Int J Stem Cells.  2018 Jun;11(1):87-95. 10.15283/ijsc18001.

Large-scale Isolation, Expansion and Characterization of Human Amniotic Epithelial Cells

• Gottipamula S
• Sridhar KN

• KMID: 2413504
• DOI: http://doi.org/10.15283/ijsc18001

Abstract

BACKGROUND AND OBJECTIVES
The human Amniotic epithelial cells (AME) derived from amniotic membrane of placenta have been considered as the potential fetal stem cell source with minimal or no ethical concerns and are important therapeutic tool for anti-fibrotic and regenerative therapies.
METHODS AND RESULTS
Here, we evaluated the isolation, media screening, scale-up and characterization of AME cells. The isolation, expansion of AMEs were performed by sequential passaging and growth kinetics studies. The AMEs were characterized using immunocytochemistry, immunophenotyping, In-vitro differentiation, and anti-fibrotic assays. The growth kinetics study revealed that the AME cultured in Ultraculture (UC) and DMEM knockout (DMEM-KO) have prominently higher growth rate compared to others. Overall, the AMEs cultured from 5 different media retained basic morphological characteristics and the functional characteristics.
CONCLUSIONS
Our result suggests that the AMEs can be successfully cultured in UC based complete media without losing its epithelial cell characteristics even after passaging for passage 2 (P2). However, a careful and methodical pre-clinical and clinical translation studies need to be conducted to show its safety and efficacy.

Keyword

Amniotic epithelial cells; Cell therapy; Cryopreservation; Stability; Growth kinetics; Tissue engineering

MeSH Terms

Amnion
Cell- and Tissue-Based Therapy
Cryopreservation
Epithelial Cells*
Fetal Stem Cells
Humans*
Immunohistochemistry
Immunophenotyping
Kinetics
Mass Screening
Methods
Placenta
Tissue Engineering

Figure

• 

  Fig. 1 Growth Kinetics of AME sub cultured in 5 different media. (A) Cumulative population doublings of AMEs plotted against the passage number. (B) Population doubling time of AMEs (Epi is significantly higher (***p<0.001) across all passages compared to all other media, except Epi vs KOSR showed *p<0.05).

• 

  Fig. 2 Immunophenotyping of AME at P1. A representative histogram of isotypes white and grey as EpCAM (A) and Cyto (B) (Cytokeratin 4, 5, 6, 10, 13, 18) of AME cultured in UC respectively. (C) Overall bar chart of Immunophenotyping of EpCAM and cyto expression of AME in all 5 different cell culture media.

• 

  Fig. 3 In-vitro differentiation of AME: (A, C, E) and (G) are un-induced AME as controls and (B, D, F) and (H) are induced with respective induction media. Scale bar – 100 microns.

• 

  Fig. 4 Conditioned media mediated fibroblast inhibition assay: Conditioned media of AME, BME and controls were added to fibroblast culture and incubated for 24 h before the proliferation was assessed via MTT assay. Both AME and BME conditioned media are able to inhibit the fibroblast proliferation at two different cell dose of 50K and 25K. Data are normalized to mean proliferation of fibroblast alone as 100% (n=3, pooled data from 3 separate experiments).

Reference

References

1. Mason C, Brindley DA, Culme-Seymour EJ, Davie NL. Cell therapy industry: billion dollar global business with unlimited potential. Regen Med. 2011; 6:265–272. DOI: 10.2217/rme.11.28. PMID: 21548728.

2. You HJ, Han SK. Cell therapy for wound healing. J Korean Med Sci. 2014; 29:311–319. DOI: 10.3346/jkms.2014.29.3.311. PMID: 24616577. PMCID: 3945123.

3. Varkey M, Ding J, Tredget EE. Advances in skin substitutes-potential of tissue engineered skin for facilitating anti-fibrotic healing. J Funct Biomater. 2015; 6:547–563. DOI: 10.3390/jfb6030547. PMID: 26184327. PMCID: 4598670.

4. Dodson BP, Levine AD. Challenges in the translation and commercialization of cell therapies. BMC Biotechnol. 2015; 15:70. DOI: 10.1186/s12896-015-0190-4. PMID: 26250902. PMCID: 4528687.

5. Ackermann K, Borgia SL, Korting HC, Mewes KR, Schäfer-Korting M. The Phenion full-thickness skin model for percutaneous absorption testing. Skin Pharmacol Physiol. 2010; 23:105–112. DOI: 10.1159/000265681.

6. Vig K, Chaudhari A, Tripathi S, Dixit S, Sahu R, Pillai S, Dennis VA, Singh SR. Advances in skin regeneration using tissue engineering. Int J Mol Sci. 2017; 18:E789. DOI: 10.3390/ijms18040789. PMID: 28387714. PMCID: 5412373.

7. Tan JL, Tan YZ, Muljadi R, Chan ST, Lau SN, Mockler JC, Wallace EM, Lim R. Amnion epithelial cells promote lung repair via lipoxin A4. Stem Cells Transl Med. 2017; 6:1085–1095. DOI: 10.5966/sctm.2016-0077. PMID: 28371562. PMCID: 5442827.

8. Fanti M, Gramignoli R, Serra M, Cadoni E, Strom SC, Marongiu F. Differentiation of amniotic epithelial cells into various liver cell types and potential therapeutic applications. Placenta. 2017; 59:139–145. DOI: 10.1016/j.placenta.2017.03.020. PMID: 28411944.

9. Yeager AM, Singer HS, Buck JR, Matalon R, Brennan S, O’Toole SO, Moser HW. A therapeutic trial of amniotic epithelial cell implantation in patients with lysosomal storage diseases. Am J Med Genet. 1985; 22:347–355. DOI: 10.1002/ajmg.1320220219. PMID: 3931477.

10. Di Germanio C, Bernier M, de Cabo R, Barboni B. Amniotic epithelial cells: a new tool to combat aging and age-related diseases? Front Cell Dev Biol. 2016; 4:135. DOI: 10.3389/fcell.2016.00135. PMID: 27921031. PMCID: 5118838.

11. Tamagawa T, Ishiwata I, Saito S. Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro. Hum Cell. 2004; 17:125–130. DOI: 10.1111/j.1749-0774.2004.tb00028.x.

12. Manuelpillai U, Lourensz D, Vaghjiani V, Tchongue J, Lacey D, Tee JY, Murthi P, Chan J, Hodge A, Sievert W. Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis. PLoS One. 2012; 7:e38631. DOI: 10.1371/journal.pone.0038631. PMID: 22719909. PMCID: 3375296.

13. Miki T, Lehmann T, Cai H, Stolz DB, Strom SC. Stem cell characteristics of amniotic epithelial cells. Stem Cells. 2005; 23:1549–1559. DOI: 10.1634/stemcells.2004-0357. PMID: 16081662.

14. McDonald CA, Payne NL, Sun G, Moussa L, Siatskas C, Lim R, Wallace EM, Jenkin G, Bernard CC. Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis. J Neuroinflammation. 2015; 12:112. DOI: 10.1186/s12974-015-0322-8. PMID: 26036872. PMCID: 4457975.

15. Parolini O, Alviano F, Bagnara GP, Bilic G, Bühring HJ, Evangelista M, Hennerbichler S, Liu B, Magatti M, Mao N, Miki T, Marongiu F, Nakajima H, Nikaido T, Portmann-Lanz CB, Sankar V, Soncini M, Stadler G, Surbek D, Takahashi TA, Redl H, Sakuragawa N, Wolbank S, Zeisberger S, Zisch A, Strom SC. Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells. 2008; 26:300–311. DOI: 10.1634/stemcells.2007-0594.

16. Miki T, Marongiu F, Dorko K, Ellis EC, Strom SC. Isolation of amniotic epithelial stem cells. Curr Protoc Stem Cell Biol. 2010; Chapter 1(Unit 1E.3):DOI: 10.1002/9780470151808.sc01e03s12.

17. Silini AR, Cargnoni A, Magatti M, Pianta S, Parolini O. The long path of human placenta, and its derivatives, in regenerative medicine. Front Bioeng Biotechnol. 2015; 3:162. DOI: 10.3389/fbioe.2015.00162. PMID: 26539433. PMCID: 4609884.

18. Matikainen T, Laine J. Placenta--an alternative source of stem cells. Toxicol Appl Pharmacol. 2005; 207(2 Suppl):544–549. DOI: 10.1016/j.taap.2005.01.039. PMID: 15990135.

19. Miki T. Amnion-derived stem cells: in quest of clinical applications. Stem Cell Res Ther. 2011; 2:25. DOI: 10.1186/scrt66. PMID: 21596003. PMCID: 3152995.

20. Wu Z, Hui G, Lu Y, Liu T, Huang Q, Guo L. Human amniotic epithelial cells express specific markers of nerve cells and migrate along the nerve fibers in the corpus callosum. Neural Regen Res. 2012; 7:41–45. PMID: 25806057. PMCID: 4354114.

21. Ochsenbein-Kölble N, Bilic G, Hall H, Huch R, Zimmermann R. Inducing proliferation of human amnion epithelial and mesenchymal cells for prospective engineering of membrane repair. J Perinat Med. 2003; 31:287–294. DOI: 10.1515/JPM.2003.040. PMID: 12951883.

22. Saito S, Yokoyama K, Tamagawa T, Ishiwata I. Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane. Hum Cell. 2005; 18:135–141. DOI: 10.1111/j.1749-0774.2005.tb00003.x.

23. Jiawen S, Jianjun Z, Jiewen D, Dedong Y, Hongbo Y, Jun S, Xudong W, Shen SG, Lihe G. Osteogenic differentiation of human amniotic epithelial cells and its application in alveolar defect restoration. Stem Cells Transl Med. 2014; 3:1504–1513. DOI: 10.5966/sctm.2014-0118. PMID: 25368378. PMCID: 4250213.

24. Murphy S, Rosli S, Acharya R, Mathias L, Lim R, Wallace E, Jenkin G. Amnion epithelial cell isolation and characterization for clinical use. Curr Protoc Stem Cell Biol. 2010; Chapter 1(Unit 1E.6):PMID: 20373516.

25. Díaz-Prado S, Muiños-López E, Hermida-Gómez T, Rendal-Vázquez ME, Fuentes-Boquete I, de Toro FJ, Blanco FJ. Multilineage differentiation potential of cells isolated from the human amniotic membrane. J Cell Biochem. 2010; 111:846–857. DOI: 10.1002/jcb.22769. PMID: 20665539.

26. Stadler G, Hennerbichler S, Lindenmair A, Peterbauer A, Hofer K, van Griensven M, Gabriel C, Redl H, Wolbank S. Phenotypic shift of human amniotic epithelial cells in culture is associated with reduced osteogenic differentiation in vitro. Cytotherapy. 2008; 10:743–752. DOI: 10.1080/14653240802345804. PMID: 18985480.

27. Ilancheran S, Michalska A, Peh G, Wallace EM, Pera M, Manuelpillai U. Stem cells derived from human fetal membranes display multilineage differentiation potential. Biol Reprod. 2007; 77:577–588. DOI: 10.1095/biolreprod.106.055244. PMID: 17494917.