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Transdifferentiation of Peripheral Blood Mononuclear Cells into Epithelial-Like Cells

2007; Elsevier BV; Volume: 171; Issue: 4 Linguagem: Inglês

10.2353/ajpath.2007.070051

1525-2191

Abelardo Medina, Ruhangiz T. Kilani, Nicholas Carr, Erin Brown, Aziz Ghahary,

Polysaccharides and Plant Cell Walls

Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3σ for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3σ and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P ≤ 0.01). The depletion of 14-3-3σ from these conditioned media by immunoprecipitation reduced the effect by 39.5% (P value, 0.05). Therefore, the releasable 14-3-3σ from PBMC-derived epithelial-like cells is involved in this process. Our findings provide new insights into the PBMC transdifferentiation to generate epithelial-like cells and subsequently release of 14-3-3σ that will disclose new therapeutic alternatives for different dermal clinical settings. Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3σ for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3σ and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P ≤ 0.01). The depletion of 14-3-3σ from these conditioned media by immunoprecipitation reduced the effect by 39.5% (P value, 0.05). Therefore, the releasable 14-3-3σ from PBMC-derived epithelial-like cells is involved in this process. Our findings provide new insights into the PBMC transdifferentiation to generate epithelial-like cells and subsequently release of 14-3-3σ that will disclose new therapeutic alternatives for different dermal clinical settings. 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69: 209-218Crossref PubMed Scopus (78) Google Scholar, 20Quesenberry P Abedi M Aliotta J Colvin J Demers D Dooner M Greer D Hebert H Menon MK Pimentel J Paggioli D Stem cell plasticity: an overview.Blood Cells Mol Dis. 2004; 32: 1-4Crossref PubMed Scopus (48) Google Scholar, 21Horwitz E Stem cell plasticity: the growing potential of cellular therapy.Arch Med Res. 2003; 34: 600-606Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Thus, circulating CD34+ bone marrow-derived adult stem cells constitute fibrocytes that rapidly infiltrate the wound bed and settle in particular locations within the dermis.22Bucala R Spiegel L Chesney J Hogan M Cerami A Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar, 23Nickoloff B The human progenitor cell antigen (CD34) is localized on endothelial cells, dermal dendritic cells, and perifollicular cells in formalin-fixed normal skin, and on proliferating endothelial cells and stromal spindle-shaped cells in Kaposi's sarcoma.Arch Dermatol. 1991; 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171: 380-389PubMed Google Scholar In addition, within resident stem cell niches of skin, located at the bulge region of hair follicles and basal layer of the interfollicular epidermis, CD34+ keratinocyte precursors have been identified.11Borue X Lee S Grove J Herzog EL Harris R Diflo T Glusac E Hyman K Theise ND Krause DS Bone marrow-derived cells contribute to epithelial engraftment during wound healing.Am J Pathol. 2004; 165: 1767-1772Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 29Brittan M Braun K Reynolds LE Conti FJ Reynolds AR Poulsom R Alison MR Wright NA Hodivala-Dilke KM Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion.J Pathol. 2005; 205: 1-13Crossref PubMed Scopus (108) Google Scholar Even though the local injection of CCL27 seems to accelerate healing process by increasing the CD34+ bone marrow-derived cell migration,30Inokuma D Abe R Fujita Y Sasaki M Shibaki A Nakamura H McMillan JR Shimizu T Shimizu H CTACK/CCL27 accelerates skin regeneration via accumulation of bone marrow-derived keratinocytes.Stem Cells. 2006; 24: 2810-2816Crossref PubMed Scopus (78) Google Scholar the source of these cells as well as their role in both normal cell renewal and tissue repair have yet to be determined. Unfortunately, there is no reliable marker that displays long-term expression for local identification and tracking of cells along the transition process of epithelial cell maturation. In this regard, the protein 14-3-3σ has been described as a specific marker for epithelial cells.31Dellambra E Golisano O Bondanza S Siviero E Lacal P Molinari M D'Atri S De Luca M Downregulation of 14-3-3σ prevents clonal evolution and leads to immortalization of primary human keratinocytes.J Cell Biol. 2000; 149: 1117-1129Crossref PubMed Scopus (194) Google Scholar, 32Pellegrini G Dellambra E Golisano O Martinelli E Fantozzi I Bondanza S Ponzin D McKeon F De Luca M p63 identifies keratinocyte stem cells.Proc Natl Acad Sci USA. 2001; 98: 3156-3161Crossref PubMed Scopus (1210) Google Scholar In addition, we have recently described a releasable form of keratinocyte-derived 14-3-3σ that induces matrix metalloproteinase (MMP)-1 expression in dermal fibroblasts.33Ghahary A Marcoux Y Karimi-Busheri F Li Y Tredget EE Kilani RT Lam E Weinfeld M Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts.J Invest Dermatol. 2005; 124: 170-177Crossref PubMed Scopus (88) Google Scholar Thus, because of its distinct expression pattern and anti-fibrotic effects on dermal fibroblasts, 14-3-3σ serves as a promising protein to elucidate further the functional commitment of PBMCs into epithelial-like cells. To assess our working hypothesis that circulating bone marrow-derived stem cells (within PBMCs) might transdifferentiate into epithelial-like cells, series of experiments were conducted, the findings of which are presented here. To characterize further the functionality of these epithelial-like cells, the release of 14-3-3σ and its effect on MMP-1 expression in dermal fibroblasts were examined. PBMCs were isolated from whole blood of volunteers by using Ficoll-Hypaque density gradient centrifugation following the manufacturer's protocol. Blood from donors was carefully layered on Ficoll solution (density 1.077; Sigma-Aldrich, St. Louis, MO). Centrifugation was performed at 931 × g for 20 minutes at 20°C (Centrifuge Allegra X-12R; Beckman Coulter, Inc., Palo Alto, CA). The mononuclear cell interphase was taken and washed three times in 1× phosphate-buffered saline (PBS) for 8 minutes at 300 × g (Centrifuge Allegra X-12R; Beckman Coulter, Inc.). Cells were counted and suspended in appropriate culture medium (see below) containing 2 × 106 cells/ml so that cell suspensions were added to either chamber slides (Lab-Tek II; Nalge Nunc International, Naperville, IL) or 75-cm2 flasks (BD Falcon; BD Biosciences, Bedford, MA). After PBMCs were isolated, circulating stem/precursor cells were isolated by using the EasySep negative selection human progenitor cell enrichment kit with CD41 depletion (StemCell Technologies, Vancouver, BC, Canada) following the manufacturer's recommendations. Cell suspension at a concentration of 5 × 107 cell/ml were prepared using 1× PBS containing 2% heat-inactivated fetal bovine serum qualified (FBS; Gibco-Invitrogen Corp., Grand Island, NY). Within a 12 × 75-mm polystyrene tube, the EasySep negative selection progenitor cell enrichment cocktail with CD41 depletion was added at 50 μl/ml cell suspension. The mixture was incubated for 15 minutes at room temperature. Then, EasySep magnetic nanoparticles (StemCell Technologies) were added at 50 μl/ml cell suspension and incubated at room temperature for another 15 minutes under constant rotation. After mixing, the sample tube was placed without cap into the magnet (EasySep Magnet 18000; StemCell Technologies) for 10 minutes. The liquid content with wanted cells was subsequently poured off into a new tube. After the isolation procedure, PBMCs were resuspended in culture medium containing 49% Dulbecco's modified Eagle medium (DMEM; Gibco-Invitrogen), 49% defined keratinocyte serum-free medium (KSFM) (Gibco-Invitrogen Corp.), 2% FBS (Gibco-Invitrogen Corp.), growth supplements (Gibco-Invitrogen Corp.), penicillin G sodium (100 U/ml), and streptomycin sulfate (100 μg/ml), and amphotericin B (0.25 μg/ml) (Gibco-Invitrogen Corp.). Half of the culture medium was changed every other day. At different time points, cells were harvested by using 0.05% ethylenediaminetetraacetic acid (EDTA) and 0.1% trypsin (Gibco-Invitrogen Corp.) in 1× phosphate-buffered saline (PBS) and gentle scraping with a rubber policeman. In case of morphological studies, adherent cells were directly fixed on chamber slides with 4% paraformaldehyde. In parallel experiments, at the same time points, conditioned media from PBMC-derived epithelial-like cells were collected after a 24-hour incubation with a test media containing 50% DMEM and 50% KSFM without supplement and growth factors. The same procedure described above was used with circulating precursor cells to induce cell transdifferentiation into epithelial-like cells. After voluntary recruitment and informed consent, foreskin samples were obtained from infants undergoing elective circumcision, under local anesthesia, according to a protocol approved by the Clinical Research Ethics Board, Office of Research Services of the University of British Columbia. Skin samples were collected individually and washed several times in sterile 1× PBS supplemented with antibiotic-antimycotic preparation (100 μg/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B) (Gibco-Invitrogen Corp.). Epidermal and dermal layers were separated by treatment with 40 mg/ml dispase (Gibco-Invitrogen Corp.) during 2 hours at 37°C. Culture of fibroblasts were established as previously described.34Karimi-Busheri f Marcoux Y Tredget EE Li l Zheng J Ghoreishi M Weinfeld M Ghahary A Expression of a releasable form of annexin II by human keratinocytes.J Cell Biochem. 2002; 86: 737-747Crossref PubMed Scopus (25) Google Scholar When reaching confluence, fibroblasts were detached by trypsinization and subculture into 75-cm2 flasks (BD Falcon, BD Biosciences). Fibroblasts at passages 3 to 7 were used in co-culture system. To establish keratinocyte cultures, epidermis was treated separately in 0.05% EDTA/0.1% trypsin (Gibco-Invitrogen Corp.) at 37°C for 5 minutes to release basal keratinocytes. After neutralization with DMEM and 10% FBS and washing three times with 1× PBS, cells were cultured in KSFM (Gibco-Invitrogen Corp.) supplemented with BPE (50 μg/ml), and epidermal growth factor (5 μg/ml). Keratinocytes from passages 3 to 5 were used as positive control after inducing cell maturation with DMEM and 2% FBS. To identify the effect of releasable factors of PBMC-derived epithelial-like cells on dermal fibroblasts, PBMCs were cultured in 30-mm-diameter culture plate inserts (Millicell-PCF; Millipore Corp., Billerica, MA). At different time points (days 0, 7, 14, 21, 28, 35, 42, and 49), these inserts were placed into six-well plates containing subconfluent culture of dermal fibroblasts by using 2 ml of test media prepared with 49% DMEM and 49% KSFM plus 2% FBS with no other additives. Only releasable factors from PBMC-derived epithelial-like cells (upper chamber) can pass through the permeable membrane of inserts (0.4-μm pore size) and interact with dermal fibroblasts (lower chamber). As a negative control, a co-culture system between fibroblasts located on inserts (upper chambers) and fibroblasts located in lower chambers was used. As a positive control, differentiated keratinocytes were used on inserts (upper chambers). After a 24-hour incubation, dermal fibroblasts from lower chambers were harvested by using 0.05% EDTA and 0.1% trypsin in 1× PBS and gentle scraping with a rubber policeman. After three washing steps with 1× PBS, fibroblast lysates were obtained with cell lysis buffer [50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA (Sigma), 1 mmol/L EGTA (Sigma), 0.025% NaN3, 1% Triton X-100, 0.5% Igepal CA-630 (Sigma), and protease inhibitor cocktail (Sigma)]. Protein concentrations were measured by using the Bradford method with a Life Science UV/Vis spectrophotometer (DU 530; Beckman Coulter, Inc.). To determine the effect of PBMC-derived epithelial-like cell-released factors on expression of MMP-1 by dermal fibroblasts, total protein content was extracted from fibroblasts grown in lower chambers and MMP-1 expression was evaluated by Western blotting. At different time points, cells cultured on chambered slides were fixed in 4% paraformaldehyde for 10 minutes followed by incubation in 1× PBS within a staining chamber for 5 minutes at room temperature. To inactivate endogenous peroxidase, slides were incubated with 30% hydrogen peroxide (Sigma-Aldrich) diluted in methanol (2% v/v) for 15 minutes. After washing twice with ddH2O and rehydration with 1× PBS for 5 minutes, samples were incubated in blocking solution (1× PBS containing 10% goat serum plus 5% bovine serum albumin; Sigma) within a humidified chamber for 30 minutes. The primary antibody was then applied and incubated inside of a humidified chamber overnight at 4°C. Two epithelial markers were selected to demonstrate cell transdifferentiation from PBMCs into epithelial-like cells. Thus, rabbit anti-human 14-3-3σ (a kind gift of Dr. A. Aitken, University of Edinburgh, Edinburgh, UK) and mouse anti-human keratin-5, -8 monoclonal antibodies (Chemicon International, Temecula, CA) at 1:1000 dilution were used. After rinsing three times with 1× PBS for 5 minutes each, samples were incubated with either biotinylated goat anti-rabbit IgG (lot BA-1000; Vector Laboratories Inc., Burlingame, CA) or goat anti-mouse IgG (lot BA-9200; Vector Laboratories Inc.) at 6 μg/ml for 45 to 60 minutes at room temperature. Application of StreptoABComplex/horseradish peroxidase subsequently was performed according to the manufacturer's recommendations (DakoCytomation, Glostrup, Denmark). Samples were then exposed to peroxidase substrate solution [25 mg of 3,3′-diaminobenzidine (Sigma), 50 ml of 1× PBS, and 50 μl of 30% H2O2] for 5 minutes. In addition, counterstaining with hematoxylin was performed using a standard protocol. Finally, dehydration with graded ethanol and treatment with xylene were performed before the application of Permount (Fisher Scientific, Fair Lawn, NJ) and coverslips. Images were obtained using a Zeiss Axioplan 2 imaging microscope (Carl Zeiss, Toronto, ON, Canada) with Northern Eclipse image analysis software (Empix Imaging, Inc., Mississauga, ON, Canada). Cultured epithelial-like cells were fixed in 2% paraformaldehyde for 10 minutes and then washed in 1× PBS and graded ethanol. Nonspecific bindings were avoided by a blocking solution (1× PBS containing 10% goat serum and 5% bovine serum albumin; Sigma). For immunofluorescence microscopy, dual staining was performed using primary rabbit anti-human 14-3-3σ antibodies (kindly provided by Dr. A. Aitken, University of Edinburgh) and mouse anti-human keratin-5, -8 monoclonal antibodies (Chemicon International) at 1:500 dilution and incubated overnight in a humidified chamber at 4°C. After three washing steps with PBS-Tween 20 for 5 minutes each, samples were incubated with rhodamine-conjugated goat anti-rabbit IgG (Chemicon International) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Chemicon International) at 1:3000 dilution for 45 minutes in dark condition. Finally, after washing with PBS-Tween 20 three times for 5 minutes each, samples were mounted in Vectashield H-1200 (Vector Laboratories, Inc.) containing 4,6-diamidino-2-phenylindole (DAPI) for nuclei staining. A Zeiss Axioplan 2 microscope and Northern Eclipse image analysis software were also used to obtain the images. In addition, single staining was conducted to identify the presence of CD34 (catalog no. 555820; Pharmingen, BD Biosciences, San Jose, CA) and involucrin (catalog no. I 9018; Sigma) at 1:1000 dilution each. In both cases, FITC-conjugated goat anti-mouse IgG (Chemicon International) was used as secondary antibody as described above. In confocal microscopy, similar procedures were performed. However, a cocktail of purified mouse anti-human monoclonal cytokeratin-14, -15, -16, and -19 antibodies (catalog no. 550951; Pharmingen, BD Biosciences) at 1:1000 dilution was used instead of mouse anti-human keratin-5, -8 monoclonal antibody. A Zeiss Axiovert 200 microscope and LSM510 META analysis software were also used to obtain the images. To study intracellular details of cytokeratins, the deconvolution procedure with Volocity software (Improvision, Coventry, UK) was used. This technique removes the out-of focus information from the image plane of interest. To determine the amount of soluble 14-3-3σ protein in conditioned media from PBMCs at different stages of differentiation into epithelial-like cells, an enzyme-linked immunosorbent assay (ELISA) was performed. Briefly, each sample of conditioned media was diluted 50/50 using 5× coating buffer (0.1 mol/L NaHCO3, pH 9.3). Then, 96-well plates (pretreated ELISA plate, Nunc-Immuno Plate MaxiSorp Surface; Nalge Nunc Int.) were coated with 100 μl per well of diluted samples and incubated overnight at 4°C. After washing twice with distilled/deionized water (150 μl per well), wells were blocked by adding 2.5% bovine serum albumin (crystallized; Sigma Chemical Co.) for 1 hour at 37°C. After removing the blocking solution, mouse anti-human 14-3-3σ monoclonal antibody (catalog no. MS-1185-P; NeoMarkers, Fremont, CA) was added at 1:1000 dilution with PBS-Tween 20 (PBS-T) plus 0.5% bovine serum albumin and incubated 1 hour at 37°C. After washing three times with PBS-T, peroxidase affinity rabbit anti-mouse IgG (H+L) (catalog no. 315-035-003; Cedarline; Hornby, BC, Canada) was added in 0.5% bovine serum albumin/PBS at 100 μl per well for 45 minutes at 37°C. Finally, after removing the secondary antibody and washing wells three times with PBS-T, 50 μl per well of substrate solution (0.4 mg/ml o-phenylenediamine in 0.05 mol/L phosphate-citrate buffer, pH 5.0, plus 40 μl of 30% H2O2 per 100 ml of substrate buffer solution; Sigma) was added according to manufacturer's recommendations and incubated at 37°C. The optical density of each well was measured by using an ELISA reader (VERSAmax; Molecular Devices, Sunnyvale, CA) and the software SOFTmax Pro. A standard curve was designed with serial dilutions of recombinant 14-3-3σ protein (0, 0.15, 0.31, 0.62, 1.25, and 2.5 ng/μl). ELISA from each sample was performed in triplicate. To measure tumor necrosis factor-α and interleukin-1 in PBMC-conditioned media at different time points of epithelial-like cell differentiation, ELISA was also performed. In these cases, each sample of conditioned media was diluted 1:5 in 1× coating buffer. As primary antibodies, mouse anti-human tumor necrosis factor-α, clone 2C8 (catalog no.