The effect of amniotic membrane extract on umbilical cord blood mesenchymal stem cell expansion: is there any need to save the amniotic membrane besides the umbilical cord blood?

Document Type: Original Article


1 Laboratory for Stem Cell Research, Anatomy Department, Shiraz University of Medical Sciences, Shiraz, Iran

2 Immunology Department, Shiraz University of Medical Sciences, Shiraz, Iran


Objective(s): Umbilical cord blood is a good source of the mesenchymal stem cells that can be banked, expanded and used in regenerative medicine.  The objective of this study was to test whether amniotic membrane extract, as a rich source of growth factors such as basic-fibroblast growth factor, can promote the proliferation potential of the umbilical cord mesenchymal stem cells.
Materials and Methods: The study design was interventional. Umbilical cord mesenchymal stem cells were isolated from voluntary healthy infants from hospitals in Shiraz, Iran, cultured in the presence of basic-fibroblast growth factor and amniotic membrane extracts (from pooled - samples), and compared with control cultures. Proliferation assay was performed and duplication number and time were calculated. The expression of stem cell’s specific markers and the differentiation capacity toward osteogenic and adipogenic lineages were evaluated.
Results: Amniotic membrane extract led to a significant increase in the proliferation rate and duplication number and a decrease in the duplication time without any change in the cell morphology. Both amniotic membrane extract and basic-fibroblast growth factor altered the expressing of CD44 and CD105 in cell population. Treating basic-fibroblast growth factor but not the amniotic membrane extract favored the differentiation potential of the stem cells toward osteogenic lineage.
Conclusion: The amniotic membrane extract administration accelerated cell proliferation and modified the CD marker characteristics which may be due to the induction of differentiation toward a specific lineage.  Amniotic membrane extract may enhance the proliferation rate and duplication number of the stem cell through changing the duplication time.


1. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 1970; 3:393-403.

2. Pittenger  MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143-147.

3.Borhani-Haghighi M, Talaei-Khozani T, Ayatollahi M, Vojdani Z. Wharton's jelly-derived mesenchymal stem cells can differentiate into hepatocyte-like cells by hepg2 cell line extract. Iran J Med Sci 2015; 40:143-151.

4. Ma K, Fox L, Shi G, Shen J, Liu Q, Pappas JD, et al. Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells. Neurol Res 2011; 33:1083-1093.

5. Gabr MM, Zakaria MM, Refaie AF, Ismail AM, Abou-El-Mahasen MA, Ashamallah SA, et al. Insulin-producing cells from adult human bone marrow mesenchymal stem cells control streptozotocin-induced diabetes in nude mice . Cell Transplant  2013; 22:133-145.

6. Taghizadeh  RR, Cetrulo KJ, Cetrulo CL. Wharton’s Jelly stem cells: Future clinical applications. Placenta  2011; 32:S311eS315

7. Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, English D, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U S A 1989; 86:3828-3832.

8. Kögler G, Sensken S, Airey JA, Trapp T, Müschen M, Feldhahn N, et al. New human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 2004; 200:123-135.

9. Malgieri A, Kantzari E, Patrizi MP, Gambardella S. Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art. Int J Clin Exp Med 2010; 3:248–269.

10.Nguyen TH, Paluck SJ, McGahran AJ, Maynard HD. Poly (vinyl sulfonate) Facilitates bFGF-Induced Cell Proliferation. Biomacromolecules 2015; 16:2684-2692.

11.Sotiropoulou PA, Perez SA, Salagianni M, Baxevanis CN, Papamichail M. Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells 2006 ; 24:462-471.

12.Ramasamy R, Tong CK, Yip WK, Vellasamy S, Tan BC, Seow HF. Basic fibroblast growth factor modulates cell cycle of human umbilical cord-derived mesenchymal stem cells. Cell Prolif 2012; 45:132-139.

13.Yang ZL, Cheng K, Han ZD. Effect of bFGF on the MCF-7 Cell Cycle with CD44(+)/CD24(-): Promoting the G0/G1→G2/S Transition. J Breast Cancer 2012; 15:388-392.

14.Ponta H, Sherman L, Herrlich PA. CD44: from adhesion molecules to signaling regulators. Nat Rev Mol Cell Biol  2003; 4:33-45.

15.Marhaba R, Zöller M.  CD44 in cancer progression: adhesion, migration and growth regulation. J Mol Histol  2004; 35:211-231.

16.Jin HJ, Park SK, Oh W, Yang YS, Kim SW, Choi SJ.  Down-regulation of CD105 is associated with multi-lineage differentiation in human umbilical cord blood-derived mesenchymal stem cells. Biochem Biophys Res Commun  2009; 381:676-681.

17.Gougos A, St Jacques S, Greaves A, O'Connell PJ, d'Apice AJF, Buhring H-J, et al.  Identification of distinct epitopes of endoglin, an RGD-containing glycoprotein of endothelial cells, leukemic cells, and syncytiotrophoblasts. Int Immunol 1992; 4:83-92.

18.Fonsatti E, Del Vecchio L, Altomonte M, Sigalotti L, Nicotra MR, Coral S, et al. Endoglin: an accessory component of the TGF-beta-binding receptor-complex with diagnostic, prognostic, and bioimmunotherapeutic potential in human malignancies. J Cell Physiol 2001; 188: 1-7.

19.Sippel KC, Ma JJ, Foster CS. Amniotic membrane surgery. Curr Opin Ophthalmol  2001; 12:69-81.

20.Koizumi NJ, Inatomi TJ, Sotozono CJ, Fullwood NJ, Quantock AJ, Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 2000; 20:173-177.

21.Kim JS, Kim JC, Na BK, Jeong JM, Song CY. Amniotic membrane patching promotes healing and inhibits proteinase activity on wound healing following acute corneal alkali burn. Exp Eye Res 2000; 70:329-337.

22.Niknejad H, Yazdanpanah G, Kakavand M. Extract of fetal membrane would inhibit thrombosis and hemolysis. Med Hypotheses 2015; 85:197-202.

23.Liang L, Li W, Ling S, Sheha H, Qiu W, Li C, et al. Amniotic membrane extraction solution for ocular chemical burns. Clin Exp Ophthalmol 2009; 37:855-863.

24.Koob TJ, Lim JJ, Massee M, Zabek N, Rennert R, Gurtner G, et al. Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration. Vasc Cell 2014; 1; 6:10.

25.Li W, He H, Chen YT, Hayashida Y, Tseng SC.  Reversal of myofibroblasts by amniotic membrane stromal extract. J Cell Physiol  2008; 215:657-664.

26.Dudok DV, Nagdee I, Cheung K, Liu H, Vedovelli L, Ghinelli E, et al. Effects of amniotic membrane extract on primary human corneal epithelial and limbal cells. Clin Exp Ophthalmol 2015; 43:443-448.

27.Kordić R, Suić SP, Jandroković S, Kalauz M, Kuzman T, Skegro I, et al. Application of the amniotic membrane extract (AMX) for the persistent epithelial defect (PED) of the cornea. Coll Antropol 2013; 37:161-164.

28.Korzynska A, Zychowicz MA. Method of estimation of the cell doubling time on basis of the cell culture monitoring data. Biocybern Biomed Eng 2008; 28:75–82.

29.Li IC, Wu SC, Fu J, Chu EH. A deterministic approach for the estimation of mutation rates in cultured mammalian cells. Mutat Res 1985; 149:127-132.

30.Barbero A, Palumberi V, Wagner B, Sader R, Grote MJ, Martin I. Experimental and mathematical study of the influence of growth factors on the growth kinetics of adult human articular chondrocytes. J Cell Physiol  2005; 204:830-838.

31.Tauzin H, Robin S, Humbert P, Viennet C, Saas P, Courderot-Masuyer C, et al. Can leg ulcer fibroblasts  phenotype be influenced by human amniotic membrane extract? Cell Tissue Bank 2014; 15:251-255.

32.Nakagawa H, Akita S, Fukui M, Fujii T, Akino K .  Human mesenchymal stem cells successfully improve skin-substitute wound healing. Br J Dermatol 2005; 153:29-36.

33.Shimabukuro Y, Terashima H, Takedachi M, Maeda K, Nakamura T, Sawada K, et al. Fibroblast growth factor-2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction. J Cell Physiol  2011; 226:809-821.