Enhanced neurotrophin synthesis and molecular differentiation in non-transformed human retinal progenitor cells cultured in a rotating bioreactor.
One approach to the treatment of retinal diseases, such as retinitis pigmentosa, is to replace diseased or degenerating cells with healthy cells. Even if all of the problems associated with tissue transplant were to be resolved, the availability of tissue would remain an ongoing problem. We have previously shown that transformed human retinal cells can be grown in a NASA-developed horizontally rotating culture vessel (bioreactor) to form three-dimensional-like structures with the expression of several retinal specific proteins. In this study, we have investigated growth of non-transformed human retinal progenitors (retinal stem cells) in a rotating bioreactor. This rotating culture vessel promotes cell-cell interaction between similar and dissimilar cells. We cultured retinal progenitors (Ret 1-4) alone or as a co-culture with human retinal pigment epithelial cells (RPE, D407) in this system to determine if 3D structures can be generated from non-transformed progenitors. Our second goal was to determine if the formation of 3D structures correlates with the upregulation of neurotrophins, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGFalpha), ciliary neurotrophic factor (CNTF), and brain-delivered neurotrophic factor (BDNF). These factors have been implicated in progenitor cell proliferation, commitment, differentiation, and survival. We also investigated the expression of the following retinal specific proteins in this system: neuron specific enolase (NSE); tyrosine hydroxylase (TH); D(2)D(3), D(4) receptors; protein kinase-C alpha (PKCalpha), and calbindin. The 3D structures generated were characterized by phase and scanning transmission electron microscopy. Retinal progenitors, cultured alone or as a co-culture in the rotating bioreactor, formed 3D structures with some degree of differentiation, accompanied by the upregulation of bFGF, CNTF, and TGFalpha. Brain-derived neurotrophic factor, which is expressed in vivo in RPE (D407), was not expressed in monolayer cultures of RPE but expressed in the rotating bioreactor-cultured RPE and retinal progenitors (Ret 1-4). Upregulation of neurotrophins was noted in all rotating bioreactor-cultured cells. Also, upregulation of D(4) receptor, calbindin, and PKCalpha was noted in the rotating bioreactor-cultured cells. We conclude that non-transformed retinal progenitors can be grown in the rotating bioreactor to form 3D structures with some degree of differentiation. We relied on molecular and biochemical analysis to characterize differentiation in cells grown in the rotating bioreactor.
Kumar
Ravindra
R
Department of Pathology, Morehouse School of Medicine, Atlanta, Georgia, USA.
Dutt
Kamla
K
D019149:Bioreactors; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis / Q000235:genetics; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000096:biosynthesis / Q000235:genetics; D018920:Coculture Techniques; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008855:Microscopy, Electron, Scanning; D009414:Nerve Growth Factors / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D016211:Transforming Growth Factor alpha / Q000096:biosynthesis / Q000235:genetics
D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D016211:Transforming Growth Factor alpha; D016222:Fibroblast Growth Factor 2