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Stem Cells 2005;23:3-9 www.StemCells.com
© 2005 AlphaMed Press

RAPID COMMUNICATION

Isolation of Multipotent Cells from Human Term Placenta

B. Linju Yena, Hsing-I Huangb, Chih-Cheng Chienb,c, Hsiang-Yiang Juia, Bor-Sheng Kod, Ming Yaod, Chia-Tung Shune, Men-luh Yenf,g, Meng-Chou Leea, Yao-Chang Chena

a Stem Cell Research Center, National Health Research Institutes, Taipei, Taiwan;
b Cathay Medical Research Institute, and
c Department of Anesthesiology, Cathay General Hospital, Taipei, Taiwan;
d Department of Internal Medicine,
e Departments of Forensic Medicine and Pathology,
f Department of Primary Care Medicine, and
g Department of Obstetrics and Gynecology, National Taiwan University Hospital and National Taiwan University, College of Medicine, Taipei, Taiwan

Key Words. Stem cell • Placenta • Multilineage differentiation Stage-specific embryonic antigen—4 (SSEA-4) • Tumor rejection antigens

Correspondence: Yao-Chang Chen, M.D., Stem Cell Research Center, National Health Research Institutes, No. 161, 4F, Min-Chuan E. Road, Taipei, 114, Taiwan. Telephone 886-2-2312-3456, ext. 5489; Fax: 886-2-2321-8438; e-mail: ycchenmd{at}nhri.org.tw


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Current sources of stem cells include embryonic stem cells (ESCs) and adult stem cells (ASCs). However, concerns exist with either source: ESCs, with their significant ethical considerations, tumorigenicity, and paucity of cell lines; and ASCs, which are possibly more limited in potential. Thus, the search continues for an ethically conducive, easily accessible, and high-yielding source of stem cells. We have isolated a population of multipotent cells from the human term placenta, a temporary organ with fetal contributions that is discarded postpartum. These placenta-derived multipotent cells (PDMCs) exhibit many markers common to mesenchymal stem cells—including CD105/endoglin/SH-2, SH-3, and SH-4—and they lack hematopoietic-, endothelial-, and trophoblastic-specific cell markers. In addition, PDMCs exhibit ESC surface markers of SSEA-4, TRA-1-61, and TRA-1-80. Adipogenic, osteogenic, and neurogenic differentiation were achieved after culturing under the appropriate conditions. PDMCs could provide an ethically uncontroversial and easily accessible source of multipotent cells for future experimental and clinical applications.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The derivation of human embryonic stem cells (ESCs) in recent years is a landmark achievement [1, 2]. These plu-ripotent stem cells can be propagated indefinitely and are capable of differentiating into tissues from all three germ layers. However, numerous considerations, both ethical and technical, limit the availability of these cells, which can only be isolated from the inner cell mass of early embryos [3]. Moreover, the tumorigenicity of ESCs has yet to be resolved [4]. Adult stem cells (ASCs), previously thought to be limited in potential, have increasingly been shown to be able to differentiate into tissues of an entirely different germ layer. The hope has been for the clinical use of such versatile cells in a number of diseases [5]. One of the most extensively studied populations of multipotent ASCs has been mesenchymal stem cells (MSCs) from the bone marrow [6]. Although considerably less problematic ethically, cells from the bone marrow still must be obtained through an invasive procedure, and stem cell numbers decrease significantly with the age of the individual [7].

The search for easily accessible sources of multipotent stem cells has led various groups to look into other tissues, including mobilized peripheral blood [8], umbilical cord blood [9–12], and, more recently, deciduous tooth [13] and umbilical cord mesenchyme [14]. However, the cell volume obtained from such tissues can be limited [10, 13], and much debate exists regarding the presence of stem cells in some of these sources [15, 16]. Thus, the search continues for an ethically conducive, easily accessible, and high-yielding source of stem cells. We have investigated the possibility of a multipotent stem cell residing in the human term placenta, a temporary organ that is discarded postpartum.

The human placenta is a fetomaternal organ, formed by both fetal and maternal tissue. Its successful formation is a critical process in embryogenesis, and the normal development and function of the placenta is crucial to the well-being of the fetus. This remarkable organ is discarded postpartum, after having performed its necessary function of supporting the embryo and fetus [17].

Stem cells isolated from term, postpartum placenta have a variety of advantages. Although they are unlikely to have the differentiation and proliferative potentials of ESCs, cells derived from the placenta are still of fetal origin and may be superior to ASCs in many aspects. No invasive procedure is necessary to obtain the organ, since the placenta is expelled after the birth of the neonate. Furthermore, there are no ethical conflicts generated, since the organ would have been discarded otherwise. Using this ethically uncontroversial and easily accessible organ, we have isolated a population of multipotent cells from the human postpartum term placenta, which we have named placenta-derived multipotent cells (PDMCs).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures
Term (38–40 weeks’ gestation, n = 16) placentas from healthy donor mothers were obtained with informed consent approved according to the procedures of the institutional review board. Umbilical cord blood was allowed to drain from the placentas, which were then dissected carefully. The harvested pieces of tissue were washed several times in phosphate-buffered saline (PBS) and then mechanically minced and enzymatically digested with 0.25% trypsin-EDTA (Gibco-Invitrogen Corp., Grand Island, NY, http://www.invitrogen.com) for approximately 10 minutes at 37°C. The homogenate was subsequently pelleted by centrifugation and suspended in complete medium, which consist of Dulbecco’s modified Eagle’s medium (Gibco-Invitrogen) supplemented by 10% fetal bovine serum (FBS; selected lots; HyClone, Logan, UT, http://www.hyclone.com), 100 U/ml penicillin, and 100 g/ml streptomycin (Sigma-Aldrich, St. Louis, http://www.sigma-aldrich.com). Cell cultures were maintained at 37°C with a water-saturated atmosphere and 5% CO2. Medium was replaced one to two times every week. When cells were more than 80% confluence, they were recovered with 0.25% trypsin/EDTA and replated at a dilution of 1:3. For control, MSCs from bone marrow aspirates were cultured according to Pittenger et al. [6]. JEG-3 cells, a human choriocarcinoma cell line, were obtained from American Type Culture Collection (ATCC; Rockville, MD, http://www.atcc.org), and cultured in 90% Eagle minimum essential medium with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate (Gibco-Invitrogen), and 10% FBS (HyClone).

Immunophenotyping of PDMCs
To detect surface antigens, aliquots of PDMCs were washed with PBS containing 2% FBS after detachment with 0.25% trypsin/EDTA. To detect intracellular antigens, cells were additionally permeabilized with 70% ethanol (10 minutes at 4°C). Antibodies against human antigens CD9, CD14, CD34, CD40L, CD45, CD80, CD84, CD90/Thy-1, CD117/ c-kit, CD166, and HLA-DR were purchased from Becton Dickinson (San Diego, http://www.bd.com). Antibodies against human antigens glycophorin A, CD13, CD29, and CD44 were purchased from Dako (Glostrup, Denmark, http://www.ump.com/dako.html). Antibodies against human antigen AC/CD133/2 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany, http://www.milt-enyibiotec.com). Antibodies against human antigens von Willebrand factor and fetal liver kinase–1 (Flk-1) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com). Antibodies against human antigen CD105/endoglin/SH-2 and stage-specific embryonic antigen–4 (SSEA-4) were purchased from Developmental Studies Hybridoma Bank of Iowa University (Iowa City, IA, http://www.uiowa.edu/~dshb). Antibodies against human antigens cytokeratin 7 and the tumor rejection antigen 1-60 (TRA-1-60) and TRA-1-81 were purchased from Chemi-con International (Temecula, CA, http://www.chemicon.com). Antibodies against human antigen HLA-ABC were purchased from Serotec (Raleigh, NC, http://serotec.oxi.net/asp/). Antibodies against human antigen HLA-G were purchased from Exbio (Vestec, Czech Republic, http://www.exibio.cz/). Antibodies against human antigens SH-3 and SH-4 were purified from the respective hybridoma cell lines acquired from ATCC. The fixed cells were stained with fluorescein isothiocyanate or phycoetrythrin (PE)–conjugated antibodies and analyzed using a Becton Dickin-son FACSCalibur flow cytometry system (BD Biosciences, Mississauga, Canada, http://www.bdbiosciences.com).

Immunofluorescent and Immunocytochemical Staining

Immunofluorescence   Cultured cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature and permeabilized with 0.1% Triton-X 100 (Sigma) for 10 minutes. Samples were then incubated sequentially with primary monoclonal antibodies at 4°C for 18 hours. Primary antibodies against the human antigen vimentin (Serotec) and MAP2 (Chemicon) were stained at a dilution of 1:100. The samples were then rinsed three times with PBS and incubated for 60 minutes at room temperature with PE-conjugated secondary antibodies at a dilution of 1:100. Staining was visualized under a fluorescence microscope (Nikon, Tokyo, http://www.nikon.co.jp).

Immunocytochemistry   Cultured cells were fixed with 4% PFA for 5 minutes at room temperature and permeabilized with 0.1% Triton-X 100 for 20 minutes. Samples were then incubated sequentially first with the primary monoclonal antibodies against the human antigen neuron-specific enolase (NSE; Sigma) at a dilution of 1:25 and at 4°C overnight. The samples were then stained with biotinylated anti-rabbit antibody and an avidin-biotin conjugate of horseradish peroxidase (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

The presence of adipocytes was assessed by the cellular accumulation of neutral lipid vacuoles that stain with Oil red O [18]. Osteoblastic differentiation was evaluated by calcium accumulation with alizarin red stain [19].

ß-Human Chorionic Gonadotropin
ß-human chorionic gonadotropin (ß-hCG) in supernatant was measured by immunometric assay (Diagnostic Products, Los Angeles, http://www.dpcweb.com). Supernatants of cell cultures were aspirated and immediately frozen at –20°C, as directed by the manufacturer for batch assay. The World Health Organization Third International Standard 75/537 was used to determine ß-hCG levels.

Differentiation Studies
For adipogenic differentiation, cells were cultured in complete medium with the addition of 0.5 µM isobutyl-methyl-xanthine, 1 µM dexamethasone, 10 µM insulin, and 60 µM indomethacin [9]. Osteogenic differentiation was achieved by culturing cells in complete medium along with 0.1 µM dexamethasone, 10 mM ß-glycerol phosphate, and 50 µm ascorbate [20]. Neurogenic differentiation was induced by culturing cells in serum-free medium with the addition of 10–6 M retinoic acid.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cellcoloniesfrom10ofthe16processedspecimensbeganto appear approximately 9–14 days after initial plating. Initial cell growth consisted of cells with two different morphologies: one population with a fibroblastoid, spindle-shaped morphology (Fig. 1AGo) and another with an epitheliod, polygonal morphology (Fig. 1BGo). After passaging with trypsin, the epitheloid population rapidly disappeared from culture and could no longer be found by the second passage. The fibroblastoid population of cells continued to proliferate, even after numerous passages (Fig. 1C–EGo).



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  		Figure 1. Multipotent cells from the human term placenta. (A–B): Appearance of initial fibroblastoid and epitheloid cell growths, repectively. (C–E): Appearance and growth of fibroblastoid cells or placenta-derived multipotent cells at passage 6 on days 1, 3, and 5, respectively. (F): Appearance of bone marrow mesenchymal stem cells at confluence, for comparison. Phase contrast: magnification x50 for all figures except B (x100).

 
Immunophenotyping of PDMCs (Fig. 2Go) revealed these cells to be positive for many markers common to MSCs [6, 21]: SH-2/CD105, SH-3, SH-4, CD29, CD44, CD90/Thy-1, and CD166 (ALCAM). PDMCs were negative for the hematopoietic surface markers of CD14, CD34, CD45, CD117/c-kit, AC133/2, and glycophorin A, as well as the endothelial cell markers of von Willebrand factor and Flk-1. Expression of the costimulatory molecules CD40L, CD80, and CD86 was also not seen (data not shown). These cells were also negative for markers found in trophoblastic cells, including HLA-G and cytokeratin 7, and they were positive for the nontrophoblastic markers of CD9 and vimentin (not shown) [22]. ß-hCG levels ranged from 2.16 IU/L to undetectable levels after the second passage; in the culture media of JEG-3 cells, levels of up to 260 IU/L were detected. Immunologically, PDMCs were positive for HLA-ABC but negative for HLA-DR. PDMCs were also strongly positive for the ESC-associated cell surface markers of SSEA-4, TRA-1-60, and TRA-1-81 [1, 2, 23].



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  		Figure 2. Immunophenotyping results of placenta-derived multipotent cells. Abbreviations: CK-7, cytokeratin 7, Flk–1, fetal liver kinase–1; vWF, von Willebrand factor; TRA, tumor rejection antigen; and SSEA, stage-specific embryonic antigen.

 
Further characterization of PDMCs revealed the ability to differentiate into mesodermal-lineage cells of osteoblasts and adipocytes, as well as ectodermal, neuron-like cells. Adipogenic differentiation could be seen by day 10 with the formation and accumulation of lipid vacuoles (Fig. 3AGo). Mineralized matrix deposition by PDMCs could be seen after approximately 3 weeks of incubation with osteogenic medium (Fig. 3BGo). Differentiation was not observed in PDMC cultures grown in regular culture medium (Fig. 3CGo). Neuron-like cells could be seen after 6–14 days of neurogenic induction (Fig. 4AGo). PDMC-differentiated neuron-like cells also stain positive for the neuronal markers of MAP2 (Fig. 4BGo) and NSE (Fig. 4CGo).



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  		Figure 3. Mesodermal differentiation of PDMCs. (A, B): Oil red and alizarin red stains of PDMCs grown in adipogenic and osteogenic medium, respectively. (C): Control PDMCs grown in regular medium. Phase contrast: magnification x200. Abbreviation: PDMC, placenta-derived multipotent cell.

 


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  		Figure 4. Neurogenic differentiation of placenta-derived multipotent cells. (A): Phase contrast microscopy (x200), showing neuron-like morphology after neurogenic induction (arrowheads). (B): Immunofluorescent staining for MAP2 (x400). (C): Immunocytochemical staining for neuron-specific enolase (x400).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that multipotent, multilineage cells are present in the human term placenta. Propagation of cells from 10 out of 16 specimens (62.5%) was achieved. Using routine cell culture techniques, PDMCs can be successfully isolated and expanded in vitro. Although the initial cell culture consisted of both fibroblastoid and non-fibroblastoid cell types, only the fibroblastoid population remained after enzymatic digestion and passaging. Contamination with trophoblastic cells was excluded by the presence of CD9, CD90/Thy-1, HLA-ABC, and vimentin and by the absence of AC133/2, HLA-G, and ß-hCG production [22]. Another source of possible contamination is with umbilical cord blood cells, which contain hematopoietic and, possibly, MSCs.

Currently, the presence of MSCs in umbilical cord blood is still contested [15–16], and in reports that document isolation of such cells, low yield and interindividual variation were consistently encountered limitations [9–11]. Moreover, one report used an isolation and culturing technique that requires negative immunodepletion of specific cells [12]. Given these obstacles to culturing umbilical cord blood MSCs even in the best of hands, it is unlikely that these cells were recovered along with PDMCs, which were isolated from the placenta itself. Pieces were harvested only after drainage of umbilical cord blood from the organ, then cultured only after multiple washes in PBS to avoid cord blood contamination. In addition, PDMCs were cultured using a very simple and straightforward technique.

If any stem cell population from the umbilical cord blood were possibly to contribute to PDMCs, it would be the hematopoietic stem cells, since their presence in cord blood is well documented and comprises a high percentage of total cord blood cells [24]. The fact that PDMCs were negative for multiple leukocyte- and erythrocyte-specific markers supports the low likelihood of such contamination. Endothelial cells are another abundant cell population within the placenta, and contamination of PDMCs with such cells also possible. But the absence of endothelial-like cell morphology during the culturing process, along with the lack of endothelial markers detected in PDMCs, makes this also unlikely. While PDMCs were positive for CD105/endoglin/ SH-2, originally defined as an endothelial marker, this cell surface marker has more recently been regarded as an MSC marker [25].

The presence of CD105/endoglin/SH-2 and numerous other MSC markers—including SH-3, SH-4, and a number of integrins and matrix receptors [6, 21]—on PDMCs, along with a fibroblastoid morphology, plastic-adherance nature, and mesodermal differentiation capabilities, suggests that these cells resemble MSCs from the bone marrow. Of considerable interest is that in addition to MSC markers, three cell surface markers—SSEA-4, TRA-1-60, and TRA-1-81—found only in ESCs and embryonic germ cells [1, 2, 23], were also found on PDMCs. Currently, ESCs are the most well- accepted pluripotent stem cells; in vivo studies have shown formation of teratomas in immunocompromised mice [1, 2], and in vitro studies have demonstrated pluripotent somatic differentiation [2]. However, the lack of availability of these cells and ethical concerns regarding their source continue to be major obstacles [3].

Furthermore, ESCs are difficult to culture and keep undifferentiated, requiring a co-culture system with a feeder layer of cells, in contrast to ASCs—and PDMCs as well—which do not have such requirements. Human ESCs are even more difficult to maintain than their murine counterparts since they are unresponsive to the addition of leukemia inhibitory factor, a factor that can maintain mouse ESCs in an undifferentiated state. Hence, human ESCs depend on a feeder layer. Current practice is to use feeders derived from mouse embryonic fibroblasts [1, 2]. This use of xenogeneic cells in the culturing process is one of several concerns regarding clinical application of ESCs. Thus, the therapeutic use of ESCs, although possessing tremendous pluripotency, awaits further research to resolve this and other issues.

The detection of ESC surface markers on PDMCs suggests that these may be very primitive cells. If this is correct, it may well be that the renewal and differentiation capacity of PDMCs are more extensive than ASCs. Although some of the PDMC cultures have been passaged over 15 times (approximately 50 population doublings), the immortality of these cells remains to be demonstrated. Regarding differentiation potential, PDMCs differentiate readily into two mesodermal lineages, as well as an ectodermal, neuron-like cell type. The multilineage differentiation and proliferative capability and the presence of various MSC– and ESC–markers lend strong support to the presence of a putative stem cell population within PDMCs. However, isolation and analysis of multiple PDMC clones will be required to confirm the presence of a stem cell population within this organ. Investigation of the differentiation capability of PDMCs into endodermal cell types, as well as further characterization of these cells, is currently ongoing.

The presence of stem cells in the placenta could have tremendous implications. The initial data on the differentiation capabilities of PDMCs are promising, and there may be important therapeutic uses for these cells. Along with the ease of accessibility, lack of ethical concerns, and abundant cell number, PDMCs may be an attractive, alternative source of progenitor or stem cells for basic research and clinical applications.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. Wern-Cherng Cheng for his assistance on the ß-hCG assay. B.L.Y. and H.I.H. contributed equally to this work.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received April 28, 2004; accepted for publication August 24, 2004.



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