Comparative characterization of cultured human term amnion epithelial and mesenchymal stromal cells for application in cell therapy.
Emerging evidence suggests human amnion tissue as a valuable source of two distinct types of pluripotent cells, amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs), for applications in cell replacement therapy. For some approaches, it may be necessary to culture and differentiate these cells before they can be transplanted. No systematic attempt has been yet made to determine the quantity and quality of amnion cells after isolation and culture. We looked at amnion cell isolates from 27 term placentas. Following our optimized protocol, primary yields were 6.3 x 10(6) hAECs and 1.7 x 10(6) hAMSCs per gram amnion. All 27 cases gave vital cultures of hAMSCs, while one third of cases (9 of 27) failed to give adherent cultures of hAECs. Primary cultures contained significantly more proliferating than apoptotic cells (hAECs: 16.4% vs. 4.0%; hAMSCs: 9.5% vs. 2.4%). Neither hAECs nor hAMSCs were clonogenic. They showed slow proliferation that almost stopped beyond passage 5. Microscopic follow-up revealed that hAEC morphology gradually changed towards mesenchymal phenotype over several passages. Flow cytometric characterization of primary cultures showed expression of mesenchymal progenitor markers CD73, CD90, CD105, and CD166, as well as the embryonic stem cell markers SSEA-3 and -4 on both amnion cell types. These profiles were grossly maintained in secondary cultures. Reverse transcriptase-PCR analysis exhibited transcripts of Oct-3/4 and stem cell factor in primary and secondary cultures of all cases, but no telomerase reverse transcriptase. Immunocytochemistry confirmed translation into Oct-3/4 protein in part of hAEC cultures, but not in hAMSCs. Further, both amnion cell types stained for CD90 and SSEA-4. Osteogenic induction studies with amnion cells from four cases showed significantly stronger differentiation of hAECs than hAMSCs; this capacity to differentiate greatly varied between cases. In conclusion, hAECs and hAMSCs in culture exhibit and maintain a similar marker profile of mesenchymal progenitors. hAECs were found as a less reliable source than hAMSCs and altered morphology during subculture.
Bilic
Grozdana
G
Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland. grozdana.bilic@usz.ch
Zeisberger
Steffen M
SM
Mallik
Ajit S
AS
Zimmermann
Roland
R
Zisch
Andreas H
AH
D000650:Amnion / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D000954:Antigens, Surface / Q000032:analysis / Q000378:metabolism; D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D048430:Cell Shape / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019089:Stem Cell Factor / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*
D000954:Antigens, Surface; D015415:Biomarkers; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger; D019089:Stem Cell Factor