PPARgamma dependent PEX11beta counteracts the suppressive role of SIRT1 on neural differentiation of HESCs.

The membrane peroxisomal proteins PEX11, play a crucial role in peroxisome proliferation by regulating elongation, membrane constriction, and fission of pre-existing peroxisomes. In this study, we evaluated the function of PEX11B gene in neural differentiation of human embryonic stem cell (hESC) by inducing shRNAi-mediated knockdown of PEX11B expression. Our results demonstrate that loss of PEX11B expression led to a significant decrease in the expression of peroxisomal-related genes including ACOX1, PMP70, PEX1, and PEX7, as well as neural tube-like structures and neuronal markers. Inhibition of SIRT1 using pharmacological agents counteracted the effects of PEX11B knockdown, resulting in a relative increase in PEX11B expression and an increase in differentiated neural tube-like structures. However, the neuroprotective effects of SIRT1 were eliminated by PPAR inhibition, indicating that PPARÉ£ may mediate the interaction between PEX11B and SIRT1. Our findings suggest that both SIRT1 and PPARÉ£ have neuroprotective effects, and also this study provides the first indication for a potential interaction between PEX11B, SIRT1, and PPARÉ£ during hESC neural differentiation.

Potential therapeutic strategies for photoreceptor degeneration: the path to restore vision.

Photoreceptors (PRs), as the most abundant and light-sensing cells of the neuroretina, are responsible for converting light into electrical signals that can be interpreted by the brain. PR degeneration, including morphological and functional impairment of these cells, causes significant diminution of the retina's ability to detect light, with consequent loss of vision. Recent findings in ocular regenerative medicine have opened promising avenues to apply neuroprotective therapy, gene therapy, cell replacement therapy, and visual prostheses to the challenge of restoring vision. However, successful visual restoration in the clinical setting requires application of these therapeutic approaches at the appropriate stage of the retinal degeneration. In this review, firstly, we discuss the mechanisms of PR degeneration by focusing on the molecular mechanisms underlying cell death. Subsequently, innovations, recent developments, and promising treatments based on the stage of disorder progression are further explored. Then, the challenges to be addressed before implementation of these therapies in clinical practice are considered. Finally, potential solutions to overcome the current limitations of this growing research area are suggested. Overall, the majority of current treatment modalities are still at an early stage of development and require extensive additional studies, both pre-clinical and clinical, before full restoration of visual function in PR degeneration diseases can be realized.

Characterization of The Retinal Progenitor Cells Generated Using Co-Culture Systems.

Objective: Degeneration of the photoreceptors due to retinal disorders can affect vision, and even lead to blindness. Recently therapeutic progress in retinal degeneration, using human embryonic stem cells (hESCs), has been facing technical challenges, demanding the development of simple and standardized protocols. In addition to the designing of the protocols, characterization of the obtained cells is highly required for confirming the reliability of the applied methods for future medical applications. Previously, we showed that human stem cells from apical papilla (SCAP) have stromal cell-derived inducing activity (SDIA). Materials and Methods: In this experimental study, we developed an efficient retinal differentiation protocol, based on the co-culture of confluent hESCs and SCAP in the absence of exogenous molecules, such as activators or inhibitors of molecular signaling pathways. This experimental procedure resulted in the generation of self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs) within 4 weeks. Results: We have focused on the characterization of the derived RPCs, as a crucial step towards further verification of the efficiency of our previously suggested protocol. The differentiated cells expressed eye-field markers, PAX6, RAX, LHX2, and SIX3, and also generated neurospheres by a floating culture system for one week. Conclusion: We have reported that the treatment of hESC-derived RPCs by the Notch pathway-inhibitor induced the generation of photoreceptor precursor cells (PPCs). The presented method demonstrates the fact that a co-culture of hESCs and SCAP without exogenous molecules provides an efficient approach to produce RPCs for the treatment of retinal disease, and act as an in vitro model for the development of human retina.

Scaffold free retinal pigment epithelium sheet engineering using modified alginate-RGD hydrogel.

Tissue-specific extracellular matrix (ECM) plays a critical role in cell survival and homeostasis, which are particularly essential for directing differentiation of different complex tissues such as retina. However, ECM maintenance should be considered to design an effective therapeutic strategy for retina regeneration. To achieve this, cell sheet engineering has emerged as a growing approach to closely reconstruct basal membrane of cells through a scaffold-free manner. Several irreversible sight-threatening diseases are characterized by the dysfunction and lose of retinal pigment epithelium (RPE), leading to vision loss and eventually total blindness in patients. According to impressive developments in achievement of RPE from human embryonic stem cells (hESCs), we obtained RPE cells without any extrinsic factors in a co-culture system, and cultured them on a temporary alginate hydrogel substrate. Subsequently, Arg-Gly-Asp (RGD) peptide was superficially immobilized on the upper layer of hydrogel to improve cell attachment before harvesting sheet layer. RPE cell sheet layer was released by treating pre-seeded hydrogels with sodium citrate as a calcium chelating agent and characterized in both in vitro and in vivo models. RPE sheets formed tight junction and expressed high levels of retina structural markers such as ZO-1, Bestrophin and Collagen type IV. One week after in vivo transplantation of RPE sheet, cells survived in the subretinal space, indicating that our harvesting method is non-invasive. To sum up, we introduced a unique scaffold-free method for RPE cell sheet engineering, which can find potential use for future therapeutic purposes.

Construction and characterization of EGFP reporter plasmid harboring putative human RAX promoter for in vitro monitoring of retinal progenitor cells identity.

BACKGROUND: In retinal degenerative disease, progressive and debilitating conditions result in deterioration of retinal cells and visual loss. In human, retina lacks the inherent capacity for regeneration. Therefore, regeneration of retinal layer from human retinal progenitor cells (hRPCs) is a challenging task and restricted in vitro maintenance of hRPCs remains as the main hurdle. Retina and anterior neural fold homeobox gene (RAX) play critical roles in developing retina and maintenance of hRPCs. In this study, for the first time regulatory regions of human RAX gene with potential promoter activity were experimentally investigated. RESULTS: For this purpose, after in silico analysis of regulatory regions of human RAX gene, the expression of EGFP reporter derived by putative promoter sequences was first evaluated in 293 T cells and then in hRPCS derived from human embryonic stem cells. The candidate region (RAX-3258 bp) showed the highest EGFP expression in hRPCs. This reporter construct can be used for in vitro monitoring of hRPC identity and verification of an efficient culture medium for maintenance of these cells. CONCLUSIONS: Furthermore, our findings provide a platform for better insight into regulatory regions of human RAX gene and molecular mechanisms underlying its vital functions in retina development.

Activated platelet-rich plasma improves cartilage regeneration using adipose stem cells encapsulated in a 3D alginate scaffold.

In the current study, the effect of superimposing platelet-rich plasma (PRP) on different culture mediums in a three-dimensional alginate scaffold encapsulated with adipose-derived mesenchymal stem cells for cartilage tissue repair is reported. The three-dimensional alginate scaffolds with co-administration of PRP and/or chondrogenic supplements had a significant effect on the differentiation of adipose mesenchymal stem cells into mature cartilage, as assessed by an evaluation of the expression of cartilage-related markers of Sox9, collagen II, aggrecan and collagen, and glycosaminoglycan assays. For in vivo studies, following induction of osteochondral lesion in a rabbit model, a high degree of tissue regeneration in the alginate plus cell group (treated with PRP plus chondrogenic medium) compared with other groups of cell-free alginate and untreated groups (control) were observed. After 8 weeks, in the alginate plus cell group, functional chondrocytes were observed, which produced immature matrix, and by 16 weeks, the matrix and hyaline-like cartilage became completely homogeneous and integrated with the natural surrounding cartilage in the defect site. Similar effect was also observed in the subchondral bone. The cell-free scaffolds formed fibrocartilage tissue, and the untreated group did not form a continuous cartilage over the defect by 16 weeks.

A ground state of PPARγ activity and expression is required for appropriate neural differentiation of hESCs.

BACKGROUND: Several evidences indicate stimulation of peroxisome proliferator activated receptor γ (PPARg), promotes neuronal differentiation. This study was conducted to testify the prominence of PPARγ during neural differentiation of human embryonic stem cells (hESCs). METHODS: PPARγ expression level was assessed during neural differentiation of hESCs. Meanwhile, the level of endogenous miRNAs, which could be engaged in regulation of PPARγ expression, was measured. Next, natural and synthetic components of PPARγ agonists and antagonist were implemented on neural progenitor formation during neural differentiation of hESCs. RESULTS: Data showed an increasing wave of PPARγ expression level when human neural progenitors (NPs) were formed upon retinoic acid treatment. Interestingly, there was no significant difference in the amount of PPARγ proteins during the differentiation of hESCs that is inconsistent with what we observed for RNA level. Our results indicated that miRNAs are not involved in the regulation of PPARγ expression, while proteasome-mediated degradation may to some degree be involved in this process. Among numerous treatments, PPARγ inactivation during NPs formation significantly decreased expression of NP markers. CONCLUSIONS: We conclude that a ground state of PPARγ activity is required for NP formation of hESCs during early neural differentiation. However, high expression and activity of PPARγ could not enhance the required neural differentiation, whereas the PPARγ inactivation could negatively influence NP formation from hESCs by antagonist.