Objective
The objective of the study was to isolate and characterize a population of mesenchymal stem cells (MSCs) from human term placental membranes.
Study Design
We isolated an adherent cell population from extraembryonic membranes. Morphology, phenotype, growth characteristics, karyotype, and immnunological and differentiation properties were analyzed.
Results
The isolated placental MSCs were from maternal origin and named as decidua-derived mesenchymal stem cells (DMSCs). DMSCs differentiated into derivatives of all germ layers. It is the first report about placental MSC differentiation into alveolar type II cells. Clonally expanded DMSCs differentiated into all embryonic layers, including pulmonary cells. DMSCs showed higher life span than placental cells from fetal origin and proliferated without genomic instability.
Conclusion
The data suggest that DMSCs are true multipotent MSCs, distinguishing them from other placental MSCs. DMSCs could be safely used in the mother as a potential source of MSCs for pelvic floor dysfunctions and immunological diseases. Additionally, frozen DMSCs can be stored for both autologous and allogeneic tissue regeneration.
Mesenchymal stem cells (MSCs) have a spindle-shaped morphology, are isolated for their adherence to plastic, and phenotypically characterized for the expression of a selection of markers and the capacity of differentiation into bone, fat, and cartilage. Besides their mesodermal differentiation, MSCs have been differentiated to cells of multiple organs such as muscle, neurons, hepatocytes, and insulin-producing cells, indicating that MSCs are an important source of stem cells for regenerative medicine and tissue engineering.
Currently bone marrow (BM) represents the main source of MSCs for both experimental and clinical studies. However, BM-MSCs are present in low quantities and collected by an invasive procedure, and their proliferation and differentiation capacity decrease with donor age.
Placenta is an additional source of MSCs with several advantages, including easy isolation without invasive procedures, low risk of viral infection, and lack of ethical concerns. Human placenta is composed of fetal and maternal tissues. Stem cells (mostly MSCs but also hematopoietic and epithelial stem cells) have been obtained from multiple parts of the placenta including the umbilical cord blood, amniotic fluid, Wharton’s jelly, amniotic membrane, chorionic membrane, decidua, and chorionic villi. Placenta-derived MSCs have higher expansion and engraftment capacity than BM-MSCs.
Placental MSCs are hypoimmunogenic and exhibit immunomodulatory properties, so they can be transplanted without the need for immunosuppression. Moreover, placental MSCs display no evidence for teratoma formation. Then MSCs from a single source can be used for multiple patients in autoimmune diseases and as a vehicle for genes in gene therapy protocols.
In this study, an adherent cell population was isolated from the extraembryonic membranes of human term placenta and characterized in reference to their morphology, immunophenotype, and differentiation potential. Isolated cells belong to the maternal side of fetal membranes (ie, decidua parietalis) and are a true population of mesenchymal stem cells with higher expansion and differentiation capacity than other MSCs, distinguishing them from other placental cell types.
Decidua-derived mesenchymal stem cells (DMSCs) and clonally expanded cells differentiated into the 3 embryonic layers. Additionally, this is the first study showing differentiation of human placenta-derived MSCs to alveolar epithelial type II cells. These maternal mesenchymal-like cells are easy to isolate, have high expansion and differentiation potential while maintaining a normal karyotype, and would be suitable for establishment of either allogenic or patient-specific banks for future clinical outcomes.
Materials and Methods
Processing of placental membranes and culture of primary cells
Human placentas were obtained at the Department of Obstetrics and Gynecology from healthy mothers during natural or cesarean births under written informed consent approved by the Ethics Committee from Hospital Universitario 12 de Octubre. Placental membranes (amnion, chorion, and decidua parietalis) were dissected and washed in phosphate-buffered saline (PBS) plus antibiotics (100 IU penicillin and 100 μg/mL streptomycin) to remove red blood cells. At times, the amnion membrane was peeled off and processed separately from the chorion-decidua tissue.
Placental tissues were processed according to protocol described for amniotic membrane, except for significant modifications. Tissue was digested with trypsin-versene (Lonza, Basel, Switzerland) twice, and cell suspensions were centrifugated at 400 × g for 10 minutes, resuspended in PBS plus 2 mM EDTA and 0.5% human serum albumin, and lysed in 0.8% ammonium chloride solution for 15 minutes to remove any remaining erythrocytes.
Isolated cells were seeded at 1.16 × 10 5 cells/cm 2 and cultured at 37°C, 5% CO2, and 95% humidity in Dulbecco’s modified Eagle’s medium supplemented with 2 mM of glutamine, 0.1 mM of sodium pyruvate, 55 μM of β-mercaptoethanol, 1% nonessential amino acids, 1% penicillin/streptomycin, 10% fetal bovine serum, and 10 ng/mL of epithelial growth factor.
Nonadherent cells were washed off 5 days after seeding. Adherent cells were grown to confluence and passaged at a density of 4-5 × 10 4 cells/cm 2 . Cloning of cells was carried out by plating 1 cell per well in a 96 well plate. Four clones were selected.
Inmunophenotypic characterization of placental cells
Placental cells from early and late passages were characterized by flow cytometry with antibodies against CD45-PerCP, CD73-PE, CD29-PE, CD44-FITC, CD117-PE, and CD90-FITC CD13-PE (BD Pharmingen, San Diego, CA); CD34-FITC, and CD133/1-PE (Miltenyi, Auburn, CA); CD105-FITC (Serotec, Oxford, UK), BCRP1-FITC (Millipore, Bedford, MA); SSEA-1 PE, SSEA-3 PE, SSEA-4 PE, TRA-1-60 PE, TRA-1-81 PE, HLA-ABC-FITC, HLA-DR-FITC, CD40-FITC, CD80-FITC, and CD86-PE (eBioscience, San Diego, CA). FITC-, PE-, and PerCP-control isotypes were used as negative controls. Cell fluorescence was evaluated in a FACScan (Becton Dickinson, Lincoln Park, NJ) and analyzed with the CellQuest Pro 9.0 software (Becton Dickinson).
In vitro cell differentiation studies
Mesodermal differentiation
Adipogenic differentiation was carried out with the adipogenic differentiation bullet kit (Lonza) according to the manufacturer’s instructions and differentiation was evaluated by Oil Red O staining (Sigma, St Louis, MO).
Osteogenic differentiation was performed with the osteogenic differentiation bullet kit (Lonza) for 3 weeks. Differentiation was assessed by Alizarin Red S and Sigma Fast 5-bromo-4-chloro-3-indoyl-phosphate, 4-toluidine salt/4-nitro blue tetrazolium chloride staining (Sigma).
For chondrogenic differentiation, cells were cultured for 21 days with the chondrogenic differentiation bullet kit (Lonza) supplemented with 10 ng/mL transforming growth factor-β1 (Sigma). Differentiation was evaluated by indirect immunofluorescent staining with antibodies for aggrecan and chondroitin sulphate (Millipore). Additionally, secreted extracellular matrix proteoglycans were visualized by Alcian Blue staining.
Myogenic differentiation was analyzed on 1.9 × 10 4 cells seeded in fibronectin-coated plates. Differentiation was initiated by 10 μM 5′-azacitidine (Sigma) incubation for 24 hours and 7-14 days in skeletal muscle growth medium (Lonza) supplemented with 10 ng/mL of fibroblast growth factor. Myogenic differentiation was evaluated by immunofluorescence with antibodies for fast skeletal myosin and α-actinin (Sigma).
For cardiogenic differentiation, cells were cultured in the skeletal muscle growth medium (Lonza) for 41 days and stained with alpha atrial natriuretic polypeptide (ANP) antibody (Millipore).
Endodermal differentiation
Pulmonary epithelial differentiation was accomplished by incubation in small airway growth medium (Lonza) for 3-4 days. Differentiated cells were stained with antibodies to prosurfactant protein C (proSP-C) and surfactant protein B (SP-B) (Millipore).
Ectodermal differentiation
For neurogenic differentiation, cells were seeded in 6-multiwell plates precoated with poly-L-lysine (Nunc, Glostrup, Denmark) at a concentration of 2.29 × 10 4 cells/cm 2 . After 24 hours, neurogenic differentiation media were added. Cells were cultured for 24-72 hours and stained with anti-β III-tubulin and anti-NF200 (Sigma).
Telomerase activity (TRAP assay)
Telomerase activity was analyzed by the TRAPeze telomerase detection kit (Millipore) according to the manufacturer’s instructions. The telomeric repeat amplification protocol (TRAP) assay is a sensitive and efficient polymerase chain reaction (PCR)-based detection method to detect telomerase activity in human cells and not only the presence of ribonucleic acid (RNA) or protein components of telomerase. First, enzymatically active telomerase present in the cell protein extracts adds a number of telomeric repeats (GGTTAG) onto the 3′ end of a substrate oligonucloetide (TS).
Later the extended products are amplified by PCR generating a ladder of products with 6 base increments starting at 50 nucleotides. Telomerase-positive cells are used as a positive control. As a negative control, cell extracts were heated before the assay at 85°C for 10 minutes, according to the kit instructions. TRAP products were visualized by SYBR Gold staining.
Fluorescence in situ hybridization (FISH)
FISH analysis was carried out with Poseidon FISH Kit (Kreatech Diagnostics, Amsterdam, The Netherlands) following the manufacturer’s instructions. Analysis was performed on DMSCs isolated from 3 male placentas using a Zeiss Axioplan2 microscope (Carl Zeiss AG, Jena, Germany
Short tandem repeats (STR) analysis
Genomic deoxyribonucleic acid (DNA) was extracted from 0.5 × 10 6 cells using a resin-based procedure (Instagene Matrix; Bio-Rad Laboratories, Hercules, CA). QF-PCR for the amplification of STR markers for chromosomes X, Y, 13, 18, and 21 (AMXY, X22, HPRT, SRY, DXYS218, D13S631, D13S634, D13S258, D13S305, D21S1414, D21S1411, D21S1446, D21S1437, D18S535, D18S386, D18S391, and D18S390) was carried out by an Aneufast QF-PCR kit (Genomed Ltd, Kent, UK) following the manufacturer’s instructions. The PCR products were analyzed on ABI PRISM 3100-AVANT genetic analyzer (Applied Biosystems, Foster City, CA).
Karyotype analysis
Placental-derived cells at passages 15-20 were processed using standard cytogenetic techniques. Cells were treated with 10 μg/mL of Colcemid for 1.5 hours, trypsinized, treated briefly with 0.075 M hypotonic KCl solution, and fixed with Carnoy’s fixative. Cells were then dropped on a microscope glass slide and dried. Metaphase cells were G banded using Wright staining. At least 20 metaphases per sample were karyotyped.
Total RNA extraction and reverse transcription (RT)-PCR
Total RNA was isolated using MasterPure RNA isolation kit (Epicentre Biotechnologies, Madison, WI). One microgram of total RNA was used for reverse transcription using ImProm II reverse transcriptase (Promega, Madison, WI). Complementary DNAs were then amplified using MBL Taq DNA polymerase (Dominion MBL) and tissue-specific primers. Amplification was carried out in a TC3000 thermocycler (Techne) at 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 seconds, specific annealing temperature for 30 seconds, and 72°C for 30 seconds. PCR products were separated on 2% agarose gel and visualized by SYBR Gold staining. Human-specific PCR primers and conditions are shown in the Supplementary Table .
Immunostaining
Cells were either fixed with 10% formalin for 90 minutes or 4% paraformaldehyde for 10 minutes at room temperature and permeabilized with 0.3% Triton X-100 for 10 minutes. Nonspecific binding was blocked by PBS plus 5% horse serum (Lonza) for 90 minutes. Primary antibodies were incubated overnight at 4°C and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies for 2 hours at room temperature. The nucleus was counterstained with 4′,6′-diamino-2-phenylindole (Sigma) for 1 minute.
Cell cryopreservation and thawing
Fifty percent confluent placental cells were trypsinized, counted, resuspended in cell culture freezing medium-dimethylsulfoxide (Millipore) at a concentration of 1 × 10 6 cells/mL, and added to 2 mL cryovials (Nunc). Vials were introduced in a freezing container (Mr Frosty; Nalgene, Rochester, NY), placed overnight at –80°C, and transferred to liquid N2. Thawed cells in a water bath at 37°C were washed and then cultured and differentiated as described for fresh cell cultures.
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