ORIGINAL RESEARCH ARTICLE
Journal of
Identity and Ranking of Colonic Mesenchymal Stromal Cells
Cellular Physiology
MICHELE SIGNORE,1 ANNA MARIA CERIO,1 ALESSANDRA BOE,1 ALFREDO PAGLIUCA,1 VALENTINA ZAOTTINI,1 ILARIA SCHIAVONI,2 GIORGIO FEDELE,2 STEFANO PETTI,1 SIMONE NAVARRA,1 CLARA MARIA AUSIELLO,2 ELVIRA PELOSI,1 ALESSANDRO FATICA,3 ANTONIO SORRENTINO,1,4 AND MAURO VALTIERI1,5* 1
Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita`, Rome, Italy
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Department of Infective, Parasitic and Immuno-mediated Diseases, Istituto Superiore di Sanita`, Rome, Italy
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Department of Biology and Biotechnology Charles Darwin, ‘‘La Sapienza’’ University, Rome, Italy
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Department of Medicine, University of California, San Francisco, California
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Sbarro Institute for Cancer Research and Molecular Medicine & Center of Biotechnology, College of Science and Technology,
Temple University, Philadelphia, Pennsylvania Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn’s disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146þ MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions. J. Cell. Physiol. 227: 3291–3300, 2012. ß 2011 Wiley Periodicals, Inc.
Ongoing clinical trials utilize bone-marrow mesenchymal stromal cells (BM-MSCs) to treat Crohn’s disease (CD) (Arseneau et al., 2007; Giordano et al., 2007; Eksteen et al., 2008; Abraham and Cho, 2009; Dryden, 2009) (www.clinicaltrials.gov) a chronic inflammatory bowel disease rapidly increasing in wealthier countries. This approach stems from the immune regulatory effect exerted by BM-MSCs (Aggarwal and Pittenger, 2005; Jiang et al., 2005; Kang et al., 2005; Bocelli-Tyndall et al., 2007; Chamberlain et al., 2007; Nauta and Fibbe, 2007; Uccelli et al., 2008; Crop et al., 2009). An important piece of information missing to complete the rationale for this cellular therapy is the presence and the activity of mesenchymal stromal cells (MSCs) in the intestinal walls. The intestinal mucosa is normally in a state of ‘‘controlled’’ inflammation where resident MSCs could finely tune a delicate balance of pro-inflammatory and anti-inflammatory cytokines (Bosani et al., 2009). Classically, MSCs represent a bone marrow (BM) population, defined by five functional properties: Extensive life-span, ability to support hematopoiesis and capacity to differentiate into osteoblasts, chondrocytes and adipocytes (Prockop, 1997; Pittenger et al., 1999; Caplan, 2007; Sorrentino et al., 2008; Valtieri and Sorrentino, 2008). Additional properties of MSCs, mediated by secretion of bioactive molecules, include structuring a regenerative microenvironment and regulating the immune system. MSCs home to sites of tissue damage (Aggarwal and Pittenger, 2005; Jiang et al., 2005; Kang et al., 2005; Bocelli-Tyndall et al., 2007; Chamberlain et al., 2007; Nauta and Fibbe, 2007; Uccelli et al., 2008; Crop et al., 2009) and can repair tissues after minimal ß 2 0 1 1 W I L E Y P E R I O D I C A L S , I N C .
engraftment or (trans-) differentiation, due to paracrine activity and to immune-modulation (Aggarwal and Pittenger, 2005; Jiang et al., 2005; Bocelli-Tyndall et al., 2007; Chamberlain et al., 2007; Crop et al., 2009). MSCs immune-regulatory activities affect different cell types including T lymphocytes, NK cells and dendritic cells (DC) (Aggarwal and Pittenger, 2005; Jiang et al., 2005; Kang et al., 2005; Bocelli-Tyndall et al., 2007; Nauta and Fibbe, 2007; Crop et al., 2009). Furthermore, MSC’s low immunogenicity, allows to match them, in most cases, with immune cells from different donors (Chamberlain et al., 2007; Nauta and Fibbe, 2007; Uccelli et al., 2008; Crop et al., 2009).
Additional supporting information may be found in the online version of this article. Contract grant sponsor: Ministero della salute, Ricerca Finalizzata 1%; Contract grant number: 8AGF. *Correspondence to: Mauro Valtieri, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy. E-mail:
[email protected] Manuscript Received: 13 October 2011 Manuscript Accepted: 28 November 2011 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 14 December 2011 DOI: 10.1002/jcp.24027
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This unique array of proficiencies, coupled with low immunogenicity, oriented the use of these versatile cells in several areas of medicine as shown by ongoing clinical trials (www.clinicaltrials.gov), also in CD (Arseneau et al., 2007; Giordano et al., 2007; Eksteen et al., 2008; Abraham and Cho, 2009; Dryden, 2009). However, the lack of consistency in MSCs isolation and characterization, together with uncertainty about their categorization, still represent a limitation for their research, application and safety (Prockop, 1997; Pittenger et al., 1999; Caplan, 2007; Sacchetti et al., 2007; Caplan, 2008; Crisan et al., 2008; Sorrentino et al., 2008; Valtieri and Sorrentino, 2008; Me´ndez-Ferrer et al., 2010; Tormin et al., 2011). In fact, nothing is known about the presence and the function of MSCs in the human adult colon. We have recently isolated, extensively characterized, and banked CD146þ tetrapotent MSC sub-clonal populations from 21 human BM donors, displaying the canonical MSC four differentiation properties, including hematopoietic support (Sacchetti et al., 2007; Sorrentino et al., 2008; Valtieri and Sorrentino, 2008). In addition to their functional properties we defined their expression of 44 antigens and their miRNoma (Sorrentino et al., 2008). CD146 represents a key novel marker for the prospective isolation of a self-renewing osteo-progenitor subpopulation (Sacchetti et al., 2007) and it has been recently used by our group (Sorrentino et al., 2008; Valtieri and Sorrentino, 2008) and other laboratories (Crisan et al., 2008) to improve and extend MSC isolation from virtually all human tissues, both normal and pathological. We have applied our method to isolate MSCs from six additional human tissues, including small discarded colon diagnostic biopsies from which, after localization of CD146þ MSCs in colon walls by confocal microscopy, nine sub-clonal C-MSCs lines were obtained. According to a recent view CD146þ MSCs might partly overlap with pericytes (Sacchetti et al., 2007; Caplan, 2008; Crisan et al., 2008; Sorrentino et al., 2008; Valtieri and Sorrentino, 2008; Tormin et al., 2011), cells lining the outer wall of pre-capillary arteriolar vessels feeding all tissues, including colon lamina propria underlying the wrinkled muco-epithelial lumen of colon walls, structured in Lieberku¨hn crypts (Abraham and Cho, 2009). The biopsy pinches these structures, harvesting fragments of tissue containing many different cell types, MSCs among others, which were isolated and expanded. Given the clonal heterogeneity of MSCs (Prowse et al., 2011; Tormin et al., 2011), availability of prospective isolation markers and information about the tissue distribution of MSCs, however, does not automatically translate into cell identity after isolation from different sources. A detailed analysis of MSCs from different areas is mandatory for the understanding of their tissue-specific role and for resolving many pending issues such as the ‘‘niche function’’ of MSCs for normal versus cancer stem cell survival, proliferation, and diffusion (Karnoub et al., 2007; Rosland et al., 2009; Me´ndez-Ferrer et al., 2010; Vermeulen et al., 2010; Winkler et al., 2010). Comparative analysis of BM- and C-MSC lines at multiple levels is needed to robustly categorize them for their safer and more controlled use in premature, yet rapidly progressing, clinical settings. Materials and Methods Confocal microscopy of colon sections
Images were acquired with an Olympus FV-1000 spectral confocal microscope. The following objectives were used: UPLAPO 20 0.70 N.A. and UPLFLN 40 1.30 N.A. oil immersion objectives. Scale bars for 20 and 40 magnifications correspond to 100 and 50 mm, respectively. Normal colonic mucosa specimens were obtained from patients who signed an informed consent before undergoing surgical resection of a tumor lesion. On day 1, tissue was fixed in PFA 4% for 2 h at RT and passed overnight at JOURNAL OF CELLULAR PHYSIOLOGY
þ48C through ascending concentrations of sucrose, from 10 to 30% in the following 2 days. On day 4, the tissue was embedded in OCT, promptly frozen with liquid nitrogen and stored at 808C. Thick sections (25 mM) were cut on a cryotome and processed for immuno-fluorescence. Permeabilization was performed with 0.5% Triton X-100 in PBS, overnight at þ48C. A blocking step was performed with 3% BSA in PBS for 1 h at RT and subsequent incubation with primary antibodies was carried out for 24 h at þ48C. Mouse anti-CD146 FITC-conjugated (Biocytex, Marseille, France) and rabbit anti-vWF (Millipore, Billerica, MA) were used. AlexaFluor555-conjugated anti-Rabbit secondary antibody was incubated at RT for 4 h together with DAPI and AlexaFluor647conjugated Phalloidin for visualization of actin cytoskeleton. Images shown in Figure 1 represent maximum intensity projections of z-stack series obtained with each different objective using a pinhole size correspondent to 1 Airy Unit. 3D reconstruction was performed using Imaris v7.2.3 software (Bitplane Scientific Software, Zurich, Switzerland): Maximum intensity projection and alpha-blend methods were used, for supplementary video 1 and 2, respectively. Isolation and characterization of MSC from human colon biopsies
The isolation procedures from human colon biopsies was derived from the BM procedure (Sorrentino et al., 2008), detailed below. BM from healthy donors was purchased from CAMBREX Poietics cell systems (Gaithersburg, MD). BM samples were treated for 20 min at 208C with RosetteSep human MSC enrichment cocktail (StemCell Technologies, Vancouver, BC, Canada) composed by CD3, CD14, CD19, CD38, CD66b, Glycophorin A tetrameric antibody (Ab) complexes crosslinking unwanted cells with red blood cells, diluted, and centrifuged over Ficoll-Hypaque gradient for 25 min at 300g at 208C. Enriched cells were collected, washed, and treated with NH4Cl (StemCell) for 10 min in ice to remove residual red blood cells. CD34þ cells were removed by MACS column (Miltenyi, Bergisch Gladbach, Germany). Enriched cells were then cultured at sub-clonal density (1–10 cells/cm2) for 3 weeks in a-medium (Invitrogen, Carlsbad, CA), 20% fetal calf serum (FCS; StemCell) in T75 flasks at 378C in 5%CO2/O2 atmosphere. Half medium was replaced two times a week until MSCs reached confluence, defined as passage (P) 0. MSCs were detached by trypsin-ethylenediamine tetraacetic acid solution (Invitrogen) treatment, partly freezed and partly replated. Each weekly replating or P was defined by a progressive number. Colon and lung surgical specimens, obtained from patients who signed an informed consent before undergoing surgical resection of a tumor lesion, were thoroughly washed with PBS supplemented with 5X antibiotic/antimycotic (A/A) solution (Invitrogen), maintained o/n in PBS 5X A/A at 48C, treated with 30–45 ml 1 mM EDTA/EGTA PBS 75’ at 208C, vigorously shaked, then processed as described for BM. Human fetal liver (FL), from legal abortions, were purchased from CAMBREX Poietics cell systems (Gaithersburg, MD). Amnion was obtained, by separation, from discarded full-term placentas, after signed informed consent. Dental and tonsil specimens were obtained from extraction after signed informed consent. All the specimens, except the teeth, detailed later, were treated as for colon and lung. Dental pulp was exposed cutting the tooth under sterile conditions as follows: The enamel of the tooth crown was first partially cut around the sagittal plane using a diamond bur, and then the cut was completed by a piezoelectric ultrasonic scalpel (Piezosurgery1- Mectron, La Spezia- Italy) in order to expose the pulp without overheating the soft tissue; the pulp was then treated with type I Collagenase (3 mg/ml) and type II Dispase (4 mg/ml) for 1 h at 378C, then processed as described for BM. Cells are cultured in controlled CO2/O2 environment provided by Forma Scientific incubators customized by BioSpherix C-Chambers and ProOx 110 controller for normoxia or hypoxia settings.
C-MSCs REPRESENT STRUCTURAL MYOFIBROBLASTS
Fig. 1. CD 146R MSC distribution in colon tissue by Confocal microscopy. CD146 is displayed in green, vWF in red, DAPI in gray, and Phalloidin in blue. Panels A and C show 20T magnifications while panels B and D were acquired with a 40T objective.
MSC phenotype
Antibodies were purchased from BD Biosciences (San Diego, CA) and appropriate isotype controls were used throughout. C- and BM-MSCs were harvested, washed in PBS/BSA 1%, transferred in round-bottom tubes for antibody labeling and flow-cytometric analysis. Mean fluorescence intensity values for each antibody were normalized over control values. Comparison of antigen expression between C- and BM-MSCs was performed by Wilcoxon nonparametric test. Statistical significance was set to 0.05, where one asterisk denotes a P-value between 0.05 and 0.01, two asterisks a P-value between 0.01 and 0.001 and three asterisks a significance lower than 0.001. Statistical analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). MSC differentiation
Osteogenic. 3.1 103 MSC/cm2 were seeded in six-well/plates (BD) in MSCGM (Cambrex, Poietics Cell Systems) for 24 h at 378C JOURNAL OF CELLULAR PHYSIOLOGY
in 5% CO2/O2 atmosphere. MSCGM was replaced with Osteogenesis Induction Medium (Cambrex), and MSCs cultured for 3 weeks with re-feeding every 3–4 days. Osteoblasts were stained with ALP and Von Kossa, labeling early and late differentiation, respectively. Chondrogenic. 2.5 105 MSC were washed twice at 150g for 5 min at 208C with incomplete chondrogenic medium (Cambrex), re-suspended in 0.5 ml complete medium (Cambrex) in 15 ml polypropylene tube and cultured for 3 weeks at 378C in 5%CO2/O2 atmosphere, re-feeding every 2–days with fresh medium. Pellets were formalin fixed and paraffin embedded or prepared for frozen sectioning. Thin sections were slide-mounted and stained with Safranin O or Alcian Blue. Adipogenic
Confluent MSCs were supplemented with Adipogenesis induction medium for 3 days followed by 1–3 days of culture in Adipogenic maintenance medium (both from Cambrex). After three cycles of
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induction/maintenance, cells were cultured for an additional week with maintenance medium at 378C in 5%CO2/O2 atmosphere. Adipocytes maturation was checked in phase contrast microscopy and then stained with Oil Red O, for further microscope analysis. MSC proliferation
BM-MSCs were cultured in 75 cm2 flasks, re-fed twice a week with 12.5 ml of fresh medium, detached at confluence, usually once a week, by trypsin-ethylenediamine tetraacetic acid solution (Invitrogen) treatment, counted, partly frozen and partly reseeded for culture at 378C in 5%CO2/O2 atmosphere. Each weekly re-seeding or passage (P) was defined by a progressive number. Colon MSCs at early passage were plated for culture at 378C in 5%CO2/O2 atmosphere in duplicate 35 mm petri dishes at 105 cells/dish. After 24 h one duplicate dish was detached as described, counted to adjust for the initial number of vital adherent cells, whilst the remaining dish was allowed to reach confluence, detached, counted, recorded in the log book and re-seeded as before. Population doubling in arbitrary units was calculated according to the following formula: N(umber) (t) ¼ N0 x 2t or N(t)/N(0) ¼ 2t. Long-term cultures (LTCs)
BM- and C-MSCs were plated at the saturating density for confluence of 3.6 104/cm2 in 96-well/plates (Falcon, Franklin Lakes, NJ) in alpha-medium, 20% MCS selected FCS; 1 week before the start of the assay the medium was changed to alpha-medium with 10% FCS, 10% Horse S (both from StemCell Technologies), and 106 M hydrocortisone (Sigma, St Louis, MO) (Valtieri et al., 1994). Plates were then seeded with 2 103 CD34þ CB cells and cultured up to 5 weeks at 338C in 5%CO2–O2 atmosphere. Half volume of each culture (0.1 ml) was harvested weekly and replaced with fresh medium. Cells recovered from the harvested medium were counted and seeded in multilineage semisolid medium for clonogenetic assay (Valtieri et al., 1994). At week 5, upon termination of cultures, both cells recovered from the medium and the cells entrapped in the MSC layers were independently counted and seeded in clonogenetic assays (Valtieri et al., 1998). Hematopoietic colonies were scored after 2–3 week culture at 378C in 5%CO2–O2 atmosphere under an Olympus inverted microscope. Purification and culture of monocyte-derived dendritic cells (MDDC)
Human monocytes were purified as described (Ausiello et al., 2002) from peripheral blood of healthy blood donors (courtesy of Dr. Girelli, ‘‘Centro Trasfusionale Policlinico Umberto I’’, University La Sapienza, Rome, Italy) and cultured in RPMI 1640 medium (GIBCO Invitrogen, Paisley, UK), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, all from Hyclone Laboratories (South Logan, UT) and 0.05 mM 2-Mercaptoethanol (Sigma) (hereafter defined as complete medium) and supplemented with heat-inactivated 10% LPS-screened FCS, in the presence of GM-CSF (25 ng/ml) and IL-4 (25 ng/ml). After 6 days, immature MDDC were washed and analyzed by cytofluorometry for the expression of surface markers CD1a, CD14, CD83, and CD38. MDDC were used in the experiments if >80% CD1a and
