Human Placenta Laminin-111 as a Multifunctional Protein for Tissue Engineering and Regenerative Medicine

  • Johannes HackethalEmail author
  • Christina M. A. P. Schuh
  • Alexandra Hofer
  • Barbara Meixner
  • Simone Hennerbichler
  • Heinz Redl
  • Andreas H. Teuschl
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1077)


Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield.

Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells.

In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.


Laminin-111 Placenta Schwann cells NG 108-15 Vasculogenesis 



The authors acknowledge Red Cross Blood Transfusion Service, Linz, Upper Austria for providing the placenta tissue, Dr. Wolfgang Holnthoner, Severin Mühleder, MSc, for providing the HUVEC and Mag. med. vet. James Ferguson for reviewing the manuscript. This work was partially funded by the City of Vienna Competence Team Tissue Engineering Signaltissue (MA23 Project-#18-08).

Disclosure Statement



  1. 1.
    Aumailley M (2013) The laminin family. Cell Adhes Migr 7:48–55CrossRefGoogle Scholar
  2. 2.
    Simon-Assmann (2013) The laminin family. Cell Adhes Migr 7:44–47CrossRefGoogle Scholar
  3. 3.
    Ekblom P, Lonai P, Talts JF (2003) Expression and biological role of laminin-1. Matrix Biol 22:35–47CrossRefGoogle Scholar
  4. 4.
    Hozumi K et al (2012) Reconstitution of laminin-111 biological activity using multiple peptide coupled to chitosan scaffolds. Biomaterials 33:4241–4250CrossRefGoogle Scholar
  5. 5.
    Ponce ML, Kleinman HK (2003) Identification of redundant angiogenic sites in laminin α1 and γ1 chains. Exp Cell Res 285:189–195CrossRefGoogle Scholar
  6. 6.
    Iorio V, Troughton LD, Hamill KJ (2015) Laminins: roles and utility in wound repair. Adv Wound Care 4:250–263CrossRefGoogle Scholar
  7. 7.
    Flanagan L a, Rebaza LM, Derzic S, Schwartz PH, Monuki ES (2006) Regulation of human neural precursor cells by laminin and integrins. J Neurosci Res 83:845–856CrossRefGoogle Scholar
  8. 8.
    Madison R, da Silva CF, Dikkes P, Chiu TH, Sidman RL (1985) Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and a laminin-containing gel. Exp Neurol 88:767–772CrossRefGoogle Scholar
  9. 9.
    Bilozur ME, Hay ED (1988) Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen. Dev Biol 125:19–33CrossRefGoogle Scholar
  10. 10.
    Rangappa N, Romero A, Nelson KD, Eberhart RC, Smith GM (2000) Laminin-coated poly(L-lactide) filaments induce robust neurite growth while providing directional orientation. J Biomed Mater Res 51:625–634CrossRefGoogle Scholar
  11. 11.
    Biernaskie J et al (2007) Skin-derived precursors generate myelinating Schwann cells that promote remyelination and functional recovery after contusion spinal cord injury. J Neurosci 27:9545–9559CrossRefGoogle Scholar
  12. 12.
    Kidd KR, Williams SK (2004) Laminin-5-enriched extracellular matrix accelerates angiogenesis and neovascularization in association with ePTFE. J Biomed Mater Res A 69:294–304CrossRefGoogle Scholar
  13. 13.
    Malinda KM, Wysocki AB, Koblinski JE, Kleinman HK, Ponce ML (2008) Angiogenic laminin-derived peptides stimulate wound healing. Int J Biochem Cell Biol 40:2771–2780CrossRefGoogle Scholar
  14. 14.
    Min SK et al (2010) The effect of a laminin-5-derived peptide coated onto chitin microfibers on re-epithelialization in early-stage wound healing. Biomaterials 31:4725–4730CrossRefGoogle Scholar
  15. 15.
    Hashimoto T, Suzuki Y, Tanihara M, Kakimaru Y, Suzuki K (2004) Development of alginate wound dressings linked with hybrid peptides derived from laminin and elastin. Biomaterials 25:1407–1414CrossRefGoogle Scholar
  16. 16.
    Gjorevski N et al (2016) Designer matrices for intestinal stem cell and organoid culture. Nat Publ Gr 539:560–564CrossRefGoogle Scholar
  17. 17.
    Horejs C-M et al (2014) Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells. Proc Natl Acad Sci USA 111:5908–5913CrossRefGoogle Scholar
  18. 18.
    Timpl R, Rohde H, Robey PG, Rennard SI, Foidart JM, Martin GR (1979) Laminin-A glycoprotein from basement membranes. Biochemistry 21:6188–6193Google Scholar
  19. 19.
    Aumailley M, Smyth N (1998) The role of laminins in basement membrane function. J Anat 193(Pt 1):1–21CrossRefGoogle Scholar
  20. 20.
    Brown JC, Spragg JH, Wheeler GN, Taylor PW (1990) Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins. Biochem J 270:463–468CrossRefGoogle Scholar
  21. 21.
    Brown JC, Wiedemann H, Timpl R (1994) Protein binding and cell adhesion properties of two laminin isoforms (AmB1eB2e, AmB1sB2e) from human placenta. J Cell Sci 107(Pt 1):329–338PubMedGoogle Scholar
  22. 22.
    Wewer U et al (1983) Human laminin isolated in a nearly intact, biologically active form from placenta by limited proteolysis. J Biol Chem 258(20):12654–12660PubMedGoogle Scholar
  23. 23.
    Engvall E, Earwicker D, Haaparanta T, Ruoslahti E, Sanes JR (1990) Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits. Cell Regul 1:731–740CrossRefGoogle Scholar
  24. 24.
    Champliaud MF et al (2000) Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin-3 from placenta. Exp Cell Res 259:326–335CrossRefGoogle Scholar
  25. 25.
    Cameron K et al (2015) Recombinant laminins drive the differentiation and self-organization of hESC-derived hepatocytes. Stem Cell Rep 5:1250–1262CrossRefGoogle Scholar
  26. 26.
    Miyazaki T et al (2008) Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem Biophys Res Commun 375:27–32CrossRefGoogle Scholar
  27. 27.
    Wondimu Z et al (2006) Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (α2β1γ1), 8 (α4β1γ1), 10 (α5β1γ1). Matrix Biol 25:89–93CrossRefGoogle Scholar
  28. 28.
    Weihs AM et al (2014) Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation. J Biol Chem 289:27090–27104CrossRefGoogle Scholar
  29. 29.
    Duhamel RC (1983) Differential staining of collagens and non-collagens with Coomassie brilliant blue G and R. Coll Relat Res 3:195–204CrossRefGoogle Scholar
  30. 30.
    Hackethal J et al (2017) An effective method of Atelocollagen type 1/3 isolation from human placenta and its in vitro characterization in two-dimensional and three-dimensional cell culture applications. Tissue Eng Part C Methods 23:274–285CrossRefGoogle Scholar
  31. 31.
    Schuh CMAP et al (2016) Extracorporeal shockwave treatment: a novel tool to improve Schwann cell isolation and culture. Cytotherapy 18:760–770CrossRefGoogle Scholar
  32. 32.
    Kaewkhaw R, Scutt AM, Haycock JW (2012) Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue. Nat Protoc 7:1996–2004CrossRefGoogle Scholar
  33. 33.
    Holnthoner W et al (2012) Adipose-derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix. J Tissue Eng Regen Med 4:524–531Google Scholar
  34. 34.
    Rohringer S et al (2017) The impact of wavelengths of LED light-therapy on endothelial cells. Sci Rep 7:1–11CrossRefGoogle Scholar
  35. 35.
    Holnthoner W et al (2017) Endothelial cell-derived extracellular vesicles size-dependently exert procoagulant activity detected by thromboelastometry. Sci Rep 7:1–9CrossRefGoogle Scholar
  36. 36.
    Knezevic L et al (2017) Engineering blood and lymphatic microvascular networks in fibrin matrices. Front Bioeng Biotechnol 5:1–12CrossRefGoogle Scholar
  37. 37.
    Arnaoutova I, Kleinman HK (2010) In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc 5:628–635CrossRefGoogle Scholar
  38. 38.
    Uriel S et al (2009) Extraction and assembly of tissue-derived gels for cell culture and tissue engineering. Tissue Eng Part C Methods 15:309–321CrossRefGoogle Scholar
  39. 39.
    Goodwin AM (2007) In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents. Microvasc Res 74:172–183CrossRefGoogle Scholar
  40. 40.
    Sorkio A et al (2014) Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating. Tissue Eng Part A 20:622–634PubMedPubMedCentralGoogle Scholar
  41. 41.
    Takayama K et al (2013) Long-term self-renewal of human ES/iPS-derived hepatoblast-like cells on human laminin 111-coated dishes. Stem Cell Rep 1:332–335CrossRefGoogle Scholar
  42. 42.
    Rodin S, Antonsson L, Hovatta O, Tryggvason K (2014) Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions. Nat Protoc 9:2354–2368CrossRefGoogle Scholar
  43. 43.
    Zou K, DeLisio M (2014) Laminin-111 improves skeletal muscle stem cell quantity and function following eccentric exercise. Stem Cells Transl Med 3:1013–1022CrossRefGoogle Scholar
  44. 44.
    Goudenege S et al (2010) Laminin-111: a potential therapeutic agent for Duchenne muscular dystrophy. Mol Ther 18:2155–2163CrossRefGoogle Scholar
  45. 45.
    Flynn L, Hrabchak C, Woodhouse KA (2006) Biological skin substitutes for wound cover and closure. Expert Rev Med Devices 3(3):1–20Google Scholar
  46. 46.
    Pacak C a, MacKay A, Cowan DB (2014) An improved method for the preparation of Type I collagen from skin. J Vis Exp 83:e51011Google Scholar
  47. 47.
    Browne S, Zeugolis DI, Pandit A (2013) Collagen: finding a solution for the source. Tissue Eng Part A 19:1491–1494CrossRefGoogle Scholar
  48. 48.
    Schneider KH et al (2016) Decellularized human placenta chorion matrix as a favorable source of small-diameter vascular grafts. Acta Biomater 29:125–134CrossRefGoogle Scholar
  49. 49.
    Wang Y, Zhao S (2010) Vascular biology of the placenta, vol 2. Morgan & Claypool LIFE Sciences, San Rafael, p 1Google Scholar
  50. 50.
    Lobo SE et al (2016) The placenta as an organ and a source of stem cells and extracellular matrix: a review. Cells Tiss Org 270:239–252CrossRefGoogle Scholar
  51. 51.
    Burgos H, Herd A, Bennett JP (1989) Placental angiogenic and growth factors in the treatment of chronic varicose ulcers: preliminary communication. J R Soc Med 82:598–599CrossRefGoogle Scholar
  52. 52.
    Shukla VK, Rasheed M a, Kumar M, Gupta SK, Pandey SS (2004) A trial to determine the role of placental extract in the treatment of chronic non-healing wounds. J Wound Care 13:177–179CrossRefGoogle Scholar
  53. 53.
    Navadiya SK, Vaghani YL, Patel MP (2012) Study of topical placental extract versus povodine iodine and saline dressing in various diabetic wounds. Nat J Med Res 2:411–413Google Scholar
  54. 54.
    Place ES, Evans ND, Stevens MM (2009) Complexity in biomaterials for tissue engineering. Nat Mater 8:457–470CrossRefGoogle Scholar
  55. 55.
    Kleinman HK et al (1982) Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:6188–6193CrossRefGoogle Scholar
  56. 56.
    Kubota Y, Kleinman HK, Martin GR, Lawley TJ (1988) Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 107:1589–1598CrossRefGoogle Scholar
  57. 57.
    Timpl R, Martin GR, Bruckner P, Wick G, Wiedemann H (1978) Nature of the collagenous protein in a tumor basement membrane. Eur J Biochem 84:43–52CrossRefGoogle Scholar
  58. 58.
    MacWright RS, Benson V a, Lovello KT, van der Rest M, Fietzek PP (1983) Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides. Biochemistry 22:4940–4948CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Johannes Hackethal
    • 1
    • 2
    Email author
  • Christina M. A. P. Schuh
    • 1
    • 2
    • 3
  • Alexandra Hofer
    • 4
  • Barbara Meixner
    • 1
    • 2
  • Simone Hennerbichler
    • 2
    • 5
  • Heinz Redl
    • 1
    • 2
  • Andreas H. Teuschl
    • 2
    • 6
  1. 1.Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in AUVA Trauma Research CenterViennaAustria
  2. 2.Austrian Cluster for Tissue RegenerationViennaAustria
  3. 3.Laboratory of Nano-Regenerative Medicine, Faculty of Medicine, Cells for CellsUniversidad de Los AndesSantiagoChile
  4. 4.Research Area Biochemical EngineeringInstitute of Chemical Engineering, Vienna University of TechnologyViennaAustria
  5. 5.Red Cross Blood Transfusion Service of Upper AustriaLinzAustria
  6. 6.Department of Biochemical EngineeringUniversity of Applied Sciences Technikum WienViennaAustria

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