Adult ciliary epithelial stem cells generate functional neurons and differentiate into both early and late born retinal neurons under non-cell autonomous influences

BMC Neuroscience

Iqbal Ahmad²  Huangui Xiong¹  Xu Peng¹  Carolina Beltrame Del Debbio² 

[1] Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198-5840, USA;Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Durham Research Center 1, Room 4044, 985840 Nebraska Medical Center, Omaha, NE 68198-5840, USA

关键词: Retina;    Cell therapy;    Retinal ganglion cells;    Photoreceptors;    Ciliary epithelium;    Stem cells;   

Others  :  1139933
DOI  :  10.1186/1471-2202-14-130

 received in 2013-06-27, accepted in 2013-10-14,  发布年份 2013

【 摘 要 】

Background

The neural stem cells discovered in the adult ciliary epithelium (CE) in higher vertebrates have emerged as an accessible source of retinal progenitors; these cells can self-renew and possess retinal potential. However, recent studies have cast doubt as to whether these cells could generate functional neurons and differentiate along the retinal lineage. Here, we have systematically examined the pan neural and retinal potential of CE stem cells.

Results

Molecular and cellular analysis was carried out to examine the plasticity of CE stem cells, obtained from mice expressing green fluorescent protein (GFP) under the influence of the promoter of the rod photoreceptor-specific gene, Nrl, using the neurospheres assay. Differentiation was induced by specific culture conditions and evaluated by both transcripts and protein levels of lineage-specific regulators and markers. Temporal pattern of their levels were examined to determine the expression of genes and proteins underlying the regulatory hierarchy of cells specific differentiation in vitro. Functional attributes of differentiation were examined by the presence of current profiles and pharmacological mobilization of intracellular calcium using whole cell recordings and Fura-based calcium imaging, respectively. We demonstrate that stem cells in adult CE not only have the capacity to generate functional neurons, acquiring the expression of sodium and potassium channels, but also respond to specific cues in culture and preferentially differentiate along the lineages of retinal ganglion cells (RGCs) and rod photoreceptors, the early and late born retinal neurons, respectively. The retinal differentiation of CE stem cells was characterized by the temporal acquisition of the expression of the regulators of RGCs and rod photoreceptors, followed by the display of cell type-specific mature markers and mobilization of intracellular calcium.

Conclusions

Our study demonstrates the bonafide retinal potential of adult CE stem cells and suggests that their plasticity could be harnessed for clinical purposes once barriers associated with any lineage conversion, i.e., low efficiency and fidelity is overcome through the identification of conducive culture conditions.

【 授权许可】

   
2013 Del Debbio et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 3.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, van der Kooy D: Retinal stem cells in the adult mammalian eye. Science 2000, 287(5460):2032-2036.
• [2]Ahmad I, Tang L, Pham H: Identification of neural progenitors in the adult mammalian eye. Biochem Biophys Res Commun 2000, 270(2):517-521.
• [3]Das AV, James J, Zhao X, Rahnenführer J, Ahmad I: Identification of c-Kit receptor as a regulator of adult neural stem cells in the mammalian eye: interactions with Notch signaling. Dev Biol 2004, 273(1):87-105.
• [4]Das AV, James J, Rahnenführer J, Thoreson WB, Bhattacharya S, Zhao X, Ahmad I: Retinal properties and potential of the adult mammalian ciliary epithelium stem cells. Vision Res 2005, 45(13):1653-1666.
• [5]Das AV, Zhao X, James J, Kim M, Cowan KH, Ahmad I: Neural stem cells in the adult ciliary epithelium express GFAP and are regulated by Wnt signaling. Biochem Biophys Res Commun 2006, 339(2):708-716.
• [6]Abdouh M, Bernier G: In vivo reactivation of a quiescent cell population located in the ocular ciliary body of adult mammals. Exp Eye Res 2006, 83(1):153-164.
• [7]Kohno H, Sakai T, Kitahara K: Induction of nestin, Ki-67, and cyclin D1 expression in Müller cells after laser injury in adult rat retina. Graefes Arch Clin Exp Ophthalmol 2006, 244(1):90-95.
• [8]Lord-Grignon J, Abdouh M, Bernier G: Identification of genes expressed in retinal progenitor/stem cell colonies isolated from the ocular ciliary body of adult mice. Gene Expr Patterns 2006, 6(8):992-999.
• [9]Xu H, Sta Iglesia DD, Kielczewski JL, Valenta DF, Pease ME, Zack DJ, Quigley HA: Characteristics of progenitor cells derived from adult ciliary body in mouse, rat, and human eyes. Invest Ophthalmol Vis Sci 2007, 48(4):1674-1682.
• [10]Demontis GC, Aruta C, Comitato A, De Marzo A, Marigo V: Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium. PLoS One 2012, 7(3):e33338.
• [11]Ballios BG, Clarke L, Coles BL, Shoichet MS, Van Der Kooy D: The adult retinal stem cell is a rare cell in the ciliary epithelium whose progeny can differentiate into photoreceptors. Biol Open 2012, 1(3):237-246.
• [12]Fischer AJ, Reh TA: Identification of a proliferating marginal zone of retinal progenitors in postnatal chickens. Dev Biol 2000, 220(2):197-210.
• [13]Inoue Y, Yanagi Y, Tamaki Y, Uchida S, Kawase Y, Araie M, Okochi H: Clonogenic analysis of ciliary epithelial derived retinal progenitor cells in rabbits. Exp Eye Res 2005, 81(4):437-445.
• [14]MacNeil A, Pearson RA, MacLaren RE, Smith AJ, Sowden JC, Ali RR: Comparative analysis of progenitor cells isolated from the iris, pars plana, and ciliary body of the adult porcine eye. Stem Cells 2007, 25(10):2430-2438.
• [15]Gu P, Harwood LJ, Zhang X, Wylie M, Curry WJ, Cogliati T: Isolation of retinal progenitor and stem cells from the porcine eye. Mol Vis 2007, 13:1045-1057.
• [16]Coles BL, Angénieux B, Inoue T, Del Rio-Tsonis K, Spence JR, McInnes RR, Arsenijevic Y, van der Kooy D: Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci USA 2004, 101(44):15772-15777.
• [17]Mayer EJ, Carter DA, Ren Y, Hughes EH, Rice CM, Halfpenny CA, Scolding NJ, Dick AD: Neural progenitor cells from postmortem adult human retina. Br J Ophthalmol 2005, 89(1):102-106.
• [18]Martínez-Navarrete GC, Angulo A, Martín-Nieto J, Cuenca N: Gradual morphogenesis of retinal neurons in the peripheral retinal margin of adult monkeys and humans. J Comp Neurol 2008, 511(4):557-580.
• [19]Das AV, Edakkot S, Thoreson WB, James J, Bhattacharya S, Ahmad I: Membrane properties of retinal stem cells/progenitors. Prog Retin Eye Res 2005, 24(6):663-681.
• [20]Inoue T, Coles BL, Dorval K, Bremner R, Bessho Y, Kageyama R, Hino S, Matsuoka M, Craft CM, McInnes RR, et al.: Maximizing functional photoreceptor differentiation from adult human retinal stem cells. Stem Cells 2010, 28(3):489-500.
• [21]Chacko DM, Das AV, Zhao X, James J, Bhattacharya S, Ahmad I: Transplantation of ocular stem cells: the role of injury in incorporation and differentiation of grafted cells in the retina. Vision Res 2003, 43(8):937-946.
• [22]Ahmad I: Stem cells: new opportunities to treat eye diseases. Invest Ophthalmol Vis Sci 2001, 42(12):2743-2748.
• [23]Amato MA, Arnault E, Perron M: Retinal stem cells in vertebrates: parallels and divergences. Int J Dev Biol 2004, 48(8–9):993-1001.
• [24]Locker M, Borday C, Perron M: Stemness or not stemness? Current status and perspectives of adult retinal stem cells. Curr Stem Cell Res Ther 2009, 4(2):118-130.
• [25]Das AM, Zhao X, Ahmad I: Stem cell therapy for retinal degeneration: retinal neurons from heterologous sources. Semin Ophthalmol 2005, 20(1):3-10.
• [26]Cicero SA, Johnson D, Reyntjens S, Frase S, Connell S, Chow LM, Baker SJ, Sorrentino BP, Dyer MA: Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells. Proc Natl Acad Sci USA 2009, 106(16):6685-6690.
• [27]Gualdoni S, Baron M, Lakowski J, Decembrini S, Smith AJ, Pearson RA, Ali RR, Sowden JC: Adult ciliary epithelial cells, previously identified as retinal stem cells with potential for retinal repair, fail to differentiate into new rod photoreceptors. Stem Cells 2010, 28(6):1048-1059.
• [28]Mears AJ, Kondo M, Swain PK, Takada Y, Bush RA, Saunders TL, Sieving PA, Swaroop A: Nrl is required for rod photoreceptor development. Nat Genet 2001, 29(4):447-452.
• [29]Meyer JS, Shearer RL, Capowski EE, Wright LS, Wallace KA, McMillan EL, Zhang SC, Gamm DM: Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci USA 2009, 106(39):16698-16703.
• [30]Krafte DS, Bannon AW: Sodium channels and nociception: recent concepts and therapeutic opportunities. Curr Opin Pharmacol 2008, 8(1):50-56.
• [31]Lampert A, O’Reilly AO, Reeh P, Leffler A: Sodium channelopathies and pain. Pflugers Arch 2010, 460(2):249-263.
• [32]Koeberle PD, Wang Y, Schlichter LC: Kv1.1 and Kv1.3 channels contribute to the degeneration of retinal ganglion cells after optic nerve transection in vivo. Cell Death Differ 2010, 17(1):134-144.
• [33]Jensen CS, Rasmussen HB, Misonou H: Neuronal trafficking of voltage-gated potassium channels. Mol Cell Neurosci 2011, 48(4):288-297.
• [34]James J, Das AV, Bhattacharya S, Chacko DM, Zhao X, Ahmad I: In vitro generation of early-born neurons from late retinal progenitors. J Neurosci 2003, 23(23):8193-8203.
• [35]Hegde GV, James J, Das AV, Zhao X, Bhattacharya S, Ahmad I: Characterization of early retinal progenitor microenvironment: presence of activities selective for the differentiation of retinal ganglion cells and maintenance of progenitors. Exp Eye Res 2007, 84(3):577-590.
• [36]Parameswaran S, Balasubramanian S, Babai N, Qiu F, Eudy JD, Thoreson WB, Ahmad I: Induced pluripotent stem cells generate both retinal ganglion cells and photoreceptors: therapeutic implications in degenerative changes in glaucoma and age-related macular degeneration. Stem Cells 2010, 28(4):695-703.
• [37]Mu X, Klein WH: A gene regulatory hierarchy for retinal ganglion cell specification and differentiation. Semin Cell Dev Biol 2004, 15(1):115-123.
• [38]Sucher NJ, Lipton SA, Dreyer EB: Molecular basis of glutamate toxicity in retinal ganglion cells. Vision Res 1997, 37(24):3483-3493.
• [39]Ahmad I, Das AV, James J, Bhattacharya S, Zhao X: Neural stem cells in the mammalian eye: types and regulation. Semin Cell Dev Biol 2004, 15(1):53-62.
• [40]Del Debbio CB, Balasubramanian S, Parameswaran S, Chaudhuri A, Qiu F, Ahmad I: Notch and Wnt signaling mediated rod photoreceptor regeneration by muller cells in adult mammalian retina. Plos One 2010, 5(8):e12425.
• [41]Zhao X, Liu J, Ahmad I: Differentiation of embryonic stem cells into retinal neurons. Biochem Biophys Res Commun 2002, 297(2):177-184.
• [42]Swaroop A, Kim D, Forrest D: Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina. Nat Rev Neurosci 2010, 11(8):563-576.
• [43]MacLaren RE, Pearson RA, MacNeil A, Douglas RH, Salt TE, Akimoto M, Swaroop A, Sowden JC, Ali RR: Retinal repair by transplantation of photoreceptor precursors. Nature 2006, 444(7116):203-207.
• [44]Koulen P, Brandstätter JH: Pre- and Postsynaptic Sites of Action of mGluR8a in the mammalian retina. Invest Ophthalmol Vis Sci 2002, 43(6):1933-1940.
• [45]Zhao X, Das AV, Soto-Leon F, Ahmad I: Growth factor-responsive progenitors in the postnatal mammalian retina. Dev Dyn 2005, 232(2):349-358.
• [46]Wohl SG, Schmeer CW, Isenmann S: Neurogenic potential of stem/progenitor-like cells in the adult mammalian eye. Prog Retin Eye Res 2012, 31(3):213-242.
• [47]Slominski A, Tobin DJ, Shibahara S, Wortsman J: Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004, 84(4):1155-1228.
• [48]Cheng A, Tang H, Cai J, Zhu M, Zhang X, Rao M, Mattson MP: Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state. Dev Biol 2004, 272(1):203-216.
• [49]Götz M, Huttner WB: The cell biology of neurogenesis. Nat Rev Mol Cell Biol 2005, 6(10):777-788.
• [50]Wong RC, Pera MF, Pébay A: Role of gap junctions in embryonic and somatic stem cells. Stem Cell Rev 2008, 4(4):283-292.
• [51]Sathananthan H, Pera M, Trounson A: The fine structure of human embryonic stem cells. Reprod Biomed Online 2002, 4(1):56-61.
• [52]Baharvand H, Matthaei KI: The ultrastructure of mouse embryonic stem cells. Reprod Biomed Online 2003, 7(3):330-335.
• [53]Cotsarelis G, Sun TT, Lavker RM: Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 1990, 61(7):1329-1337.
• [54]Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, et al.: Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007, 449(7165):1003-1007.
• [55]Ahmad I, Del Debbio CB, Das AV, Parameswaran S: Muller Glia: a promising target for therapeutic regeneration. Invest Ophthalmol Vis Sci 2011, 52(8):5758-5764.
• [56]Xu S, Sunderland ME, Coles BL, Kam A, Holowacz T, Ashery-Padan R, Marquardt T, McInnes RR, van der Kooy D: The proliferation and expansion of retinal stem cells require functional Pax6. Dev Biol 2007, 304(2):713-721.
• [57]Chua SJ, Casper RF, Rogers IM: Toward transgene-free induced pluripotent stem cells: lessons from transdifferentiation studies. Cell Reprogram 2011, 13(4):273-280.
• [58]Jopling C, Boue S, Izpisua Belmonte JC: Dedifferentiation, transdifferentiation and reprogramming: three routes to regeneration. Nat Rev Mol Cell Biol 2011, 12(2):79-89.
• [59]Okita K, Yamanaka S: Induced pluripotent stem cells: opportunities and challenges. Philos Trans R Soc Lond B Biol Sci 2011, 366(1575):2198-2207.
• [60]Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichisaka T, Kawamura Y, Mochizuki H, Goshima N, Yamanaka S: Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature 2011, 474(7350):225-229.
• [61]Burke ZD, Shen CN, Ralphs KL, Tosh D: Characterization of liver function in transdifferentiated hepatocytes. J Cell Physiol 2006, 206(1):147-159.
• [62]Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Aryee MJ, Ji H, Ehrlich LI, et al.: Epigenetic memory in induced pluripotent stem cells. Nature 2010, 467(7313):285-290.
• [63]Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, Apostolou E, Stadtfeld M, Li Y, Shioda T, et al.: Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol 2010, 28(8):848-855.
• [64]Zhao S, Thornquist SC, Barnstable CJ: In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural retina. Brain Res 1995, 677(2):300-310.
• [65]Eberhard D, Tosh D: Transdifferentiation and metaplasia as a paradigm for understanding development and disease. Cell Mol Life Sci 2008, 65(1):33-40.