Rat adult stem cell differentiation into immature retinal cells


Cell therapy has been proposed as an alternative treatment for retinal diseases. Applications involving stem cells have shown that undifferentiated cells fail to engraft and cannot convert to retinal cells. However, positive results have been reported for retinal precursor cells, suggesting that this approach is the best option. Unfortunately, the source of this cell type is controversial. Predifferentiated adult stem cells may provide an alternative source of cells. The present study proposes a sequential culture media aimed at inducing cells from this source into a preretinal-like lineage. Rat bone marrow stem cells were cultivated in a neuroinduction mix medium for 24 h. The sequence involves immunocytochemistry to detect nestin and tubulin III to demonstrate the cell’s neuronal lineage, followed by incubation in retinal-induction mixed medium for 24 h. RT-PCR was performed to detect expression of Brn3b, Pax6, THY1.1, Opn4, and Ath5 genes. Immunocytochemistry results showed increased expression of nestin and tubulin III after 24 h of incubation in the neuroinduction medium. RT-PCR showed slightly increased expression of Pax6, THY1.1, and Opn4 after 48 h of sequential incubation in the neuroinduction and predifferentiation media. Brn3b and Ath5 gene expression increased markedly. These results suggest that mesenchymal stem cells have a high predisposition to differentiate into preretinal-like cells with minimal time in culture. These cells may provide a viable alternative for restoring damaged retinas.

Share and Cite:

Teresa González-Garza, M. and E. Moreno-Cuevas, J. (2012) Rat adult stem cell differentiation into immature retinal cells. Stem Cell Discovery, 2, 62-69. doi: 10.4236/scd.2012.22010.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Quigley, H.A. and Broman, A.T. (2006) The number of people with glaucoma worldwide in 2010 and 2020. British Journal of Ophthalmology, 90, 262-267. doi:10.1136/bjo.2005.081224
[2] Morgan, J.E. (2000) Optic nerve head structure in glaucoma: Astrocytes as mediators of axonal damage. Eye, 14, 437-444. doi:10.1038/eye.2000.128
[3] Quigley, H.A., Nickells, R.W., Kerrigan, L.A, Pease, ME, Thibault, D.J. and Zack, D.J. (1995) Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Investigative Ophthalmology and Visual Science, 36, 774-786.
[4] Soto, I., Pease, M.E., Son, J.L., Shi, X., Quigley, H.A. and Marsh-Armstrong, N. (2011) Retinal ganglion cell loss in a rat ocular hypertension model is sectorial and involves early optic nerve axon loss. Investigative Ophthalmology and Visual Science, 52, 434-441. doi:10.1167/iovs.10-5856
[5] Hess, D.C and Borlongan, C.V. (2008) Stem cells and neurological conditions. Cell Proliferation, 41, 94-114. doi:10.1111/j.1365-2184.2008.00486.x
[6] Daley, G.Q. and Scadden, D.T. (2008) Prospects for stem cell-based therapy. Cell, 132, 544-548. doi:10.1016/j.cell.2008.02.009
[7] Enzmann, V., Yolcu, E., Kaplan, H.J, and Ildstad, S.T. (2009) Stem cells as tools in regenerative therapy for retinal degeneration. Archives of Ophthalmology, 127, 563-571. doi:10.1001/archophthalmol.2009.65
[8] Ballios, B.G. and van der Kooy, D. (2010) Biology and therapeutic potential of adult retinal stem cells. Canadian Journal of Ophthalmology, 45, 342-351.
[9] Adolph, A.R., Zucker, C.L., Ehinger, B. and Bergstrom, A. (1994) Function and structure in retinal transplants. Journal of Neural Transplantation and Plasticity, 5, 147-161. doi:10.1155/NP.1994.147
[10] Lamba, D.A., Karl, M.O., Ware, C.B. and Reh, T.A. (2006) Efficient generation of retinal progenitor cells from human embryonic stem cells. Proceedings of the National Academy of Sciences of USA, 103, 12769-12774. doi:10.1073/pnas.0601990103
[11] Meyer, J.S., Katz, M.L., Maruniak, J.A. and Kirk, M.D. (2006) Embryonic stem cell-derived neural progenitors incorporate into degenerating retina and enhance survival of host photoreceptors. Stem Cells, 24, 274-283. doi:10.1634/stemcells.2005-0059
[12] Banin, E., Obolensky, A., Idelson, M., Hemo, I., Reinhardtz, E., Pijarsky, E., Ben-Hur, T., and Reubinoff, B. (2006) Retinal incorporation and differentiation of neural precursors derived from human embryonic stem cells. Stem Cells, 24, 246-257. doi:10.1634/stemcells.2005-0009
[13] Lamba, D.A., Gust, J. and Reh, T.A. (2009) Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice. Cell Stem Cell, 4, 73-79. doi:10.1016/j.stem.2008.10.015
[14] Aoki, H., Hara, A., Niwa, M., Yamada, Y. and Kunisada, T. (2009) In vitro and in vivo differentiation of human embryonic stem cells into retina-like organs and comparison with that from mouse pluripotent epiblast stem cells. Development Dynamics, 238, 2266-2279. doi:10.1002/dvdy.22008
[15] Francis, P.J., Wang, S., Zhang, Y., Brown, A., Hwang, T., McFarland, T.J., Jeffrey, B.G., Lu, B., Wright, L., Appukuttan, B., Wilson, D.J, Stout. J.T., Neuringer, M., Gamm, D.M. and Lund, R.D. (2009) Subretinal transplantation of forebrain progenitor cells in nonhuman primates: survival and intact retinal function. Investigative Ophthalmology and Visual Science, 50, 3425-3431. doi:10.1167/iovs.08-2908
[16] Wang, S., Girman, S., Lu, B., Bischoff, N., Holmes, T., Shearer, R., Wright, L.S., Svendsen, C.N., Gamm, D.M. and Lund, R.D. (2008) Long-term vision rescue by human neural progenitors in a rat model of photoreceptor degeneration. Investigative Ophthalmology and Visual Science, 49, 3201-3206. doi:10.1167/iovs.08-1831
[17] Lamba, D.A., McUsic, A., Hirata, R.K., Wang, P-R., Russell, D. and Reh, T.A. (2010) Generation, Purification and transplantation of photoreceptors derived from human induced pluripotent stem cells. PLoS ONE, 5, e8763. doi:10.1371/journal.pone.0008763
[18] Kokkinaki, M., Sahibzada, N. and Golestaneh, N. (2011) Human induced pluripotent stem-derived retinal pigment epithelium (RPE) cells exhibition transport, membrane potential, polarized VEGF secretion and gene expression pattern similar to native RPE. Stem Cells, 29, 825-835. doi:10.1002/stem.635
[19] Mao, W., Yan, R.T. and Wang, S.Z. (2008) Reprogramming chick RPE progeny cells to differentiate towards retinal neurons by ash1. Molecular Vision, 14, 2309-2320.
[20] Belmonte, J.C., Ellis, J., Hochedlinger, K. and Yamanaka S. (2009) Induced pluripotent stem cells and reprogramming: seeing the science through the hype. Nature Reviews Genetics, 10, 878-883.
[21] Wang, S.Z, Ma, W., Yan, R.T, and Mao, W. (2010) Generating retinal neurons by reprogramming retinal pigment epithelial cells. Expert Opinion on Biological Therapy, 10, 1227-1239.
[22] Li, S., Hu, B., Tay, D., So, K.F. and Yip, H.K. (2004) Intravitreal transplants of Schwann cells and fibroblasts promote the survival of axotomized retinal ganglion cells in rats. Brain Research, 1029, 56-64. doi:10.1016/j.brainres.2004.09.038
[23] Takahashi, M., Palmer, T.D., Takahashi, J. and Gage, F.H. (1998) Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Molecular and Cellular Neuroscience, 12, 340-348. doi:10.1006/mcne.1998.0721
[24] Nishida, A., Takahashi, M., Tanihara, H., Nakano, I., Takahashi, J.B., Mizoguchi, A., Ide ,C. and Honda, Y. (2000) Incorporation and differentiation of hippocampus-derived neural stem cells transplanted in injured adult rat retina. Investigative Ophthalmology and Visual Science, 41, 4268-4274.
[25] Kurimoto, Y., Shibuki, H., Kaneko, Y., Ichikawa, M., Kurokawa, T., Takahashi, M. and Yoshimura, N. (2001) Transplantation of adult rat hippocampus-derived neural stem cells into retina injured by transient ischemia. Neuroscience Letter, 306, 57-60. doi:10.1016/S0304-3940(01)01857-2
[26] Guo, Y., Saloupis, P., Shaw, S.J. and Rickman, D.W. (2003) Engraftment of adult neural progenitor cells transplanted to rat retina injured by transient ischemia. Investigative Ophthalmology and Visual Science, 44, 3194-3201. doi:10.1167/iovs.02-0875
[27] Sheedlo, H.J., Li, L.X. and Turner, J.E. (1989) Functional and structural characteristics of photoreceptor cells rescued in RPE-cell grafted retinas of RCS dystrophic rats. Experimental Eye Research, 48, 841-854. doi:10.1016/0014-4835(89)90067-5
[28] Sauve, Y., Klassen, H., Whiteley, S.J.O. and Lund R.D. (1998) Visual field loss in RCS rats and the effect of RPE cell transplantation. Experimental Neurology, 152, 243-250. doi:10.1006/exnr.1998.6849
[29] Klassen, H., Whiteley, S.J.O., Young, M.J. and Lund R.D. (2001) Graft location affects functional rescue following RPE cell transplantation in the RCS rat. Experimental Neurology, 169, 114-121. doi:10.1006/exnr.2000.7617
[30] Gouras, P., Lopez, R., Kjeldbye, H., Sullivan, B. and Brittis, M. (1989) Transplantation of retinal epithelium prevents photoreceptor degeneration in the RCS rat. Progress in Clinical and Biological Research, 314, 659-671.
[31] Phillips, S.J., Sadda, S.R., Tso, M.O., Humayan, M.S., de Juan, E. Jr. and Binder, S. (2003) Autologous transplantation of retinal pigment epithelium after mechanical debridement of Bruch’s membrane. Current Eye Research, 26, 81-88.
[32] Hu, Y., Zhang, T., Wu, J., Li, Y., Lu, X., Qian, F., Yin, Z. and Ma, Z. (2008) Autologous transplantation of RPE with partial-thickness choroid after mechanical debridement of Bruch membrane in the rabbit. Investigative Ophthalmology and Visual Science, 49, 3185-3192. doi:10.1167/iovs.07-1299
[33] Algvere, P.V., Gouras, P. and Dafgard Kopp, E. (1999) Long-term outcome of RPE allografts in non-immunosuppressed patients with AMD. European Journal of Ophthalmology, 9, 217-230.
[34] Tomita, M., Adachi, Y., Yamada, H., Takahashi, K., Kiuchi, K., Oyaizu, H., Ikebukuro, K., Kaneda, H., Matsumura, M. and Ikehara, S. (2002) Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells, 20, 279-283. doi:10.1634/stemcells.20-4-279
[35] Tomita, M., Yamada, H., Adachi, Y., Cui, Y., Yamada, E., Higuchi, A., Minamino, K., Suzuki, Y., Matsumura, M. and kehara, S. (2004) Choroidal neovascularization is provided by bone marrow cells. Stem Cells, 22, 21-26. doi:10.1634/stemcells.22-1-21
[36] Inoue, Y., Iriyama, A., Ueno, S., Takahashi, H., Kondo, M., Tamaki, Y., Araie, M. and Yanagi, Y. (2007) Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration. Experimental Eye Research, 85, 234-241. doi:10.1016/j.exer.2007.04.007
[37] Lund, R.D., Wang, S., Lu, B., Girman, S., Holmes, T., Sauvé, Y., Messina, D.J., Harris, I.R., Kihm, A.J., Harmon, A.M., Chin, F.Y., Gosiewska, A. and Mistry, S.K. (2007) Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease. Stem Cells, 25, 602-611. doi:10.1634/stemcells.2006-0308
[38] Canola, K., Angenieux, B., Tekaya, M., Quiambao, A., Naash, M.I., Munier, F.L,, Schorderet, D.F. and Arsenijevic, Y. (2007) Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate. Investigative Ophthalmology and Visual Science, 48, 446-454. doi:10.1167/iovs.06-0190
[39] MacLaren, R.E., Pearson, R.A., MacNeil, A., Douglas, R.H., Salt, T.E., Akimoto, M., Swaroop, A., Sowden, J.C. and Ali, R.R. (2006) Retinal repair by transplantation of photoreceptor precursors. Nature, 444, 203-207.
[40] Coles, B.L., Angénieux, B., Inoue, T., Del Rio-Tsonis, K., Spence, J.R. McInnes, R.R., Arsenijevic, Y. and van der Kooy, D. (2004) Facile isolation and the characterization of human retinal stem cells. Proceedings of the National Academic of Science USA, 101, 15772-15777. doi:10.1073/pnas.0401596101
[41] Yang, J., Klassen, H., Pries, M., Wang, W. and Nissen, M.H. (2006) Aqueous humor enhances the proliferation of rat retinal precursor cells in culture, and this effect is partially reproduced by ascorbic acid. Stem Cells, 24, 2766-2775. doi:10.1634/stemcells.2006-0103
[42] Merhi-Soussi, F., Angénieux, B., Canola, K., Kostic, C., Tekaya, M., Hornfeld, D. and Arsenijevic Y. (2006) High yield of cells committed to the photoreceptor fate from expanded mouse retinal stem cells. Stem Cells, 24, 2060-2070. doi:10.1634/stemcells.2005-0311
[43] Mori, T., Kiyono, T., Imabayashi, H., Takeda, Y., Tsuchiya, K., Miyoshi, S,, Makino, H., Matsumoto, K., Saito, H., Ogawa, S., Sakamoto, M., Hata, J. and Umezawa, A. (2005) Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential. Molecular and Cellular Biology, 25, 5183-5195. doi:10.1128/MCB.25.12.5183-5195.2005
[44] Ahmad, I., Tang, L. and Pham, H. (2000) Identification of neural progenitors in the adult mammalian eye. Biochemical and Biophysical Research Communications, 270, 517-521. doi:10.1006/bbrc.2000.2473
[45] Mayer, E.J., Hughes, E.H., Carter, D.A. and Dick, A.D. (2003) Nestin positive cells in adult human retina and in epiretinal membranes. British Journal of Ophthalmology, 87, 1154-1158. doi:10.1136/bjo.87.9.1154
[46] Mukhopadhyay, M., Shtrom, S., Rodriguez-Esteban, C., Chen, L., Tsukui, T., Gomer, L., Dorward, D.W., Glinka, A., Grinberg, A., Huang, S.P., Niehrs, C., Izpisúa Belmonte, J.C. and Westphal, H. (2001) Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse. Developmental Cell, 1, 423-434. doi:10.1016/S1534-5807(01)00041-7
[47] Mukhopadhyay, M., Gorivodsky, M., Shtrom, S., Grinberg, A., Niehrs, C., Morasso, M.I. and Westphal, H. (2006) Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium. Development, 133, 2149-2154. doi:10.1242/dev.02381
[48] Glinka, A., Wu, W., Delius, H., Monaghan, A.P., Blumenstock, C. and Niehrs, C. (1998) Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature, 391, 357-362.
[49] Pera, E.M., Wessely, O., Li, S.Y. and De Robertis, E, M. (2001) Neural and head induction by insulin-like growth factor signals. Developmental Cell, 1, 655-665. doi:10.1016/S1534-5807(01)00069-7
[50] Lim, D.A., Tramontin, A.D., Trevejo, J.M., Herrera, D.G., García-Verdugo, J.M. and Alvarez-Buylla, A. (2000) Noggin antagonizes BMP signaling to create a niche for adult neurogenesis. Neuron, 28, 713-726. doi:10.1016/S0896-6273(00)00148-3
[51] Hollis, E.R. 2nd, Lu, P., Blesch, A. and Tuszynski, M.H. (2009) IGF-I gene delivery promotes corticospinal neuronal survival but not regeneration after adult CNS injury. Experimental Neurology, 215, 53-59. doi:10.1016/j.expneurol.2008.09.014
[52] Xu. S., Sunderland, M.E., Coles, BL, Kam, A., Holowacz, T., Ashery-Padan, R., Marquardt, T., McInnes, R.R, and van der Kooy, D. (2007) The proliferation and expansion of retinal stem cells require functional pax6. Development Biology, 304, 713-721. doi:10.1016/j.ydbio.2007.01.021
[53] Zaghloul, N.A. and Moody, S.A. (2007) Alterations of rx1 and pax6 expression levels at neural plate stages differentially affect the production of retinal cell types and maintenance of retinal stem cell qualities. Developmental Biology, 306, 222-240. doi:10.1016/j.ydbio.2007.03.017
[54] Gu, P., Yang, J., Wang, J., Young, M.J. and Klassen, H. (2009) Sequential changes in the gene expression profile of murine retinal progenitor cells during the induction of differentiation. Molecular Vision, 15, 2111-2122.
[55] Schmitt, S., Aftab, U., Jiang, C., Redenti, S., Klassen, H., Miljan, E., Sinden, J. and Young, M. (2009) Molecular characterization of human retinal progenitor cells. Investigative Ophthalmology and Visual Science, 50, 5901-5908. doi:10.1167/iovs.08-3067
[56] Klassen, H., Kiilgaard, J.F., Zahir, T., Ziaeian, B., Kirov, I., Scherfig, E., Warfvinge, K. and Young, M.J. (2007) Progenitor cells from the porcine neural retina express photoreceptor markers after transplantation to the subretinal space of allorecipients. Stem Cells, 25, 1222-1230. doi:10.1634/stemcells.2006-0541
[57] Klassen, H., Sakaguchi, S., Young, M. (2004) Stem cells and retinal repair. Progress in Retinal and Eye Research, 23, 149-181.doi:10.1016/j.preteyeres.2004.01.002
[58] Qiu, F., Jiang, H., Xiang, M. (2008) A comprehensive negative regulatory program controlled by Brn3b to ensure ganglion cell specification from multipotential retinal precursors. Journal of Neuroscience, 28, 3392-3403. doi:10.1523/JNEUROSCI.0043-08.2008
[59] Liu. W., Mo, Z. and Xiang M. (2001) The Ath5 proneural genes function upstream of Brn3 POU domain transcripttion factor genes to promote retinal ganglion cell development. Proceeding of National Academic Science USA. 98, 1649-1654. doi:10.1073/pnas.98.4.1649
[60] Ma, W., Yan, R.T., Xie, W. and Wang, S.Z. (2004) A role of ath5 in inducing neuroD and the photoreceptor pathway. Journal of Neuroscience, 24, 7150-7158. doi:10.1523/JNEUROSCI.2266-04.2004
[61] Tondreau, T., Lagneaux, L., Dejeneffe, M., Massy, M., Mortier, C., Delforge, A. and Bron, D. (2004) Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation, 72, 319-326. doi:10.1111/j.1432-0436.2004.07207003.x
[62] Mareschi, K., Novara, M., Rustichelli, D., Ferrero, I., Guido, D., Carbone, E., Medico, E., Madon, E., Vercelli, A. and Fagioli, F. (2006) Neural differentiation of human mesenchymal stem cells: Evidence of neuronal markers and eag K+ channel types. Experimental Hematology, 34, 1563-1575. doi:10.1016/j.exphem.2006.06.020

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.