The Role of Some Molecular Factors in the Generation of Insulin-Producing Cells from Stem Cells

Abstract

The field of regenerative medicine is rapidly evolving, paving the way for novel therapeutic interventions through cellular therapies which are reshaping the biomedical field. As for DM, cell replacement strategies via islet transplantation offer potential therapeutic options for diabetic patients. However, the discrepancy between the limited number of donor islets and the high number of patients who could benefit from such a treatment reflects the severe need for renewable sources of high-quality β-cells. Interestingly, MSCs have made their mark in the evolving field of regenerative medicine, and have been suggested as a potential weapon to develop therapies for DM. Furthermore, genetic manipulation of key transcription factors can serve to promote trans-differentiation and increase plasticity of MSCs. REST has been reported as a master negative regulator of neurogenesis. Furthermore, it has been shown to be associated with the core selfrenewal genes in ESCs, and also has been reported to play important roles in β-cells. Accordingly, in the current study, we sought to further investigate WJ-MSCs being a readily accessible potential source for cell therapy of DM together with the genetic manipulation of the transcription factor REST. In an attempt to gain more insight into its role to generate functioning IPCs from WJ-MSCs, and also to study the potential of its modulation as a suggested new modality to improve the therapeutic outcome of cell therapy for DM. To fulfil the aims of the current study, we isolated WJ-MSCs from human UCs, and further characterized them according to the defined criteria by ISCT. Afterwards, we induced them to differentiate to IPCs using various protocols. Meanwhile, we assessed the levels of both stem cells and β-cells markers side by Summary and Conclusions 142 side in all the studied differentiation protocols. Finally, we carried out knock down of REST using specific targeting siRNAs, and studied the effect of such genetic manipulation on WJ-MSCs under both control and differentiation conditions. The findings of the current study are summarized as follows:  The isolation of WJ-MSCs was relatively easy, and provided a homogenous population, together with large expansion potential, which come in accordance with all the previous reports highlighting the importance of WJ-MSCs for regenerative medicine.  During the induction of differentiation of WJ-MSCs to IPCs, surprisingly, the expression levels of both stemness and β-cells markers were induced in the derived IPCs from various protocols. The sustained expression of these pluripotency markers might be associated with the incomplete differentiation and inability of the derived IPCs to attain mature β-cell state. Nevertheless, Oct-4 and Sox-2 in the current study could play some unknown important role during the differentiation process. The current findings imply that the strategy of focusing on the induction of β-cell markers alone might not be sufficient to attain complete differentiation of MSCs to mature functioning IPCs. The pluripotency markers’ levels during the differentiation process should be considered carefully, and further stem cell markers directed investigations are warranted to provide more evidence of this finding.  Various extrinsic inducing factors incorporated in differentiation protocols like; nicotinamide and exendin-4, besides their inducing effects on differentiation markers, they are also having inducing effects on pluripotency markers. That interesting finding needs much further investigations and unravelling the mechanism of action of various extrinsic inducing factors is indeed warranted. Summary and Conclusions 143  Unfortunately, the IPCs derived from all the studied protocols could not attain mature glucose-responsive β-cell state, which reflects their incomplete differentiation. This can be at least partially explained by the sustained expression of stemness markers together with the induced expression of β-cells markers. Besides, the remaining detected levels of REST protein in all the derived IPCs could also be an important contributing factor to the impaired GSIS.  The knock down of REST via siREST unexpectedly negatively affected the differentiation outcome of WJ-MSCs to IPCs. The existence of a tightly controlled temporal regulation that requires REST to be present in the initial induction early differentiation stages, and to be absent in mature functioning β-cells could at least partially explain such unexpected finding.  The genetic manipulation of REST as a putative new modality to improve the differentiation outcome of WJ-MSCs to IPCs appears to be far more complex than what we thought previously.  Some sort of interplay might exist between REST, Oct-4, Sox-2, and other unknown factors during the induction of differentiation of WJ-MSCs to IPCs. That interplay might be extended to other types of stem cells differentiating to IPCs. The mechanism of such interplay if unraveled could provide putative therapeutic targets for DM.