Methodology of neuronal pluripotent stem cell of in vitro difference process as an alternative therapy of neurodegenerative disease


  • Wahyuni Wulansari Universitas 'Asiyiyah Yogyakarta
  • Fuad Gandhi Torizal Tokyo Medical and Dental University
  • Annisa Khumaira Universitas 'Asiyiyah Yogyakarta
  • Ika Afifah Nugraheni Universitas 'Asiyiyah Yogyakarta



Neuronal stem cell, pluripotent stem cell, differentiation technique


Neurodegenerative is diseases which occur with age but no therapeutic approach is efficient enough to inhibit neurodegenerative progression which results from aging of the cells themselves. PSC is a stem cell which has the potential to differentiate into various types of cells in the body. In the expansion process, PSC encountered problems, one of the common obstacles. This research was conducted using a literature study method that aims to obtain information about the appropriate differentiation method so that it can be used as an alternative treatment for neurodegenerative disease in the future. The result showed that 3D technique is neuronal differentiation technique that has a good output and can resemble the conditions of its development in vivo. The use of growth factors such as FGF2, PDGF, IGF1, T3, FGF, EGF, and CNTF can also overcome the problem of differentiation of several target cells in neurodegenerative therapy such as neurons, astrocytes, and oligodendrocytes. 


Alghuwainem, A., Alshareeda, A. T., & Alsowayan, B. (2019, Oct 4). Scaffold-Free 3-D Cell Sheet Technique Bridges the Gap between 2-D Cell Culture and Animal Models. Int J Mol Sci, 20(19).

Amira Ragab El Barky, E. A., Tarek M Mohamed. (2017). Stem Cells, Classifications and their Clinical Applications. . Stem cells and development.

Antoni, D., Burckel, H., Josset, E., & Noel, G. (2015, Mar 11). Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci, 16(3), 5517-5527.

Araki, T., Ikegaya, Y., & Koyama, R. (2021, Sep). The effects of microglia- and astrocyte-derived factors on neurogenesis in health and disease. Eur J Neurosci, 54(5), 5880-5901.

Aurélie de Rus Jacquet, H. L. D., Francesca Cicchetti, Melanie Alpaugh. (2021). Current and future applications of induced pluripotent stem cell-based models to study pathological proteins in neurodegenerative disorders. Molecular Psychiatry, 26, pages2685–2706.

Avila, A., Vidal, P. M., Tielens, S., Morelli, G., Laguesse, S., Harvey, R. J., Rigo, J. M., & Nguyen, L. (2014, Nov). Glycine receptors control the generation of projection neurons in the developing cerebral cortex. Cell Death Differ, 21(11), 1696-1708.

Bourgognon, J. M., Spiers, J. G., Scheiblich, H., Antonov, A., Bradley, S. J., Tobin, A. B., & Steinert, J. R. (2018, Aug). Alterations in neuronal metabolism contribute to the pathogenesis of prion disease. Cell Death Differ, 25(8), 1408-1425.

Cabezas, R., Avila-Rodriguez, M., Vega-Vela, N. E., Echeverria, V., Gonzalez, J., Hidalgo, O. A., Santos, A. B., Aliev, G., & Barreto, G. E. (2016). Growth Factors and Astrocytes Metabolism: Possible Roles for Platelet Derived Growth Factor. Med Chem, 12(3), 204-210.

Carpenter, M. K., Inokuma, M. S., Denham, J., Mujtaba, T., Chiu, C. P., & Rao, M. S. (2001, Dec). Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol, 172(2), 383-397.

Centeno, E. G. Z., Cimarosti, H., & Bithell, A. (2018, May 22). 2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegenerative disease modelling. Mol Neurodegener, 13(1), 27.

Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009, Mar). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol, 27(3), 275-280.

Chandrasekaran, A., Avci, H. X., Leist, M., Kobolak, J., & Dinnyes, A. (2016). Astrocyte Differentiation of Human Pluripotent Stem Cells: New Tools for Neurological Disorder Research. Front Cell Neurosci, 10, 215.

Chang, C. Y., Ting, H. C., Liu, C. A., Su, H. L., Chiou, T. W., Harn, H. J., & Lin, S. Z. (2018, Nov). Induced Pluripotent Stem Cells: A Powerful Neurodegenerative Disease Modeling Tool for Mechanism Study and Drug Discovery. Cell Transplant, 27(11), 1588-1602.

Cunha, C., Panseri, S., & Antonini, S. (2011, Feb). Emerging nanotechnology approaches in tissue engineering for peripheral nerve regeneration. Nanomedicine, 7(1), 50-59.

de la Torre-Ubieta, L., & Bonni, A. (2011, Oct 6). Transcriptional regulation of neuronal polarity and morphogenesis in the mammalian brain. Neuron, 72(1), 22-40.

Dhara, S. K., & Stice, S. L. (2008, Oct 15). Neural differentiation of human embryonic stem cells. J Cell Biochem, 105(3), 633-640.

Doss, M. X., & Sachinidis, A. (2019, Apr 30). Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications. Cells, 8(5).

Douvaras, P., Wang, J., Zimmer, M., Hanchuk, S., O'Bara, M. A., Sadiq, S., Sim, F. J., Goldman, J., & Fossati, V. (2014, Aug 12). Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Reports, 3(2), 250-259.

Ehrlich, M., Mozafari, S., Glatza, M., Starost, L., Velychko, S., Hallmann, A. L., Cui, Q. L., Schambach, A., Kim, K. P., Bachelin, C., Marteyn, A., Hargus, G., Johnson, R. M., Antel, J., Sterneckert, J., Zaehres, H., Scholer, H. R., Baron-Van Evercooren, A., & Kuhlmann, T. (2017, Mar 14). Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors. Proc Natl Acad Sci U S A, 114(11), E2243-E2252.

Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., Kawada, M., Yonemura, S., Matsumura, M., Wataya, T., Nishiyama, A., Muguruma, K., & Sasai, Y. (2008, Nov 6). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell, 3(5), 519-532.

Emdad, L., D'Souza, S. L., Kothari, H. P., Qadeer, Z. A., & Germano, I. M. (2012, Feb 10). Efficient differentiation of human embryonic and induced pluripotent stem cells into functional astrocytes. Stem Cells Dev, 21(3), 404-410.

Feliciano, D. M., Bordey, A., & Bonfanti, L. (2015, Sep 18). Noncanonical Sites of Adult Neurogenesis in the Mammalian Brain. Cold Spring Harb Perspect Biol, 7(10), a018846.

Gordeeva, O. (2019, Nov 23). TGFbeta Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells' Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer. Cells, 8(12).

Gorris, R., Fischer, J., Erwes, K. L., Kesavan, J., Peterson, D. A., Alexander, M., Nothen, M. M., Peitz, M., Quandel, T., Karus, M., & Brustle, O. (2015, Dec). Pluripotent stem cell-derived radial glia-like cells as stable intermediate for efficient generation of human oligodendrocytes. Glia, 63(12), 2152-2167.

Gupta, P. K., Das, A. K., Chullikana, A., & Majumdar, A. S. (2012, Jul 9). Mesenchymal stem cells for cartilage repair in osteoarthritis. Stem Cell Res Ther, 3(4), 25.

Her, G. J., Wu, H. C., Chen, M. H., Chen, M. Y., Chang, S. C., & Wang, T. W. (2013, Feb). Control of three-dimensional substrate stiffness to manipulate mesenchymal stem cell fate toward neuronal or glial lineages. Acta Biomater, 9(2), 5170-5180.

Hopkins, A. M., De Laporte, L., Tortelli, F., Spedden, E., Staii, C., Atherton, T. J., Hubbell, J. A., & Kaplan, D. L. (2013). Silk Hydrogels as Soft Substrates for Neural Tissue Engineering. Advanced Functional Materials, 23(41), 5140-5149.

Hu, B. Y., Du, Z. W., & Zhang, S. C. (2009). Differentiation of human oligodendrocytes from pluripotent stem cells. Nat Protoc, 4(11), 1614-1622.

Hung, C. W., Liou, Y. J., Lu, S. W., Tseng, L. M., Kao, C. L., Chen, S. J., Chiou, S. H., & Chang, C. J. (2010, May 5). Stem cell-based neuroprotective and neurorestorative strategies. Int J Mol Sci, 11(5), 2039-2055.

Izrael, M., Zhang, P., Kaufman, R., Shinder, V., Ella, R., Amit, M., Itskovitz-Eldor, J., Chebath, J., & Revel, M. (2007, Mar). Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. Mol Cell Neurosci, 34(3), 310-323.

Jimenez-Moreno, N., Stathakos, P., Caldwell, M. A., & Lane, J. D. (2017, Aug 11). Induced Pluripotent Stem Cell Neuronal Models for the Study of Autophagy Pathways in Human Neurodegenerative Disease. Cells, 6(3).

Jinghua Piao, T. M., Gordon Auyeung, Edelweiss Policarpio, Jayanthi Menon, Leif Droms, Philip Gutin, Kunihiro Uryu, Jason Tchieu, Denis Soulet, Viviane Tabar. (2015). Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitors Remyelinate the Brain and Rescue Behavioral Deficits following Radiation. Cell Stem Cell, Pages 198-210.

Keirstead, H. S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K., & Steward, O. (2005, May 11). Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci, 25(19), 4694-4705.

Kempermann, G., Jessberger, S., Steiner, B., & Kronenberg, G. (2004). Milestones of neuronal development in the adult hippocampus. Trends in Neurosciences, 27(8), 447–452.

Kim, C., Ho, D. H., Suk, J. E., You, S., Michael, S., Kang, J., Joong Lee, S., Masliah, E., Hwang, D., Lee, H. J., & Lee, S. J. (2013). Neuron-released oligomeric alpha-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat Commun, 4, 1562.

Kirkeby, A., Nelander, J., & Parmar, M. (2012). Generating regionalized neuronal cells from pluripotency, a step-by-step protocol. Front Cell Neurosci, 6, 64.

Ko, K. R., & Frampton, J. P. (2016, Jul). Developments in 3D neural cell culture models: the future of neurotherapeutics testing? Expert Rev Neurother, 16(7), 739-741.

Krencik, R., & Zhang, S. C. (2011, Oct 13). Directed differentiation of functional astroglial subtypes from human pluripotent stem cells. Nat Protoc, 6(11), 1710-1717.

Kristiansen, M., & Ham, J. (2014, Jul). Programmed cell death during neuronal development: the sympathetic neuron model. Cell Death Differ, 21(7), 1025-1035.

Lafaille, F. G., Pessach, I. M., Zhang, S. Y., Ciancanelli, M. J., Herman, M., Abhyankar, A., Ying, S. W., Keros, S., Goldstein, P. A., Mostoslavsky, G., Ordovas-Montanes, J., Jouanguy, E., Plancoulaine, S., Tu, E., Elkabetz, Y., Al-Muhsen, S., Tardieu, M., Schlaeger, T. M., Daley, G. Q., Abel, L., Casanova, J. L., Studer, L., & Notarangelo, L. D. (2012, Nov 29). Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells. Nature, 491(7426), 769-773.

Lancaster, M. A., & Knoblich, J. A. (2014, Jul 18). Organogenesis in a dish: modeling development and disease using organoid technologies. Science, 345(6194), 1247125.

Larijani, B., Esfahani, E. N., Amini, P., Nikbin, B., Alimoghaddam, K., Amiri, S., Malekzadeh, R., Yazdi, N. M., Ghodsi, M., Dowlati, Y., Sahraian, M. A., & Ghavamzadeh, A. (2012). Stem cell therapy in treatment of different diseases. Acta Med Iran, 50(2), 79-96.

Lee, H. K., Velazquez Sanchez, C., Chen, M., Morin, P. J., Wells, J. M., Hanlon, E. B., & Xia, W. (2016). Three Dimensional Human Neuro-Spheroid Model of Alzheimer's Disease Based on Differentiated Induced Pluripotent Stem Cells. PLOS ONE, 11(9), e0163072.

Li, X. J., Du, Z. W., Zarnowska, E. D., Pankratz, M., Hansen, L. O., Pearce, R. A., & Zhang, S. C. (2005, Feb). Specification of motoneurons from human embryonic stem cells. Nat Biotechnol, 23(2), 215-221.

Li, Y., Liu, M., Yan, Y., & Yang, S. T. (2014, Jan 26). Neural differentiation from pluripotent stem cells: The role of natural and synthetic extracellular matrix. World J Stem Cells, 6(1), 11-23.

Liddelow, S. A., Guttenplan, K. A., Clarke, L. E., Bennett, F. C., Bohlen, C. J., Schirmer, L., Bennett, M. L., Munch, A. E., Chung, W. S., Peterson, T. C., Wilton, D. K., Frouin, A., Napier, B. A., Panicker, N., Kumar, M., Buckwalter, M. S., Rowitch, D. H., Dawson, V. L., Dawson, T. M., Stevens, B., & Barres, B. A. (2017, Jan 26). Neurotoxic reactive astrocytes are induced by activated microglia. Nature, 541(7638), 481-487.

Liu, G., David, B. T., Trawczynski, M., & Fessler, R. G. (2020, Feb). Advances in Pluripotent Stem Cells: History, Mechanisms, Technologies, and Applications. Stem Cell Rev Rep, 16(1), 3-32.

Livesey, M. R., Magnani, D., Cleary, E. M., Vasistha, N. A., James, O. T., Selvaraj, B. T., Burr, K., Story, D., Shaw, C. E., Kind, P. C., Hardingham, G. E., Wyllie, D. J., & Chandran, S. (2016, Apr). Maturation and electrophysiological properties of human pluripotent stem cell-derived oligodendrocytes. Stem Cells, 34(4), 1040-1053.

Lobsiger, C. S., & Cleveland, D. W. (2007, Nov). Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat Neurosci, 10(11), 1355-1360.

Lou, Y. R., & Leung, A. W. (2018, Jan - Feb). Next generation organoids for biomedical research and applications. Biotechnol Adv, 36(1), 132-149.

McComish, S. F., & Caldwell, M. A. (2018, Jul 5). Generation of defined neural populations from pluripotent stem cells. Philos Trans R Soc Lond B Biol Sci, 373(1750).

Mercurio, S., Serra, L., & Nicolis, S. K. (2019, Sep 13). More than just Stem Cells: Functional Roles of the Transcription Factor Sox2 in Differentiated Glia and Neurons. Int J Mol Sci, 20(18).

Ming, G. L., & Song, H. (2011, May 26). Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron, 70(4), 687-702.

Mira, H., & Lie, D. C. (2017, Nov 9). Regulation of Adult Neurogenesis 2.0 - Beyond Signaling Pathways and Transcriptional Regulators. Brain Plast, 3(1), 1-3.

Miyazaki, T., Miyauchi, S., Matsuzaka, S., Yamagishi, C., & Kobayashi, K. (2010, May). Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors. Tissue Eng Part A, 16(5), 1575-1584.

Mormone, E., D'Sousa, S., Alexeeva, V., Bederson, M. M., & Germano, I. M. (2014, Nov 1). "Footprint-free" human induced pluripotent stem cell-derived astrocytes for in vivo cell-based therapy. Stem Cells Dev, 23(21), 2626-2636.

Nolbrant, S., Heuer, A., Parmar, M., & Kirkeby, A. (2017, Sep). Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat Protoc, 12(9), 1962-1979.

Oh, Y., & Jang, J. (2019, Mar 31). Directed Differentiation of Pluripotent Stem Cells by Transcription Factors. Mol Cells, 42(3), 200-209.

Oliva, J., Florentino, A., Bardag-Gorce, F., & Niihara, Y. (2019, Mar). Engineering, differentiation and harvesting of human adipose-derived stem cell multilayer cell sheets. Regen Med, 14(3), 151-163.

Osborn, L. M., Kamphuis, W., Wadman, W. J., & Hol, E. M. (2016, Sep). Astrogliosis: An integral player in the pathogenesis of Alzheimer's disease. Prog Neurobiol, 144, 121-141.

Pankratz, M. T., Li, X. J., Lavaute, T. M., Lyons, E. A., Chen, X., & Zhang, S. C. (2007, Jun). Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage. Stem Cells, 25(6), 1511-1520.

Rosenzweig, E. S., Brock, J. H., Lu, P., Kumamaru, H., Salegio, E. A., Kadoya, K., Weber, J. L., Liang, J. J., Moseanko, R., Hawbecker, S., Huie, J. R., Havton, L. A., Nout-Lomas, Y. S., Ferguson, A. R., Beattie, M. S., Bresnahan, J. C., & Tuszynski, M. H. (2018, May). Restorative effects of human neural stem cell grafts on the primate spinal cord. Nat Med, 24(4), 484-490.

Sakthiswary, R., & Raymond, A. A. (2012, Aug 15). Stem cell therapy in neurodegenerative diseases: From principles to practice. Neural Regen Res, 7(23), 1822-1831.

Secondo, A., Esposito, A., Petrozziello, T., Boscia, F., Molinaro, P., Tedeschi, V., Pannaccione, A., Ciccone, R., Guida, N., Di Renzo, G., & Annunziato, L. (2018, Dec). Na(+)/Ca(2+) exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca(2+) regulation during neuronal differentiation. Cell Death Discov, 4, 12.

Serio, A., Bilican, B., Barmada, S. J., Ando, D. M., Zhao, C., Siller, R., Burr, K., Haghi, G., Story, D., Nishimura, A. L., Carrasco, M. A., Phatnani, H. P., Shum, C., Wilmut, I., Maniatis, T., Shaw, C. E., Finkbeiner, S., & Chandran, S. (2013, Mar 19). Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy. Proc Natl Acad Sci U S A, 110(12), 4697-4702.

Shi, Y., Kirwan, P., & Livesey, F. J. (2012, Oct). Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc, 7(10), 1836-1846.

Shirazi, R., Zarnani, A. H., Soleimani, M., Abdolvahabi, M. A., Nayernia, K., & Ragerdi Kashani, I. (2012). BMP4 can generate primordial germ cells from bone-marrow-derived pluripotent stem cells. Cell Biol Int, 36(12), 1185-1193.

Simunovic, M., & Brivanlou, A. H. (2017, Mar 15). Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis. Development, 144(6), 976-985.

Singh, V. K., Kumar, N., Kalsan, M., Saini, A., & Chandra, R. (2015). Mechanism of Induction: Induced Pluripotent Stem Cells (iPSCs). J Stem Cells, 10(1), 43-62.

Slanzi, A., Iannoto, G., Rossi, B., Zenaro, E., & Constantin, G. (2020). In vitro Models of Neurodegenerative Diseases. Front Cell Dev Biol, 8, 328.

Stacpoole, S. R., Spitzer, S., Bilican, B., Compston, A., Karadottir, R., Chandran, S., & Franklin, R. J. (2013). High yields of oligodendrocyte lineage cells from human embryonic stem cells at physiological oxygen tensions for evaluation of translational biology. Stem Cell Reports, 1(5), 437-450.

Stappert, L., Klaus, F., & Brustle, O. (2018). MicroRNAs Engage in Complex Circuits Regulating Adult Neurogenesis. Front Neurosci, 12, 707.

Stover, A. E., Brick, D. J., Nethercott, H. E., Banuelos, M. G., Sun, L., O'Dowd, D. K., & Schwartz, P. H. (2013, Oct). Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling. J Neurosci Res, 91(10), 1247-1262.

Tao, Y., & Zhang, S. C. (2016, Nov 3). Neural Subtype Specification from Human Pluripotent Stem Cells. Cell Stem Cell, 19(5), 573-586.

Ulrich, D., Muralitharan, R., & Gargett, C. E. (2013, Oct). Toward the use of endometrial and menstrual blood mesenchymal stem cells for cell-based therapies. Expert Opin Biol Ther, 13(10), 1387-1400.

Wang, S., Bates, J., Li, X., Schanz, S., Chandler-Militello, D., Levine, C., Maherali, N., Studer, L., Hochedlinger, K., Windrem, M., & Goldman, S. A. (2013, Feb 7). Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell, 12(2), 252-264.

Watanabe, K., Kamiya, D., Nishiyama, A., Katayama, T., Nozaki, S., Kawasaki, H., Watanabe, Y., Mizuseki, K., & Sasai, Y. (2005, Mar). Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci, 8(3), 288-296.

Yap, M. S., Nathan, K. R., Yeo, Y., Lim, L. W., Poh, C. L., Richards, M., Lim, W. L., Othman, I., & Heng, B. C. (2015). Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling. Stem Cells Int, 2015, 105172.

Yin, X., Mead, B. E., Safaee, H., Langer, R., Karp, J. M., & Levy, O. (2016, Jan 7). Engineering Stem Cell Organoids. Cell Stem Cell, 18(1), 25-38.

Zakrzewski, W., Dobrzynski, M., Szymonowicz, M., & Rybak, Z. (2019, Feb 26). Stem cells: past, present, and future. Stem Cell Res Ther, 10(1), 68.

Zhang, S. C., Wernig, M., Duncan, I. D., Brüstle, O., & Thomson, J. A. (2011). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature Biotechnology, 19(12), 1129–1133.






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