Differentiation of mesenchymal stem cells isolated from the amniotic membrane and umbilical cord to osteocytes and the expression of RunX2, Osteonectin, ALP genes

Nooshin Barikrow, Amin Tabasi


Mesenchymal stem cells (MSCs) are multipotent cells and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. They show to be very potent tools for tissue engineering and regenerative medicine. Several researches have shown that amniotic membrane mesenchymal stem cells (AM-MSCs) and umbilical cord mesenchymal stem cells (UCB-MSCs) are both multipotent in nature differentiating into several cell types such as adipocytes and osteoblasts. In this study, mesenchymal stem cells were derived from the human amniotic membrane (hAM-dMSCs) and umbilical cord then characterized with their surface antigens using flow cytometry. These cells differentiated to osteocyte and adipocyte in induction medium then the expression of RunX2, Osteonectin, and ALP genes were calculated by Real-Time PCR. We showed that AM-MSCs and UCB-MSCs can discriminate to osteogenic and adipogenic cells in the specific induction medium. The capability of AM-MSCs and UCB-MSCs differentiation to osteogenic cells was confirmed by enhanced expression of RUNX2, ALP and Osteonectin gene and deposition calcium shown by alzerin staning. Given the available evidence, we conclude that AM-MSCs and UCB-MSCs have suitable access, low immunization and lack of medical ethics problems are one of the appropriate sources for differentiation in to osteogenic and adipogenic cells. Also, they can be considered as good choices for treatment of mesenchymal tissue injuries and tissue engineering.


Amniotic membrane, Umblical cord, Osteocyst, Osteonectin, RunX2


Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med. 2003; 349(3):275-86.

Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 1968; 6(2):230-47.

In 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells. 2004; 22(7):1338-45.

Hendrijantini N, Hartono P. Phenotype Characteristics and Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells Derived from Amnion Membrane (HAMSCs) and Umbilical Cord (HUC-MSCs). Acta Inform Med. 2019; 27(2):72-7.

Li J, Zhou Z, Wen J, Jiang F, Xia Y. Human Amniotic Mesenchymal Stem Cells Promote Endogenous Bone Regeneration. Front Endocrinol (Lausanne). 2020; 11:543623.

Huang Z, Nelson ER, Smith RL, Goodman SB. The sequential expression profiles of growth factors from osteoprogenitors [correction of osteroprogenitors] to osteoblasts in vitro. Tissue Eng. 2007; 13(9):2311-20.

Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells. 2004; 22(7):1330-7.

Bieback K, Netsch P. Isolation, Culture, and Characterization of Human Umbilical Cord Blood-Derived Mesenchymal Stromal Cells. Methods Mol Biol. 2016; 1416:245-58.

Ma J, Wu J, Han L, Jiang X, Yan L, Hao J, et al. Comparative analysis of mesenchymal stem cells derived from amniotic membrane, umbilical cord, and chorionic plate under serum-free condition. Stem Cell Res Ther. 2019; 10(1):19.

Mennan C, Wright K, Bhattacharjee A, Balain B, Richardson J, Roberts S. Isolation and characterisation of mesenchymal stem cells from different regions of the human umbilical cord. Biomed Res Int. 2013; 2013:916136.

Wang L, Ott L, Seshareddy K, Weiss ML, Detamore MS. Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells. Regen Med. 2011; 6(1):95-109.

Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K, Hara Y, et al. Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells. 2003; 21(2):217-27.

Chao YH, Wu HP, Chan CK, Tsai C, Peng CT, Wu KH. Umbilical cord-derived mesenchymal stem cells for hematopoietic stem cell transplantation. J Biomed Biotechnol. 2012; 2012:759503.

Lee KO, Gan SU, Calne RY. Stem cell therapy for diabetes. Indian J Endocrinol Metab. 2012; 16(Suppl 2):S227-9.

Shrestha C, Zhao L, Chen K, He H, Mo Z. Enhanced healing of diabetic wounds by subcutaneous administration of human umbilical cord derived stem cells and their conditioned media. Int J Endocrinol. 2013; 2013:592454.

Chicha L, Smith T, Guzman R. Stem cells for brain repair in neonatal hypoxia-ischemia. Childs Nerv Syst. 2014; 30(1):37-46.

Jin YZ, Lee JH. Mesenchymal Stem Cell Therapy for Bone Regeneration. Clin Orthop Surg. 2018; 10(3):271-8.

Li M, Gao J, Feng R, Wang Y, Chen X, Sun J, et al. Generation of monoclonal antibody MS17-57 targeting secreted alkaline phosphatase ectopically expressed on the surface of gastrointestinal cancer cells. PLoS One. 2013; 8(10):e77398.

Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ, VanderWerff I, et al. Immune properties of human umbilical cord Wharton's jelly-derived cells. Stem Cells. 2008; 26(11):2865-74.

Wang Y, Jiang F, Liang Y, Shen M, Chen N. Human Amnion-Derived Mesenchymal Stem Cells Promote Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells by Influencing the ERK1/2 Signaling Pathway. Stem Cells Int. 2016; 2016:4851081.

Sharma RR, Pollock K, Hubel A, McKenna D. Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices. Transfusion. 2014; 54(5):1418-37.

Aubin JE. Regulation of osteoblast formation and function. Rev Endocr Metab Disord. 2001; 2(1):81-94.

Hoemann CD, El-Gabalawy H, McKee MD. In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization. Pathol Biol (Paris). 2009; 57(4):318-23.

Komori T. Regulation of Proliferation, Differentiation and Functions of Osteoblasts by Runx2. Int J Mol Sci. 2019; 20(7):1694.

Huang B, Qian J, Ma J, Huang Z, Shen Y, Chen X, et al. Myocardial transfection of hypoxia-inducible factor-1α and co-transplantation of mesenchymal stem cells enhance cardiac repair in rats with experimental myocardial infarction. Stem Cell Res Ther. 2014; 5(1):22.

Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992; 7(6):683-92.

Bradshaw AD. Diverse biological functions of the SPARC family of proteins. Int J Biochem Cell Biol. 2012; 44(3):480-8.

Murphy-Ullrich JE, Sage EH. Revisiting the matricellular concept. Matrix Biol. 2014; 37:1-14.

Ryan JM, Barry FP, Murphy JM, Mahon BP. Mesenchymal stem cells avoid allogeneic rejection. J Inflamm (Lond). 2005; 2:8.

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

Ji Y, Zhang P, Xing Y, Jia L, Zhang Y, Jia T, et al. Effect of 1α, 25-dihydroxyvitamin D3 on the osteogenic differentiation of human periodontal ligament stem cells and the underlying regulatory mechanism. Int J Mol Med. 2019; 43(1):167-76.

Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 2007; 25(11):2739-49.

Zhang L, Su P, Xu C, Chen C, Liang A, Du K, et al. Melatonin inhibits adipogenesis and enhances osteogenesis of human mesenchymal stem cells by suppressing PPARγ expression and enhancing Runx2 expression. J Pineal Res. 2010; 49(4):364-72.

Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods. 2000; 46(1-2):69-81.

von Bahr L, Batsis I, Moll G, Hägg M, Szakos A, Sundberg B, et al. Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation. Stem Cells. 2012; 30(7):1575-8.

Kalinina N, Kharlampieva D, Loguinova M, Butenko I, Pobeguts O, Efimenko A, et al. Characterization of secretomes provides evidence for adipose-derived mesenchymal stromal cells subtypes. Stem Cell Res Ther. 2015; 6:221.

Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 2008; 8(9):726-36.

Rey A, Manen D, Rizzoli R, Ferrari SL, Caverzasio J. Evidences for a role of p38 MAP kinase in the stimulation of alkaline phosphatase and matrix mineralization induced by parathyroid hormone in osteoblastic cells. Bone. 2007; 41(1):59-67.

Wang Y, Wu H, Shen M, Ding S, Miao J, Chen N. Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells. Exp Cell Res. 2017; 350(1):41-9.

Rafii H, Ruggeri A, Volt F, Brunstein CG, Carreras J, Eapen M, et al. Changing Trends of Unrelated Umbilical Cord Blood Transplantation for Hematologic Diseases in Patients Older than Fifty Years: A Eurocord-Center for International Blood and Marrow Transplant Research Survey. Biol Blood Marrow Transplant. 2016; 22(9):1717-20.

Frank O, Heim M, Jakob M, Barbero A, Schäfer D, Bendik I, et al. Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro. J Cell Biochem. 2002; 85(4):737-46.

Planat-Benard V, Silvestre JS, Cousin B, André M, Nibbelink M, Tamarat R, et al. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004; 109(5):656-63.

Valenti MT, Garbin U, Pasini A, Zanatta M, Stranieri C, Manfro S, et al. Role of ox-PAPCs in the differentiation of mesenchymal stem cells (MSCs) and Runx2 and PPARγ2 expression in MSCs-like of osteoporotic patients. PLoS One. 2011; 6(6):e20363.

Mueller SM, Glowacki J. Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem. 2001; 82(4):583-90.

Rodríguez JP, Astudillo P, Ríos S, Pino AM. Involvement of adipogenic potential of human bone marrow mesenchymal stem cells (MSCs) in osteoporosis. Curr Stem Cell Res Ther. 2008; 3(3):208-18.

Machado do Reis L, Kessler CB, Adams DJ, Lorenzo J, Jorgetti V, Delany AM. Accentuated osteoclastic response to parathyroid hormone undermines bone mass acquisition in osteonectin-null mice. Bone. 2008; 43(2):264-73.

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.

Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem. 1997; 64(2):295-312.

Zhou YS, Liu YS, Tan JG. Is 1, 25-dihydroxyvitamin D3 an ideal substitute for dexamethasone for inducing osteogenic differentiation of human adipose tissue-derived stromal cells in vitro? Chin Med J (Engl). 2006; 119(15):1278-86.

Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci. 2000; 113 ( Pt 7):1161-6.

Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002; 418(6893):41-9.

Maeno T, Moriishi T, Yoshida CA, Komori H, Kanatani N, Izumi S, et al. Early onset of Runx2 expression caused craniosynostosis, ectopic bone formation, and limb defects. Bone. 2011; 49(4):673-82.

Komori T. Roles of Runx2 in Skeletal Development. Adv Exp Med Biol. 2017; 962:83-93.

Kawane T, Qin X, Jiang Q, Miyazaki T, Komori H, Yoshida CA, et al. Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation by regulating Fgfr2 and Fgfr3. Sci Rep. 2018; 8(1):13551.

Pawłowska E, Wójcik KA, Synowiec E, Szczepańska J, Błasiak J. Expression of RUNX2 and its signaling partners TCF7, FGFR1/2 in cleidocranial dysplasia. Acta Biochim Pol. 2015; 62(1):123-6.

Komori T. Molecular Mechanism of Runx2-Dependent Bone Development. Mol Cells. 2020; 43(2):168-75.

Qin X, Jiang Q, Miyazaki T, Komori T. Runx2 regulates cranial suture closure by inducing hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in suture mesenchymal cells. Hum Mol Genet. 2019; 28(6):896-911.

Štefková K, Procházková J, Pacherník J. Alkaline phosphatase in stem cells. Stem Cells Int. 2015; 2015:628368.

Kermer V, Ritter M, Albuquerque B, Leib C, Stanke M, Zimmermann H. Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation. Neurosci Lett. 2010; 485(3):208-11.

Nguyen L, Malgrange B, Breuskin I, Bettendorff L, Moonen G, Belachew S, et al. Autocrine/paracrine activation of the GABA(A) receptor inhibits the proliferation of neurogenic polysialylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum. J Neurosci. 2003; 23(8):3278-94.

Andäng M, Hjerling-Leffler J, Moliner A, Lundgren TK, Castelo-Branco G, Nanou E, et al. Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation. Nature. 2008; 451(7177):460-4.

Yang K, Tang XD, Guo W, Xu XL, Ren TT, Ren CM, et al. BMPR2-pSMAD1/5 signaling pathway regulates RUNX2 expression and impacts the progression of dedifferentiated chondrosarcoma. Am J Cancer Res. 2016; 6(6):1302-16.

Franceschi RT, Xiao G. Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J Cell Biochem. 2003; 88(3):446-54.

Gaur T, Lengner CJ, Hovhannisyan H, Bhat RA, Bodine PV, Komm BS, et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem. 2005; 280(39):33132-40.

Datta P, Ghosh P, Ghosh K, Maity P, Samanta SK, Ghosh SK, et al. In vitro ALP and osteocalcin gene expression analysis and in vivo biocompatibility of N-methylene phosphonic chitosan nanofibers for bone regeneration. J Biomed Nanotechnol. 2013; 9(5):870-9.

Hassan MQ, Tare R, Lee SH, Mandeville M, Weiner B, Montecino M, et al. HOXA10 controls osteoblastogenesis by directly activating bone regulatory and phenotypic genes. Mol Cell Biol. 2007; 27(9):3337-52.

Golub EE, Boesze-Battaglia K. The role of alkaline phosphatase in mineralization. Curr Opin Orthop. 2007; 18(5):444-8.

Hassan MQ, Tare RS, Lee SH, Mandeville M, Morasso MI, Javed A, et al. BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network. J Biol Chem. 2006; 281(52):40515-26.

Li K, Han J, Wang Z. Histone modifications centric-regulation in osteogenic differentiation. Cell Death Discov. 2021; 7(1):91.

Khorshied MM, Gouda HM, Shaheen IA, Al Bolkeny TN. The osteogenic differentiation potentials of umbilical cord blood hematopoietic stem cells. Comp Clin Pathol. 2012; 21(4):441-7.

Krukiewicz K, Putzer D, Stuendl N, Lohberger B, Awaja F. Enhanced Osteogenic Differentiation of Human Primary Mesenchymal Stem and Progenitor Cultures on Graphene Oxide/Poly(methyl methacrylate) Composite Scaffolds. Materials (Basel). 2020; 13(13).

Hankenson KD, James IE, Apone S, Stroup GB, Blake SM, Liang X, et al. Increased osteoblastogenesis and decreased bone resorption protect against ovariectomy-induced bone loss in thrombospondin-2-null mice. Matrix Biol. 2005; 24(5):362-70.

Delany AM, Hankenson KD. Thrombospondin-2 and SPARC/osteonectin are critical regulators of bone remodeling. J Cell Commun Signal. 2009; 3(3-4):227-38.

Mansergh FC, Wells T, Elford C, Evans SL, Perry MJ, Evans MJ, et al. Osteopenia in Sparc (osteonectin)-deficient mice: characterization of phenotypic determinants of femoral strength and changes in gene expression. Physiol Genomics. 2007; 32(1):64-73.

Phimphilai M, Zhao Z, Boules H, Roca H, Franceschi RT. BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype. J Bone Miner Res. 2006; 21(4):637-46.

Delany AM, Amling M, Priemel M, Howe C, Baron R, Canalis E. Osteopenia and decreased bone formation in osteonectin-deficient mice. J Clin Invest. 2000; 105(7):915-23.

Delany AM, Kalajzic I, Bradshaw AD, Sage EH, Canalis E. Osteonectin-null mutation compromises osteoblast formation, maturation, and survival. Endocrinology. 2003; 144(6):2588-96.

Lin GL, Hankenson KD. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem. 2011; 112(12):3491-501.


  • There are currently no refbacks.

Copyright (c) 2022 © The Author(s)

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.