Advertisement

Journal of Molecular Neuroscience

, Volume 69, Issue 4, pp 597–607 | Cite as

Application of Hanging Drop Culture for Retinal Precursor-Like Cells Differentiation of Human Adipose-Derived Stem Cells Using Small Molecules

  • Hossein Salehi
  • Shahnaz Razavi
  • Ebrahim Esfandiari
  • Mohammad Kazemi
  • Shahram Amini
  • Noushin AmirpourEmail author
Article

Abstract

Retinal degenerative diseases lead to blindness due to poorly regenerative potential of the retina. Recently, cell therapy is more considered for degenerative diseases. Autologous mesenchymal stem cells derived from adipose tissue are a suitable source for this purpose. Therefore, we conducted a stepwise efficient method to differentiate human adipose-derived stem cells (hADSCs) into retinal precursor-like cells in vitro. We compared two differentiation protocols, monolayer and hanging drop cultures. Through the defined medium and 3D hanging drop culture method, we could achieve up to 75% retinal precursor gene expression profile (PAX6, RAX, CHX10, and CRX) from hADSCs. By imitation of in vivo development, for direct conversion of stem cells into retinal cells, the suppression of the BMP, Nodal, and Wnt signaling pathways was carried out by using three small molecules. The hADSCs were primarily differentiated into anterior neuroectodermal cells by expression of OTX2, SIX3, and Β-TUB III and then the differentiated cells were propelled into the retinal cells. According to our data from real-time PCR, RT-PCR, immunocytochemistry, and functional assay, it seems that the hanging drop method improved retinal precursor differentiation yield which these precursor-like cells respond to glutamate neurotransmitter. Regarding the easy accessibility and immunosuppressive properties of hADSCs and more efficient hanging drop method, this study may be useful for future autologous cell therapy of retinal degenerative disorders.

Keywords

hADSCs Retinal precursor Small molecules Hanging drop Differentiation 

Notes

Funding Information

This study was funded by grants from Iran National Science Foundation (Grant no. 92018501).

Compliance with Ethical Standards

The samples were harvested with informed consent which was approved by the Care Committee of Isfahan University of Medical Sciences.

Conflict of Interest

The authors declare that they have no conflicts of interest.

References

  1. Amirpour N, Razavi S, Esfandiari E, Hashemibeni B, Kazemi M, Salehi H (2017) Hanging drop culture enhances differentiation of human adipose-derived stem cells into anterior neuroectodermal cells using small molecules. Int J Dev Neurosci 59:21–30.  https://doi.org/10.1016/j.ijdevneu.2017.03.002 CrossRefPubMedGoogle Scholar
  2. Amirpour N, Amirizade S, Hashemibeni B, Kazemi M, Hadian M, Salehi H (2018) Differentiation of eye field neuroectoderm from human adipose-derived stem cells by using small-molecules and hADSC-conditioned medium. Ann Anat In pressGoogle Scholar
  3. Andoniadou CL, Martinez-Barbera JP (2013) Developmental mechanisms directing early anterior forebrain specification in vertebrates. Cell Mol Life Sci 70:3739–3752.  https://doi.org/10.1007/s00018-013-1269-5 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Banerjee M, Bhonde RR (2006) Application of hanging drop technique for stem cell differentiation and cytotoxicity studies. Cytotechnology 51:1–5.  https://doi.org/10.1007/s10616-006-9001-z CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cardozo AJ, Gómez DE, Argibay PF (2011) Transcriptional characterization of Wnt and Notch signaling pathways in neuronal differentiation of human adipose tissue-derived stem cells. J Mol Neurosci 44:186–194.  https://doi.org/10.1007/s12031-011-9503-9 CrossRefPubMedGoogle Scholar
  6. Castanheira P, Torquetti L, Nehemy MB, Goes AM (2008) Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats. Arq Bras Oftalmol 71:644–650CrossRefGoogle Scholar
  7. Chen S, Wang QL, Nie Z, Sun H, Lennon G, Copeland NG, Gilbert DJ, Jenkins NA, Zack DJ (1997) Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19:1017–1030CrossRefGoogle Scholar
  8. Connaughton V (1995) Glutamate and glutamate receptors in the vertebrate retina. In: Kolb H, Fernandez E, Nelson R (eds) Webvision: The Organization of the Retina and Visual System, Salt Lake CityGoogle Scholar
  9. Dirk J, Iris H, Michael F, Verdon T, Rolf K (2005) β-Catenin–mediated cell-adhesion is vital for embryonic forebrain development. Dev Dyn 233:528–539.  https://doi.org/10.1002/dvdy.20365 CrossRefGoogle Scholar
  10. Fekany-Lee K, Gonzalez E, Miller-Bertoglio V, Solnica-Krezel L (2000) The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of BMP2/4 and Wnt pathways. Development 127:2333–2345PubMedPubMedCentralGoogle Scholar
  11. Gibb S et al (2009) Interfering with Wnt signalling alters the periodicity of the segmentation clock. Dev Biol 330:21–31.  https://doi.org/10.1016/j.ydbio.2009.02.035 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hatakeyama J, Tomita K, Inoue T, Kageyama R (2001) Roles of homeobox and bHLH genes in specification of a retinal cell type. Development 128:1313–1322PubMedGoogle Scholar
  13. Heavner W, Pevny L (2012) Eye development and retinogenesis. Cold Spring Harb Perspect Biol:4.  https://doi.org/10.1101/cshperspect.a008391 CrossRefGoogle Scholar
  14. Horie N, Moriya T, Mitome M, Kitagawa N, Nagata I, Shinohara K (2004) Lowered glucose suppressed the proliferation and increased the differentiation of murine neural stem cells in vitro. FEBS Lett 571:237–242.  https://doi.org/10.1016/j.febslet.2004.06.085 CrossRefPubMedGoogle Scholar
  15. Hunt NC, Hallam D, Karimi A, Mellough CB, Chen J, Steel DHW, Lako M (2017) 3D culture of human pluripotent stem cells in RGD-alginate hydrogel improves retinal tissue development. Acta Biomater 49:329–343.  https://doi.org/10.1016/j.actbio.2016.11.016 CrossRefPubMedGoogle Scholar
  16. Kelm JM, Ehler E, Nielsen LK, Schlatter S, Perriard JC, Fussenegger M (2004) Design of artificial myocardial microtissues. Tissue Eng 10:201–214.  https://doi.org/10.1089/107632704322791853 CrossRefPubMedGoogle Scholar
  17. Koike C et al (2007) Functional roles of Otx2 transcription factor in postnatal mouse retinal development. Mol Cell Biol 27:8318–8329.  https://doi.org/10.1128/MCB.01209-07 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kurokawa D, Ohmura T, Sakurai Y, Inoue K, Suda Y, Aizawa S (2014) Otx2 expression in anterior neuroectoderm and forebrain/midbrain is directed by more than six enhancers. Dev Biol 387:203–213.  https://doi.org/10.1016/j.ydbio.2014.01.011 CrossRefPubMedGoogle Scholar
  19. Lagutin OV et al (2003) Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development. Genes Dev 17:368–379.  https://doi.org/10.1101/gad.1059403 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lamba DA, Karl MO, Ware CB, Reh TA (2006) Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A 103:12769–12774.  https://doi.org/10.1073/pnas.0601990103 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Leow SN, Luu CD, Hairul Nizam MH, Mok PL, Ruhaslizan R, Wong HS, Wan Abdul Halim WH, Ng MH, Ruszymah BHI, Chowdhury SR, Bastion MLC, Then KY (2015) Safety and efficacy of human Wharton’s jelly-derived mesenchymal stem cells therapy for retinal degeneration. PLoS One 10:e0128973.  https://doi.org/10.1371/journal.pone.0128973 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lindroos B, Suuronen R, Miettinen S (2011) The potential of adipose stem cells in regenerative medicine. Stem Cell Rev 7:269–291.  https://doi.org/10.1007/s12015-010-9193-7 CrossRefGoogle Scholar
  23. Liyang G, Abdullah S, Rosli R, Nordin N (2014) Neural commitment of embryonic stem cells through the formation of embryoid bodies (EBs). Malays J Med Sci 21:8–16PubMedPubMedCentralGoogle Scholar
  24. Lupo G et al (2013) Multiple roles of activin/nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/beta-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures. Open Biol 3:120167.  https://doi.org/10.1098/rsob.120167 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Madhu V, Dighe AS, Cui Q, Deal DN (2016) Dual inhibition of activin/nodal/TGF-beta and BMP signaling pathways by SB431542 and dorsomorphin induces neuronal differentiation of human adipose derived stem cells. Stem Cells Int 2016:1035374.  https://doi.org/10.1155/2016/1035374 CrossRefPubMedGoogle Scholar
  26. Mathers PH, Jamrich M (2000) Regulation of eye formation by the Rx and pax6 homeobox genes. Cell Mol Life Sci 57:186–194.  https://doi.org/10.1007/PL00000683 CrossRefPubMedGoogle Scholar
  27. Mochizuki H, Ohnuki Y, Kurosawa H (2011) Effect of glucose concentration during embryoid body (EB) formation from mouse embryonic stem cells on EB growth and cell differentiation. J Biosci Bioeng 111:92–97.  https://doi.org/10.1016/j.jbiosc.2010.09.001 CrossRefPubMedGoogle Scholar
  28. Nieuwkoop PD (1952) Activation and organization of the central nervous system in amphibians. Part II. Differentiation and organization. J Exp Zool 120:33–81.  https://doi.org/10.1002/jez.1401200103 CrossRefGoogle Scholar
  29. Nishida A, Furukawa A, Koike C, Tano Y, Aizawa S, Matsuo I, Furukawa T (2003) Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nat Neurosci 6:1255–1263.  https://doi.org/10.1038/nn1155 CrossRefPubMedGoogle Scholar
  30. Osakada F et al (2008) Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol 26:215–224.  https://doi.org/10.1038/nbt1384 CrossRefPubMedGoogle Scholar
  31. Osakada F et al (2009) In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J Cell Sci 122:3169–3179.  https://doi.org/10.1242/jcs.050393 CrossRefPubMedGoogle Scholar
  32. Pettinato G, Wen X, Zhang N (2014) Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays. Sci Rep 4:7402.  https://doi.org/10.1038/srep07402 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Rezanejad H, Soheili ZS, Haddad F, Matin MM, Samiei S, Manafi A, Ahmadieh H (2014) In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene. Cell Tissue Res 356:65–75.  https://doi.org/10.1007/s00441-014-1795-y CrossRefPubMedGoogle Scholar
  34. Rowan S, Chen C-MA, Young TL, Fisher DE, Cepko CL (2004) Transdifferentiation of the retina into pigmented cells in ocular retardation mice defines a new function of the homeodomain gene <em>Chx10</em>. Development 131:5139–5152.  https://doi.org/10.1242/dev.01300 CrossRefPubMedGoogle Scholar
  35. Salehi H, Amirpour N, Niapour A, Razavi S (2016) An overview of neural differentiation potential of human adipose derived stem cells. Stem Cell Rev 12:26–41.  https://doi.org/10.1007/s12015-015-9631-7 CrossRefGoogle Scholar
  36. Schmitt S et al (2009) Molecular characterization of human retinal progenitor cells. Invest Ophthalmol Vis Sci 50:5901–5908.  https://doi.org/10.1167/iovs.08-3067 CrossRefPubMedGoogle Scholar
  37. Shen MM (2007) Nodal signaling: developmental roles and regulation. Development 134:1023–1034.  https://doi.org/10.1242/dev.000166 CrossRefPubMedGoogle Scholar
  38. Sivan PP, Syed S, Mok PL, Higuchi A, Murugan K, Alarfaj AA, Munusamy MA, Awang Hamat R, Umezawa A, Kumar S (2016) Stem cell therapy for treatment of ocular disorders. Stem Cells Int 2016:8304879.  https://doi.org/10.1155/2016/8304879 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Smith JR, Vallier L, Lupo G, Alexander M, Harris WA, Pedersen RA (2008) Inhibition of activin/nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev Biol 313:107–117.  https://doi.org/10.1016/j.ydbio.2007.10.003 CrossRefPubMedGoogle Scholar
  40. Teraoka ME, Paschaki M, Muta Y, Ladher RK (2009) Rostral paraxial mesoderm regulates refinement of the eye field through the bone morphogenetic protein (BMP) pathway. Dev Biol 330:389–398.  https://doi.org/10.1016/j.ydbio.2009.04.008 CrossRefPubMedGoogle Scholar
  41. Voorneveld PW et al (2015) The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC. Br J Cancer 112:122–130.  https://doi.org/10.1038/bjc.2014.560 CrossRefPubMedGoogle Scholar
  42. Wilson L, Maden M (2005) The mechanisms of dorsoventral patterning in the vertebrate neural tube. Dev Biol 282:1–13.  https://doi.org/10.1016/j.ydbio.2005.02.027 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Anatomical Sciences and Molecular Biology, School of MedicineIsfahan University of Medical SciencesIsfahanIran
  2. 2.Department of Genetic, School of MedicineIsfahan University of Medical SciencesIsfahanIran

Personalised recommendations