A Rapid and Cost-Effective Protocol for Isolating Mesenchymal Stem Cells from the Human Amniotic Membrane

Seyed Mohammad Taghi Razavi Tousi, Naser Amirizadeh, Farinaz Nasirinezhad, Mahin Nikougoftar, Meysam Ganjibakhsh, Nahid Aboutaleb

Background: Regarding the role of mesenchymal stem cells (MSCs) in regenerative medicine, many studies have been conducted to isolate these cells from various sources. In this study, a method was developed which will use only one enzyme and in the shortest time MSCs will be isolated from the amniotic membranes and expanded. Materials and Methods: The amniotic membrane (AM) was mechanically separated from the underlying membrane called chorionic. Then, the AM was sliced into tiny pieces and to isolate MSCs, it was digested only using collagens I instead of applying various enzymes. The isolated cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% fetal bovine serum. The expression profiles of several markers at isolated cells were determined by flow cytometry. To assess the differentiation potential, the isolated cells were cultured in osteogenic and adipogenic induction media. Results: The results indicated that cells isolated from the AM expressed markers of CD44, CD105 and CD166 mesenchymal cells, but did not express CD34 and CD45 hematopoietic markers. The osteoblastic differentiation of the isolated cells was proven by alizarin red and alkaline phosphatase staining methods, whereas the adipogenic differentiation of the isolated cells was proven by Oil Red-O staining. Conclusion: The results of the study indicated that the isolated cells were of the MSCs family. Furthermore, it was demonstrated that MSCs can be obtained easily only by spending a short time and using one enzyme.[GMJ.2017;6(3):217-225] DOI: 10.22086/gmj.v0i0.670

Mesenchymal Stem Cell; Amniotic Membrane; Protocol

Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int. J. Biochem. Cell Biol. 2004; 36(4):568-84.

Lv F-J, Tuan RS, Cheung K, Leung VY. Concise review: the surface markers and identity of human mesenchymal stem cells. Stem Cells. 2014; 32(6):1408-19.

Lu L-L, Liu Y-j, Yang S-G, Zhao Q-J, Wang X, Gong W, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica. 2006; 91(8):1017-26.

Miao Z, Jin J, Chen L, Zhu J, Huang W, Zhao J, et al. Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells. Cell Biol Int. 2006; 30(9):681-7.

Scherjon SA, Kleijburg‐van der Keur C, de Groot‐Swings GM, Claas FH, Fibbe WE, Kanhai HH. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells. 2004; 22(7):1338-45.

Cao Y, Sun Z, Liao L, Meng Y, Han Q, Zhao RC. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem Biophys Res Commun. 2005; 332(2):370-9.

Tsuji H, Miyoshi S, Ikegami Y, Hida N, Asada H, Togashi I, et al. Xenografted human amniotic membrane–derived mesenchymal stem cells are immunologically tolerated and transdifferentiated into cardiomyocytes. Circul Res. 2010; 106(10):1613-23.

Issaragrisil S. Isolation, characterization and neural differentiation potential of amnion derived mesenchymal stem cells. J Med Assoc Thai. 2010; 93(7):S183-S91.

Soncini M, Vertua E, Gibelli L, Zorzi F, Denegri M, Albertini A, et al. Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med. 2007; 1(4):296-305.

Mihu CM, Rus Ciuca D, Soritau O, Susman S, Mihu D. Isolation and characterization of mesenchymal stem cells from the amniotic membrane. Rom J Morphol Embryol. 2009; 50(1):73-7.

Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284(5411):143-7.

Sudo K, Kanno M, Miharada K, Ogawa S, Hiroyama T, Saijo K, et al. Mesenchymal progenitors able to differentiate into osteogenic, chondrogenic, and/or adipogenic cells in vitro are present in most primary fibroblast‐like cell populations. Stem Cells. 2007; 25(7):1610-7.

Paracchini V, Carbone A, Colombo F, Castellani S, Mazzucchelli S, Di Gioia S, et al. Amniotic mesenchymal stem cells: a new source for hepatocyte-like cells and induction of CFTR expression by coculture with cystic fibrosis airway epithelial cells. BioMed Research International. 2012; 2012.

Alviano F, Fossati V, Marchionni C, Arpinati M, Bonsi L, Franchina M, et al. Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro. BMC Dev Biol. 2007; 7(1):11.

Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B. Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 2006; 7(1):14.

Nöth U, Steinert AF, Tuan RS. Technology insight: adult mesenchymal stem cells for osteoarthritis therapy. Nat Rev Rheumatol. 2008;4(7): 371-80.

Chen FH, Tuan RS. Mesenchymal stem cells in arthritic diseases. Arthritis Res Ther. 2008; 10(5):223.

Zhang D, Jiang M, Miao D. Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse. PLoS One. 2011; 6(2):e16789.

Bačenková D, Rosocha J, Tóthová T, Rosocha L, Šarisský M. Isolation and basic characterization of human term amnion and chorion mesenchymal stromal cells. Cytotherapy. 2011; 13(9):1047-56.

Lisi A, Briganti E, Ledda M, Losi P, Grimaldi S, Marchese R, et al. A combined synthetic-fibrin scaffold supports growth and cardiomyogenic commitment of human placental derived stem cells. PLoS One. 2012; 7(4):e34284.

Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8(4):315-7.

Lee KD, Kuo TKC, Whang‐Peng J, Chung YF, Lin CT, Chou SH, et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology. 2004; 40(6):1275-84.


  • There are currently no refbacks.