; EVALUATION OF MULTIPOTENT MESENCHYMAL STEM CELLS FROM HUMAN PLACENTA
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EVALUATION OF MULTIPOTENT MESENCHYMAL STEM CELLS FROM HUMAN PLACENTA

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EVALUATION OF MULTIPOTENT MESENCHYMAL STEM CELLS FROM HUMAN PLACENTA

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									      LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (2), 2010 TIMIŞOARA

EVALUATION OF MULTIPOTENT MESENCHYMAL STEM CELLS
              FROM HUMAN PLACENTA
                     1                  1                   2                     3
         I. GROZA , EMİKE PALL , DARIA GROZA , OLGA SORIłĂU ,
                  3           1                    1              4
    C. TOMULEASA , M. CENARIU , IOANA CRISTINA ILEA , EMILIA GROZA
1
    University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary
     Medicine, Department of Veterinary Reproduction, Obstetrics and Gynecology,
                 400372, Manastur Street No. 3-5, Cluj-Napoca, Romania
         2
          “Iuliu HaŃieganu“ University of Medicine and Pharmacy, Cluj-Napoca
                3
                  ”Prof.dr.Ioan ChiricuŃă“ Oncological Institute, Cluj-Napoca
       4
        University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca
                                 E-mail: isgroza@yahoo.com

                                            Summary

          Several types of progenitor cells can be isolated from various human adult tissues
such as bone marrow, adipose tissues, placenta and umbilical cord. Placental tissue
collected after labour and delivery can provide a valuable source for adult stem cells. These
progenitor cells, termed placenta-derived multipotent cells (PDMCs), are fibroblast-like cells
which can attach on the culture vessels. PDMCs are capable of differentiating into various
cells lines. The aim of this study was to isolate mesenchymal stem cells from human
placentas. Our data demonstrate that we successfully isolated, culture-expanded CD 44
positive cells from human placentas.
          Key words: mesenchymal stem cells, culture expansion, CD 44 positive cells

         Human mesenchymal stem cells (hMSCs) from various sources are able to
differentiate into different cell lineages under specific culture conditions (1,5) and
have generated a great deal of interest because of their potential use in
regenerative medicine. Recently the human placenta, umbilical cord, and amnion
have received attention as possible sources of hMSCs because of their easy
acquisition with few ethical problems compared to other types of cells (3,6,7). 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 wellbeing of the fetus.
This remarkable organ is discarded postpartum, after having performed its
necessary function of supporting the embryo and fetus (5,9). 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 foetal origin and may be superior to ASCs in many
aspects (8). 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

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   LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (2), 2010, TIMIŞOARA

population of multipotent cells from the human postpartum term placenta, which we
have named placenta derived multipotent cells (PDMCs) (2,4).
         The aim of this study was to harvest and identify the mesenchymal stem
cells from human term placental tissue.

                              Materials and methods

          Biological material, placenta was obtained at the Department of Obstetrics
and Gynecology II "Dominic Stanca" from women undergoing elective caesarean
section under the approval of the ethics committee of the University of Medicine
and Pharmacy Iuliu Hatieganu Cluj –Napoca.
          Term placentas (38-40 weeks gestation, n = 5) from healthy donor mothers
were obtained with informed consent approved according to the procedures of the
institutional review board. 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) for 30 min at 37° C. After
centrifugation the cell suspension was filtered to eliminate undigested fragments.
For lysis the erythrocytes, cells suspensions were treated with FACS Lysing
Solution 10x (BD Biosciences) for 15 min. The suspension pelleted by
centrifugation (1500 rpm/7 min) and suspended in propagation medium, which
consist of Dulbecco’s modified Eagle’s medium (Gibco) supplemented by 10 %
fetal calf serum (FCS), 100 U/ml penicillin-streptomycin (Gibco). Cell cultures were
maintained at 37° C with a water-saturated atmosphere and 5% CO2. Medium was
replaced two times every week. Three to 5 days after initiating incubation, the small
digested residues were removed and the culture was continued. Approximately 3 -
4 weeks later, there were some colonies that contained 20-50 fibroblast like cells
that were more than 80% confluent; then they were recovered with 0.25% trypsin-
EDTA and replanted at a dilution of 1:4. Identification of cell phenotypic markers by
FACS (Fluorescence-Activated Cell Sorter) passage 5. After the second passage,
the cells were trypsinised (0.25% trypsine EDTA), washed twice with PBS and
stained according to the recommendation of the manufacturer with the monoclonal
antibodies, FITC-CD44, examined with a FACS CantoII apparatus (Becton–
Dickinson).

                             Results and discussions

         We successfully isolated, cultured and expanded placenta-derived
mesenchymal stem cells using routine methods. After the initial 3 days of primary
culture, human placental mesenchymal stem cells adhered to a plastic surface and
presented a small population of single cells with spindle shape.




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   LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (2), 2010 TIMIŞOARA




 Fig. 1. Multipotent cells from the human term placenta A-D, D – confluent culture,
                                       PC 20x

       After several days of culture morphology of these cells is usually
heterogeneous.




Fig. 2. Placental mesenchymal stem cells - fibroblastoid and epitheloid morphology


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   LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (2), 2010, TIMIŞOARA

         Initially in primary culture most cells have two different types of features:
one population with a fibroblastoid, spindle-shaped morphology (Fig.2A) and
another with an epitheliod, polygonal morphology (Fig.2B).
         The time for 1st passage was about 8 days. After the cells were continually
passaged for more than 3 months (about 20 passages), they continued to retain
their characteristics.
         Immunophenotyping of cells suspensions (Fig. 3) revealed these cells were
positive for marker CD44 (98,2%) which shows that these cells are mesenchymal
stem cells, and identification of the multilinear ability of this cells will be the main
purpose of future research.




      Fig. 3. Immunophenotyping results of placenta-derived multipotent cells

         In addition to haematopoietic stem cells, placenta has been reported to
contain a population of multipotent stem cells demonstrating some of the
characteristics of pluripotent stem cells including expression of stem cell markers c-
kit, Thy-1, OCT-4, SOX2, hTERT, SSEA1, SSEA3, SSEA4, TRA1-60 and TRA1-
81. These cells resemble mesenchymal stem cells and can be induced to
differentiate into hepatocyte-, vascular endothelial-, pancreatic- and neurallike cells
(6). Should this finding be confirmed, these placental derived stem cells offer a
promising source of stem cells for both therapeutic and toxicological applications.




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    LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (2), 2010 TIMIŞOARA

                                     Conclusions

          Originally, MSCs were derived from plastic-adherent bone marrow (BM)
cells (1, 2), but more recent studies have identified a number of other sources that
contain MSC at varying frequencies and varying differentiation capacities.
          Mesenchymal stem cells (MSCs) are widely distributed in a variety of
tissues in the adult human body (e.g., bone marrow, kidney, lung, and liver). These
cells are also present in the fetal environment (e.g., blood, liver, bone marrow, and
kidney). However, MSCs are a rare population in these tissues. The most well
studied and accessible source of MSCs is bone marrow, although even in this
tissue the cells are present in a low frequency.
          The human placenta is an attractive new source of mesenchymal stem
cells (MSCs), but the biological characteristics of placenta-derived MSCs have not
yet been characterized (3). Our results show that mesenchymal stem cells are
present in the human term placenta. Using routine cell culture techniques, placental
derived mesenchymal stem cells can be successfully isolated and expanded in
vitro. Initial cell culture consisted of two different cells morphology: fibroblastoid and
non-fibroblastoid cell types but after enzymatic digestion and passaging only the
fibroblastoid population remained. In their undifferentiated state MSCs are spindle-
shaped and resemble fibroblasts. There are no markers which specifically and
uniquely identify MSCs and therefore they are defined by their immunophenotypic
profile, by their characteristic morphology; and by their extensive capacity for self-
renewal.
          This study showed that the placenta-derived MSCs cells could be easily
isolated and expanded without morphological and characteristic changes in
medium supplemented only with FBS. Therefore, the placenta may prove to be an
attractive and rich source of MSCs and further studies are required to better
understand the precise nature of placenta-derived cells and to explore their
potential clinical applications.

                                      References

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       normal and irradiated mouse hematopoietic organs. Exp. Hematol, 1976, 4,
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       Tobias, P., Bertram, G., Günter, E., Lorenz, T., Mesengenic Progenitor
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    3. Mark, F.P., Alastair, M.M., Stephen, C.B., Jaiswal, Rama K., Robin, D.,
       Joseph, D.M., Mark, A.M., Donald, W.S., Stewart, C., Daniel, R.M.,
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LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XLIII (2), 2010, TIMIŞOARA

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