Bizzoco, D.

Cignini, P.

Giorlandino, C.

Grossi, S.

Mesoraca, A.

Mobili, L.

Wouters, G.

PubMed

Mesoraca, Alvaro

Bizzocco, Domenico

Mobili, Luisa

Cignini, Pietro

Giorlandino, Claudio

Scientific Open-access Literature Archive and Repository

Journal of Prenatal Medicine 2007; 1 (3): 39-40 39G. Wouters1S. Grossi2A. Mesoraca3D. Bizzoco3L. Mobili4P. Cignini4C. Giorlandino41 Cryo-Save Labs, Generaal de Wittelaan19 C-B13. B-2800 Mechelen - Belgium2 Cryo-Save Italia - Roma3“ARTEMISIA” Fetal-Maternal Medical Centre, Department of Genetics and Molecular Biology, Rome, Italy4“ARTEMISIA” Fetal-Maternal Medical Centre, Department Prenatal Diagnosis, Rome, ItalyReprint requests to: Alvaro Mesoraca“ARTEMISIA” Fetal-Maternal Medical CentreDepartment of Genetics and Molecular Biology, Viale Liegi, 45 - 00198 Rome - ItalyTel.: +39068505805E-mail: alvaro.mesoraca@artemisia.itIntroductionMesenchymal stem cells (MSC) are capable of differen-tiating into different mesenchymal lineages, includingadipose and connective tissue, bone and cartilage. Themost common source of MSCs for clinical use is humanadult bone marrow. Because the frequency and differen-tiating capacity of MSCs are decreasing with age, differ-ent fetal tissues have been studied as an alternativesource for MSCs. Recently, in ‘t Anker et al. (2003) andTsai et al. (2004) reported that second-trimester amniot-ic fluid (AF) is a novel and rich source of fetal MSCs use-ful for clinical applications. AF contains a heterogeneouspopulation of cells from fetal origin. The fetal MSCs inAF are probably deriving from the amnion membrane orother embryonic and extra-embryonic tissues during theprocess of fetal development and growth (in ‘t Anker etal., 2004). Aim of the studyThe purpose of this study was to investigate whether wewere able to isolate and cultivate MSC from fresh sec-ond-trimester amniotic fluid samples. Materials and methodsAmniotic fluid was collected from 8 second-trimesterpregnancies (mean gestational age, 16 weeks [range16-18 weeks]). Four mL of the AF samples were cen-trifugated for 10 minutes at 300g. Pellets were resus-pended and cultured in DMEM with low glucose supple-mented with 15% Fetal bovine serum and antibiotics.Culture flasks were incubated at 37°C, 5% CO2 and100% humidity. Medium was changed every 3-4 daysuntil day 14. At that moment, treatment with trypsin/ED-TA was applied to detach the cells from the bottom of theflasks (Passage 0). Cell number and vitality was deter-mined using a Bürker counting chamber combined witha trypan blue stain. After cell count, cells were reculturedand further passaged to determine the population dou-bling level of the AF derived MSCs. Mesenchymal stemcell characteristics were analysed by flow cytometry us-ing specific CD73+/CD44+/CD45- cell surface markers.  Results6156 ± 9056 (mean ± SD) flat, spindle shaped cellswere found attached to the bottom of the plate after a 14day culture period out of 4mL of fresh AF (n = 8). The re-sults varied strongly among the 8 different test samples(minimum = 2083 cells, maximum = 52083 cells), 1sample (gestational week 16) could not give any MSC. In Passage 1 (after 2 trypsine treatments) 75 ± 11%(mean ± SD) of the cells showed MSC surface markerswhereas in Passage 2 this percentage increased to 83±7% (mean ± SD). The vitality of the MSCs remained high (>90%) over thedifferent passages (Fig. 1).In Figure 2, the doubling levels over the different pas-sages in time are depicted.Whereas from passage 0 to passage 1 the MSCs, de-Isolation of amniotic fluid-derived mesenchymalstem cells Figure 1 - Vitality of AFMSCs.ISOLATION_Wouters  3-01-2008  14:49  Pagina 39rived from the AF, possessed a high expansion potencywith a doubling level of 5.5, this growth rate decreaseddrastically to passage 2 with a remaining doubling levelof 0.7 while decreasing futher to the next passage.After 55 days, the initial population would be doubled 8times, from 6156 cells at the start to 1.5 x 106 cells.ConclusionAmniotic fluid may be an alternative fetal stem cellsource. The expansion potency is extremely high in thefirst passage (a doubling level of 5.5 compared with ourfindings for umbilical cord and adipose tissue derivedMSCs showing a doubling level of 3). Whereas theMSCs in these latter tissues keep their growth charac-teristics in time, the amniotic fluid derived MSCs losetheir expansion potency drastically. Furhter studies areneeded to overcome this problem (e.g. changing mediacomposition).Studies for an optimal cryopreservation of amniotic fluidare undergoing.References1. Kim BG, Hwang DH, Lee SI, Kim EJ, Kim SU. Stem cell-based cell therapy for spinal cord injury. Cell Transplant2007;16:355-364.2. Rao MS, Mattson MP. Stem cells and aging: expanding thepossibilities. Mech Ageing Dev 2001;122:713-714. Review.3. Anker PS, Scherjon SA, Kleijburg-van der Keur C et al.Amniotic fluid as a novel source of mesenchymal stemcells for therapeutic transplantation. Blood 2003;121:368-374.4. Tsai MS, Lee JL, Chang YJ, Hwang SM. Isolation of hu-man multipotent mesenchymal stem cells from second-tri-master amniotic fluid using a novel two-stage culture pro-tocol. Human Reproduction 2004;19:145-1456.5. Kim J, Lee Y, Hwang KJ et al. Human amniotic fluid-de-rived stem cells have characteristic of multipotent stemcells. Cell Prolif. 2007;40:75-90.6. Anker PS, Scherjon SA, Kleijburg-van der Keur C et al.Isolation of mesenchymal stem cells of fetal or maternalorigin from human placenta. Stem Cells 2004;22:1338-1345.7. Kunisaki SM, Armant M, Kao GS et al. Tissue engineeringfrom human mesenchymal amniocytes: a prelude to clini-cal trials. J Pediatr Surg 2007;42:974-979.8. Kolambkar YM, Peister A, Soker S, Atala A, Guldberg RE.Chondrogenic differentiation of amniotic fluid-derived stemcells. J Mol Histol 2007; in press.G. Wouters et al.40 Journal of Prenatal Medicine 2007; 1 (3): 39-40Figure 2 - Doubling population level of AFMSCs.ISOLATION_Wouters  3-01-2008  14:49  Pagina 40

Isolation of amniotic fluid-derived mesenchymal

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Isolation of amniotic fluid-derived mesenchymal stem cells

Isolation of amniotic fluid-derived mesenchymal stem cells

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