Motor neurons differentiation of encapsulated human endometrial stem cells in collagen without HLA-DR expression

  • Aazam Rahimi
  • Amir Nahanmoghadam
  • Jafar Ai
  • Navid Gholami
  • Robab Ebrahimi-Barough
  • Somayeh Ebrahimi-Barough
Keywords: Human endometrial stem cell, Purmorphamine, Collagen, Scaffold, HLA-DR


Background: Human endometrial stem cells (hEnSCs) are attractive cells in regenerative medicine and neurons derived from EnSCs are one of the best targets for cell therapy due to the fact that they do not stimulate immune system. Small molecules as useful chemical tools can effectively change cell fate. Thus, these small molecules provide a promising strategy for differentiation of cells in regenerative medicine. Purmorphamine (PMA) is a small molecule that possesses certain differentiation effects by activating SHH signaling pathway.  In this study, we used PMA as a small molecule to differentiate motor neurons from hEnSCs by using collagen polymer.

Methods and Materials: hEnSCs were cultured in differentiation medium containing 1µM PMA in 2D and 3D environments. Scanning electron microscope (SEM) was used for cell attachment and cell viability was assessed by and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT). Immunocytochemistry was performed to confirm the expression of islet-1 and Acetylcholine transferase (Chat). Real-time PCR also confirmed for expression of neural markers such as NF, Chat, islet-1, HB9.

Results: Therefore, our data revealed that induced hEnSCs with PMA could significantly express motor neuron markers in RNA and protein levels. Further, flow cytometry was done for the expression of the cell surface antigen HLA-DR in endometrial stem cells and motor neurons derived from these cells. The results confirmed that hEnSCs and motor neurons derived from them do not express HLA-DR. Our motor neurons differentiation protocol provides an accessible system to provide motor neurons that do not stimulate immune system.

Conclusion: In conclusion, encapsulation of hEnSCs in collagen along with induction media containing PMA have potential for being used in neural tissue engineering through activation of the SHH signaling pathway.


Key words: Human endometrial stem cell, Purmorphamine, Collagen, Scaffold, HLA-DR


Wagers AJ, Weissman IL. Plasticity of Adult Stem Cells. J Cell. 2004;116: 639-648.

Henningson CT, Stanislaus MA, Gewirtz MA. Embryonic and adult stem cell therapy. Allergy Clin Immunol. 2003; 111:745-753.

Fernandez VB, Romaniuk MA, Choi H, Labovsky V, Otaegui J, Chasseing NA. Mesenchymal stem cells and their use in therapy: what has been achieved? Differentiation.2013; 85:1-10.

Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 2012;284:143-147.

Hass H, Kasper C, Bohm S, Jacobs R. Different populations and sources of human mesenchymal stem cells: A comparison of adult and neonatal tissue-derived MSC. Cell Communi Signal. 2011;9:1-14.

Faghihi F, Mirzaei E, Ai J, Lotfi A, Sayahpour FA, Ebrahimi-Barough S, Joghataei MT. Differentiation Potential of Human Chorion-Derived Mesenchymal Stem Cells into Motor Neuron-Like Cells in Two- and Three-Dimensional Culture Systems. Mol Neurobiol. 2016 Apr;53(3):1873.

Lee HJ, Kim KS, Ahn J, Bae HM, Lim J, Kim SU. Homan motor neurons generated from neural stem cells delay clinical onset and prolong life in ALS mouse model. PLOS ONE. 2014; 9:1-9.

Santamaria X, Mas A, Cervelló I, Taylor H, Simon C.Uterine stem cells: from basic research to advanced cell therapies. Hum Reprod Update. 2018 Nov 1;24(6):673-693.

Mutlu L, Hufnagel D, Taylor H.S. The endometrium as a source of mesenchymal stem cells for regenerative medicine. Biology of Reprod. 2015; 138: 1-11.

Darzi S, Werkmeiser JA, Deane JA, Gargett C E. Identification and characterization of human endometrial mesenchymal stem cells and their potential for cellular therapy. Stem cells translational medicine. 2016; 5: 1127-1132.

Figueira PGM, Abrao M.S, Krikun, G, Taylor H. Stem cell in endometrium and their role in the pathogenesis of endometriosis. National Institutes of Health. 2011; 1221:10-17.

Park J, Lee N, Lee J, Choi EK, Kim MK, Lee J, Byun MS, Chon MW, Kim SW, Lee C J, Kim J.H, Kwon J.S, Chang M.S. Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons. Scientific Reports. 2017; 7:1-14.

Ebrahimi-Barough S, Hoveizi E, Norouzi Javidan A, Ai J. Investigating the neuroglial differentiation effect of neuroblastoma conditioned medium in human endometrial stem cells cultured on 3D nanofibrous scaffold. JBMR A. 2015; 103(8):2621-7.

Drannik A, Martin J, Peterson R, Ma X, Jiang, F. Cerebrospinal fluid from patient with amyotrophic lateral sclerosis inhibits sonic hedgehog function. PLOS ONE.2017; 12:1-20.

Bahrami N, Bayat M, Mohamadnia A, Khakbiz M, Yazdankhah M, Ai J, Ebrahimi-Barough S. Purmorphamine as a Shh Signaling Activator Small Molecule Promotes Motor Neuron Differentiation of Mesenchymal Stem Cells Cultured on Nanofibrous PCL Scaffold.Mol Neurobiol. 2017;54(7):5668-5675.

Sanchez MA, Armstrong RC.Postnatal Sonic hedgehog (Shh) responsive cells give rise to oligodendrocyte lineage cells during myelination and in adulthood contribute to remyelination. Exp Neurol. 2018;299(Pt A):122-136.

Castillo-Azofeifa D, Losacco JT, Salcedo E, Golden EJ, Finger TE, Barlow LA. Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance. Development. 2017; 1:144(17):3054-3065.

Tempest N, Maclean A, Hapangama DK. Endometrial Stem Cell Markers: Current Concepts and Unresolved Questions.Int J Mol Sci. 2018; 19:19(10).

Engel M, Do-Ha D, Munoz SS, Oie L. Common pitfalls of stem cell differentiation: a guide to improving protocols for neurodegenerative disease models and research. Cellular and Molecular Life Sciences. 2016;73: 3693-3709.

Epstein S, Gadaria-Rathod N, Wei Y, Maguire MG, Asbell PA. HLA-DR expression as a biomarker of inflammation for multicenter clinical trials of ocular surface disease. National Institutes of Health. 2013;11:95-104.

Jucaude V. The immunogenicity of HLA class II mismatches: the predicted presentation of nonself allo-HLA-derived peptide by the HLA-DR Phenotype of the recipient is associated with the formation of DSA. Journal of Immunology Research. 2017; 14: 1-12.

Wong FS, Wong CC, Chan BP, Lo AC. Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model. PLoS One. 2016 Jul 21;11(7):e0159342.

Ugbode CI, Smith I, Walley BJ, Hirst WD, Rattray M. Sonic hedgehog signaling mediates astrocyte crosstalk with neurons to comfer neuroprotection. Journal of Neurochemistry. 2017; 142:429-443.

Li X, Bai J, Ji X, Li R, Xuan Y., Wang, Y, Wang Y. Comprehensive characterization of four different populations of human mesenchymal stem cells as regards their immune properties, proliferation and differentiation. International Journal of molecular medicine. 2014; 34:695-704.

Lucas DP, Leffell M, Zachary A. Differences in immunogenicity of HLA antigens and the impact of cross-reactivity on the humoral response. Transplantation. 2015; 99:78-86.

Wieczorek M, Abualrous E, Sticht J, Alvaro-benito M, Stolzenberg S, Noe F, Freund C. Major Histocampatibility complex (MHC) class I and class II proteins: conformational plasticity in antigen presentation. Frontiers in Immunology. 2017; 8:1-16.

Zachary A, Leffell M. HLA Mismatching sterategies for solid organ transplantation-a balancing act. Frontiers in Immunology. 2016; 7: 1-14.

Original Articles