Objective
Preeclampsia is associated with perinatal brain injury. Autologous placenta stem cell transplantation represents a promising future treatment option for neuroregeneration. The aim of this study was to compare the neuroregenerative capacity of preeclampsia-placenta stem cells to previously characterized placentas from uncomplicated pregnancies.
Study Design
Placenta stem cells from amnion (epithelium, mesenchyme) and chorion were assessed for cell surface markers and the formation of neuronal-like cells, oligodendrocytes and their progenitors in culture.
Results
Markers of preeclampsia-placenta stem cells were different from uncomplicated pregnancies-placenta stem cells in amnion epithelium and chorion, but not in amnion mesenchyme. Similarly to uncomplicated pregnancies-placenta stem cells, preeclampsia-placenta stem cells derived from amnion and chorion differentiated preferably into nestin-positive stem/progenitor cells and Tuj-1-positive neurons. However, other important markers were varying after neurogenic differentiation of uncomplicated pregnancies- and preeclampsia-placenta stem cells.
Conclusion
Surface marker expression patterns of preeclampsia-placenta stem cell’s and uncomplicated pregnancies-placenta stem cell’s differ. In vitro differentiation assays, however, provide evidence that both preeclampsia-placenta stem cells and uncomplicated pregnancies-placenta stem cells are comparably suitable for neuroregeneration purposes.
Preeclampsia (PE), a pregnancy-specific disease, is characterized by hypertension and proteinuria. Affecting 5-10% of all pregnancies PE contributes substantially to increased perinatal mobidity and mortality of both the mother and child. The exact underlying pathomechanisms leading to PE are far from being understood. It is widely accepted, however, that the placenta plays a central role in the pathogenesis of the disease, because after delivery of the child and its placenta, the mother recovers within days. Regarding the PE treatment, no therapeutic intervention has been proven to be successful yet. PE, and particularly early-onset PE, have an important impact on the increased rate of preterm deliveries and their sequelae. In countries of the industrial world, such as the United States, preterm and extreme preterm birth (before 32 completed weeks of gestation) account for up to 12% and 2%, respectively, of all deliveries. Preterm deliveries are associated with low birthweight, encephalopathy, and subsequent occurrence of cognitive, behavioral, attentional, or major motor deficits in at least 5-10%. A large number of these affected infants need a high level of specialized care, which bears a heavy burden on the society.
Various therapeutic interventions have been proposed to improve the long-term outcome in preterm brain damage: efforts to replace necrotized neurons have involved transplantation of stem cell populations in various animal models. Stem cells can be obtained from adult tissues (somatic or adult stem cells), embryonic tissue (embryonic stem cells), or from extra-embryonic tissues (placental stem cells). The placenta is usually discarded at birth. Thus, ethical concerns are less important when stem cells are isolated from extraembryonic than from embryonic tissue. It has been shown that placental tissue is a rich source of placental stem cells (PSCs). We reported previously on isolation of stem cells from umbilical cord blood, as well as from placental tissue. In a previous study, we proposed the transplantation of placental mesenchymal stem cells for potential pre- and perinatal neuroregeneration : after isolation of mesenchymal cells from amnion, chorion, and villous stroma, we let these cells differentiate into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineages. Thus, placental tissue might represent an ideal source for an autologous stem cell graft, which, in turn, is a promising tool regarding neuroregeneration in peripartal encephalopathy.
In an earlier investigation, we studied umbilical cord blood hematopoietic progenitor-stem cells from preeclamptic patients and showed that these were different with regard to numbers as well as proliferative capacity. However, these cells are fetally derived, and it remains unknown if stem cells derived from extraembryonic tissue would change in PE.
To our knowledge, no data are available on PSCs, derived from extraembryonic mesodermal tissue, in PE. Yet, it would be of high interest to know more about the characterization of these cells. Because children from PE pregnancies with preterm delivery, are often having peripartum encephalopathy, they represent a potential target population for stem cell therapies for neuroregeneration.
We conducted this study to address the questions, whether PSCs are present in PE placentae, and if yes, whether these isolated cells share similar characteristics with PSCs obtained from uncomplicated pregnancies, focusing on neural differention.
Materials and Methods
Isolation technique and culture conditions of placenta-derived stem cells
Third-trimester placentae were obtained after cesarean sections from healthy donor mothers, including cases with isolated cervical insuffiency (34-39 weeks’ gestational age [GA], n = 10) and pregnant women affected by PE (29-38 weeks’ GA, n = 10). Tissue was collected after informed consent, and all experiments were approved by the local institutional review board.
Amnion and chorion laeve tissue were separated by blunt dissection. Human amnion epithelial cells (HAECs) were obtained by gently scraping the epithelial side of amnion after a treatment with dispase II (Roche Diagnostics, Basel, Switzerland) and cultivation of the released cells in Dulbecco’s modified eagle medium (DMEM, high-glucose, glutamine, 10% fetal bovine serum [FBS], inactivated, antibiotic/antimycotic solution: 100 U/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin B; all from Invitrogen, Carlsbad, CA). Human amnion mesenchymal cells (HAMCs) were released from the remaining amniotic mesenchymal layer after digestion with collagenase 2 (Worthington, Lakewood, NJ). HAMCs were propagated in minimum essential medium alpha (α-MEM; Invitrogen, 20% FBS, antimycotic/antibiotic solution). Human chorion mesenchymal cells (HCMCs) were obtained from the reticular layer of the chorion. First, surrounding layers were mechanically and enzymatically removed (dispase II), then the reticular matrix was degraded with collagenase 2. HCMCs were cultivated in α-MEM/20% FBS.
Flow cytometric analysis of cell surface antigens
HAMCs, HAECs, and HCMCs (for each cell subtype: n = 10, ie, originating from 10 individual placentae) were detached with 0.05%Trypsin/0.53 mM EDTA (Invitrogen), then resuspended in cell culture medium (DMEM high-glucose, 10% FBS) and incubated on ice for 10 minutes. After washing with Dulbecco’s phosphate-buffered saline (PBS, pH 7.2, 10% FBS; Invitrogen), they were labeled with 1 of 10 different primary antibodies, ie, CD166 (Acris, Hiddenhausen, Germany), CD105 (Serotec, Oxford, UK), CD90 (Acris), CD73 (Becton Dickinson, Franklin Lakes, NJ), CD44 (Chemicon, Temecula, CA), CD14 (Chemicon) CD34 (Becton Dickinson), CD45 (Becton Dickinson), HLA-ABC (MHC class I; Chemicon), and HLA-DR (MHC class II; Becton Dickinson), following the manufacturer’s instructions. All antibodies were labeled with fluoroisothiocyanate (FITC), except for the 1 against CD73, which was supplemented with a secondary FITC-conjugated antibody.
Cells were fixed with 1% formaldehyde (Sigma-Aldrich, St. Louis, MO) and analyzed 2-3 days later by fluorescence-activated cell sorting (FACScan; Becton Dickinson; quantification with FlowJo software version 5.7.1.; TreeStar, Ashland, OR).
Neural differentiation
Procedure for HAMCs and HCMCs
Cells were trypsinized and seeded in low-attachment plastic tissue culture dishes (Thermo Scientific Nunc Lab-Tek Sterile Petri Dishes; Cole-Parmer, Vernon Hills, IL) at a concentration of 2 × 10 5 cells/cm 2 in neurobasal medium containing 2% B27 supplement, 2 mM glutamax, antibiotic/antimycotic solution (all Invitrogen), and 2 μg/mL Heparin (Fluka/Sigma-Aldrich, Buchs, Switzerland). Induction of neurospheres was achieved by adding daily 20 ng/mL human epidermal growth factor (hEGF), 20 ng/mL human fibroblast growth factor-2 (hFGF-2) (both from BD Biosciences), and keeping them in a 95% humidified atmosphere at 37°C and 5% CO 2 . Three passages by mechanical trituration were needed to proceed to the final step of neural differentiation.
Neurogenesis was initiated by seeding triturated cells on collagen-I-coated dishes (Biocoat; BD Biosciences) at a concentration of 1 × 10 4 cells/cm 2 in neurobasal medium (10% FBS; Invitrogen; 2% B27 supplement, 2 mM glutamax-I, antibiotic/antimycotic solution) with 1 μM all-transretinoic acid (RA; Sigma-Aldrich) and 15 ng/mL human brain-derived neurotrophic factor (hBDNF; Invitrogen). Cells were cultivated for 1 week, changing the medium 3 times.
Procedure for HAECs
HAECs of passage 1 were seeded on collagen-I-coated dishes at a concentration of 1.5-2 × 10 4 cells/cm 2 in neurobasal medium (10% FBS, 2% B27 supplement, 2 mM glutamax-I, antibiotic/antimycotic solution). Cells were cultured for 10 days, the medium was changed 3 times, and supplemented with hEGF and hFGF, 20 ng/mL of each.
Immunocytochemistry of neural differentiation markers
PSCs from normal (n = 10) and PE pregnancies (n = 10) were stained for different neural markers before and after neurogenic differentiation. Triplicates were performed for each individual marker staining. Cells were fixed in fresh 4% (w/v) paraformaldehyde (Sigma-Aldrich), washed in 1× PBS and blocked in DMEM-high glucose with 0.1% (w/v) BSA, 0.1% (w/v) saponin (both Sigma-Aldrich) and antibiotic/antimycotic solution. The primary antibody was applied for 1 hour at room temperature. After washing with 1 × PBS, the secondary antibody was added for 30 minutes. Finally, samples were mounted in Mowiol (Calbiochem, Merck Biosciences, Darmstadt, Germany). A semiquantitive scoring of marker positive cells was performed microscopically estimating the percentage of fluorescent positive cells in randomly chosen visual fields (6 per culture).
Primary antibodies and dilutions
Nestin (monoclonal, 1:100; Acris, Hiddenhausen, Germany); β-tubulin III (Tuj-1, monoclonal, 1:200; Neuromics, Edina, MN); NeuN (monoclonal, 1:100; Chemicon); microtubule associated proteins MAP2 (2a+2b; monoclonal, 1:500; Acris); O1 (monoclonal, 1:100; Chemicon); O4 (monoclonal, 1:100; Chemicon); chondroitin sulfate proteoglycan NG2 (polyclonal, 1:150; Chemicon); myelin basic protein (MBP polyclonal, 1:200; Chemicon); galactocerebroside (GalC polyclonal, 1:100; Acris); and glial fibrillary acidic protein (GFAP; polyclonal, 1:1000; Chemicon).
Secondary antibodies and dilutions
Alexa 488 or Alexa 594-labeled secondary antibodies were then applied (1:200; Molecular Probes/Invitrogen).
Results
General observations
Isolation of primary stem cells from amnion and chorion of normal placentae has been described by our group previously. The same isolation procedure could also be applied on PE complicated placentae. Tryptan blue assay showed similar viability, ie, 85-90%, of stem cells in PE and control tissue.
Comparison of surface markers by FACS analysis
Flow cytometry analysis of PE-PSC cultures from amnion mesenchyme, amnion epithelium and chorion stained positive for the representative MSC markers CD166, CD105, CD90, CD73, and CD44 but were negative for the hematopoietic stem cell markers CD45 and CD34 or the monocytic marker CD14 Further, they expressed HLA-ABC (MHC class I) but no HLA-DR (MHC class II). All these markers were investigated on all 10 samples for each group. This surface marker profile was consistent with the profile of UP-PSCs; however, the following quantitative differences were found ( Figure 1 ).
