Comparison of chromosomal instability of human amniocytes in primary and long-term cultures in AmnioMAX II and DMEM media: A cross-sectional study


Background: The genomic stability of stem cells to be used in cell therapy and other clinical applications is absolutely critical. In this regard, the relationship between in vitro expansion and the chromosomal instability (CIN), especially in human amniotic fluid cells (hAFCs) has not yet been completely elucidated.

Objective: To investigate the CIN of hAFCs in primary and long-term cultures and two different culture mediums.

Materials and Methods: After completing prenatal genetic diagnoses (PND) using karyotype technique and chromosomal analysis, a total of 15 samples of hAFCs from 650 samples were randomly selected and cultured in two different mediums as AmnioMAX II and DMEM. Then, proliferative cells were fixed on the slide to be used in standard chromosome G-banding analysis. Also, the senescent cells were screened for aneuploidy considering 8 chromosomes by FISH technique using two probe sets including PID I (X-13-18-21) & PID II (Y-15-16-22).

Results: Karyotype and interphase fluorescence in situ hybridization (iFISH) results from 650 patients who were referred for prenatal genetic diagnosis showed that only 6 out of them had culture- derived CIN as polyploidy, including mosaic diploidtriploid and diploid-tetraploid. Moreover, the investigation of aneuploidies in senesced hAFCs demonstrated the rate of total chromosomal abnormalities as 4.3% and 9.9% in AmnioMAX- and DMEM-cultured hAFCs, respectively.

Conclusion: hAFCs showed a low rate of CIN in two AmnioMAX II and DMEM mediums and also in the proliferative and senescent phases. Therefore, they could be considered as an attractive stem cell source with therapeutic potential in regenerative medicine.

Key words: Human amniotic fluid cells, Chromosomal instability, Pseudomosaicism, Amniocentesis, Replicative senescence.

[1] Klemmt PAB, Vafaizadeh V, Groner B. The potential of amniotic fluid stem cells for cellular therapy and tissue engineering. Expert Opin Biol Ther 2011; 11: 1297–1314.

[2] Oliveira PH, da Silva CL, Cabral JMS. Concise review: Genomic instability in human stem cells: current status and future challenges. Stem Cells 2014; 32: 2824–2832.

[3] Hoseini S, Kalantar F, Kalantar SM, Bahrami AR, Zareien F, Moghadam-Matin M. Mesenchymal stem cells: Interactions with immune cells and immunosuppressive-immunomodulatory properties. Sci J Iran Blood Transfus Organ 2020; 17: 147–169.

[4] Hoseini SM, Moghaddam-Matin M, Bahrami AR, Kalantar SM. [Human amniotic fluid stem cells: general characteristics and potential therapeutic applications]. J Shahid Sadoughi Uni Med Sci 2020; in press. (in Persian)

[5] Fauza DO, Bani M. Fetal stem cells in regenerative medicine: Principles and Translational Strategies. New York: Springer; 2016.

[6] Hoseini SM, Kalantar SM, Bahrami AR, Moghadam- Matin M. Human amniocytes: A comprehensive study on morphology, frequency and growth properties of subpopulations from a single clone to the senescence. Cell and Tissue Biology 2020; 14: 102–112.

[7] Mamede AC, Boteiho MF. Amniotic membrane: origin, characterization and medical applications. New York, London: Springer Dordrecht Heidelberg; 2015.

[8] Hoseini SM, Montazeri F, Bahrami AR, Kalantar SM, Rahmani S, Zarein F, et al. Investigating the expression of pluripotency-related genes in human amniotic fluid cells: A semi-quantitative comparison between different subpopulations, from primary to cultured amniocytes. Reproductive Biology 2020; 20: 338–347.

[9] Stultz BG, McGinnis K, Thompson EE, Lo Surdo JL, Bauer SR, Hursh DA. Chromosomal stability of mesenchymal stromal cells during in vitro culture. Cytotherapy 2016; 18: 336–343.

[10] Harrison NJ. Genetic instability in neural stem cells: an inconvenient truth? J Clin Invest 2012; 122: 484– 486.

[11] Nguyen HT, Geens M, Spits C. Genetic and epigenetic instability in human pluripotent stem cells. Hum Reprod Update 2013; 19: 187–205.

[12] Neri S. Genetic stability of mesenchymal stromal cells for regenerative medicine applications: A fundamental biosafety aspect. Int J Mol Sci 2019; 20: 2406–2431.

[13] Agostini F, Rossi FM, Aldinucci D, Battiston M, Lombardi E, Zanolin S, et al. Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media. Stem Cell Res Ther 2018; 9: 130–145.

[14] McGowan-Jordan J, Simons A, Schmid M. ISCN 2016: an international system for human cytogenomic nomenclature. New York: Karger; 2016.

[15] Montazeri F, Foroughmand AM, Kalantar SM, Aflatoonian A, Khalilli MA. Tips and Tricks in Fluorescence In-situ Hybridization (FISH)- based Preimplantation Genetic Diagnosis /Screening (PGD/PGS). International Journal of Medical Laboratory 2018; 5: 84–98.

[16] Rajamani K, Li YS, Hsieh DK, Lin SZ, Harn HJ, Chiou TW. Genetic and epigenetic instability of stem cells. Cell Transplant 2014; 23: 417–433.

[17] Peakman DC, Moreton MF, Corn BJ, Robinson A. Chromosomal mosaicism in amniotic fluid cell cultures. Am J Hum Genet 1979; 31: 149–155.

[18] Hsu LY, Perlis TE. United States survey on chromosome mosaicism and pseudomosaicism in prenatal diagnosis. Prenat Diagn 1984; 4: 97–130.

[19] Garcia-Martinez J, Bakker B, Schukken KM, Simon JE, Foijer F. Aneuploidy in stem cells. World J Stem Cells 2016; 8: 216–222.

[20] Rebuzzini P, Zuccotti M, Redi CA, Garagna S. Chromosomal abnormalities in embryonic and somatic stem cells. Cytogenet Genome Res 2015; 147: 1–9.

[21] Sensebe L, Tarte K, Galipeau J, Krampera M, Martin I, Phinney DG, et al. Limited acquisition of chromosomal aberrations in human adult mesenchymal stromal cells. Cell Stem Cell 2012; 10: 9–10.

[22] Ben-David U, Mayshar Y, Benvenisty N. Largescale analysis reveals acquisition of lineage-specific chromosomal aberrations in human adult stem cells. Cell Stem Cell 2011; 9: 97–102.

[23] Nikitina VA, Osipova EY, Katosova LD, Rumyantsev SA, Skorobogatova EV, Shamanskaya TV, et al. Study of genetic stability of human bone marrow multipotent mesenchymal stromal cells. Bull Exp Biol Med 2011; 150: 627–631.

[24] Grigorian AS, Kruglyakov PV, Taminkina UA, Efimova OA, Pendina AA, Voskresenskaya AV, et al. Alterations of cytological and karyological profile of human mesenchymal stem cells during in vitro culturing. Bull Exp Biol Med 2010; 150: 125–130.

[25] Estrada JC, Torres Y, Benguria A, Dopazo A, Roche E, Carrera-Quintanar L, et al. Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy. Cell Death Dis 2013; 4: e691: 1–13.

[26] Corselli M, Parodi A, Mogni M, Sessarego N, Kunkl A, Dagna-Bricarelli F, et al. Clinical scale ex vivo expansion of cord blood-derived outgrowth endothelial progenitor cells is associated with high incidence of karyotype aberrations. Exp Hematol 2008; 36: 340–349.

[27] Macedo JC, Vaz S, Bakker B, Ribeiro R, Bakker PL, Escandell JM, et al. FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence. Nat Commun 2018; 9: 2834–2850.

[28] Burton DGA, Faragher RGA. Cellular senescence: from growth arrest to immunogenic conversion. Age 2015; 37: 27–45.

[29] Mukherjee AB, Thomas S. A longitudinal study of human age-related chromosomal analysis in skin fibroblasts. Exp Cell Res 1997; 235: 161–169.

[30] Mosquera A, Fernandez JL, Campos A, Goyanes VJ, Ramiro-Diaz J, Gosalvez J. Simultaneous decrease of telomere length and telomerase activity with ageing of human amniotic fluid cells. J Med Genet 1999; 36: 494–496.

[31] Harley CB, Villeponteau B. Telomeres and telomerase in aging and cancer. Curr Opin Genet Dev 1995; 5: 249–255.

[32] Bartnitzke S, Skarbek H, Lackmann C, Bullerdiek J. Chang medium raises the chromatin instability of pericentromeric areas of chromosome 1 in amniotic fluid cells. Prenat Diagn 1992; 12: 310–311.

[33] Bui TH, Iselius L, Lindsten J. European collaborative study on prenatal diagnosis: mosaicism, pseudomosaicism and single abnormal cells in amniotic fluid cell cultures. Prenat Diagn 1984; 4: 145–162.

[34] Krawczun MS, Jenkins EC, Masia A, Kunaporn S, Stark SL, Duncan CJ, et al. Chromosomal abnormalities in amniotic fluid cell cultures: a comparison of apparent pseudomosaicism in Chang and RPMI-1640 media. Clin Genet 1989; 35: 139– 145.

[35] Blázquez-Prunera A, Díez JM, Gajardo R, Grancha S. Human mesenchymal stem cells maintain their phenotype, multipotentiality, and genetic stability when cultured using a defined xeno-free human plasma fraction. Stem cell Res Ther 2017; 8: 103– 113.

[36] Garitaonandia I, Amir H, Boscolo FS, Wambua GK, Schultheisz HL, Sabatini K, et al. Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions. PloS One 2015; 10: e0118307: 1– 25.