Objective
This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from term human placenta (placenta-derived mesenchymal stem cells, PD-MSCs).
Study Design
Sequential enzymatic digestion was used to isolate PD-MSCs in which trypsin removes the trophoblast layer, followed by collagenase treatment of remaining placental tissue. Karyotype, phenotype, growth kinetics, and differentiability of PD-MSC isolates from collagenase digests were analyzed.
Results
PD-MSC isolation was successful in 14 of 17 cases. Karyotyping of PD-MSC isolates from deliveries with a male fetus revealed that these cells are of maternal origin. Flow cytometry and immunocytochemistry confirmed the mesenchymal stem cell phenotype. Proliferation rates of PD-MSCs remained constantly high up to passage 20. These cells could be differentiated toward mesodermal lineage in vitro up to passage 20. Nonconfluent culture was critical to maintain the MSC stemness during long-term culture.
Conclusion
Term placenta constitutes a rich, very reliable source of maternal mesenchymal stem cells that remain differentiable, even at high passage numbers.
Mesenchymal stem cells (MSCs) represent an interesting cell type for research and therapy because of their ability to differentiate into mesodermal lineage cells, such as osteocytes, chondrocytes, cardiac muscle, or endothelial cells. In addition, they secrete large amounts of proangiogenic and antiapoptotic cytokines and possess remarkable immunosuppressive properties.
MSCs have been derived from many different organs and tissues. Evidence has emerged that also different parts of human placenta, umbilical cord, and amniotic membrane, as well as umbilical cord blood, harbor MSCs. These tissues are normally discarded after birth, avoiding ethical concerns. Mechanical, as well as enzymatic, methods for MSC isolation from different regions of human placenta of different gestational ages were reported ( Table 1 ).
| Stem cell source | Gestational age | Isolation method | Reference |
|---|---|---|---|
| Whole placental tissue | First trimester | Collagenase plus trypsin | Genbacev et al |
| Whole placental tissue | First trimester | Trypsin plus collagenase II plus dispase II | Portmann-Lanz et al |
| Whole placental tissue | First trimester | Chorionic villi sampling | Poloni et al |
| Whole placental tissue | Term | Explant culture method |
|
| Central placental lobules | Term | Trypsin | Fukuchi et al |
| Whole placental tissue | Term | Trypsin-EDTA |
|
| Whole placental tissue | Term | Collagenase I |
|
| Whole placental tissue | Term | Collagenase P |
|
| Placental tissue without stem villi and amniotic fetal membranes | Term | Collagenase plus dispase II |
|
| Decidua basalis | Second trimester | Mechanical mincing (no enzymatic digestion) | In’t Anker et al |
| Decidua parietalis | Second trimester | Mechanical mincing (no enzymatic digestion) | In’t Anker et al |
| Decidua parietalis | Term | Collagenase plus hyaluronidase plus pronase | Strakova et al |
Knowledge about vitality, karyotype, phenotype, and expandability of such placenta-derived MSC isolates is a prerequisite for therapeutic application; however, systematic investigations into reliability of this MSC source and phenotypic stability have not yet been attempted. Furthermore, former reports on placenta-derived MSCs often lack information about the karyotype of the cell isolates.
In this article, we describe enzymatic fractionation of term human placenta that allows recovery of multipotent, fibroblast-like cells, which we tentatively term as placenta-derived mesenchymal stem cells (PD-MSCs) with high fidelity. Unexpectedly, as demonstrated by genotypic analyses of cell isolates from male deliveries, the resulting isolates were of maternal, not fetal, origin.
Our systematic characterization of cell isolates from multiple cases showed that these cell isolates reproducibly fulfill the general definition of MSCs by both phenotypic and functional criteria. We demonstrate that maternally derived PD-MSCs can be greatly expanded and maintain their differentiation capacity and stable phenotype up to passage 20.
Materials and Methods
Placenta collection
The Ethical Committee of the District of Zurich approved the protocol (study Stv22/2006). Following written consent, placentas were collected from 17 women donors immediately after elective caesarean section in the absence of labor, preterm rupture of membranes, chorioamnionitis, or chromosomal abnormalities. Mean maternal age was 32 years (between 28–39 years) and mean gestational age was 38 ± 1 weeks. Mean placental weight was 573 ± 113 g.
Cell isolation
Figure 1 depicts the isolation procedure. After removal of decidua and fetal membranes, approximately 30 g of placental tissue was minced and washed 3 times in physiologic saline. Blood vessels and clots were removed mechanically.
The minced placental tissue was subjected to sequential digests with trypsin and collagenase I. First, to remove the trophoblastic epithelial cell layer, tissue was incubated in 50 mL of 0.25% trypsin solution containing 80 U/mL of DNase I (Roche AG, Basel, Switzerland) for 1 h at 37°C. The remaining placental fragments were separated in a 250 μm metal sieve from the trypsin cell suspension. Approximately 15 g of placental fragments were subjected to a second digest with collagenase. For that, tissue fragments were incubated with 50 mL of 12.5 U/mL collagenase I (Sigma-Aldrich AG, Buchs, Switzerland) and 80 U/mL DNase I for 1 hour at 37°C.
Cell suspensions from both trypsin and collagen digests were filtered twice through 100 μm cell strainers (BD Bioscience, San Jose, CA), and then the cells were collected by centrifugation for 5 min at 300 × g . The cell pellets were shortly resuspended in hypotonic red blood cell lysis buffer (physiologic saline with 2 mm EDTA, 0.5% bovine serum albumin, without calcium and magnesium, diluted 1:10 with distilled water), pelleted again by 5 minutes of centrifugation at 300 × g . Finally, the cells were suspended in 10 mL nonhematopoietic stem cell expansion medium (NH expansion medium; Miltenyi Biotec GmBH, Bergisch-Gladbach, Germany) and plated into a single 75 cm 2 tissue culture flask (TPP AG, Trasadingen, Switzerland) and cultured at 37°C.
Colony assay
Freshly isolated PD-MSCs, passage 0, were replated at low density (ie, 50 cells per well of 6-well plates [TPPAG]) and cultured at 37°C and 5% CO 2 in nonhematopoietic stem cell expansion medium (NH expansion medium). Outgrowing colonies of spread cells were visualized and counted by fluorescence microscopy using rhodamine-labeled phalloidin to stain actin cytoskeleton (Invitrogen, Basel, Switzerland), and 4′6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Eugene, OR) to stain cell nuclei. Images were acquired with a Zeiss Axiovert 200M (Carl Zeiss AG, Feldbach, Switzerland) equipped with a digital camera AxioCam MRc (Carl Zeiss AG).
Growth kinetics
PD-MSCs of passages 1, 10, and 20 taken from 4 different cases were plated at 5 × 10 3 cells per well in 12-well plates (TPPAG) and cultured at 37°C and 5% CO 2 in nonhematopoietic stem cell expansion medium (NH expansion medium). All experiments were performed in triplicate. Cell counts were determined after 24, 48, and 72 hours of culture. For that, cells were detached with 0.25% trypsin solution (GIBCO-Invitrogen AG, Basel, Switzerland) and counted with a Coulter Z1 cell counter (Instrumenten Gesellschaft AG, Zurich, Switzerland). Dead cells were identified by staining with 0.4% trypan blue staining solution (Sigma-Aldrich AG).
Antibodies
Information about primary and secondary antibodies used for flow cytometry and immunochemistry is provided in Table 2 .
| Antigen | Clone no. | Conjugate | Host | Company |
|---|---|---|---|---|
| CD11b | ICRF44 | PE | M | BioLegend |
| CD14 | H5E2 | — | M | BioLegend |
| CD19 | HIB19 | PE | M | BioLegend |
| CD34 | AC136 | FITC | M | Miltenyi Biotec GmbH |
| CD44 | G44-26 | FITC | M | BD Pharmingen |
| CD45 | 5B1 | FITC | M | Miltenyi Biotec GmbH |
| CD54 | HA58 | PE | M | BD Pharmingen |
| CD73 | AD2 | PE | M | BD Pharmingen |
| CD79a | HM47 | PE | M | BioLegend |
| CD90 a | 5E10 | — | M | BD Pharmingen |
| CD105 a | 266 | — | M | BD Pharmingen |
| CD117 | YB5.B8 | PE | M | BD Pharmingen |
| CD133/1 | AC133 | APC | M | Miltenyi Biotec GmbH |
| CD163 | GHI/61 | PE | M | BD Pharmingen |
| CD166 | 3A6 | PE | M | BD Pharmingen |
| CD271 | ME20.4-1H4 | PE | M | Miltenyi Biotec GmbH |
| HLA-ABC | G46-2.6 | FITC | M | BD Pharmingen |
| HLA-DR | G46-6 | FITC | M | BD Pharmingen |
| Cytokeratin-7 (CK-7) | CAM 5.2 | FITC | M | BD Pharmingen |
| Cytokeratin-18 (CK-18) | CY-90 | FITC | M | Sigma-Aldrich AG |
| α-SMA | 1A4 | PE | M | R&D Systems |
| vWF | Sheep polyclonal | FITC | S | Abcam plc |
| KDR/VEGF receptor-2 | 89106 | PE | M | R&D Systems |
| Placental alkaline phosphatase (PLAP) a | H17E2 | — | M | AbD Serotec |
| Hepatocyte-specific antigen (HSA) a | OCH1E5 | — | M | Abcam plc |
| SSEA-3 b | MC-631 | — | R | R&D Systems |
| SSEA-4 | MC813-70 | PE | M | R&D Systems |
| Oct-3/4 b | 240408 | — | R | R&D Systems |
| Vimentin | V9 | PE | M | Sigma-Aldrich AG |
| Nestin a | 10C2 | — | M | Abcam plc |
| E-cadherin | 36 | PE | M | BD Pharmingen |
| Stro-1 a | STRO-1 | — | M | R&D Systems |
| Embryonic stem cell marker Tra-1-60 a | Tra-1-60 | — | M | Abcam plc |
| Embryonic stem cell marker Tra-1-81 a | Tra-1-60 | — | M | Abcam plc |
| ZO-1 | ZO-1-1A12 | FITC | M | Zymed Laboratories |
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