SCD  Vol.2 No.4 , October 2012
Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells
ABSTRACT
Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.

Cite this paper
Hongisto, H. , Mikhailova, A. , Hiidenmaa, H. , Ilmarinen, T. and Skottman, H. (2012) Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells. Stem Cell Discovery, 2, 176-186. doi: 10.4236/scd.2012.24022.
References
[1]   Binder, S., Stanzel, B.V., Krebs, I. and Glittenberg, C. (2007) Transplantation of the RPE in AMD. Progress in Retinal and Eye Research, 26, 516-554. doi:10.1016/j.preteyeres.2007.02.002

[2]   Klassen, H., Sakaguchi, D.S. and Young, M.J. (2004) Stem cells and retinal repair. Progress in Retinal and Eye Research, 23, 149-181. doi:10.1016/j.preteyeres.2004.01.002

[3]   Binder, S., Krebs, I., Hilgers, R.D., Abri, A., Stolba, U., Assadoulina, A., Kellner, L., Stanzel, B.V., Jahn, C. and Feichtinger, H. (2004) Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: A prospective trial. Investigative Ophthalmology & Visual Science, 45, 4151-4160. doi:10.1167/iovs.04-0118

[4]   Chen, F.K., Uppal, G.S., MacLaren, R.E., Coffey, P.J., Rubin, G.S., Tufail, A., Aylward, G.W. and Da Cruz, L. (2009) Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration. Clinical & Experimental Ophthalmology, 37, 275-285. doi:10.1111/j.1442-9071.2009.01915.xs

[5]   Radtke, N.D., Aramant, R.B., Seiler, M.J., Petry, H.M. and Pidwell, D. (2004) Vision change after sheet transplant of fetal retina with retinal pigment epithelium to a patient with retinitis pigmentosa. Archives of Ophthalmology, 122, 1159-1165. doi:10.1001/archopht.122.8.1159

[6]   Carr, A.J., Vugler, A.A., Hikita, S.T., Lawrence, J.M., Gias, C., Chen, L.L., Buchholz, D.E., Ahmado, A., Semo, M., Smart, M.J., Hasan, S., Da Cruz, L., Johnson, L.V., Clegg, D.O. and Coffey, P.J. (2009) Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS One, 4, e8152. doi:10.1371/journal.pone.0008152

[7]   Klimanskaya, I., Hipp, J., Rezai, K.A., West, M., Atala, A. and Lanza, R. (2004) Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning and Stem Cells, 6, 217-245.

[8]   Lu, B., Malcuit, C., Wang, S., Girman, S., Francis, P., Lemieux, L., Lanza, R. and Lund, R. (2009) Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells, 27, 2126-2135. doi:10.1002/stem.149

[9]   Lund, R.D., Wang, S., Klimanskaya, I., Holmes, T., Ramos-Kelsey, R., Lu, B., Girman, S., Bischoff, N., Sauve, Y. and Lanza, R. (2006) Human embryonic stem cellderived cells rescue visual function in dystrophic RCS rats. Cloning and Stem Cells, 8, 189-199. doi:10.1089/clo.2006.8.189

[10]   Vugler, A., Lawrence, J., Walsh, J., Carr, A., Gias, C., Semo, M., Ahmado, A., Da Cruz, L., Andrews, P. and Coffey, P. (2007) Embryonic stem cells and retinal repair. Mechanisms of Development, 124, 807-829. doi:10.1016/j.mod.2007.08.002

[11]   Schwartz, S.D., Hubschman, J.P., Heilwell, G., FrancoCardenas, V., Pan, C.K., Ostrick, R.M., Mickunas, E., Gay, R., Klimanskaya, I. and Lanza, R. (2012) Embryonic stem cell trials for macular degeneration: A preliminary report. The Lancet, 379, 713-720. doi:10.1016/S0140-6736(12)60028-2

[12]   Westenskow, P., Piccolo, S. and Fuhrmann, S. (2009) Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression. Development, 136, 2505-2510. doi:10.1242/dev.032136

[13]   Eiraku, M. and Sasai, Y. (2012) Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues. Nature Protocols, 7, 69-79. doi:10.1038/nprot.2011.429

[14]   Fuhrmann, S. (2008) Wnt signaling in eye organogenesis. Organogenesis, 4, 60-67. doi:10.4161/org.4.2.5850

[15]   Yang, X.J. (2004) Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis. Semin Cell and Developmental Biology, 15, 91-103. doi:10.1016/j.semcdb.2003.09.004

[16]   Smukler, S.R., Runciman, S.B., Xu, S. and Van Der Kooy, D. (2006) Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences. The Journal of Cell Biology, 172, 79-90. doi:10.1083/jcb.200508085

[17]   Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., Cohen, M.A., Even-Ram, S., Berman-Zaken, Y., Matzrafi, L., Rechavi, G., Banin, E. and Reubinoff, B. (2009) Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell, 5, 396-408. doi:10.1016/j.stem.2009.07.002

[18]   Osakada, F., Jin, Z.B., Hirami, Y., Ikeda, H., Danjyo, T., Watanabe, K., Sasai, Y. and Takahashi, M. (2009) In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. Journal of Cell Science, 122, 3169-3179. doi:10.1242/jcs.050393

[19]   Osakada, F., Ikeda, H., Mandai, M., Wataya, T., Watanabe, K., Yoshimura, N., Akaike, A., Sasai, Y. and Takahashi, M. (2008) Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nature Biotechnology, 26, 215-224. doi:10.1038/nbt1384

[20]   Rowland, T.J., Buchholz, D.E. and Clegg, D.O. (2012) Pluripotent human stem cells for the treatment of retinal disease. Journal of Cellular Physiology, 227, 457-466. doi:10.1002/jcp.22814

[21]   Vaajasaari, H., Imarinen, T., Juuti-Uusitalo, K., Rajala, K., Onnela, N., Narkilahti, S., Suuronen, R., Hyttinen, J., Uusitalo, H. and Skottman, H. (2011) Towards defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelium cells. Molecular Vision, 22, 558-575.

[22]   Clarke, L., Ballios, B.G. and Van Der Kooy, D. (2012) Generation and clonal isolation of retinal stem cells from human embryonic stem cells. European Journal of Neuroscience, 36, 1951-1959. doi:10.1111/j.1460-9568.2012.08123.x

[23]   Gong, J., Sagiv, O., Cai, H., Tsang, S.H. and Del Priore, L.V. (2008) Effects of extracellular matrix and neighboring cells on induction of human embryonic stem cells into retinal or retinal pigment epithelial progenitors. Experimental Eye Research, 86, 957-965. doi:10.1016/j.exer.2008.03.014

[24]   Okamoto, S. and Takahashi, M. (2011) Induction of retinal pigment epithelial cells from monkey iPS cells. Investigative Ophthalmology & Visual Science, 52, 8785-8790. doi:10.1167/iovs.11-8129

[25]   Martinez-Morales, J.R., Rodrigo, I. and Bovolenta, P. (2004) Eye development: A view from the retina pigmented epithelium. Bioessays, 26, 766-777. doi:10.1002/bies.20064

[26]   Fuhrmann, S., Levine, E.M. and Reh, T.A. (2000) Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development, 127, 4599-4609.

[27]   Skottman, H. (2010) Derivation and characterization of three new human embryonic stem cell lines in Finland. In Vitro Cellular & Developmental Biology: Animal, 46, 206-209. doi:10.1007/s11626-010-9286-2

[28]   Rajala, K., Lindroos, B., Hussein, S.M., Lappalainen, R.S., Pekkanen-Mattila, M., Inzunza, J., Rozell, B., Miettinen, S., Narkilahti, S., Kerkela, E., Aalto-Setala, K., Otonkoski, T., Suuronen, R., Hovatta, O. and Skottman, H. (2010) A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells. PLoS One, 5, e10246. doi:10.1371/journal.pone.0010246

[29]   Hussein, S.M., Batada, N.N., Vuoristo, S., Ching, R.W., Autio, R., Narva, E., Ng, S., Sourour, M., Hamalainen, R., Olsson, C., Lundin, K., Mikkola, M., Trokovic, R., Peitz, M., Brustle, O., Bazett-Jones, D.P., Alitalo, K., Lahesmaa, R., Nagy, A. and Otonkoski, T. (2011) Copy number variation and selection during reprogramming to pluripotency. Nature, 471, 58-62. doi:10.1038/nature09871

[30]   Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408.

[31]   http://imagej.nih.gov/ij/index.html

[32]   Eiselleova, L., Peterkova, I., Neradil, J., Slaninova, I., Hampl, A. and Dvorak, P. (2008) Comparative study of mouse and human feeder cells for human embryonic stem cells. International Journal of Developmental Biology, 52, 353-363. doi:10.1387/ijdb.082590le

[33]   Prowse, A.B., McQuade, L.R., Bryant, K.J., Marcal, H. and Gray, P.P. (2007) Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media. Journal of Proteome Research, 6, 3796-3807. doi:10.1021/pr0702262

[34]   Lim, J.W. and Bodnar, A. (2002) Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics, 2, 1187-1203. doi:10.1002/1615-9861(200209)2:9<1187::AID-PROT1187>3.0.CO;2-T

[35]   Prowse, A.B., McQuade, L.R., Bryant, K.J., Van Dyk, D.D., Tuch, B.E. and Gray, P.P. (2005) A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells. Proteomics, 5, 978-989. doi:10.1002/pmic.200401087

[36]   Bendall, S.C., Hughes, C., Campbell, J.L., Stewart, M.H., Pittock, P., Liu, S., Bonneil, E., Thibault, P., Bhatia, M. and Lajoie, G.A. (2009) An enhanced mass spectrometry approach reveals human embryonic stem cell growth factors in culture. Molecular & Cellular Proteomics, 8, 421-432. doi:10.1074/mcp.M800190-MCP200

[37]   Bendall, S.C., Stewart, M.H., Menendez, P., George, D., Vijayaragavan, K., Werbowetski-Ogilvie, T., RamosMejia, V., Rouleau, A., Yang, J., Bosse, M., Lajoie, G., and Bhatia, M. (2007) IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature, 448, 1015-1021. doi:10.1038/nature06027

[38]   Hongisto, H., Vuoristo, S., Mikhailova, A., Suuronen, R., Virtanen, I., Otonkoski, T. and Skottman, H. (2012) Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture. Stem Cell Research, 8, 97-108. doi:10.1016/j.scr.2011.08.005

[39]   Kokkinaki, M., Sahibzada, N. and Golestaneh, N. (2011) Human induced pluripotent stem-derived retinal pigment epithelium (RPE) cells exhibit ion transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression pattern similar to native RPE. Stem Cells, 29, 825-835. doi:10.1002/stem.635

[40]   Meyer, J.S., Howden, S.E., Wallace, K.A., Verhoeven, A.D., Wright, L.S., Capowski, E.E., Pinilla, I., Martin, J.M., Tian, S., Stewart, R., Pattnaik, B., Thomson, J.A. and Gamm, D.M. (2011) Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells, 29, 1206-1218. doi:10.1002/stem.674

 
 
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