Cell and Tissue Banking

, Volume 17, Issue 3, pp 517–529 | Cite as

Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing

  • E. Katsiani
  • A. Garas
  • C. Skentou
  • A. Tsezou
  • C. I. Messini
  • K. Dafopoulos
  • A. Daponte
  • I. E. Messinis
Original Paper


Mesenchymal stem cells (MSCs) can be obtained from a variety of human tissues. MSCs derived from placental chorionic villi of the first trimester are likely to resemble, biologically, embryonic stem cells (ESC), due to the earlier development stage of placenta. In the present study long-term cultures of MSC-like cells were assessed in order to evaluate MSCs multipotent characteristics and molecular features during the period of culture. CV-cells obtained from 10 samples of chorionic villus displayed typical fibroblastoid morphology, undergone 20 passages during a period of 120 days, maintaining a stable karyotype throughout long term expansion. The cells were positive, for CD90, CD73, CD105, CD29, CD44, HLA ABC antigens and negative for CD14, CD34, AC133, and HLA DR antigens as resulted from the flow cytometry analysis. CV-cells were differentiated in adipocytes, osteoblasts, chondrocytes and neuronal cells under specific culture conditions. The expression of the ESC-gene markers POU5F1 (Oct-4) and NANOG was observed at earliest stages (4–12 passages) and not at the late stages (14–20 passages) by RT-PCR analysis. ZFP42 and SOX2 expression were not detected. Moreover, CV-cells were found to express GATA4 but not NES (Nestin). Chorionic villi-derived cells possess multipotent properties, display high proliferation rate and self-renew capacity, share common surface antigens with adult MSCs and express certain embryonics stem cells gene markers. These characteristics highlight chorionic villi as an attractive source of MSCs for the needs of regenerative medicine.


Chorionic villi mesenchymal like cells (CV-MSCs) Adult mesenchymal stem cells (MSCs) Embryonic stem cells (ESCs) Long-term culturing Multipotency Pluripotency genes 


Authors’ contribution

E. Katsiani contribution to the paper was the conception, the design and the first drafting of the article. A. Garas and C. Skentou contribution was the acquisition of chorionic villi samples and participation in the analysis of data. A. Tsezou and K. Dafopoulos participated in the analysis and interpretation of data. CI Messini contributed to the drafting, A. Daponte contributed to the shortening of the revised manuscript and I.E. Messinis contributions were at the conception of study and the final approval.

Compliance with ethical standards

Conflict of interest

There are no conflicts of interests to be declared.


  1. Abdulrazzak H, Moschidou D, Jones G, Guillot PV (2010) Biological characteristics of stem cells from foetal, cord blood and extraembryonic tissues. J R Soc Interface 7:689–706. doi: 10.1098/rsif.2010.0347 CrossRefGoogle Scholar
  2. Aluigi M, Foglii M, Curti A, Isidori A, Grupioni E, Chiodoni C, Colombo MP, Versura P, Grigioni AD, Ferri E et al (2006) Nucleofaction is an efficient non-viral transfection technique for bone marrow-derived mesenchymal stem cells. Stem Cells 24(2):454–461. doi: 10.1634/stemcells.2005-0198 PubMedCrossRefGoogle Scholar
  3. Alviano F, Fossati V, Marchionni C, Arpinati M, Bonsi L, Franchina M, Lanzoni G, Cantoni S, Cavallini C, Bianchi F et al (2007) Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro. BMC Dev Biol 21:7–11. doi: 10.1186/1471-213X-7-11 Google Scholar
  4. Arakawa R, Aoki R, Arakawa M, Saito K (2012) Human first-trimester chorionic villi have a myogenic potential. Cell Tissue Res 348(1):189–197. doi: 10.1007/s00441-012-1340-9 PubMedPubMedCentralCrossRefGoogle Scholar
  5. Avanzini MA, Bernardo ME, Cometa AM, Perotti C, Zaffaroni N, Novara F, Visai L, Moretta A, Del Fante C, Villa R, Ball LM (2009) Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood-and bone marrow-derived progenitors. Haematologica 94(12):1649–1660PubMedPubMedCentralCrossRefGoogle Scholar
  6. Bailo M, Soncini M, Vertua E, Signoroni PB, Sanzone S, Lombardi G, Arienti D, Calamani F, Zatti D, Paul P et al (2004) Engraftment potential of human amnion and chorion cells derived from term placenta. Transplantation 78(10):1439–1448. doi: 10.1097/01.TP.0000144606.84234.49 PubMedCrossRefGoogle Scholar
  7. Barlow S, Brooke G, Chatterjee K, Price G, Pelekanos R, Rossetti T, Doody M, Venter D, Pain S, Gilshenan K et al (2008) Comparison of human placenta- and bone marrow-derived multipotent mesenchymal stem cells. Stem Cells Dev 17(6):1095–1107. doi: 10.1089/scd.2007.0154 PubMedCrossRefGoogle Scholar
  8. Bhandari DR, Seo KW, Roh KH, Jung JW, Kang SK, Kang KS (2010) REX-1 expression and p38 MAPK activation status can determine proliferation/differentiation fates in human mesenchymal stem cells. PLoS ONE 5(5):e10493. doi: 10.1371/journal.pone.0010493 PubMedPubMedCentralCrossRefGoogle Scholar
  9. Blum B, Benvenisty N (2009) The tumorigenicity of diploid and aneuploid human pluripotent stem cells. Cell Cycle 8(23):3822–3830. doi: 10.4161/cc.8.23.10067 PubMedCrossRefGoogle Scholar
  10. Boiani M, Schöler HR (2005) Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 6(11):872–884. doi: 10.1038/nrm1744 PubMedCrossRefGoogle Scholar
  11. Bossolasco P, Montemurro T, Cova L, Zangrossi S, Calzarossa C, Buiatiotis S, Soligo D, Bosari S, Silani V, Deliliers GL et al (2006) Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential. Cell Res 16(4):329–336. doi: 10.1038/sj.cr.7310043 PubMedCrossRefGoogle Scholar
  12. Campbell PA, Perez-Iratxeta C, Andrade-Navarro MA, Rudnicki MA (2007) Oct4 targets regulatory nodes to modulate stem cell function. PLoS ONE 2(6):e553. doi: 10.1371/journal.pone.0000553 PubMedPubMedCentralCrossRefGoogle Scholar
  13. Can A, Karahuseyinoglu S (2007) Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells 25(11):2886–2895. doi: 10.1634/stemcells.2007-0417 PubMedCrossRefGoogle Scholar
  14. Castrechini NM, Murthi P, Gude NM, Erwich JJ, Gronthos S, Zannettino A, Brennecke SP, Kalionis B (2010) Mesenchymal stem cells in human placental chorionic villi reside in a vascular Niche. Placenta 31(3):203–212. doi: 10.1016/j.placenta.2009.12.006 PubMedCrossRefGoogle Scholar
  15. Chien CC, Yen BL, Lee FK, Lai TH, Chen YC, Chan SH, Huang HI (2006) In vitro differentiation of human placenta-derived multipotent cells into hepatocyte-like cells. Stem Cells 24(7):1759–1768. doi: 10.1634/stemcells.2005-0521 PubMedCrossRefGoogle Scholar
  16. Colter DC, Sekiya I, Prockop DJ (2001) Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA 98(14):7841–7845. doi: 10.1073/pnas.141221698 PubMedPubMedCentralCrossRefGoogle Scholar
  17. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ et al (2007) Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 25(1):100–106. doi: 10.1038/nbt1274 PubMedCrossRefGoogle Scholar
  18. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317. doi: 10.1080/14653240600855905 PubMedCrossRefGoogle Scholar
  19. Episkopou V (2005) SOX2 functions in adult neural stem cells. Trends Neurosci 28(5):219–221. doi: 10.1016/j.tins.2005.03.003 PubMedCrossRefGoogle Scholar
  20. Fong CY, Richards M, Manasi N, Biswas A, Bongso A (2007) Comparative growth behaviour and characterization of stem cells from human Wharton’s jelly. Reprod Biomed Online 15(6):708–718. doi: 10.1016/S1472-6483(10)60539-1 PubMedCrossRefGoogle Scholar
  21. Fukuchi Y, Nakajima H, Sugiyama D, Hirose I, Kitamura T, Tsuji K (2004) Human placenta-derived cells have mesenchymal stem/progenitor cell potential. Stem Cells 22(5):649–658. doi: 10.1634/stemcells.22-5-649 PubMedCrossRefGoogle Scholar
  22. Gonzalez R, Maki CB, Pacchiarotti J, Csontos S, Pham JK, Slepko N, Patel A, Silva F (2007) Pluripotent marker expression and differentiation of human second trimester mesenchymal stem cells. Biochem Biophys Res Commun 362(2):491–497. doi: 10.1016/j.bbrc.2007.08.033 PubMedCrossRefGoogle Scholar
  23. Götherström C, West A, Liden J, Uzunel M, Lahesmaa R, Le Blanc K (2005) Difference in gene expression between human fetal liver and adult bone marrow mesenchymal stem cells. Haematologica 90(8):1017–1026Google Scholar
  24. Greco SJ, Liu K, Rameshwar P (2007) Functional similarities among genes regulated by OCT4 in human mesenchymal and embryonic stem cells. Stem Cells 25(12):3143–3154. doi: 10.1634/stemcells.2007-0351 PubMedCrossRefGoogle Scholar
  25. Guillot PV, O’Donoghue K, Kurata H, Fisk NM (2006) Fetal stem cells: betwixt and between. Semin Reprod Med 24(5):340–347. doi: 10.1055/s-2006-952149 PubMedCrossRefGoogle Scholar
  26. Guillot PV, Cui W, Fisk NM, Polak DJ (2007a) Stem cell differentiation and expansion for clinical applications of tissue engineering. J Cell Mol Med 11(5):935–944. doi: 10.1111/j.1582-4934.2007.00106.x PubMedPubMedCentralCrossRefGoogle Scholar
  27. Guillot PV, Gotherstrom C, Chan J, Kurata H, Fisk NM (2007b) Human first-trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC. Stem Cells 25(3):646–654. doi: 10.1634/stemcells.2006-0208 PubMedCrossRefGoogle Scholar
  28. Guo B, Rooney P, Slevin M, Li C, Parameshwar S, Liu D, Kumar P, Bernabeu C, Kumar S (2004) Overexpression of CD105 in rat myoblasts: role of CD105 in cell attachment, spreading and survival. Int J Oncol 25(2):285–291. doi: 10.3892/ijo.25.2.285 PubMedGoogle Scholar
  29. Horwitz EM (2003) Stem cell plasticity: the growing potential of cellular therapy. Arch Med Res 34(6):600–606. doi: 10.1016/j.arcmed.2003.09.006 PubMedCrossRefGoogle Scholar
  30. Hua J, Yu H, Dong W, Yang C, Gao Z, Lei A, Sun Y, Pan S, Wu Y, Dou Z (2009) Characterization of mesenchymal stem cells (MSCs) from human fetal lung: potential differentiation of germ cells. Tissue Cell 41:448–455. doi: 10.1016/j.tice.2009.05.004 PubMedCrossRefGoogle Scholar
  31. Hung CN, Mar K, Chang HC, Chiang YL, Hu HY, Lai CC, Chu RM, Ma CM (2011) A comparison between adipose tissue and dental pulp as sources of MSCs for tooth regeneration. Biomaterials 32:6995–7005. doi: 10.1016/j.biomaterials.2011.05.086 PubMedCrossRefGoogle Scholar
  32. Hwang HS, Cho NH, Maeng YS, Kang MH, Park YW, Kim YH (2007) Differential expression of nestin in normal and pre-eclamptic human placentas. Acta Obstet Gynecol Scand 86(8):909–914. doi: 10.1080/00016340701417018 PubMedCrossRefGoogle Scholar
  33. Igura K, Zhang X, Takahashi K, Mitsuru A, Yamaguchi S, Takashi TA (2004) Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta. Cytotherapy 6(6):543–553. doi: 10.1080/14653240410005366 PubMedCrossRefGoogle Scholar
  34. Krause DS (2002) BM-derived stem cells for the treatment of non hematopoietic diseases. Cytotherapy 4(6):503–506. doi: 10.1080/146532402761624629 PubMedCrossRefGoogle Scholar
  35. Lankford L, Selby T, Becker J, Ryzhuk V, Long C, Farmer D, Wang A (2015) Early gestation chorionic villi-derived stromal cells for fetal tissue engineering. World J Stem Cells 20157(1):195–207. doi: 10.4252/wjsc.v7.i1.195 CrossRefGoogle Scholar
  36. Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60(4):585–595. doi: 10.1016/0092-8674(90)90662-X CrossRefGoogle Scholar
  37. Lo B, Parham L (2009) Ethical issues in stem cell research. Endocr Rev 30(3):204–213. doi: 10.1210/er.2008-0031 PubMedPubMedCentralCrossRefGoogle Scholar
  38. Marcus AJ, Woodbury D (2008) Fetal stem cells from extra-embryonic tissues: do not discard. J Cell Mol Med 12(3):730–742. doi: 10.1111/j.1582-4934.2008.00221.x PubMedCentralCrossRefGoogle Scholar
  39. Meraviglia V, Vecellio M, Grasselli A, Baccarin M, Farsetti A, Capogrossi MC, Pompilio G, Coviello DA, Gaetano C, Di Segni M, Rossini A (2012) Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties. Differentiation 83(5):260–270PubMedCrossRefGoogle Scholar
  40. Montzka K, Lassonczyk N, Tschöke B, Neuss S, Führmann T, Franzen R, Smeets R, Brook GA, Wöltje M (2009) Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression. BMC Neurosci 10:16. doi: 10.1186/1471-2202-10-16 PubMedPubMedCentralCrossRefGoogle Scholar
  41. Moon JH, Lee JR, Jee BC, Suh CS, Kim SH, Lim HJ, Kim HK (2008) Successful vitrification of human amnion-derived mesenchymal stem cells. Hum Reprod 23(8):1760–1770. doi: 10.1093/humrep/den202 PubMedCrossRefGoogle Scholar
  42. Mousiolis AV, Kollia P, Skentou C, Messinis IE (2012) Effects of leptin on the expression of fatty acid-binding proteins in human placental cell cultures. Mol Med Report 5(2):497–502. doi: 10.3892/mmr.2011.686 Google Scholar
  43. Papa K, Anagnou N (2009) Novel sources of fetal stem cells: where do they fit on the developmental continuum? Regen Med 4(3):423–433. doi: 10.2217/rme.09.12 CrossRefGoogle Scholar
  44. Parolini O, Soncini M, Evangelista M, Schmidt D (2009) Amniotic membrane and amniotic fluid-derived cells: potential tools for regenerative medicine? Regen Med 4(2):275–291. doi: 10.2217/17460751.4.2.275 PubMedCrossRefGoogle Scholar
  45. Paul G, Özen I, Christophersen NS, Reinbothe T, Bengzon J, Visse E, Jansson K, Dannaeus K, Henriques-Oliveira C, Roybon L et al (2012) The adult human brain harbors multipotent perivascular mesenchymal stem cells. PLoS ONE 7(4):e35577. doi: 10.1371/journal.pone.0035577 PubMedPubMedCentralCrossRefGoogle Scholar
  46. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147. doi: 10.1126/science.284.5411.143 PubMedCrossRefGoogle Scholar
  47. Poloni A, Rosini V, Mondini E, Maurizi G, Mancini S, Discepoli G, Biasio S, Battaglini G, Berardinelli E, Serrani F, Leoni P (2008) Characterization and expansion of mesenchymal progenitor cells from first-trimester chorionic villi of human placenta. Cytotherapy 10(7):690–697. doi: 10.1080/14653240802419310 PubMedCrossRefGoogle Scholar
  48. Poloni A, Maurizi G, Babini L, Serrani F, Berardinelli E, Mancini S, Costantini B, Discepoli G, Leoni P (2011) Human mesenchymal stem cells from chorionic villi and amniotic fluid are not susceptible to transformation after extensive in vitro expansion. Cell Transplant 20(5):643–654. doi: 10.3727/096368910X536518 PubMedCrossRefGoogle Scholar
  49. Poloni A, Maurizi G, Serrani F, Mancini S, Discepoli G, Tranquilli AL, Bencivenga R, Leoni P (2012) Human AB serum for generation of mesenchymal stem cells from human chorionic villi: comparison with other source and other media including platelet lysate. Cell Prolif 45(1):66–75. doi: 10.1111/j.1365-2184.2011.00799.x PubMedCrossRefGoogle Scholar
  50. Porada CD, Zanjani ED, Almeida-Porad G (2006) Adult mesenchymal stem cells: a pluripotent population with multiple applications. Curr Stem Cell Res Ther 1(3):365–369. doi: 10.2174/157488806778226821 PubMedCrossRefGoogle Scholar
  51. Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P (2005) Transcriptional regulation of Nanog, by OCT4 and SOX2. J Biol Chem 280(26):24731–24737. doi: 10.1074/jbc.M502573200 PubMedCrossRefGoogle Scholar
  52. Roselli EA, Lazzati S, Iseppon F, Manganini M, Marcato L, Gariboldi MB, Maggi F, Grati FR, Simoni G (2013) Fetal mesenchymal stromal cells from cryopreserved human chorionic villi: cytogenetic and molecular analysis of genome stability in long-term cultures. Cytotherapy 15(11):1340–1351. doi: 10.1016/j.jcyt.2013.06.019 PubMedCrossRefGoogle Scholar
  53. Sethe S, Scutt A, Stolzing A (2006) Aging of mesenchymal stem cells. Ageing Res Rev 5:91–116. doi: 10.1016/j.arr.2005.10.001 PubMedCrossRefGoogle Scholar
  54. Spitalieri P, Cortese G, Pietropolli A, Filareto A, Dolci S, Klinger FG, Giardina E, Di Cesare S, Bernardini L, Lauro D et al (2009) Identification of multipotent cytotrophoblast cells from human first trimester chorionic villi. Cloning Stem Cells 11(4):535–556. doi: 10.1089/clo.2009.0046 PubMedCrossRefGoogle Scholar
  55. Stolzing A, Jones E, McGonagle D, Scutt A (2008) Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 129:163–173. doi: 10.1016/j.mad.2007.12.002 PubMedCrossRefGoogle Scholar
  56. Theise ND, Krause DS (2002) Bone marrow to liver: the blood of Prometheus. Semin Cell Dev Biol 13(6):411–417. doi: 10.1016/S1084952102001283 PubMedCrossRefGoogle Scholar
  57. Tondreau T, Meuleman N, Delforge A, Dejeneffe M, Leroy R, Massy M, Mortier C, Bron D, Lagneaux L (2005) Mesenchymal stem cells derived from CD133-positive cells in mobilized peripheral blood and cord blood: proliferation, Oct4 expression, and plasticity. Stem Cells 23(8):1105–1112. doi: 10.1634/stemcells.2004-0330 PubMedCrossRefGoogle Scholar
  58. Tsagias N, Koliakos I, Karagiannis V, Eleftheriadou M, Koliakos GG (2011) Isolation of mesenchymal stem cells using the total length of umbilical cord for transplantation purposes. Transfus Med 21:253–261. doi: 10.1111/j.1365-3148.2011.01076.x PubMedCrossRefGoogle Scholar
  59. Tsai MS, Lee JL, Chang YJ, Hwang SM (2004) Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod 19(6):1450–1456. doi: 10.1093/humrep/deh279 PubMedCrossRefGoogle Scholar
  60. Wulf GG, Jackson KA, Goodell MA (2001) Somatic stem cell plasticity: current evidence and emerging concepts. Exp Hematol 29(12):1361–1370. doi: 10.1016/S0301-472X(01)00752-4 PubMedCrossRefGoogle Scholar
  61. Zaim M, Karaman S, Cetin G, Isik S (2012) Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells. Ann Hematol 91(8):1175–1186. doi: 10.1007/s00277-012-1438-x PubMedCrossRefGoogle Scholar
  62. Zheng C, Yang S, Guo Z, Liao W, Zhang L, Yang R, Han ZC (2009) Human multipotent mesenchymal stromal cells from fetal lung expressing pluripotent markers and differentiating into cell types of three germ layers. Cell Transplant 18(10):1093–1109. doi: 10.3727/096368909X12483162197042 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • E. Katsiani
    • 1
  • A. Garas
    • 1
  • C. Skentou
    • 1
  • A. Tsezou
    • 2
  • C. I. Messini
    • 1
  • K. Dafopoulos
    • 1
  • A. Daponte
    • 1
  • I. E. Messinis
    • 1
    • 3
  1. 1.Department of Obstetrics and Gynaecology, Medical School, University HospitalUniversity of ThessalyLarissaGreece
  2. 2.Department of Biology and Laboratory of Cytogenetics and Molecular Genetics, Medical School, University HospitalUniversity of ThessalyLarissaGreece
  3. 3.Department of Obstetrics and Gynaecology, School of Health Sciences, Faculty of MedicineUniversity of ThessalyViopolis, LarissaGreece

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