Expression of a Hoechst 33342 efflux phenomenon and common characteristics of pluripotent stem cells in a side population of amniotic fluid cells.
OBJECTIVE
The aim of this study was to verify the existence of a side population (SP) of cells in second-trimester amniotic fluid.


METHODS
Amniotic fluid samples (n = 35) were obtained, and the number and size of viable amniotic fluid cells (AFCs) were analyzed. Small AFCs (SAFCs) and large AFCs (LAFCs) were isolated using a sterile 10-microm pore size strainer. Hoechst 33342 dye exclusion assay, flow cytometry analysis, reverse transcriptase polymerase chain reaction and immunocytochemistry were used to analyze the characteristics of SAFCs and LAFCs.


RESULTS
The mean concentration of viable AFCs from 16 to 21 weeks of gestation was 0.3 x 10(5), 0.8 x 10(5), 1.1 x 10(5), 1.3 x 10(5), 1.0 x 10(5) and 1.0 x 10(5) cells/mL respectively. The mean percentage of SAFCs from 16 to 21 weeks of gestation was 27.3%, 40.5%, 49.7%, 60.2%, 41.0% and 58.2%, respectively. The Hoechst 33342 efflux phenomenon was obvious among SAFCs but was rare in the LAFC population. Flow cytometry analyses showed that cell surface antigen expression on LAFCs and SAFCs were positive for CD29, CD44, CD73, CD90, CD166 and HLA-I, but negative for CD31, CD34, CD45, CD117 and HLA-II. Importantly, Nanog, Oct-4, ABCG2 and SOX2 expression in cells was easily detectable among the SAFC population. Expression of Nanog and ABCG2 was not observed among LAFCs.


CONCLUSIONS
Amniotic fluid contains a SP that was found mostly among the SAFCs. Enriched SP cells isolated by the efflux of Hoechst 33342 could be a novel and promising source of pluripotent-like amniotic derived stem cells for cellular therapy in the near future.
Tsai
Yieh-Loong
YL
Shin Kong Wu Ho Su Memorial Hospital, Taipei, Taiwan.
Chang
Yu-Jen
YJ
Chou
Ching-Yu
CY
Cheong
Mei-Leng
ML
Tsai
Ming-Song
MS
D015720:5'-Nucleotidase / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000653:Amniotic Fluid / Q000166:cytology*; D015703:Antigens, CD / Q000378:metabolism; D001562:Benzimidazoles; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism; D002470:Cell Survival; D005260:Female; D005326:Fetal Proteins / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins / Q000378:metabolism; D006680:HLA Antigens / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D018800:Thy-1 Antigens / Q000378:metabolism; D020410:Activated-Leukocyte Cell Adhesion Molecule
D015720:5'-Nucleotidase; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015703:Antigens, CD; D001562:Benzimidazoles; D015816:Cell Adhesion Molecules, Neuronal; D005326:Fetal Proteins; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins; D006680:HLA Antigens; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; D019012:Integrin beta1; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; D018800:Thy-1 Antigens; C116614:ABCG2 protein, human; C105613:ALCAM protein, human; D020410:Activated-Leukocyte Cell Adhesion Molecule; C486780:NANOG protein, human; C527782:SOX2 protein, human; C548241:NT5E protein, human; C017807:bisbenzimide ethoxide trihydrochloride