isolation of stem cell populations with trophic and immunoregulatory functions from human intestinal...
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Cytotherapy (2009) Vol. 11, No. 8, 1020–1031
Isolation of stem cell populations with trophic and immunoregulatory functions from human intestinal tissues: potential for cell therapy in infl ammatory
bowel disease Giacomo Lanzoni1, Francesco Alviano 1, Cosetta Marchionni 1, Laura Bonsi1,
Roberta Costa1, Laura Foroni 2, Giulia Roda 3, Andrea Belluzzi3, Alessandra Caponi3, Francesca Ricci4, Pier Luigi Tazzari4, Pasqualepaolo Pagliaro4, Roberta Rizzo 5,
Francesco Lanza6, Olavio Roberto Baricordi5, Gianandrea Pasquinelli7,8, Enrico Roda3 and Gian Paolo Bagnara1,8,9
1Department of Histology, Embryology and Applied Biology, and 2Department of Specialistic Surgical and Anaesthesiological Sciences, University
of Bologna, Bologna, Italy, 3Department of Clinical Medicine, Gastroenterology Unit, S. Orsola Hospital, University of Bologna, Bologna, Italy, 4Transfusion Medicine Service, Department of Hematology, Oncology and Laboratory Medicine, S. Orsola-Malpighi Hospital, Bologna, Italy, 5Department of Experimental and Diagnostic Medicine, Laboratory of Immunogenetics, Section of Medical Genetics, University of Ferrara,
Ferrara, Italy, 6Section of Hematology, BMT Unit, St Anna Hospital, University of Ferrara, Ferrara, Italy, 7Department of Radiological and
Histocytopathological Sciences, S. Orsola Hospital, Bologna, Italy, 8INBB, Biostructures and Biosystems National Institute, Interuniversity
Consortium, Rome, Italy, and 9‘Giorgio Prodi’ Interdepartmental Research Centre for Cancer Research, University of Bologna, Bologna, Italy
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Background aims
Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal
stromal cells (MSC) are currently under evaluation in phase III
clinical trials for infl ammatory bowel disease and other intestinal
disease manifestations. The therapeutic effi cacy of these treatments
may derive from a combination of the differentiation, trophic and
immunomodulatory abilities of the transplanted cells. We investi-
gated intestinal tissues as sources of MSC: such cells may support
tissue-specifi c functions and hold advantages for engraftment and
contribution in the gastrointestinal environment.
Methods
Intestinal specimens were collected, and the mucosa and submucosa
mechanically separated and enzymatically digested. Mesenchymal
stromal populations were isolated, expanded and characterized under
conditions commonly used for MSC. The differentiation potential,
trophic effect and immunomodulatory ability were investigated.
Results
We successfully isolated and extensively expanded populations showing
the typical MSC profi le: CD29� , CD44 � , CD73 � , CD105 � and
© 2009 ISCT
Correspondence to: Giacomo Lanzoni , Department of Histology, Embr 8-Bologna 40126, Italy. E-mail: [email protected]
CD166� , and CD14−, CD34− and CD45−. Intestinal mucosal (IM)
MSC were also CD117� , while submucosal cultures (ISM MSC)
showed CD34� subsets. The cells differentiated toward osteogenic,
adipogenic and angiogenic commitments. Intestinal-derived MSC
were able to induce differentiation and organization of intestinal
epithelial cells (Caco-2) in three-dimensional collagen cultures.
Immunomodulatory activity was evidenced in co-cultures with
normal heterologous phytohemagglutinin-stimulated peripheral
blood mononuclear cells.
Conclusions
Multipotent MSC can be isolated from intestinal mucosal and
submucosal tissues. IM MSC and ISM MSC are able to per-
form trophic and immunomodulatory functions. These fi ndings
could open a pathway for novel approaches to intestinal disease
treatment.
Keywords
cell therapy , immunomodulation , infl ammatory bowel disease ,
intestinal stem cell , mesenchymal stromal cells , stem cells , trophic
function .
DOI: 10.3109/14653240903253840
yology and Applied Biology, University of Bologna , Via Belmeloro ,
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Introduction Multipotent mesenchymal stromal cells (MSC) are a rare population of cells possessing stem characteristics [ 1– 3 ] initially found in the bone marrow (BM) environment [ 4 ]. Evidence regarding the multipotent nature has occurred both in vitro and in vivo : BM-derived MSC were found to be able to differentiate toward multiple mesodermal commit-ments, such as osteogenesis, chondrogenesis, adipogenesis and angiogenesis [ 5 ]. The differentiation potential seems to encompass endodermal and ectodermal commitments as well, widening the biologic signifi cance of these cells and the range of practicable approaches to tissue regeneration [ 6 ].
MSC seem to be distributed diversely in vivo and may occupy a ubiquitous stem cell niche, playing a part in physiologic tissue kinetics, tissue regeneration and remodeling [ 7 ]. Evidence is mounting that such multipotent progenitors may reside within the perivascular niche of several organs [ 8 ].
MSC are candidates for cell therapy in several clinical settings because they possess a number of appealing properties: along with their multidifferentiation potential, MSC exhibit the ability to modulate the immune response, act as trophic mediators, persist in tissues of allogeneic transplanted receivers [ 9 ] and migrate and engraft in sites of wound healing [ 10 , 11 ]. Clinical trials are under way to evaluate their effect in conditions requiring regeneration and paracrine stimulation (e.g. myocardial infarction and cardiovascular diseases [ 12] ) and immune suppression (e.g. allogeneic transplantation and graft-versus-host disease [ 13] ). Moreover, MSC are attracting the attention of investigators and clinicians in the fi eld of infl ammatory bowel disease (IBD). IBD is thought to be the result of an abnormal immune response to commensal bacteria and luminal antigens in a susceptible host. The intermittent and aggressive presenta-tion of the pathology profoundly affects the quality of life of patients. Poor disease prognosis, the inadequacy of conventional therapies and the current understanding of IBD biology are motivating investigators to develop novel approaches, so advancement in cell-based therapies could drive a major change. MSC may afford a therapeutic approach targeted at the site of injury, aiming at tissue regeneration and local immune modulation.
To this end, BM- and adipose tissue (AT)-derived MSC are currently being evaluated in phase III clinical trials as therapeutic agents for IBD manifestations [ 14 ]. However, it is becoming clear that the differentiation yield, spontaneous in vivo differentiation and trophic potential differ profoundly
among MSC from alternative sources [ 15 – 17 ]. Thus, it should be expected that resident MSC could be ontogenically privileged for supporting specifi c functions of the tissue of origin, and engraftment may be preferential when used in a clinical context.
We describe a method for the isolation and culture of human MSC with stem characteristics from intestinal mucosal (IM) and submucosal (ISM) specimens; the popu-lations show an immunophenotype comparable with that found in BM-derived MSC; furthermore, they are able to differentiate in vitro toward osteogenic, adipogenic and angiogenic commitments following protocols established for MSC. These cells also have a remarkable ability to instruct differentiation and three-dimensional (3-D) organization of epithelial cell lines as well as modulate the proliferative response of phytohemagglutinin-stimulated lymphocytes. In line with the recommendation of the International Society for Cellular Therapy [ 18 ], we have named the populations intestinal mucosal mesenchymal stromal cells (IM MSC) and intestinal submucosal mesenchymal stromal cells (ISM MSC).
Methods Isolation and culture expansion of IM MSC and ISM MSC Clinical specimens were obtained after informed consent, as approved by the local ethics committee (S. Orsola-Malpighi University Hospital, Bologna, Italy). Seven colon cancer patients undergoing tumor resection gave informed consent for the study. We obtained surgical specimens from unaffected regions of colonic and ileal segments (average size 2 � 3 cm) and these were rapidly transferred to the laboratory in phosphate-buffered saline (PBS; Sigma Aldrich Co., St Louis, MO, USA) containing penicillin 100 U/mL (Sigma Aldrich Co.) and streptomycin 100 μg/mL (Sigma Aldrich Co.). The mucosa and submucosa were separated mechanically, washed with PBS and maintained at 37°C, 5% CO2 for 24 h in a disinfection medium, i.e. Dulbecco’s modifi ed Eagle medium (DMEM; Sigma Aldrich Co.) containing penicillin 100 U/mL (Sigma Aldrich Co.), streptomycin 100 μg/mL (Sigma Aldrich Co.), amphotericin B 25 μg/mL (Sigma Aldrich Co.), cefazolin 240 μg/mL (Pharmacia, Milan, Italy), lincomycin 120 μg/mL (Pharmacia), colimycin 100 μg/mL (UCB Pharma, Brussels, Belgium) and vanco-mycin 50 μg/mL (Hospira, Lake Forest, IL, USA). Tissues were then washed in PBS, minced and enzymatically digested by 1.5 mL trypsin 500 mg/L–EDTA 200 mg/L (Sigma Aldrich Co.) and 1.5 mL collagenase IV 10 mg/mL
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(Sigma Aldrich Co.) for 45 min at 37°C, 5% CO2. The sus-pensions were passed through a 100-μm strainer and the enzymatic digestion was blocked by adding an equal volume of fetal bovine serum (FBS; Lonza, Basel, Switzerland). The cell suspensions were then washed in PBS, resuspended in DMEM with 20% FBS, seeded into 75-cm 2 culture fl asks (BD, Franklin Lakes, NJ, USA) and incubated at 37°C, 5% CO2. Non-adherent cells were removed at day 7 of culture, and the medium replaced with fresh DMEM–10% FBS every 4 days. When the culture reached 90% confl uence, cells were harvested using trypsin–EDTA 1X solution (Sigma Aldrich Co.) and expanded or cryopreserved. Each population was culture-expanded for at least 15 passages. BM MSC, isolated as reported previously, [ 19 ] were used as positive controls.
Immunophenotypic characterization For fl ow cytometric analysis, the adherent populations obtained from mucosal and submucosal samples were harvested at the fourth culture passage and 10 5 cells/test were incubated with monoclonal antibodies (MAb) accord-ing to the manufacturers’ suggestion. The antibodies (Ab) used were: CD14–fl uorescein isothiocyanate (FITC), CD29–FITC, CD34–phycoerythrin (PE), CD44–FITC, CD45–allophycocyanin (APC), CD105–PE, CD117–FITC, CD166–PE, HLA class I–FITC, HLA class II–FITC (all from Beckman-Coulter, Fullerton, CA, USA) and CD73–FITC (BD-Pharmingen, San Diego, CA, USA). After washing, cells were analyzed using a Cytomics FC500 fl ow cytometer (Beckman-Coulter). Results were analyzed using CXP software (Beckman-Coulter).
In vitro differentiation studies At the third culture passage, IM MSC and ISM MSC were induced to differentiate toward adipogenic, osteogenic and angiogenic commitments. As positive controls, differentiation studies were performed in parallel with BM MSC (data not shown). Parallel cultures in DMEM–10% FBS were considered as negative controls. In vitro osteogenic, adipo-genic and angiogenic inductions were conducted following protocols widely used for BM MSC as described previously [ 20 ]. For osteogenesis evaluation, cultures were fi xed in ethanol and von Kossa-stained to detect calcium phosphate deposition. Adipogenesis was evaluated by Red Oil staining. Angiogenesis was tested by matrigel assay (BD) and fl ow cytometry. After angiogenic induction, the cells’ ability to self-organize in tube-like structures was tested over a
Matrigel matrix; the expression of FLT-1, KDR and von Willebrand Factor (vWF) was evaluated by fl ow cytometry. The following MAb were used according to the manufac-turers’ suggestions: FLT-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), KDR (RD System, Minneapolis, MN, USA) and vWF (DakoCytomation, Glostrup, Denmark); for vWF detection, permeabilization was performed with a Intraprep Kit (Beckman-Coulter). Data were acquired using a Cytomics FC500 fl ow cytometer and analyzed using CXP software.
Trophic effect in 3-D collagen cultures Three-dimensional collagen cultures were arranged in order to investigate the trophic effect of IM MSC, ISM MSC and BM MSC on intestinal epithelial cells. The epithelial colorectal adenocarcinoma cell line Caco-2 was used. Caco-2 cells were cultured in DMEM–10% FBS and kept subconfl uent. Passage three IM MSC (three lines), ISM MSC (three lines) and BM MSC (three lines) were seeded at 1.5 � 10 4 cells/cm 2 in T25 fl asks and cultured in DMEM–10% FBS. After 72 h the conditioned medium was collected, and the cells harvested and resuspended at a concentration of 3 � 10 5 cells/mL in DMEM–10% FBS. Caco-2 were harvested contemporaneously and resuspended at 3 � 10 5 cells/mL in DMEM–10% FBS. In 96-well plates, 50 μL epithelial cell suspension were combined with 50 μL IM MSC suspension (1:1 MSC to epithelial cell ratio), ISM MSC suspension, BM MSC suspension, IM MSC conditioned medium, ISM MSC conditioned medium, BM MSC conditioned medium or DMEM–10% FBS as a control; 25 μL collagen solution (type I/type III bovine collagen; StemCell Technologies, Vancouver, Canada) were added rapidly to each well and resuspended. Plates were incubated at 37°C 5% CO2. Polymerization occurred in 30 min; 50 μL fresh medium were then added over the collagen gels and changed every 24 h: either DMEM–10% FBS (for co-cultures and controls) or the respective condi-tioned medium was used. After 7 days of culture, the gels were fi xed in 2.5% phosphate-buffered glutaraldehyde for 24 h at 4°C. Samples were processed for either histologic analysis or transmission electron microscopy.
Transmission electron microscopy Glutaraldehyde fi xation was followed by post-fi xation with 1% buffered OsO4 for 1 h at 20°C. The cells were then washed with 0.15 m phosphate buffer, dehydrated in a graded series of ethanol and embedded in epoxy resin.
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Ultra-thin sections were counterstained with lead citrate and uranyl acetate and observed in a transmission electron microscope, Tecnai 12 (FEI Company, Hillsboro, OR, USA).
Immune modulation The immune-modulating activity of IM and ISM MSC was evaluated in co-culture experiments with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) and compared with that of BM MSC following protocols reported previously [ 19 ]. This experimental setting overcomes interferences by HLA class I and II mismatches that may occur in the context of mixed lymphocyte reactions (MLR). Briefl y, PBMC were obtained from two healthy donors after informed consent and isolated from whole blood by Ficoll gradient (Cederlane, Burlington, Canada). Third-passage IM MSC (two samples), ISM MSC (two samples) and BM MSC (two samples) were harvested, and 8 � 10 3 MSC co-cultured in quadruplicate in a 96-well cell culture cluster (Corning Inc., Corning, NY, USA) with 4 � 10 4 PBMC (fi nal ratio MSC/PBMC � 1:5) and purifi ed PHA 1 μg/mL. As a control, PBMC and MSC were cultured alone and with or without PHA. The lymphoproliferative response was measured after 72 h of culture by overnight incubation with 1 μCi/well [methyl- 3 H]thymidine (3.7 � 10 4 Bq; Amersham, Amersham, UK). Radioactivity was measured using a multiple cell harvester (Skatron, Oslo, Norway) and a beta-counter (LKB, Uppsala, Sweden).The results were obtained as counts per minute of quadruplicate cultures (mean c.p.m. � standard deviation). In order to compare the response obtained from the two different PBMC, data were expressed as the percentage residual response.
Results MSC can be derived from mucosal and submucosal tissues After 2 weeks of culture, homogeneous populations of MSC were isolated from IM and ISM tissues. The cultures were then maintained in a subconfl uent condition, corresponding to the exponential phase of growth, and expanded at a 1:3 ratio every 3 days. Like BM-derived MSC, the populations displayed a fi broblast-like morphology and were easily expanded for at least 10 culture passages ( Figure 1 ).
IM and ISM MSC express MSC markers The immunophenotypic profi le of IM and ISM MSC was consistent with that reported for BM MSC. Characterization
by fl ow cytometry revealed that fourth passage IM MSC were positive for the markers CD29, CD44, CD73, CD105 and CD166, and negative for CD14, CD34 and CD45 ( Figure 1 ). ISM MSC were positive for the markers CD29, CD44, CD73, CD105 and CD166, possessed a positive subset for CD34, and were negative for CD14 and CD45. Of note, IM MSC appeared CD117/c-kit positive to a wide extent (65% of cells), whereas ISM MSC were found to be CD117/c-kit negative. IM and ISM MSC were positive for HLA class I and negative for HLA class II ( Figure 1 ).
IM and ISM MSC are able to differentiate toward multiple mesenchymal commitments Third-passage IM and ISM MSC were induced toward osteogenic, adipogenic and angiogenic commitments ( Figures 2 and 3 ).Osteogenic differentiation was evident after 2 weeks of induction in both IM and ISM MSC: morphologic changes were accompanied by widespread deposition of a mineralized matrix. The abundant mineralization was positive upon von Kossa staining ( Figure 2 ). No signs of osteogenic differentiation were found in control cultures.
Following adipogenic induction, IM and ISM MSC accumulated neutral lipid droplets and were found to be positive upon Oil Red O staining ( Figure 2 ). IM MSC gave a globally lower yield in adipogenic differentiation. No signs of adipogenic differentiation were found in control cultures.
Remarkably again, both IM and ISM MSC showed features of angiogenic differentiation potential. Cultured with the specifi c medium and seeded on top of matrigel, IM and ISM MSC started organizing after 4 h and capillary-like structures were evident after 6 h ( Figure 3 , matrigel panel); cells maintained a tube-like organization for at least 24 h. Unstimulated cells showed few signs of organization. Flow cytometry analysis of FLT-1, KDR and vWF expression yielded interesting observations. IM MSC showed a con-spicuous increase in angiogenic marker expression after induction, whereas in ISM MSC the increase was less prominent ( Figure 3 , fl ow cytometry panel).
IM and ISM MSC have trophic functions on intestinal epithelial cells We set up 3-D collagen cultures in order to investigate the trophic effect of IM MSC, ISM MSC and BM MSC upon intestinal epithelial cells (Caco-2 line). Light and transmission electron microscopy observations revealed that IM and
1024 G. Lanzoni et al.
IM-MSC
CD29 CD44 CD73 CD105 CD166 CD14 CD34 CD45 CD117 HLA class I HLA class II
IM-MSC
ISM-MSC
ISM-MSC
BM-MSC
BM-MSC
Figure 1. MSC were isolated from IM and ISM; fourth passage cultures in active proliferation are shown. These populations displayed a
homogeneous fi broblast-like morphology, similar to that of BM-derived MSC. The immunophenotypic profi le of IM and ISM MSC was
consistent with that obtained for BM MSC. Representative histograms of fl ow cytometric evaluations are shown; white areas represent
background log fl uorescence relative to isotype control Ab. IM MSC expressed CD29, CD44, CD73, CD105, CD166, CD117 and HLA
class I and were negative for CD14, CD34, CD45 and HLA class II. Similarly ISM MSC were positive for CD29, CD44, CD73, CD105,
CD166 and HLA class I, possessed a positive subset for CD34 and were negative for CD14, CD45, CD117 and HLA class II.
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ISM MSC, but not BM MSC, were able to induce Caco-2 differentiation and 3-D organization. When cultured alone in collagen, Caco-2 cells proliferated as discrete cell aggregates with minimal ultrastructural features of epithelial differen-tiation, i.e. the presence of rows of small well-developed desmosomes; no signs of cell polarization could be seen ( Figure 4 ). When co-cultured with IM or ISM MSC,
von Kossa stainingOsteogenic induction
IM-MSCs
ISM-MSCs
Control
Figure 2. IM and ISM MSC were induced toward osteogenic comm
induced cultures indicated that IM and ISM MSC can differentiate int
Kossa staining panel). IM and ISM MSC were induced toward adipog
accumulated neutral lipid droplets, positive at Oil Red O staining. Control
Caco-2 formed distinct tubular structures still evident with light microscopy; with hematoxylin-eosin staining, nearly 100% of the Caco-2 cell clusters showed a glandular lumen. Enterocyte differentiation and polarized cytoplasm organization were clearly evident upon ultrastructural investigation: specialized microvilli, with inner contractile fi laments and outer glycocaliceal fi laments, projected from
ControlAdipogenic inductionOil Red O staining
itment and von Kossa-stained. The widespread positivity found in
o osteocytes. Control cultures showed no signs of differentiation (von
enic commitment and stained with Oil Red O. IM and ISM MSC
cultures showed no signs of differentiation (Oil Red O staining panel).
Intestinal MSC 1025
IM-MSCs
Angiogenic induction
Mat
rigel
_Flo
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Control Angiogenic induction Control
ISM-MSCs
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4h
6h
2h
4h
6h
FLT-1 KDR vWF FLT-1 KDR vWF
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FL4 Log FL4 LogFL2 Log FL2 LogFL1 Log FL1 Log
027
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023
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032
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Figure 3. In order to investigate the angiogenic potential of IM and ISM MSC, cells were cultured in induction or control media and seeded
on matrigel. Induced cells organized into capillary-like structures; controls showed few signs of spontaneous organization. The time–course
was reported at 2, 4 and 6 h (matrigel panel). The expression of angiogenic markers was analyzed by fl ow cytometry: FLT-1, KDR and
vWF increased sharply in IM MSC after angiogenic induction, while the shift in expression for angiogenic ISM MSC was less prominent.
Black areas represent fl uorescence relative to uninduced cells, light areas represent angiogenic induced cells (fl ow cytometry panel).
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the apical plasma membrane into the secretory lumen. Moreover, tight junctions and desmosomes separated the apical side from the basolateral surface ( Figure 4 ). Particu-larly in IM MSC/Caco-2 co-cultures, electron microscopy disclosed many immature secretory intracytoplasmatic lumina that were characterized by the presence of tightly packed well-structured microvilli. Considering the ultra-structural appearance of induced glandular differentiation, ISM MSC seem to have a greater pro-differentiative effect than IM MSC. BM MSC were able to induce a limited dif-ferentiation, without architectural organization, on Caco-2 cells: well-developed desmosomes and sporadic closed lumina were the only features of differentiation observed ( Figure 4 ).
To test the hypothesis that IM and ISM MSC can release soluble factors involved in this induction, we set up colla-gen cultures with MSC-conditioned media; the treatment partially replicated the induction observed in co-cultures, thus confi rming the involvement (data not shown).
IM and ISM MSC can have a remarkable immune modulatory role Intestinal-derived MSC displayed the ability to inhibit proliferation of PHA-stimulated PBMC. The results obtained from six different MSC/PBMC/PHA experiments are reported in Figure 5 . The lymphoproliferative response was evaluated after 72 h of culture and [methyl- 3 H]thymidine incorporation;
1026 G. Lanzoni et al.
Caco-2 Caco-2�IM-MSCs Caco-2�ISM-MSCs Caco-2�BM-MSCs
4µm 4µm 4µm 2µm
0.5µm0.5µm0.5 µm0.5 µm 1µm1µm1µm 1 µm
Figure 4. Three-dimensional collagen cultures were set up to investigate the trophic effect of IM, ISM and BM MSC upon intestinal
epithelial cells (Caco-2 line). Transmission electron microscopy observations revealed that Caco-2 cultured alone in collagen proliferated as
cell aggregates with no signs of cell polarization. In 3-D co-cultures with IM or ISM MSC, Caco-2 showed features of enterocytic differentiation,
i.e. secretory lumina lined with specialized microvilli and junctional complexes separating the apical side from the basolateral surface. Caco-2 �
IM MSC co-cultures at high magnifi cation: microvilli showing surface glycocaliceal fi laments (arrowheads) and actin-fi lament rootlets
(arrow). Caco-2 � ISM MSC co-cultures at high magnifi cation: detailed structure of a junctional complex showing tight junction (large
arrowhead), intermediate junction (small arrowhead) and desmosome (arrow); the thin arrows show cross-sectioned microvilli with evident
inner contractile fi laments. Overall ISM MSC seemed to have a greater pro-differentiative effect than IM MSC. BM MSC were able to
induce a limited differentiation, without architectural organization, on Caco-2 cells. Caco-2� BM MSC at high magnifi cation: one of the
sporadic closed lumina, with well-developed desmosomes.
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it is reported as a percentage residual response, i.e. the ratio of MSC/PBMC/PHA proliferation compared with the corresponding PHA-activated PBMC culture response considered as 100%. IM and ISM MSC showed a strong inhibitory effect on the proliferation of PHA-stimulated PBMC, comparable to that of BM MSC .
120
100
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40
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Immunomodulatory effect of MSCs on PHA-stimulated PBMCs
0PBMC�PHA PBMC�PHA
�IM-MSC_1PBMC�PHA�IM-MSC_2
PBMC�PHA�ISM-MSC_1
PBMC�PHA�ISM-MSC_2
PBMC�PHA�BM-MSC_1
PBMC�PHA�BM-MSC_2
Figure 5. The MSC ability to modulate the proliferation of
PHA-stimulated PBMC was investigated. The lymphoproliferative
response of six different MSC/PBMC/PHA culture combinations,
measured as [methyl-3H]thymidine incorporation, is reported as a
% residual response Like BM MSC, IM and ISM MSC showed a
strong inhibitory effect on PBMC proliferation.
Discussion MSC have a number of attractive properties for clinical application: they exhibit important stem characteristics and have trophic and immunomodulatory functions. Pro-tocols based on MSC are among the fi rst cell-therapy approaches to have reached an advanced phase for the treatment of IBD: BM-derived MSC and AT-derived MSC are currently under evaluation. A protocol based on systemic infusion of allogeneic BM MSC has been designed for the treatment of high-grade steroid-resistant Crohn’s disease; phase I and II trials have obtained intrigu-ing results, and the Food and Drugs Administration recently allowed it to advance to phase III. AT MSC have been shown to possess promising potential for local treatment of Crohn’s disease manifestations, and a protocol for isolation, expansion and in situ delivery of autologous AT MSC in ulcerations recently moved to phase III [ 14 , 21 ]. The cell source is a key parameter to consider in the process of designing targeted cell therapies. We investigated intestinal tissues as sources of MSC: such cells may support tissue-specifi c functions and hold advantages for engraftment and contribution in the gastrointestinal environment.
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MSC seem to be diversely distributed in vivo and may occupy a ubiquitous stem cell niche, with functions in physiologic tissue kinetics, tissue regeneration and remodeling [ 7 ]. Evidence is mounting that such multipotent progenitors may reside within the perivascular niche of several organs [ 8 ]. The best characterized MSC population is the one found in BM, but cells with similar properties have been isolated from various sources, such as AT [ 22 ], placenta [ 23 ], amniotic membrane [ 20 ], umbilical cord [ 24 ], dental pulp [ 19 ], skeletal muscle [ 25 ] and arterial and venous vessels [ 26 , 27 ]. Because of the lack of a single defi nitive marker, prospective isolation from fresh samples is still impossible; MSC are retrospectively characterized by the presence of a combination of markers (CD29 � , CD44 � , CD73 � , CD105 � and CD166 �) and the absence of hematopoietic markers (CD14−, CD34− and CD45−).
MSC possess a multipotent nature: they have been found to be able to differentiate toward multiple mesodermal commitments [ 5 ]. The differentiation potential may also encompass endodermal and ectodermal commitments, widening the biologic signifi cance of these cells and the range of practicable approaches to tissue regeneration [ 6 ].
Moreover, MSC play remarkable immunomodulatory roles [ 28 ]. They express intermediate levels of HLA class I, low levels of HLA class II and do not activate allogeneic T cells, a behavior connected with the lack of expression of co-stimulatory molecules. MSC also suppress allogeneic T-cell proliferation and do not elicit an immune response after transplantation into immunocompetent recipients [ 29 , 30 ]. The suppression of lymphocyte proliferation has been shown in primary MLR and mitogen responses to PHA, concanava-lin and tuberculin [ 31 ]. Again, MSC inhibit the T-lymphocyte activation mediated by anti-CD3 and -CD28 Ab in primary and in vitro expanded cultures [ 32 ]. The molecular mecha-nisms that enable MSC to abrogate lymphoproliferation are still widely unknown, but several reports claim the impor-tance of soluble factors; modifi cations in the cytokine balance, such as an increase in interleukin (IL)-2, IL-10 and soluble HLA-G (sHLA-G) levels, are suggested to have a lympho-suppressive property [ 33 , 34 ]. MSC have been shown to improve the clinical outcome of autoimmune disease, as a result of suppressive modulation of the T-cell pathogenic autoimmune response [ 35 ]; interesting data obtained in a mouse model of autoimmune enteropathy corroborate the hypothesis that MSC can induce immune tolerance target-ing gut-associated lymph nodes [ 36 ]. MSC can be safely administered in autologous and allogeneic settings [ 12 ].
Furthermore, MSC have been shown to produce a number of growth factors and cytokines with a key role in tissue repair and remodeling. Intense studies on the paracrine activity of these cells suggest they are able to participate in tissue healing and long-term repair as trophic mediators, stimulating endogenous populations [ 11 , 37 ].
MSC have been isolated from several tissues and a large amount of work converges in describing shared features among these populations, but still differences exist in strict relation to the tissue of origin [ 38 ]. BM-derived MSC have been investigated extensively and clinical protocols have already been established for conditions requiring immune modulation and tissue regeneration. Evidence has accrued regarding their ability to contribute to intestinal lineages under stress conditions; they have been shown to engraft in the mucosa, giving rise to both mesenchymal and epithelial cytotypes [ 39 , 40 ]. Nevertheless, the use of BM-derived MSC could represent a troublesome and low-yield approach. AT-derived MSC are an attractive autologous source, but rather ectopic if the application is intended for intestinal tissues. Moreover, concern has been raised over MSC spontaneous differentiation and trophic potential, as they may be critically related to the tissue of origin [ 16 , 38 ]. Evidence from stringent in vivo assays shows that populations derived from different sources are not functionally equivalent. Kaltz et al. [ 16 ] showed that BM-derived MSC do possess the ability to form spontaneously a heterotopic ossicle and a hematopoiesis-supporting stroma in vivo , whereas umbilical vein-derived MSC do not. The ontogenetical history that BM MSC have undergone enables a type of tissue differentiation that has not been reproduced by MSC from a different source. It is possible to speculate that tissue-resident MSC could possess a developmental privilege for supporting specifi c functions of the tissue of origin and, thereafter, for engrafting it. Yet little is known about the presence and role of cells with stem characteristics in the intestinal stromal compartment. Mucosal tissue kinetics have been investigated deeply on the epithelial side, although an exhaustive depiction of an intestinal epithelial stem cell is still lacking, whereas the subepithelial stroma remains a rather obscure milieu. In the intestinal environment, mesenchymal cell populations play key roles in both normal and pathologic conditions. Mucosal mesenchyme coordinates the differentiation of the epithelium derived from the endoderm during embryogenesis and throughout adulthood. Subepithelial mesenchymal cells constitute the epithelial stem cell niche and promote mucosal remodeling by instructing the proliferation and
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migration of epithelial cells over denuded areas in ulceration recovery. Again, they are in charge of extracellular matrix (ECM) deposition and remodeling, representing the main source of matrix metalloproteinases, proteolytic enzymes ultimately responsible for ECM degradation and tissue destruction during infl ammation. They respond to the chronic infl ammatory environment, causing the fi brosis complication frequently found in Crohn’s disease. Moreover, mesenchymal populations participate in infl ammation management via the secretion of immunoregulatory cytokines [ 41 ]. A specialized mesenchymal differentiation, intestinal microvasculature, is critically involved in IBD pathogenesis [ 42 , 43] ; not only does it superintend the egression of migrating leukocytes to the infl amed areas, but it is crucial in ensuring perfusion for wound healing.
The submucosa still deserves consideration: the mucosa and submucosa cooperate during infl ammation [ 44 ] and the layer is a site for hemorrhage and edema in high-grade Crohn’s disease. Moreover, gastrointestinal stromal tumors (GIST; representing 1–3% of all gastrointestinal malig-nancies) originate here, presumably from cells related to interstitial cells of Cajal [ 45 ].
Mucosal biopsies can be obtained easily by endoscopy but, because of the nature of the tissue, primary cultures are at high risk of bacterial and mycotic contamination. The submucosa is less accessible by endoscopy, but is easily obtainable from tissue resection. Submucosa is located in a sterile environment and the risk of culture contamination is low.
Our investigation focused on intestinal resident mesen-chymal stromal populations, as they may possess advantages for applications targeted at the intestine. We succeeded in isolating multipotent mesenchymal cell populations from intestinal mucosa and submucosa. After overnight culture in disinfection medium, cells were isolated through mechanical and enzymatic digestion. Plastic-adherent mesenchymal cells appeared morphologically similar to BM-derived MSC, exhibited a high proliferative potential and expressed typical MSC markers, such as CD29, CD44, CD73, CD105 and CD166, while expressing limited or no hematopoietic markers such as CD14, CD34 and CD45. Of note, ISM mesenchymal cultures possessed CD34-positive subsets, while IM MSC expressed c-kit, the stem cell factor receptor. These cells may be mesenchymal precursors of interstitial cells of Cajal and thus be correlated with cells giving rise to GIST [ 45 ]. Intestinal MSC expressed HLA class I but not HLA class II. In vitro induction experiments indicate that
these cells possess multiple differentiation potential: they are able to differentiate toward the osteogenic, adipogenic and angiogenic lineages. In adipogenic-induced cultures an unusual differentiation pattern occurs: several cells accumulating droplets detach from the surface and remain in suspension. This behavior has never been described for MSC deriving from other sources. ISM MSC display a higher adipogenic yield than IM MSC.
Again, human IM and ISM MSC show the ability to undergo angiogenic commitment: induced populations increase the expression of VEGF receptor 1 and 2 (FLT-1 and KDR, respectively) and vWF. Moreover they acquire the ability to self-organize and stabilize tube-like networks over matrigel, a basement membrane matrix. The micro-circulatory system is involved in IBD establishment and protraction, reduced mucosal perfusion accompanies poor wound healing and the maintenance of infl ammation [ 42 ]. Microvascular cells regulate leukocyte transmigration via the secretion of chemokines and expression of adhesion mole-cules. Disregulated activation of the CD40/CD40 ligand system could lead to a loop perpetuating infl ammation [ 46 ]. Again, microvascular dysfunction may be connected with a loss of nitric oxide-mediated vasodilation [ 47 ]. Khalil et al. [ 43 ] have reported that engineered murine CD34− cells, derived from BM and peripheral blood, are able to home in the intestinal environment and differ-entiate toward an endothelial fate therein. Therapeutically infused in dextran sulfate sodium-colitic mice, the cells improved intestinal mucosal perfusion and enhanced microcirculation. The treatment resulted in improved tissue regeneration and lowered mortality. The angiogenic potential that we found in intestinal MSC may be of central importance for cell-based approaches to ameliorate perfusion and offset vascular-mediated impairments. Our results pave the way for in vivo evaluation of IM and ISM MSC differentiation.
Three-dimensional collagen cultures provided interesting insights into IM and ISM MSC biology, showing their ability to induce differentiation and support architectural organization of intestinal epithelial cells. We documented this trophic effect in co-culture experiments with Caco-2 cells, and have further observed that soluble factors released by IM and ISM MSC are involved in this induction. Inter-estingly, BM MSC were able to induce only a limited differentiation, without architectural organization, on Caco-2 cells. These data strengthen our hypothesis regarding the ability of intestinal MSC to support tissue-specifi c functions,
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such as creating the microenvironment for instructing epithelial cells toward commitment and organization. This may be of great relevance for approaches intended to stimulate intestinal wound re-epithelialization.
Co-culture experiments with PHA-stimulated PBMC indicated remarkable immunomodulatory properties for these populations. These fi ndings warrant future research: intestinal MSC and their progeny may play roles in the maintenance of the physiologic minimal persistent infl am-matory state, acting as immune regulators and tolerogenic mediators. The immune-modulating ability of intestinal MSC may be benefi cial in the treatment of IBD and other immune-based disorders.
MSC are under evaluation in clinical trials in both autologous and allogeneic settings. Intestinal MSC could be isolated from mucosal biopsies obtained by endoscopy; this process, routinely performed by endoscopists, involves minimal discomfort for the patient and could allow the expansion of IM MSC for autologous transplantation. Submucosal resections could be obtained with limited discomfort by endoscopic pinching after local adrenaline injection, allowing the expansion of autologous ISM MSC. Tissues collected during appendectomies and obtained from multiorgan donors could be considered as sources of intestinal MSC for allogeneic applications. Such cells could be characterized for HLA compatibility and banked.
Finding cells with a broad stem potential in the intestine supports the existence of a stem continuum in mesenchy-mal tissues and may be helpful in the clarifi cation of intes-tinal tissue kinetics. Moreover, the isolation of these stem cells may open new possibilities for therapeutic approaches targeting intestinal tissue regeneration.
Authors’ contributions GL, FA, CM and RC carried out MSC isolation, differen-tiation and cultures. GL, LB, RR, FL and ORL performed immune modulation assays. FR and PLT performed the cytometric analysis. LF and GP performed electron microscopy analysis. GR, AB, AC, PP, GP and ER contrib-uted to study design and coordination. GL and GPB designed and coordinated the study. All authors read and approved the fi nal manuscript.
Acknowledgment We thank Professor Mario Taffurelli, Dr Giampaolo Ugolini and Dr Giancarlo Rosati for providing tissue samples, Dr Gino Ferioli and Dr Gabriella Mattei for technical
assistance in histologic staining, and Dr Ralph Nisbet for checking the manuscript.
These studies were supported by grants from the University of Bologna (RFO 2006) and the Cassa di Risparmio in Bologna CARISBO Foundation.
Declaration of Interest: The authors report no confl icts of interest. The authors alone are responsible for the content and writing of the paper.
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