Correspondence: J á n Rosocha , DVM, PhD, Tissue Bank, Faculty of Medicine, P. J. Š af á rik University, Trieda SNP1, SK-040 11 Ko š ice, Slovakia. E-mail: jan.rosocha@upjs.sk.

(Received 26 November 2010; accepted 23 May 2011)

Isolation and basic characterization of human term amnion and chorion mesenchymal stromal cells

D ARINA BA C ENKOV Á 1 , J Á N ROSOCHA 1 , T IMEA T Ó THOV Á 1 , LADISLAV ROSOCHA 2 & MAREK Š ARISSK Ý 3

1 Tissue Bank, Faculty of Medicine, P. J. Š af á rik University, Trieda SNP 1, Ko š ice, Slovakia, 2 2nd Department of Gynaecology and Obstetrics, Faculty of Medicine, P. J. Š af á rik University, and L. Pasteur Faculty Hospital Ko š ice, Rastislavova, Ko š ice, Slovakia, and 3 Department of Pharmacology, Faculty of Medicine, P. J. Š af á rik University, Trieda SNP 1, Ko š ice, Slovakia

Abstract Background aims. Emerging evidence suggests human placental membrane is a valuable source of mesenchymal stromal cells (MSC). Amnion and chorion are tissues of early embryologic origin that may entail progenitor potential. These tissues are abundantly available and ethically unobjectionable and, because they are discarded post-partum, they can be widely used for extensive research and eventually for therapeutic studies. Methods. We looked at the cells isolated from the six amnions and chorions of term placentas of gestational weeks 39 � 1. Isolated cells were characterized by morphologic and immunophenotypic analysis. Results. With fl ow cytometry immunophenotype analysis, amnion- and chorion-derived cells were positive for MSC markers, and negative for hematopoietic markers. Immunocytochemical staining was positive for the embryonic cell markers Oct-3/4 and Rex-1. Oct-3/4 is a POU transcription factor that is expressed in embryonic stem (ES) cells and germ cells, and its expression is required to sustain cell self-renewal and pluripotency. Oct-3/4 is the most recognized marker for totipotent ES cells. Rex-1 is a zinc fi nger family transcription factor that is highly expressed in embryonic stem cells. It is one of several gene markers used to identify undifferentiated stem cells, and its expression is downregulated upon stem cell differentiation. Amnion- and chorion-derived cells were capable, under differentiation con-ditions, to differentiate into to mesoderm lineages. Conclusions. Phenotypic studies indicate MSC-like profi les in both amnion- and chorion-derived cells. Cells in vitro had fi broblastoid morphology. The in vitro growth behavior of such pla-centa-derived progenitor cells was similar to that of bone marrow MSC. Our results indicate that MSC can be easily isolated from the human term placenta. The human amniotic and chorion MSC maintained a marker profi le similar to the mes-enchymal progenitors and could be used for studies as an alternative source of MSC for further application in cellular therapy.

Key Words: amnion , chorion , human term placenta , mesenchymal stromal cells

Introduction

Multipotent mesenchymal stromal cells (MSC) are a promising cell resource for cell-based therapeutics because of their ability to self-renew and differenti-ate into specifi c functional cell types (1). There are many reports showing that bone marrow (BM)-de-rived MSC are able to differentiate into all mesoder-mic lineages: adipose and connective tissue, as well as bone and cartilage (2). Cell therapy with MSC has been used to treat a wide range of diseases (3). The procedures required to obtain the BM may be invasive and associated with appreciable morbidity, the cell numbers obtained can be low, and the dif-ferentiation potential may be dependent on the age of the donor (4). As an alternative to BM as a poten-tial source of multipotent MSC are fetal membranes,

the amnion and chorion of term human placenta. Cells that are phenotypically similar to BM MSC (2) have been isolated from unfractionated placenta (5 – 7), villous stroma of the para/umbilical area (8), the internal area of placental lobules (9), and from amnion and chorion membranes (10,11).

The human placenta is a fetomaternal entity that consists of a fetal component (the chorionic plate) and a maternal component (the deciduas). The amnion is the inner layer and is comprised primarily of two cell types: the epithelial cuboid cells and the columnar cells. The amniotic epithelial cells (AEC) on one side of the amnion create a continuous lining adjacent to the amniotic fl uid, while on the other side of the amni-otic epithelium is a thin layer of amniotic mesoderm (AM), throughout which a few fetal macrophages are

Cytotherapy, 2011; 13: 1047–1056

ISSN 1465-3249 print/ISSN 1477-2566 online © 2011 Informa HealthcareDOI: 10.3109/14653249.2011.592522

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

1048 D. Ba c enkov á et al.

sporadically distributed (12). The chorion is com-posed of the inner chorionic mesoderm, similar to the mesenchymal region of the amnion, and an outer highly variable trophoblastic layer, with extravillous cytotrophoblast cells that represent the only residues of the former villi of the chorion frondosum (13).

MSC are primarily from the BM. There are fi ve minimum criteria for defi ning human amniotic MSC (hAMSC) and human chorionic MSC (hCMSC) that originate from the extra-embryonic mesoderm of the amnion or chorion: (i) fetal origin (maternal contamination of 1% or less); (ii) formation of fi bro-blast colony-forming units; (iii) a specifi c surface antigen pattern, CD90 � CD73 � CD105 � CD45 – CD34 – CD14 – and HLA-DR – ; (iv) a differentiation potential to one or more lineages, osteogenic, adipo-genic, chondrogenic or vascular/endothelial; and (v) adherence to plastic (14).

Transmission electron microscopy of hAMSC shows mesenchymal and epithelial characteristics. This hybrid phenotype is interpreted as a sign of multipo-tentiality and is not found in hCMSC, which are more primitive and metabolically quiescent. Specifi cally, if compared with hAMSC, transmission electron micros-copy of hCMSC shows a simpler cytoplasmic organiza-tion. The most relevant features include the presence of stacks of rough endoplasmic reticulum cisternae, dis-persed mitochondria and glycogen lakes. Features of higher specialization, such as the presence of assembled contractile fi laments, prominence of endocytotic traf-fi c and junctional communications, are lacking (14). Mixed populations of fetal membrane-derived cells express numerous cell-surface antigens and intracel-lular antigens similar to of BM-derived MSC (10). All express typical mesenchymal markers, but are negative for hematopoietic-, endothelial- and trophoblastic-specifi c cell markers, while they express stage-specifi c embryonic antigens (SSEA)-3 and �4 (15,16).

Both hAMSC and hCMSC express low HLA-ABC and no HLA-DR, indicating their immunoprivileged status (16,17). Several studies have revealed that MSC are not immunogenic and also exhibit immunomodula-tory properties (18). The low immunogenicity of these cells is confi rmed by clinical applications of amniotic membranes in surgical procedures, where they can be used as a biologic dressing (19) and for the treatment of corneal or conjunctiva destructive loss (20).

Knowledge about yields, phenotype and expan-sion of hAMSC and hCMSC is a prerequisite for their therapeutic application in regenerative medi-cine. We report on a protocol for the isolation and culture of hAMSC and hCMSC from term pla-centa. The aim of our study was to characterize and compare the differentiation potential of isolated amnion- and chorion-derived cells after culturing under appropriate conditions.

Methods

Isolation and primary culture of human amnion and chorion cells

Term (38 – 40-week gestation, n � 6) placentas from healthy donor mothers were obtained. Part of the amnion and chorion measuring 10 � 10 cm was then separated manually and washed extensively in phosphate-buffered saline (PBS), containing 100 IU penicillin/mL, 100 μ g streptomycin/mL and 0.25 μ g amphotericin B/mL (Invitrogen, Carlsbad, California, USA), before being cut into small pieces ( c. 1.5 � 1.5 cm). Amnion fragments were incubated for 20 min at 37 ° C in Dulbecco ’ s Minimal Essential Medium (MEM) (Biochrom AG, Berlin, Germany) containing 2.4 U/mL dispase (Invitrogen, GIBCO ® , Carlsbad, California, USA) and 1% antibiotic – antimycotic solu-tion. Cells were collected by centrifugation at 150 g for 15 min. Then they were digested with 1.0 mg/mL collagenase type II (Invitrogen, GIBCO) for approxi-mately 2 h at 37 ° C in Dulbecco ’ s MEM containing 100 U/mL penicillin, 100 μ g/mL streptomycin and 0.25 μ g amphotericin (Invitrogen, GIBCO). Amnion fragments were later removed and the released cells were passed through a 40- μ m cell strainer (BD Fal-con ™ , Biosciences, Bedford, MA, USA). Cells were collected by centrifugation at 150 g for 15 min. We refer to these cells as hAMSC.

Chorion fragments, after mechanical removal of decidua, were incubated for 20 min at 37 ° C in Dulbecco ’ s MEM supplemented with additives as described above. Dispersed chorion cells, called hCMSC, after fi ltration through a 40- μ m cell strainer, were collected by centrifugation.

The cells were cultured with alpha-MEM medium (Biochrom AG) supplemented with 10% fetal bovine serum (FBS; Invitrogen, GIBCO) and 1% antibi-otic – antimycotic solution in 75-cm 2 culture fl asks (Sarstedt AG & Co., Nümbrecht, Germany). Cell cultures were maintained at 37 ° C in a humidifi ed atmosphere with 5% CO 2 atmosphere. Three to fi ve days after initiating incubation, the small digested residues were removed and the culture was contin-ued with fresh media added twice weekly. When cells were more than 80% confl uent, they were recovered with 0.25% trypsin/ethylene diamine tetra acetic acid (EDTA) (Invitrogen, GIBCO). The cells were expanded for several passages (2 – 4).

Enrichment of CD105 � cells

hAMSC and hCMSC were incubated with colloidal MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) coated with CD105 monoclonal antibodies for 15 min at 10 ° C. CD105 � cells were then enriched using a MACS System (Miltenyi Biotec) according

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

Isolation and characterization of amnion and chorion MSC 1049

to the manufacturer ’ s specifi cations. The percentages of CD105 � cells were then used for further analysis. CD105 � hAMSC and hCMSC after the second pas-sage were used for immunophenotypic and differen-tiation analysis.

In vitro differentiation of CD105 � hAMSC and hCMSC and their characterization

Differentiation studies were performed with cells obtained after two passages by immunomagnetic separa-tion. For that, cells were seeded at a density of 2.0 � 10 4 cells/cm 2 for adipogenic differentiation, at a density of 4.2 � 10 3 cells/cm 2 for osteogenic differentiation, and at density of 2.5 � 10 5 cells/per well for chondrogenic differentiation, in glass chamber slides (Lab-Tec ® ; Nalgene Nunc International, Naperville, IL, USA). To evaluate multilineage differentiation, cells were allowed to become near-confl uent and then cultured for 2 – 3 weeks in appropriate induction medium.

Osteogenic differentiation was performed as rec-ommended by the manufacturer of the osteogenic induction medium, with additional supplements of dex-amethasone, ascorbate-phosphate and β -glycerolphos-phate (human MSC functional identifi cation kit; R&D Systems, Minneapolis, USA). hAMSC and hCMSC were maintained in osteogenic induction medium for 2 weeks, with fresh medium added every third day. Cal-cium deposits characteristic of osteogenic differentia-tion in cultures were visualized by von Kossa staining.

Chondrogenic differentiation was assessed in a monolayer culture using chondrogenic induction media, with additional supplements of dexametha-sone, ascorbate-phosphate, proline, sodium pyru-vate and transforming growth factor (TGF)- β 3, and (Insulin-Transferrin-Selenium-X (ITS) Sup-plement, Invitrogen, Carlsbad, California, USA) supplement (a solution containing insulin, transfer-rin, selenious acid, bovine serum albumin and lino-leic acid) (human MSC functional identifi cation kit; R&D Systems). Alcian blue staining was used to assess the formation of the extracellular matrix, which is the mark of chondrogenic differentiation.

hAMSC and hCMSC were exposed to adipo-genic induction media, with additional supplements of hydrocortisone, isobutylmethylxanthine and indo-methacin (human MSC functional identifi cation kit; R&D Systems), according to the manufacturer ’ s instructions. Cytoplasmic inclusions of neutral lipids were then stained with Scharlach Sudan S.

Immunocytochemical staining of hAMSC and hCMSC

To detect the antigens, after detachment with 0.5% trypsin/EDTA, cells from passage 1 were washed

with medium without FBS. To detect intracellular antigens, amnion and chorion cells were initiated by placement in individual slide 8 chambers (LabTekTM, NUNC A/S, Roskilde, Denmark) at a concentration of 10 000/1 chamber. Next, they were cultured for 3 – 4 days in complete culture medium to form a mono-layer. Cells were fi xed with 4% paraformaldehyde for 20 min at room temperature (RT) and permeabi-lized with 0.1% Triton and 1% bovine serum albu-min (BSA) for 45 min at RT. Amnion and chorion cell samples were then incubated overnight at 4°C with primary monoclonal antibodies rat anti-human Oct-3/4 (R&D Systems, Mineapolis, USA ), rat anti-human Rex-1 (R&D Systems, Mineapolis, USA) and mouse anti-human CD45 (R&D Systems, Mineapolis, USA) at a dilution of 1:25, and mouse anti-human CD105 (Dako Cytomation, Glostrup, Denmark) at a dilution of 1:50. The samples were incubated with biotinylated anti-rat resp. anti-mouse antibody and visualized by streptavidin - HRP conjugate (Cell and tissue staining kit, R&D Systems, Mineapolis, USA resp. with Dako EnVisionTM + System-HRP (DAB) (Glostrup, Denmark).

Flow cytometry analysis of CD105 � hAMSC and hCMSC

For evaluation of surface-marker expression, cell sus-pensions were incubated for 15 min with phycoeryth-rin (PE)-conjugated antibodies against the following human antigens, CD105 clone 43A3 (BioLegend, San Diego, California, USA), CD44 – PE clone G44-26 and CD90 – PE clone 5E10 (BD Biosciences, Franklin Lakes, New Jersey, USA), and fl uorescein isothiocyanate (FITC)-conjugated antibodies against human antigens CD146 clone 541-10B2 (Miltenyi Biotec), CD45 – FITC clone HI30 (BD Pharmingen, Franklin Lakes, New Jersey, USA), CD34 – PE clone 8612 (BD Biosciences), HLA-ABC clone G46-2.6 and HLA-DR clone G46-6. The fi xed cells were stained with goat anti-mouse Ig – FITC (BD Biosci-ence). Samples were analyzed with a fl uorescent- activated cell sorter (FACS) Calibur (BD Biosciences) and CellQuest software (BD Biosciences).

Results

Isolation and phenotype characterization of hAMSC and hCMSC

Searching for alternative sources of MSC, we attempted to prepare human term placentas ( n � 6) and isolate fi broblast-like cells dissected from the defl ected part of the fetal membranes. After their mechanical separa-tion, the amnion and chorion were digested with dis-pase and collagenase separately. hAMSC and hCMSC cultures were successful in all six cases. Both cell types

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

1050 D. Ba c enkov á et al.

Figure 1. Morphology of hAMSC and hCMSC. (A) hAMSC 1 day after isolation (200 � magnifi cation); (B) adherent hAMSC 8 days after isolation (200 � magnifi cation); (C) CD105 � hAMSC confl uent at the third passage (200 � magnifi cation); (D) hCMSC and non-adherent cells 1 day after isolation (200 � magnifi cation); (E) adherent hCMSC 8 days after isolation (200 � magnifi cation); (F) CD105 � hCMSC confl uent at the fourth passage of chorionic cells, Giemsa – Romanowski stain (200 � magnifi cation). Scale bar: 50 μ m.

adhered within 24 h and could be expanded further. We isolated 6 � 10 6 hAMSC and 11 � 10 6 hCMSC from six membrane samples. The amniotic cells were initially plated on 75-cm 2 culture fl asks at a density of 0.8 � 10 5 nucleated cells/cm 2 (Figure 1A), and the chorion cells at a higher density, 0.14 � 10 6 nucleated cells/cm 2 (Figure 1D).

Both hAMSC (Figure 1B) and hCMSC (Figure 1E) adhered and proliferated on tissue culture

plastic and could be kept until passages 4 – 5 after immunomagnetic separation (Figure 1C,F). Adher-ent cells had heterogeneous shapes: most with a spindle-shaped morphology (some cells showing dendrites) and lesser amounts of highly refl ective small, rounded, rapidly proliferating cells. After the fi rst passage, cells from both samples expanded in the same monolayer manner, with homogeneous fi broblast-like cells.

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

Isolation and characterization of amnion and chorion MSC 1051

Enrichment of CD105 1 cells

A subpopulation of CD105 1 cells was isolated by immunomagnetic separation from cells pre-adherent to the fl asks. From the initial sorting of six samples, the average percentage of CD105 1 cells was 63% in the case of hAMSC and 57% in case of hCMSC. After cultivation of CD105 1 cells, cell morphologies became more uniformly spindle-shaped and a confl uent cell layer with fi broblastoid morphology was obtained.

Figure 2. Multilineage differentiation in vitro of CD105 � hAMSC and hCMSC (A) hAMSC, adipogenic differentiation by Scharlach Sudan stain (200 � magnifi cation); (B) hAMSC, chondrogenic differentiation by Alcian blue stain (200 � magnifi cation); (C) hAMSC, osteogenic differentiation by von Kossa staining (100 � magnifi cation); (D) hCMSC, adipogenic differentiation by Scharlach Sudan stain (100 � magnifi cation); (E) hCMSC, chondrogenic differentiation by Alcian blue stain (200 � magnifi cation); (F) hCMSC, osteogenic differentiation by von Kossa staining (100 � magnifi cation).

Differentiation potential of hAMSC and hCMSC

After 3 weeks of culture in adipogenic induction medium, CD105 � hAMSC (Figure 2A) and hCMSC (Figure 2D) were able to undergo adipogenic differ-entiation, as demonstrated by the development of positive staining with Scharlach Sudan S. Our analysis revealed weak osteogenic differentiation of CD105 � hAMSC (Figure 2C) and hCMSC (Figure 2F), which was visualized by positive von Kossa staining. The

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

1052 D. Ba c enkov á et al.

Figure 3. Immunophenotypic CD105 � hAMSC. Immunopheno-typic characteristics were examined by fl ow cytometry: positive for CD105, CD44 and CD90, weakly positive for CD146 and HLA-ABC, and negative for CD34, CD45 and HLA-DR.

Figure 4. Immunophenotypic CD105 � hCMSC. Immunopheno-typic characteristics were examined by fl ow cytometry: positive for CD105, CD90, CD44 and HLA-ABC, weakly positive for CD146, and negative for CD34, CD45 and HLA-DR.

ability of CD105 � hAMSC (Figure 2B) and hCMSC (Figure 2E) to undergo chondrogenic differentiation was shown by Alcian blue staining.

Immunophenotyping of hAMSC and hCMSC

Immunophenotyping of CD105 � hAMSC and hCMSC cells revealed that they were positive for many

markers common to MSC. The cell- surface markers analyzed using FACS revealed that the hAMSC had a positive expression of CD105, CD90 and CD44, a weak positive expression of CD146, HLA-ABC, and negative expression of HLA-DR, CD34 and CD45 (Figure 3); hCMSC cells had a positive expression of CD105, CD90 , CD44 and HLA-ABC, a weak posi-tive expression of CD146, and negative expression of

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

Isolation and characterization of amnion and chorion MSC 1053

HLA-DR, CD34 and CD45 (Figure 4). Immunolog-ically, hAMSC were weakly positive for HLA-ABC, hCMSC were positive for HLA-ABC, while both hAMSC and hCMSC were negative for HLA-DR. Both cells were negative for the hematopoietic surface markers CD34 and CD45.

Figure 5. Immunohistophenotyping of hAMSC. (A) CD45 antibody staining of hAMSC with HRP-DAB, no signal detected; (B) negative control, no signal detected; (C) CD105 antibody staining of hAMSC with HRP-DAB, highly immunoreactive; (D) negative control, no signal detected; (E) Rex-1 antibody staining of hAMSC with HRP-DAB, very low immunoreaction; (F) negative control, no signal detected; (G) Oct-3/4 antibody staining of hAMSC with HRP-DAB, immunoreactive; (H) negative control, no signal detected. Scale bar: 50 μ m.

Immunocytochemical staining of hAMSC and hCMSC

Iimmunocytochemical analysis showed that both hAMSC (Figure 5C) and hCMSC (Figure 6C) were strongly positive for CD105 and the pluripotency marker Oct-3/4 (Figures 5G and 6G), very weakly

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

1054 D. Ba c enkov á et al.

Figure 6. Immunohistophenotyping of hCMSC. (A) CD45 antibody staining of hCMSC with HRP-DAB, no signal detected; (B) negative control, no signal detected; (C) CD105 antibody staining of hCMSC with HRP-DAB, highly immunoreactive; (D) negative control, no signal detected; (E) Rex-1 antibody staining of hCMSC with HRP-DAB, very low immunoreaction; (F) negative control, no signal detected; (G) OCT-¾ staining of hCMSCs with HRP-DAB, immunoreactive (H) negative control for OCT-¾, no signal detected. Scale bar: 50 μ m.

positive for Rex-1 (Figures 5E and 6E) and negative for hematopoietic marker CD45 (Figures 5A and 6A). The results are summarized in Table I.

Discussion

Cells of amniochorion membrane origin are receiving recently renewed interest in the fi elds of regenerative

medicine and cell transplantation. In this study we have demonstrated that hAMSC and hCMSC obtained from normal term placentas represent populations of cells with properties of MSC. MSC-like cells were iso-lated from human term placenta (chorion and amnion) using a routine cell-culture technique. Our results indi-cate that hAMSC and hCMSC can be expanded suc-cessfully in vitro . The initial cell culture consisted of

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

Isolation and characterization of amnion and chorion MSC 1055

specifi cally expressed in embryonic cells and germ cells (26). Rex-1 is a marker for a subpopulation of undif-ferentiated embryonic cells (27). Rex-1 is regulated by the pluripotency-regulating genes Sox-2, Oct-4 and Nanog. Sox is a transcription factor that is essential to maintain self-renewal of undifferentiated embryonic stem cells. Nanog binds Rex-1 via its C-terminal (28). Sox-2 and Oct-4 augment the up-regulation of Rex-1 by Nanog.

Using immunocytochemistry studies, we con-fi rmed the expression of the embryonic marker Oct-4 and low expression of Rex-1 by hAMSC and hCMSC cells. Cells obtained from normal term pregnan-cies express markers usually referring to stem cells. hAMSC and hCMSC are plastic adherent, fi bro-blastoid cells that are defi ned functionally by their capacity to undergo multilineage differentiation into connective tissue lineages. The presence of CD105 and numerous other MSC markers, including CD90, CD44, fi broblastoid morphology, plastic-adherence and mesodermal differentiation capabilities, sug-gests that these cells resemble MSC from the BM. The detection of Embryonic stem cell (ESC) surface markers on hAMSC and hCMSC suggests that these may be very primitive cells.

In summary, our results describe the successful isolation of MSC from the amnion and chorion of human term placenta. The morphologic and dif-ferentiation characteristics of isolated hAMSC and hCMSC indicate that they exhibit characteristics of mesodermal lineages. The current results add to the rapidly expanding fi eld of interest in the potential research and therapeutic application of fetal amnio-chorion membranes. These observations implicate the postpartum human placenta as an important and easily accessible and ethically acceptable source of multipotent stem cells for further experimental and clinical applications.

Acknowledgments

This investigation was supported by VEGA Grant No 1/4223/07.

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.

References

Mannello F. Multipotent mesenchymal stromal cell recruit-1. ment, migration, and differentiation: what have matrix metalloproteinases got to do with it? Stem Cells. 2006;24:1904 – 7. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, 2. Mosca JD, et al. Multilineage potential of adult human mes-enchymal stem cells. Science. 1999;284:143 – 7.

both fi broblastoids and a very small mixture of epi-theloid cell types from the amnion-derived cell popu-lation. There were also non-adherent cells present in both cell cultures, which were effi ciently eliminated by medium exchanges. The isolation procedure using dispase removes more epithelial cells, as demonstrated by the morphologic analysis using phase – contrast microscopy. The initial cell cultures consisted of both fi broblastoid and non-fi broblastoid cell types. After passaging and CD105 immunomagnetic isolation, only the fi broblastoid cell population remained. Cells obtained from amnions and chorions after cultivation were similar. hAMSC and hCMSC showed a homoge-neous population of spindle-shaped cells after the fi rst passage. After imunomagnetic selection, CD105 � cells of hAMSC and hCMSC formed a homogeneous layer of fi broblastoid cells and they were capable of multilin-eage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes.

Several different protocols have been described to isolate MSC from the placental membrane, including cultures of unfractionated placenta and of cells recov-ered after rinsing placental vessels, mechanical separa-tion and enzymatic digestion (7,9,21). The morphologic features of the isolated MSC of fetal origin were similar to those described for BM MSC, and included plas-tic adherence and fi broblast-like growth (11). It was concluded that hAMSC and hCMSC could not be cultured beyond fi ve passages. The proliferation rates and other stem cell-like properties of the human mes-enchymal stem cells (hMSC) gradually changed during expansion and, therefore, it is advisable to not expand them beyond four or fi ve passages (22). The expression profi le conformed to the criteria recently defi ned for multipotent MSC (23). These cells presented a mesen-chymal phenotype and were also positive for embryonic stem cell markers such as SSEA-4 and Oct-4, while they lacked endothelial and trophoblastic markers (12). Oct-4 is involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is frequently used as a marker for undifferentiated cells (24). Several studies suggest a role for Oct-4 in sustaining the self-renewal capacity of adult somatic stem cells from epithelium, BM and liver (25). Previously, several reports have demonstrated that human amniotic MSC express several stem cell markers, such as Oct-4, which is

Table I. Results of immunocytochemical staining of hAMSC and hCMSC.

Immunocytochemical staining of hAMSC and hCMSC CD45 CD105 Rex-1 Oct-3/4

hAMSC – � � � hCMSC – � � �

– , negative expression; � , positive expression; � , low expression.

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.

1056 D. Ba c enkov á et al.

with the ability to differentiate into endothelial cells in vitro. BMC Dev Biol. 2007;7:11. Wolbank S, Peterbauer A, Fahrner M, Hennerbichler S, 16. van Griensven M, Stadler G, et al. Dose-dependent immu-nomodulatory effect of human stem cells from amniotic mem-brane: a comparison with human mesenchymal stem cells from adipose tissue. Tissue Eng. 2007;13:1173 – 83. Kubo M, Sonoda Y, Muramatsu R, Usui M. Immunogenicity 17. of human amniotic membrane in experimental xenotrans-plantation. Invest Ophthalmol Vis Sci. 2001;42:1539 – 46. Barry FP, Murphy JM, English K, Mahon, BP. Immunogenic-18. ity of adult mesenchymal stem cells: lessons from the fetal allograft. Stem Cells Dev. 2005;14:252 – 65. Trelford JD, Trelford-Sauder M. The amnion in surgery, past 19. and present. Am J Obstet Gynecol. 1979;134:833 – 45. Solomon A, Espana EM, Tseng SC. Amniotic membrane 20. transplantation for reconstruction of the conjunctival fornices. Ophthalmology. 2003;110:93 – 100. Li H, Niederkorn JY, Neelam S, Mayhew E, Word AR, 21. McCulley JP, Alizadeh H. Immunosuppressive factors secreted by human amniotic epithelial cells. Invest Ophthalmol Vis Sci. 2005;46:900 – 7. Prockop DJ, Phinney DG, Bunnell BA. Methods in molecular 22. biology. Mesen Stem Cells Meth Proto. 2008;449:109 – 11. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, 23. Marini FC, Krause DS, et al. Minimal criteria for defi ning multipotent mesenchymal stromal cells. Cytotherapy. 2006;8:315 – 17. Niwa H, Miyazaki J, Smith AG. Quantitative expression of 24. Oct-3/4 defi nes differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000;24:372 – 6. Tai MH, Chang CC, Kiupel M, Webster JD, Olson LK, 25. Trosko JE. Oct4 expression in adult human stem cells: evi-dence in support of the stem cell theory of carcinogenesis. Carcinogenesis. 2005;26:495 – 502. Toda A, Okabe M, Yoshida T, Nikaido T. The potential of 26. amniotic membrane/amnion-derived cells for regeneration of various tissues. J Pharmacol Sci. 2007;105:215 – 28. Henderson JK, Draper JS, Baillie, HS, Fishel S, Thomson 27. JA, Moore H, Andrews PW. Preimplantation human embryos and embryonic stem cells show comparable expres-sion of stage-specifi c embryonic antigens. Stem Cells. 2002;20:329 – 37. Shi W, Wang H, Pan G, Gang Y, Guo Y, Pei D. Regulation of 28. the pluripotency marker Rex-1 by Nanog and Sox2. J Biol Chem. 2006;281:23319 – 25.

Sze SK, de Kleijn DP, Lai RC, Tan EKY, Zhao H, Yeo KS, 3. et al. Elucidating the secretion proteome of human ESC-derived mesenchymal stem cells. Mol Cell Proteom. 2007;6:1680 – 9. D ’ Ippolito G, Howard GA, Roos BA, Schiller PC. Sustained 4. stromal stem cell self-renewal and osteoblastic differentiation during aging. Rejuvenation Res. 2006;9:10 – 19. Miao Z, Jin J, Chen L, Zhu J, Huang W, Zhao J, et al. Isolation 5. of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells. Cell Biol Int. 2006;30:681 – 7. Yen BJ, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, et al. 6. Isolation of multipotent cells from human term placenta. Stem Cells. 2005;23:3 – 9. Zhang Y, Li C, Jiang X, Zhang S, Wu Y, Liu B, et al. Human 7. placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34 � cells. Exp Hematol. 2004;32:657 – 64. Wulf GG, Viereck V, Hemmerlein B, Haase D, Vehmeyer K, 8. Pukrop T, et al. Mesengenic progenitor cells derived from human placenta. Tissue Eng. 2004;10:1136 – 47. Fukuchi Y, Nakajima H, Sugiyama D, Hirose I, Kitamura T, 9. Tsuji K. Human placenta-derived cells have mesenchymal stem/progenitor cell potential. Stem Cells . 2004; 22:649 – 58. Bailo M, Soncini M, Vertua E, Signoroni PB, Sanzone S, 10. Lombardi G, et al. Engraftment potential of human amnion and chorion cells derived from term placenta. Transplanta-tion. 2004;78:1439 – 48. Soncini M, Vertua E, Gibelli L, Zorzi F, Denegri M, Albertini 11. A, et al. Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med. 2007;1:296 – 305. Parolini O, Soncini M. Human placenta: a source of pro-12. genitor/stem cells? J Reprod Med. 2006;3:117 – 26. Benirschke K, Kaufmann P. Pathology of Human Placenta. 13. New York: Springer-Verlag; 2005. Parolini O, Alviano F, Bagnara GP, Bilic G, B ü hring HJ, 14. Evangelista M, et al. Isolation and characterization of cells from human term placenta: outcome of the fi rst international workshop on placenta derived stem cells. Stem Cells. 2008;26:300 – 11. Alviano F, Fossati V, Marchionni C, Arpinati M, Bonsi L, 15. Franchina M, et al. Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells

Cyt

othe

rapy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

193.

87.7

5.25

0 on

11/

11/1

1Fo

r pe

rson

al u

se o

nly.