Journal of Molecular Histology

, Volume 49, Issue 3, pp 289–301 | Cite as

Development of decellularized amniotic membrane as a bioscaffold for bone marrow-derived mesenchymal stem cells: ultrastructural study

  • Radwa Ayman Salah
  • Ihab K. Mohamed
  • Nagwa El-Badri
Original Paper


Developing effective stem cell-based therapies requires the design of complex in vitro culture systems for accurate representation of the physiological stem cell niche. Human amniotic membrane (hAM) has been successfully used in clinical grafting applications due to its unique biological and regenerative properties. Decellularized hAM (d-hAM) has been previously applied to the culture of human bone marrow mesenchymal stem cells (hMSCs), promoting their expansion and differentiation into adipogenic and osteogenic lineages. In the present study, hAM was decellularized by NaOH-treatment, to provide the three-dimensional (3D) bioscaffold for culturing hMSCs. The ultrastructural differences between intact hAM and decellularized hAM were characterized using the transmission electron microscope (TEM), as well as the 3D interaction between d-hAM and hMSCs cultured on the membrane. TEM examination of the intact hAM showed many microvilli on the epithelial layer cells, active Golgi apparatus, smooth endolplasmic reticulum and the characteristic pinocytic vesicles. The epithelial layer with its structures was absent in the d-hAM. However, no observable difference was detected in the ultrastructural characteristics of the compact stromal layer of d-hAM compared to intact hAM. Both contained bundles of extra cellular matrix (ECM) proteins, and scattered elastic fibres. Cultured human mesenchymal stem cells (hMSCs) examined by TEM appeared oval to spherical in shape and had a rough and non-uniform surface with distinct protrusions or irregular fillopodia. Their diameter ranged from 20.49 to 21.6 µm. Most of the cellular organelles were also noticed. SEM examination of the prepared samples revealed unique 3D interaction between the hMSC and d-hAM, where the latter seems to envelop the segments of the hMSCs lying on the surrounding membrane. This study shows that the decellularization process affected the epithelial layer only of hAM and had no effect on altering the presence of ECM components present in the stromal layer of the d-hAM. The interaction between hMSCs and d-hAM maybe mediated by hAM components other than human amniotic epithelial cells, such as ECM components or MSCs present in the deeper spongy layer of the membrane or/and the adhesive components of the basement membrane of the removed epithelial layer.


Human amniotic membrane Ultrastructure Mesenchymal stem cells Developmental biology Decellularization Regenerative medicine 



We thank the Electron Microscopy Unit of Theodor Bilharz Research Institute (TBRI), Giza, Egypt for using their Electron Microscopy imaging facility. We thank the Sheikh Zayed Hospital team for facilitating the process of obtaining the amniotic membrane samples and the consent of patients. This work was supported by Science and Technology Development Fund Grant 5300.


  1. Ab Hamid SS et al (2014) Scanning electron microscopic assessment on surface morphology of preserved human amniotic membrane after gamma sterilisation. Cell Tissue Bank 15(1):15–24CrossRefPubMedGoogle Scholar
  2. Al-Yahya ARA, Makhlouf MM (2013) Characterization of the human amniotic membrane: histological, immunohistochemical and ultrastructural studies. Life Sci J 4:10Google Scholar
  3. Aplin JD, Campbell S, Allen TD (1985) The extracellular matrix of human amniotic epithelium: ultrastructure, composition and deposition. J Cell Sci 79:119–136PubMedGoogle Scholar
  4. Brown EJ, Goodwin JL (1988) Fibronectin receptors of phagocytes. Characterization of the Arg-Gly-Asp binding proteins of human monocytes and polymorphonuclear leukocytes. J Exp Med 167(3):777–793CrossRefPubMedGoogle Scholar
  5. Buhimschi IA et al (2004) The novel antimicrobial peptide beta3-defensin is produced by the amnion: a possible role of the fetal membranes in innate immunity of the amniotic cavity. Am J Obstet Gynecol 191(5):1678–1687CrossRefPubMedGoogle Scholar
  6. Chen C et al (2018) Transplantation of amniotic scaffold-seeded mesenchymal stem cells and/or endothelial progenitor cells from bone marrow to efficiently repair 3-cm circumferential urethral defect in model dogs. Tissue Eng A 24(1–2):47–56CrossRefGoogle Scholar
  7. Clark RA (1993) Biology of dermal wound repair. Dermatol Clin 11(4):647–666PubMedGoogle Scholar
  8. Clark RA et al (1988) Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment. J Biol Chem 263(24):12115–12123PubMedGoogle Scholar
  9. Cooper LJ et al (2005) An investigation into the composition of amniotic membrane used for ocular surface reconstruction. Cornea 24(6):722–729CrossRefPubMedGoogle Scholar
  10. Du Y et al (2016) The angiogenic variation of skeletal site-specific human BMSCs from same alveolar cleft patients: a comparative study. J Mol Histol 47(2):153–168CrossRefPubMedGoogle Scholar
  11. Eggenhofer E et al (2012) Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion. Front Immunol 3:297CrossRefPubMedPubMedCentralGoogle Scholar
  12. El-Badawy A et al (2017) Cancer cell-soluble factors reprogram mesenchymal stromal cells to slow cycling, chemoresistant cells with a more stem-like state. Stem Cell Res Ther 8(1):254CrossRefPubMedPubMedCentralGoogle Scholar
  13. Felix H et al (1982) Interactions of tumor cells with human amnion membrane: a model for studying tumor invasion in vitro. Scan Electron Microsc 1982(Pt 2):741–749Google Scholar
  14. Gholipourmalekabadi M et al (2015) Development of a cost-effective and simple protocol for decellularization and preservation of human amniotic membrane as a soft tissue replacement and delivery system for bone marrow stromal cells. Adv Healthc Mater 4(6):918–926CrossRefPubMedGoogle Scholar
  15. Gholipourmalekabadi M et al (2017) Silk fibroin/amniotic membrane 3D bi-layered artificial skin. Biomed Mater. Google Scholar
  16. Guilak F et al (2009) Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5(1):17–26CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hao Y et al (2000) Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea 19(3):348–352CrossRefPubMedGoogle Scholar
  18. Hu J, Cai Z, Zhou Z (2009) Progress in studies on the characteristics of human amnion mesenchymal cells. Prog Nat Sci 19(9):1047–1052CrossRefGoogle Scholar
  19. Janicki P, Schmidmaier G (2011) What should be the characteristics of the ideal bone graft substitute? Combining scaffolds with growth factors and/or stem cells. Injury 42(Suppl 2):S77–S81CrossRefPubMedGoogle Scholar
  20. Jiang F et al (2015) Amniotic mesenchymal stem cells can enhance angiogenic capacity via mmps in vitro and in vivo. Biomed Res Int 2015:324014PubMedPubMedCentralGoogle Scholar
  21. Khalil S et al (2016) A cost-effective method to assemble biomimetic 3D cell culture platforms. PLoS ONE 11(12):e0167116CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kim JS et al (2000) Amniotic membrane patching promotes healing and inhibits proteinase activity on wound healing following acute corneal alkali burn. Exp Eye Res 70(3):329–337CrossRefPubMedGoogle Scholar
  23. King AE et al (2007) Expression of natural antimicrobials by human placenta and fetal membranes. Placenta 28(2–3):161–169CrossRefPubMedGoogle Scholar
  24. Lawler PR, Lawler J (2012) Molecular basis for the regulation of angiogenesis by thrombospondin-1 and -2. Cold Spring Harb Perspect Med 2(5):a006627CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lee SB et al (2000) Suppression of TGF-beta signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane. Curr Eye Res 20(4):325–334CrossRefPubMedGoogle Scholar
  26. Lim LS et al (2009) Effect of dispase denudation on amniotic membrane. Mol Vis 15:1962–1970PubMedPubMedCentralGoogle Scholar
  27. Lin F et al (2005) Three-dimensional migration of human adult dermal fibroblasts from collagen lattices into fibrin/fibronectin gels requires syndecan-4 proteoglycan. J Invest Dermatol 124(5):906–913CrossRefPubMedGoogle Scholar
  28. Matsubara, Sato BK (2000) Pathology of the human placenta. Springer, New YorkGoogle Scholar
  29. Miko M et al (2015) Ultrastructural analysis of different human mesenchymal stem cells after in vitro expansion: a technical review. Eur J Histochem 59(4):2528CrossRefPubMedPubMedCentralGoogle Scholar
  30. Motamed M et al (2017) Tissue engineered human amniotic membrane application in mouse ovarian follicular culture. Ann Biomed Eng 45(7):1664–1675CrossRefPubMedGoogle Scholar
  31. Niknejad H et al (2008) Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater 15:88–99CrossRefPubMedGoogle Scholar
  32. Nubile M et al (2008) Amniotic membrane transplantation for the management of corneal epithelial defects: an in vivo confocal microscopic study. Br J Ophthalmol 92(1):54–60CrossRefPubMedGoogle Scholar
  33. Pappa KI, Anagnou NP (2009) Novel sources of fetal stem cells: where do they fit on the developmental continuum? Regen Med 4(3):423–433CrossRefPubMedGoogle Scholar
  34. Parolini O et al (2008) Concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells. Stem Cells 26(2):300–311CrossRefPubMedGoogle Scholar
  35. Pasquinelli G et al (2007) Ultrastructural characteristics of human mesenchymal stromal (stem) cells derived from bone marrow and term placenta. Ultrastruct Pathol 31(1):23–31CrossRefPubMedGoogle Scholar
  36. Plaks V, Kong N, Werb Z (2015) The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells? Cell Stem Cell 16(3):225–238CrossRefPubMedPubMedCentralGoogle Scholar
  37. Ravi M et al (2015) 3D cell culture systems: advantages and applications. J Cell Physiol 230(1):16–26CrossRefPubMedGoogle Scholar
  38. Saghizadeh M et al (2013) A simple alkaline method for decellularizing human amniotic membrane for cell culture. PLoS ONE 8(11):e79632CrossRefPubMedPubMedCentralGoogle Scholar
  39. Scadden DT (2014) Nice neighborhood: emerging concepts of the stem cell niche. Cell 157(1):41–50CrossRefPubMedPubMedCentralGoogle Scholar
  40. Schleich A et al (1981) Interaction of human carcinoma cells with an epithelial layer and the underlying basement membrane. A new model. Arch Geschwulstforsch 51(1):40–44PubMedGoogle Scholar
  41. Schultz GS, Wysocki A (2009) Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen 17(2):153–162CrossRefPubMedGoogle Scholar
  42. Shaw RJ et al (1990) Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA. J Cell Biol 111(5 Pt 1):2139–2148CrossRefPubMedGoogle Scholar
  43. Shimazaki J, Yang HY, Tsubota K (1997) Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmology 104(12):2068–2076CrossRefPubMedGoogle Scholar
  44. Singh RK et al (1996) Tumor cell invasion of basement membrane in vitro is regulated by amino acids. Cancer Invest 14(1):6–18CrossRefPubMedGoogle Scholar
  45. Solomon A et al (2001) Suppression of interleukin 1alpha and interleukin 1beta in human limbal epithelial cells cultured on the amniotic membrane stromal matrix. Br J Ophthalmol 85(4):444–449CrossRefPubMedPubMedCentralGoogle Scholar
  46. Soncini M et al (2007) Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med 1(4):296–305CrossRefPubMedGoogle Scholar
  47. Talmi YP et al (1991) Antibacterial properties of human amniotic membranes. Placenta 12(3):285–288CrossRefPubMedGoogle Scholar
  48. Toda A et al (2007) The potential of amniotic membrane/amnion-derived cells for regeneration of various tissues. J Pharmacol Sci 105(3):215–228CrossRefPubMedGoogle Scholar
  49. Tseng SC, Li DQ, Ma X (1999) Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J Cell Physiol 179(3):325–335CrossRefPubMedGoogle Scholar
  50. Tsuboi R, Rifkin DB (1990) Bimodal relationship between invasion of the amniotic membrane and plasminogen activator activity. Int J Cancer 46(1):56–60CrossRefPubMedGoogle Scholar
  51. Walasek MA, van Os R, de Haan G (2012) Hematopoietic stem cell expansion: challenges and opportunities. Ann N Y Acad Sci 1266:138–150CrossRefPubMedGoogle Scholar
  52. Wells WJC (1946) Amniotic membrane for corneal grafting. Br Med J 2(4477):624–625CrossRefPubMedCentralGoogle Scholar
  53. Westekemper H et al (2017) Clinical outcomes of amniotic membrane transplantation in the management of acute ocular chemical injury. Br J Ophthalmol 101(2):103–107CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Radwa Ayman Salah
    • 1
  • Ihab K. Mohamed
    • 2
  • Nagwa El-Badri
    • 1
  1. 1.Center of Excellence for Stem Cells and Regenerative MedicineZewail City of Science and TechnologyGizaEgypt
  2. 2.Department of Zoology, Faculty of ScienceAin Shams UniversityCairoEgypt

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