastrointestinal ontogenesis [6, 261. A notable feature was that the majority of pericryptal myofibroblasts were of a human origin, whereas the rest of the lamina propria consisted of a chimeric mixture (Fig. 3D). Additional immunocytochemical steps demonstrated that human CD45+ leukocytes were present both within the epithelium and lamina propria in developed xenografts (Fig. 3E). In situ electron microscopy showed the human intraepithelial lymphocytes (IELs) to possess a classical lymphocytic appearance (Fig. 4A). The vasculature supplying the xenografts was shown to have a chimeric cellular comp6sition. The endothelial cells were exclusively of murine origin and were encompassed by vimentinpositive human pericytes (Fig. 3F). This feature was corroborated further using in situ hybridisation at the electron microscopical level (Fig. 4B). Temporal and spatial distribution of epithelial cell lineages in intestinal xenografs

Light and electron microscopy demonstrated that differentiated forms of all the major intestinal epithelial cell lineages i.e. columnar absorptive, goblet, Paneth and enteroendocrine cells, were present within developed xenografts. Absorptive cells

Genetic determination of host and donor cell types in intestinal xenografs

Non-isotopic double-label in situ hybridisation was used to determine the genetic origin of the cellular components comprising the intestinal xenografts. Specifically, the nature and extent of murine cell infiltration was assessed by simultaneous detection of DNA molecules from human and mouse species, coupled to biotin and digoxigenin reporter molecules, respectively. In situ hybridisation performed on pre-transplanted fetal gut as control demonstrated all cell types to be of a human origin (Fig. 3A). The epithelium and muscularis mucosa

Absorptive cells represented the major epithelial cell lineage in developed xenografts, and appeared highly differentiated compared with cells in pre-transplanted fetal gut. Typically, these had a well developed glycocalyx and brush border membrane with microvilli projecting from the underlying terminal web (Fig. 4A). Neighbouring epithelial cells were interconnected by apical zonula occludens and desmosomes, which were interspersed along the lateral plasma membrane. These cells also possessed a large number of apical mitochondria, a well organised network of rough endoplasmic reticulum and golgi apparatus situated just above the basally positioned nucleus. The differentiation state of absorptive cells was

366

Fig. 6a-c. Epithelial cell lineages in developed small intestinal xenograft tissue. A PAS-positive goblet cells (arrows),also showing the well-developed PAS-stained brush border membrane (arrowheads) of enterocytes, B Paneth cells within H&E-stained intesti-

nal crypts (arrows). C Chromogranin-positive enteroendocrine cell identified using an immunoperoxidase staining technique with diaminobenzidine substrate (arrow). Scale bars represent 25 pm for A and C, 50 ym for B

367

Fig. 4A, B. Ultrastructural in situ hybridisation utilising either (A) the human DNA probe with 10nm gold particles or (B) the mouse DNA probe with 15-nm gold particles. A Section through small intestinal xenograft villus epithelium (010 weeks after transplantation. A human origin for the intraepithelial lymphocyte ( / E L ) is demonstrated by the nuclear labelling with 10-nm gold particles as shown in the insert (arrowheads);Mi, mitochondria; Mv, microvilli. B Part of a blood vessel within the lamina propria showing large clumps of 15-nm gold particles (armwheads) within the nucleus (N)of an endothelial cell (En); R, erythrocytes. Scale hars represent 1 p n and 0.5 pm for panels and inserts, respectively

Fig. 3A-F. Identification of host and donor cell types within intestinal xenografts using a non-isotopic double-label in situ hybridisation technique. Species-specific DNA probes, coupled to biotin and digoxigenin, permit the identification of human (brown nuclei) and murine (black/purple nuclei) cell types in situ, respectively. The double-labelling method was applied to: pre-transplanted fetal small intestine of 14.2 weeks gestational age (A) (e, epithelium; lp, lamina propria); developing xenograft small intestine 6 weeks after transplantation (B); and developed xenograft small intestine 10 weeks after transplantation (C-F). C demonstrates the murine cell infiltration to be largely restricted to the lamina propria and submucosa. The epithelium ( e ) and muscularis mucosa and externa (m)are of a human 0rigin.D Intestinal crypts are embraced by human pericryptal myofibroblasts (arrows);arrowhead, mitotic figure in crypt. Additional immunocytochemical steps for the human common-leukocyte antigen (CD45) and vimentin are shown to positively identify human leukocytes (E arrows; including IELs) and blood vessels (F arrnws), respectively. Small intestinal villi are outlined in (C-E) as a consequence of non-specific brush border alkaline phosphatase activity during the APAAP reaction. F The xenograft vasculature consists of a chimeric mixture, possessing murine endothelial cells (arrowheads), which are closely associated with vimentin-expressing human pericytes (arrows). The intestinal xenografts, therefore, exploit the host vasculature in a fashion akin to tumour angiogenesis. Scale bars represent I50 pm in C, 75 pm in A, 25 pm in B, D and E; I5 pm in F

also investigated by measuring brush border alkaline phosphatase (AP) activity along the crypt-villus axis in fetal, xenograft and paediatric intestine. AP is a maturation marker for absorptive cells and is found primarily in the proximal small intestine [2, 331. A clear cephalocaudal gradient in enzyme activity was recorded in developed xenograft tissues, with highest and lowest levels measured in proximal small intestine and large bowel, respectively (Fig. 5A). A direct comparison of xenograft and paediatric proximal small intestine demonstrated both tissues to possess similar levels of AP enzyme activity, as well as distribution gradient along the crypt-villus axis (Fig. 1K and 5B). Developed xenografts expressed a cellular AP activity which was 5-fold higher compared with pre-transplanted fetal gut, demonstrating an advanced maturational state in the former (Fig. 5B). Goblet cells

Mature goblet cells were abundant in xenografted intestine and harboured large numbers of homogenous mucus granules positioned in between the basally situated cres-

368 n70

-

50

w 0 40 -

r

GOBLET CELLS

60

W

50

z

U 30 m

-

a 20 0 10 -

z5r

v)

m

a

0 -

PSI

DSI

LI **

n 80

? 0

z 0

In

PANETH CELLS

70

::o

60

$1

!L&-

-** -**

1

L

ENTEROENDOCRINE CELLS

50

4r

40 30 20 10

5 0 \

-

-50

0

50

100 150 200 250 300 350 400 450 500

DISTANCE FROM C:V JUNCTION ( p m ) Fig. SA, B. Cephalocaudal differences in villus brush border alkaline phosphatase (AP) activity. A Mean AP activities for proximal small intestine (PSI), distal small intestine (DSI) and large intestine (LI) in developed xenografts are compared statistically with corresponding segments of pre-transplanted fetal intestine. Data for paediatric gut is restricted to measurements from proximal small intestine (* P
cent shaped nucleus and the apical brush border. The most apically positioned granules were occasionally seen to discharge into the luminal environment. A number of organelles, including mitochondria and free ribosomes, were located in between the mucus granules and in the vicinity of the lateral membranes. At a light microscopical level, goblet cells were readily identified in periodic acid-schiff (PAS)-stained paraffin sections (Figs. 1 and 6A). Mature goblet cells were present in all tissues investigated and demonstrated a marked abundance in the distal small and large, as compared with the proximal small intestine (Fig. 7). Goblet cells were, however, present at reduced levels in fetal and developing xenograft tissues compared with developed xenograft and paediatric gut (Fig. 7). Paneth cells Paneth cells were collectively located in the bottom of intestinal xenograft crypts and showed only occasional evidence of dysplasia. Ultrastructurally, the characteristic Paneth cell secretory granules were situated in the supranuclear region and showed a highly homogeneous and electron-dense matrix. The cytoplasm around the basally positioned nucleus contained the rough endoplasmic reticulum and golgi apparatus. Light microscopical analysis of the distribution of Paneth cells in hematoxy-

PSI

DSI

LI

Fig. 7. Temporal and spatial distribution of epithelial cell lineages during intestinal xenograft development. Cephalocaudal distribution of goblet, Paneth and enteroendocrine cells in fetal (A), developing (less than 8 weeks, 0)and developed (more than 10 weeks, 0 )xenograft intestine. Values for developed xenograft tissues are compared statistically with corresponding segments of pre-transplanted fetal and developing xenograft intestine. Data for paediatric gut (0)is restricted to measurements from proximal small intestine (EC, epithelial cells; * P
lin and eosin (H&E)-stained paraffin sections demonstrated them to be confined to small intestinal crypts, as they were largely absent in the large bowel (Fig. 6B and 7). Mature Paneth cells were occasionally present in the proximal small intestine in pretransplanted fetal gut, but were consistently absent in distal small and large bowel. This cephalocaudal gradient in Paneth cell distribution also formed a prominent feature in developing and developed xenograft intestine, although they were present in significantly higher numbers in the latter (Fig. 7). Enteroendocrine cells Enteroendocrine cells were triangular in shape, narrowing at the apical membrane domain. The irregularly shaped nucleus tended to be situated centrally within the cell, with the characteristic cytoplamic granules located in the basal cell portion. Cytoplasmic mitochondria, free ribosomes and a well organised golgi complex were also consistently present. Enteroendocrine cells were identified at the light microscopical level using a chromogranin monoclonal antibody [7] which stains the entire cell lineage in human intestine (Fig. 6C). Chromagranin-positive enteroendocrine cells represented the minority cell type in all tissues investigated, constituting approximately 1% of the epithelial cell population in developed xenograft intestine (Fig. 7). Cells were present throughout

369

the crypt-villus axis and were often seen to cluster within tissue sections. In contrast to goblet and Paneth cells, enteroendocrine cells were more numerous in fetal and developing tissues compared with matured xenograft intestine.

Discussion Cell division during gastrointestinal ontogenesis gives rise to several characteristic epithelial cell lineages [ 11, which emerge according to a specific chronology [19, 351. The inductive epithelial-mesenchymal interactions which are essential during this developmental process are usually reciprocal with respect to time and exert a specific local regulation between cell proliferation, differentiation and morphogenesis [ 121. These effects also extent into adulthood where they play an important role in establishing and maintaining structural and functional gradients along the gastrointestinal tract, as well as along the crypt-villus axis[ 131. The present work demonstrates that immature human fetal gut retains a ‘positional memory’ when transplanted subcutaneously into scid mice. This results in xenografted tissues growing and developing in an idiosyncratic manner reflecting the characteristics of the initial donor tissue i.e. proximal and distal small and large bowel. A normal complement of mature intestinal epithelial cell lineages is recorded once the xenografts develop and mature. Brush border AP activity, used as a differentiation marker for columnar absorptive cells, displayed a strong proximal to distal gradient in developed xenograft tissues, as is normally observed in human gut [2, 211. In addition, the pattern of AP activity along the crypt-villus axis closely resembled that of paediatric intestine, indicating an advanced maturation state for absorptive cells on the villi compared with actively proliferating MIB-I -positive cells confined to the crypts. Goblet cells displayed a characteristic proximal to distal increase in numbers, being most abundant in xenografted large bowel. A similar gradient was also recorded in fetal and developing xenograft tissues, although the goblet cell representation in these tissues was lower than that in developed xenografts. Mature Paneth cells were largely absent in pre-transplanted fetal tissues and appeared only in small numbers during xenograft development, where they were confined to the mucosal folds forming the primitive crypts of Lieberkuhn. Paneth cells were rarely detected in xenografted large bowel, but were present in normal numbers in developed small intestinal xenografts. Chromogranin-positive enteroendocrine cells were present in all tissues studied, often occurring in clusters along the entire crypt-villus axis. Enteroendocrine cells were more abundant in fetal and developing xenograft tissues when compared with developed xenograft mucosa, suggesting a putative role for the products of these cells to be particularly important during gastrointestinal ontogenesis. Several reports have demonstrated marked effects of enteroendocrine - derived products e.g. enteroglucagon on gastrointestinal differentiation and func-

tion [40, 421. Work is currently in progress to ascertain whether specific functional subclasses of enteroendocrine cells andor growth factors are implicated in xenograft development, as has been suggested for human fetal gut developing in utero [29]. The above findings demonstrate position-dependent differences in the terminal differentiation of human intestinal epithelial cells during xenograft development. Transplanted human intestinal stem cells, therefore, not only retain a prolonged proliferative capacity in vivo [35], but may also possess positional information which permits the regeneration of a morphologically and functionally distinct epithelial cell population [5, 381. These suggestions have been made from work using other intestinal grafting models [9, 37, 38, 431. Juxtaposed nonepithelial cells could also potentially relay such positional information, by providing inductive information either directly to epithelial stem cells, or to immature crypt cells which have yet to undergo terminal differentiation, thereby bypassing a direct stem cell involvement [32]. The double-label in situ hybridisation studies demonstrated that the xenograft mesenchyme had a chimeric composition due to murine cell infiltration shortly after transplantation. As the intestinal xenografts differentiated, the extent of murine cell infiltration within the lamina propria more than halved. This was due to a reduced presence of both murine inflammatory and connective tissue cells within the developed mucosa, indicating the existence of, possible negative selection mechanisms and/or a lack of infiltration into developed tissues and/or a preferential expansion of human cells within the lamina propria. In developed xenografts, human mesenchymal cells consisted primarily of fibroblasts and smooth muscle cells, although the latter may have been transformed from the former as a consequence of an inductive influence by the human endoderm [3]. A notable feature was that although the lamina propria consisted of a chimeric cell mixture, the intestinal crypts were embraced predominantly by human pericryptal myofibroblasts. It has been suggested that these cells mediate cross-talk between the lamina propria and epithelium, thus regulating epithelial cell gene expression, proliferation and differentiation during fetal development and adulthood [ 17, 361. To date it is still unclear whether mesenchymal stem cells exist, although the present work has demonstrated the existence of human mesenchymal cells 6 months after transplantation. Consequently, these cells are either long-lived surviving mesenchymal cells or daughter cells derived from an undefined stem cell lineage. A local control is, therefore, exerted on epithelial-pericryptal myofibroblast interactions in vivo, and is most likely to be mediated by preferential cell-associations between the epithelium and human cells within the lamina propria. Alternatively, in a response to locally derived signals, altered differentiation pathways could be proposed to act on epithelial stem cells, to generate a human pericryptal myofibroblast lineage. This latter view seems unlikely, however, when regarding the chimeric epithelialpericryptal myofibroblast interactions described by other authors [3, 121.

370

Epithelial differentiation pathways are likely to be influenced by local trans-acting factors produced by nonepithelial cells i.e. mesenchymal, neuronal and immune cells or from luminal stimuli present in gastrointestinal secretions. The detection of human IELs in the developed intestinal xenografts was intriguing as these must have survived the initial tissue degeneration and subsequently homed back into the epithelium. A putative role for such cells in regulating intestinal xenograft development may, therefore, be suggested, especially as human IELs have been shown to express genes which are implicated in the development and maintenance of gastrointestinal epithelia [4,27, 391. A notable feature was that xenograft development occurred without high systemic levels of hormones which are normally associated with pregnancy e.g. progesterone. In addition, conventional luminal stimuli e.g. amniotic fluid, bile salts and pancreatic secretions, all of which have been shown to influence epithelial proliferation and differentiation, were not essential for tissue-specific development [31]. In view of these findings, supported by a relative lack of pancreatic and biliary secretions during intrauterine life, it is unlikely that these particular factors play a major role in gastrointestinal ontogenesis. Post-natally, such factors are undoubtedly important, as are additional components made available as a consequence of food, indigenous flora and luminal antigens, all of which profoundly regulate intestinal epithelial proliferation and differentiation pathways [ 161. As a consequence, we are now studying the physiological effects that specific luminal stimuli e.g. purified and/or recombinant growth factors and cytokines, have on human gastrointestinal development and function, by utilising the xenograft model system as a new approach to critically evaluate such events under stringent experimental conditions. Acknowledgements. The authors thank Drs T.C. Freeman and L. Wong for helpful comments. This work was funded by the Biotechnology and Biological Sciences Research Council (T.C. Savidge), the Royal Society (A.N. Shmakov) and a Queen Elizabeth Hospital for Children Research Appeal Trust (T.C. Savidge).

References 1. Cheng H, Leblond CP (1974) Origin, differentiation and re-

newal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am J Anat 141537-562 2. Dahlqvist A, Lindberg T (1966) Development of the intestinal disaccharidase and alkaline phosphatase activities in the human fetus. Clin Sci 3 0 5 17-528 3. del Bouno R, Fleming KA, Morey AL, Hall PA, Wright NA ( 1992) A nude mouse xenograft model of fetal intestine development and differentiation. Development 1 14:67-73 4. Deutsch K, Schollhorn K, Butz C . Wagner F, Classen M (1993) Human intestinal intraepithc4i.il lymphocytes constitutively express genes that maintain cpiihclial growth and function. Gastroenterology 104:A691 5. Duluc I, Freund J, Leberquier C, Kediii!p M (1994) Fetal endoderm primarily holds the temporal ; i i d positional information required for mammalian intestinill tlcvelopment. J Cell Biol 126:211-221

6. Grand RJ, Watkins JB, Torti FM (1976) Development of the human gastrointestinal tract: A review. Gastroenterology 70:79O-8 10 7. Facer P, Bishop AE, Lloyd RV, Wilson BS, Hennessy RJ, Polak JM (1985) Chromogranin: A newly recognised marker for endocrine cells of the human gastrointestinal tract. Gastroenterology 89: 1366-1 373 8. Ferguson A, Parrot DMV (1972) Growth and development of ‘antigen-free’ grafts of fetal mouse intstine. J Pathol 106:95-102 9. Ferguson A, Gerskowitch VP, Russel RI (1973) Pre- and postweaning disaccharidase patterns in isografts of fetal mouse intestine. Gastroenterology 64:292-297 10. Friedberg JS, Ryan DP, Driscoll SG, Folkman J (1985) Human small bowel transplants into athymic mice and rats. Surg Forum 36:375-378 1 1. Grumpel-Pinot M, Yasugi S, Mizuno T (1978) Differentiation d’epitheliums endodermiques associes au mesoderme splanchnique. CR Acad Sci Paris 286: I 17-121 12. Haffen K, Kedinger M, Bouziges F, Simon-Assmann P (1989) Mesenchyme-endoderm interactions and enterocyte development. In: Smith MW, Sepulveda FV (eds) Adaptation and development of gastrointestinal function. Manchester University Press, Manchester, pp. 92-102 13. Haffen K, Kedinger M, Simon-Assmann P (1989) Cell contact dependent regulation of enterocytic differentiation. In: Lebenthal E (ed) Human Gastrointestinal Development. Raven Press, New York, pp 19-39 14. Hendrickson EA (1993) The SCID mouse: relevance as an animal model system for studying human disease. Am J Pathol 143:1511-1 522 15. Henning SJ (1987) Functional development of the gastrointestinal tract. In: Johnson LR (ed) Physiology of the gastrointestinal tract. Raven Press, New York, pp. 285-300 16. Jankowski JA, Wright NA (1992) Epithelial stem cells in gastrointestinal morphogenesis, adaptation and carcinogenesis. Semin Cell Biol 3:445456 17. Kaye GI, Lane N, Pascal RR (1968) Colonic pericryptal fibroblast sheath: replication, migration, and cytodifferentation of a mesenchymal cell system in adult tissue. II. Fine structural aspects of normal rabbit and human colon. Gastroenterology 54:852-865 18. Kedinger M, Simon PM, Grenier JF, Haffen K (1981) Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes. Dev Biol 86:339-347 19. Klein RM, McKenzie JC (1983) The role of cell renewal in the ontogeny of the intestine. 1. Cell proliferation patterns in adult, fetal, and neonatal intestine. J Pediat Gastroenterol Nutr 2:10-43 20. Lacroix B, Kedinger M, Simon-Assmann PM, Haffen K (1984) Effects of human fetal gastroenteric mesenchymal cells on some developmental aspects of animal gut endoderm. Differentiation 28: 129-135 21. Lacroix B, Kedinger M, Simon-assmann P, Haffen K (1984) Early organogenesis of human small intestine: scanning electron microscopy and brush border enzymology. Gut 25~925-930 22. Leapman SB, Deutsch AA, Grand RJ, Folkman J (1974) Transplantation of fetal intestine: survival and function in a subcutaneous location in adult animals. Ann Surg 179:109-114 23. Le Dourain N, Bussonnet C, Chaumont F (1968) Etude des capacitCs de differenciation et du r6le morphogkne de I’endoderme pharyngien chez I’embryon d’oiseau. Ann Embryo1 Movhog 1 :29-39 24. Louvard D, Kedinger M, Hauri HP (1992) The differentiating intestinal epithelial cell: Establishment and maintenance of functions through interactions between cellular structures. Annu Rev Cell Biol 8: 157-1 95 25. Lynch DAF, Clarke AMT, Jackson P, Axon ATR, Dixon MF, Quirke P (1994) Comparison of labelling by bromodeoxyuri-

37 I dine, MIB-I, and proliferating cell nuclear antigen in gastric mucosal biopsy specimens. J Clin Pathol47:122-125 26. Mathan M, Hermos JA, Trier JS (1972)Structural features of the epithelio-mesenchymal interface of rat duodenal mucosa during development. J Cell Biol52:577-588 27. Marsh MN, Cummins AG (1993)The interactive role of mucosal T lymphocytes in intestinal growth, development and enteropathy. J Gastroent Hepatol 8:270-278 28.Morey AL, Ferguson DJP, Leslie KO, Taatjes DJ, Fleming KA (1993)Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridisation with digoxigenin-labelled probes. Histochem J 25:421429 29. Moxey PC, Trier JS ( I 977)Endocrine cells in the human fetal small intestine. Cell Tissue Res 183:33-50 30.Moxey PC, Trier JS (1978)Specialised cell types in the human small intestine. Anat Rec 191:269-286 31. Mulvihill SJ, Stone MM, Debas HT, Fonkalsrud EW (1985) The role of amniotic fluid in fetal nutrition. J Paed Surg

20:668-672 32. Paulus U, Loeffler M, Zeidler J, Owen G, Potten CS (1993) The differentiation and lineage development of goblet cells in the murine small intestinal crypt: experimental and modelling studies. J Cell Sci 106:473484 33. Phillips AD, Smith MW, Walker-Smith JA (1988)Selective alteration of brush-border hydrolases in intestinal diseases in childhood. Clin Sci 74:193-200 34. Polkinghorne Report (1989)Review of the Guidance on the Research Use of Fetuses and Fetal Material. CM 762 HMSO, London 35. Potten CS, Loeffler M (1990)Stem cells: attributes, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development I10:1101-1020 36. Richman PI, Tilley R. Jass JR, Bodmer WF (1987)Colonic pericrypt sheath cells: characterisation of cell type with a new monoclonal antibody. J Clin Pathol40:593-600 37. Rubin DC, Roth KA, Birkenmeier EH, Gordon JI (1991)Epithelial cell differentiation in normal and transgenic mouse intestinal isografts. J Cell Biol I 13:1183-1 192

38.Rubin DC, Swietlicki E, Roth KA. Gordon JI (1992)Use of fetal intestinal isografts from normal and transgenic mice to study the programming of positional information along the duodenal-to-colonic axis. J Biol Chem 267:I5 122-15 133 39. Shmakov AN, Trufakin VA (1989)The effect of lymphocytesT on the proliferation of jejunal mucosa epithelium in thymectomized mice. Tsitologiya 3 1 : 1074-1 079 40.Simonpoulos C, Gaffen JD, Bennet A (1989)Effects of gastrointestinal hormones on the growth of human intestinal epithelial cells in vitro. Gut 30:600-604 41.Smith MW (1991)Cell biology and molecular genetics of enterocyte differentiation. Curr Top Memb 39:153-1 79 42.Solicia E, Capella C, Buffa R, Vsellini L, Fiocca R, Sessa F (1987)Endocrine cells of the digestive system. In: Johnson LR (ed) Physiology of the gastrointestinal tract. Raven Press, New York, pp 1 1 1-130 43.Tait IS,Flint N, Campbell FC, Evans GS (1994)Generation of neomucosa in vivo by transplantation of dissociated rat postnatal small intestinal epithelium. Differentiation 56:91-100 44.Thulin JD, Kuhlenschmidt MS, Gelberg HB (1991)Development, characterisation and utilisation of an intestinal xenograft model for infectious disease research. Lab Invest 65719-73 1 45.Verstijnen CPHJ, Kate JT, Arends JW, Schutte B, Bosman FT (1988) Xenografting of normal colonic mucosa in athymic mice. J Pathol 15577-85 46.Winter HS, Hendren RB, Fox CH, Russel GJ, Perez-Atayde A, Bhan AK, Folkman J (1991)Human intestine matures as nude mouse xenograft. Gastroenterology IOO:89-98 47.Yasugi S (1993)Role of epithelial-mesenchymal interactions in differentiation of epithelium of vertebrate digestive organs. Dev Growth Differ 35:1-9 48.Zinzar SN, Leitina BI, Tumyan BG, Svet-Moldavsky GJ ( I 971) Very large organlike structures formed by syngeneic foetal alimentary tract transplanted as a whole or in parts. Rev Europ Etudes Clin Et Biol XVI:455458