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


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.


hADSCs Retinal precursor Small molecules Hanging drop Differentiation 


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.


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© 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

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