Korean J Obstet Gynecol.  2010 Sep;53(9):825-832. 10.5468/kjog.2010.53.9.825.

Characteristics of amniotic fluid derived stem cells with trisomy 21

• Choi KM
• Im EH
• Cho JA
• Lee SJ
• Park H
• Shin BS
• Lee JH
• Choi JS
• Lee KW

• KMID: 2078000
• DOI: http://doi.org/10.5468/kjog.2010.53.9.825

Abstract

OBJECTIVE
To assess molecular markers of amniotic fluid derived stem cells (AFSCs) in aspects of increased neurological deficit in Down syndrome.
METHODS
Amniotic fluid samples through amniocentesis for prenatal diagnosis from four mid trimester pregnancies; by routine chromosomal analysis, two of them were trisomy 21 (Down syndrome) and others were normal, were selected after informed consent. Cells from two-stage culture protocol were assayed; morphology through phase contrast microscopy, chromosomal analysis, reverse transcriptase-polymerase chain reaction and Western blot analysis.
RESULTS
AFSCs were highly proliferative in subcultures and most of them were mononuclear, fibroblast-like, fusiform cells. There were also a few ovoid cells. The chromosomal analysis of amniotic fluid stem cells was identical to that of amniotic fluid cells. Two of four samples were 47,XX,+21, others were 46,XX. Of the proteins related to Down syndrome, the expression of S100beta were increased in AFSCs of Down syndrome, COL6A1 (Collagen IV, alpha 1) was down-regulated in them and insulin like growth factor binding protein-1 was expressed in all AFSCs. Stem cell markers were expressed heterogeneously. Oct4 (POU5F1), nanog, and SOX2 (sex determining region Y) were expressed in both groups. But c-Kit was not expressed in AFSCs of Down syndrome. The neural cell marker, neuron specific enolase was detected in both groups. Other neural cell markers, microtubule associated protein 2, glial fibrillary acidic protein were undetectable in ASFCs of Down syndrome. Bcl-2 gene family proteins related with apoptosis were assayed. The expression of Bcl-XL was increased in Down syndrome more than in normal pregnancy. Bcl-2 and BID were expressed in all AFSCs and Bax was down-regulated in Down syndrome.
CONCLUSION
AFSCs are an excellent choice for many future tissue engineering strategies and cell based therapies. Analysis of molecular features of AFSCs from normal and Down syndrome will provide the basis of further experimental study.

Keyword

Amniotic fluid derived stem cell; Down syndrome

MeSH Terms

Amniocentesis
Amniotic Fluid
Apoptosis
Blotting, Western
Down Syndrome
Female
Genes, bcl-2
Glial Fibrillary Acidic Protein
Humans
Informed Consent
Insulin
Microscopy, Phase-Contrast
Microtubule-Associated Proteins
Phosphopyruvate Hydratase
Pregnancy
Prenatal Diagnosis
Proteins
Stem Cells
Tissue Engineering
Trisomy
Glial Fibrillary Acidic Protein
Insulin
Microtubule-Associated Proteins
Phosphopyruvate Hydratase
Proteins

Figure

• 

  Figure 1 Morphology of amniotic fluid derived stem cells. The cultured amniotic fluid derived stem cells were observed by optical microscopy without stain at the 100 fold magnitude. The morphology was heterogenous; most mononuclear fusiform cells and a few mononuclear ovoid cells. (A) Normal, (B) Down syndrome.

• 

  Figure 2 Chromosomal analysis of amniotic fluid derived stem cells. The culture amniotic fluid derived stem cells were observed through system microscope after Giemsa stain at the 1,000 fold magnitude. Chromosomal analysis reveals trisomy 21 (47,XX,+21) and normal (46,XX). (A) Normal, (B) Down syndrome.

• 

  Figure 3 Molecular marker analysis by RT-PCR & Western blot analysis. (A) Proteins related to Down syndrome. (B) Stem cell markers expression. (C) Neural cell markers expression (D) Bcl-2 gene family proteins. RT-PCR: Reverse transcriptase-polymerase chain reaction, IGFBP-1: insulin like growth factor binding protein-1, NSE: neuron specific enolase, GFAP: glial fibrillary acidic protein, MAP2: microtubule associated protein 2.

Reference

1. Bossolasco P, Montemurro T, Cova L, Zangrossi S, Calzarossa C, Buiatiotis S, et al. Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential. Cell Res. 2006. 16:329–336.

2. Kim HK, Im EH, Park H, Cho JA, Yang DY, Kim KH, et al. Characterization and differentiation into adipocytes of mesenchymal stem cells (MSCs) from human adipose tissue and amniotic fluid. Korean J Obstet Gynecol. 2009. 52:447–455.

3. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol. 2007. 25:100–106.

4. Slavin S, Kurkalli BG, Karussis D. The potential use of adult stem cells for the treatment of multiple sclerosis and other neurodegenerative disorders. Clin Neurol Neurosurg. 2008. 110:943–946.

5. Perin L, Sedrakyan S, Da Sacco S, De Filippo R. Characterization of human amniotic fluid stem cells and their pluripotential capability. Methods Cell Biol. 2008. 86:85–99.

6. Jezierski A, Gruslin A, Tremblay R, Ly D, Smith C, Turksen K, et al. Probing Stemness and Neural Commitment in Human Amniotic Fluid Cells. Stem Cell Rev. 2010. 6:199–214.

7. Tsai MS, Hwang SM, Tsai YL, Cheng FC, Lee JL, Chang YJ. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol Reprod. 2006. 74:545–551.

8. Tsai MS, Lee JL, Chang YJ, Hwang SM. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod. 2004. 19:1450–1456.

9. Epstein CJ, Korenberg JR, Anneren G, Antonarakis SE, Ayme S, Courchesne E, et al. Protocols to establish genotype-phenotype correlations in Down syndrome. Am J Hum Genet. 1991. 49:207–235.

10. Vicari S. Motor development and neuropsychological patterns in persons with Down syndrome. Behav Genet. 2006. 36:355–364.

11. Glasson EJ, Sullivan SG, Hussain R, Petterson BA, Montgomery PD, Bittles AH. The changing survival profile of people with Down's syndrome: implications for genetic counselling. Clin Genet. 2002. 62:390–393.

12. Stoll C, Alembik Y, Dott Bt, Roth MP. Epidemiology of Down syndrome in 118,265 consecutive births. Am J Med Genet Suppl. 1990. 37:79–83.

13. Choi JH, Han HJ, Hwang JH, Chung SR, Moon H, Park MI, et al. Meta Analysis of Clinical Studies of Pregnancy and Delivery in Elderly Gravida. Korean J Obstet Gynecol. 2006. 49:293–308.

14. Griffin WST, Sheng JG, McKenzie JE, Royston MC, Gentleman SM, Brumback RA, et al. Life-long overexpression of S100[beta] in Down's syndrome: implications for Alzheimer pathogenesis. Neurobiology of Aging. 1998. 19:401–405.

15. Engidawork E, Baiic N, Fountoulakis M, Dierssen M, Greber-Platzer S, Lubec G. Beta-amyloid precursor protein, ETS-2 and collagen alpha 1 (VI) chain precursor, encoded on chromosome 21, are not overexpressed in fetal Down syndrome: further evidence against gene dosage effect. J Neural Transm Suppl. 2001. (61):335–346.

16. Fernandez F, Morishita W, Zuniga E, Nguyen J, Blank M, Malenka RC, et al. Pharmacotherapy for cognitive impairment in a mouse model of Down syndrome. Nat Neurosci. 2007. 10:411–413.

17. Delabar JM. New perspectives on molecular and genic therapies in Down syndrome. Med Sci (Paris). 2010. 26:371–376.

18. Davies GE, Howard CM, Farrer MJ, Coleman MM, Bennett LB, Cullen LM, et al. Genetic variation in the COL6A1 region is associated with congenital heart defects in trisomy 21 (Down's syndrome). Ann Hum Genet. 1995. 59:253–269.

19. Moghadam S, Engel W, Bougoussa M, Hennen G, Igout A, Sancken U. Maternal serum placental growth hormone and insulinlike growth factor binding proteins 1 and 3 in pregnancies affected by fetal aneuploidy and other abnormalities: implications for prenatal diagnosis of trisomy 21. Fetal Diagn Ther. 1998. 13:291–297.

20. Mann DM. The pathological association between Down syndrome and Alzheimer disease. Mech Ageing Dev. 1988. 43:99–136.

21. Cataldo AM, Mathews PM, Boiteau AB, Hassinger LC, Peterhoff CM, Jiang Y, et al. Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects. Am J Pathol. 2008. 173:370–384.

22. Chung IH, Lee SH, Lee KW, Park SH, Cha KY, Kim NS, et al. Gene expression analysis of cultured amniotic fluid cell with Down syndrome by DNA microarray. J Korean Med Sci. 2005. 20:82–87.

23. Quarello E, Guimiot F, Moalic JM, Simoneau M, Ville Y, Delezoide AL. Quantitative evaluation of collagen type VI and SOD gene expression in the nuchal skin of human fetuses with trisomy 21. Prenat Diagn. 2007. 27:926–931.

24. Edling CE, Hallberg B. c-Kit: a hematopoietic cell essential receptor tyrosine kinase. Int J Biochem Cell Biol. 2007. 39:1995–1998.

25. Miettinen M, Lasota J. KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation. Appl Immunohistochem Mol Morphol. 2005. 13:205–220.

26. Miliaras D, Karasavvidou F, Papanikolaou A, Sioutopoulou D. KIT expression in fetal, normal adult, and neoplastic renal tissues. J Clin Pathol. 2004. 57:463–466.

27. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003. 114:763–776.

28. Shi H, Zhu F, Xiao AQ, Zhang ZR, Zhang R. [Clinical significance of CD117/CD34 co-expression in adult patients with acute leukemia]. Ai Zheng. 2006. 25:762–764.

29. Steward O, Wallace CS. mRNA distribution within dendrites: relationship to afferent innervation. J Neurobiol. 1995. 26:447–449.

30. Sawa A, Oyama F, Cairns NJ, Amano N, Matsushita M. Aberrant expression of bcl-2 gene family in Down's syndrome brains. Brain Res Mol Brain Res. 1997. 48:53–59.

31. Cairney CJ, Sanguinetti G, Ranghini E, Chantry AD, Nostro MC, Bhattacharyya A, et al. A systems biology approach to Down syndrome: identification of Notch/Wnt dysregulation in a model of stem cells aging. Biochim Biophys Acta. 2009. 1792:353–363.