Optimization and application of encapsulation technology for cell therapy in diabetes.

189 p.El eje central de esta tesis doctoral consiste en la búsqueda del conjunto de técnicas más adecuadas para la obtención de células productoras de insulina diferenciadas a partir de células mesenquimales en el interior de microcápsulas, cuya composición se habrá optimizado para recrear el ambiente de estos tipos celulares con la mayor fidelidad posible. Para ello, se han llevado a cabo los siguientes estudios: (I) Obtención de un biomaterial compuesto de alginato y ácido hialurónico apto para microencapsulación con similares propiedades físicas y químicas a las microcápsulas de alginato.(II) Estudios in vitro con células encapsuladas para comprobar que el biomaterial recrea el ambiente in vivo de las células mesenquimales.(III) Comparativa del comportamiento biológico de células productoras de insulina encapsuladas en matrices de alginato y alginato-hialurónico.(IV) Diferenciación de células mesenquimales procedentes de distintos tejidos encapsuladas en mezclas de alginato y alginato-hialurónico para la obtención de células ß productoras de insulina

Characterization of retinal cells derived from iPSCs of a patient with PRPF31 associated retinitis pigmentosa

Motivation: Retinitis pigmentosa (RP) is a group of hereditary retinal dystrophies caused by mutations in different genes with a prevalence of 1 in 4000. It is an untreatable disease with a variable clinical evolution in which patients develop severe visual impairment or total blindness. Mutations in pre-mRNA splicing PRPF31 gene have been described as the second most common cause of autosomal dominant RP. Previous studies relate mutations in PRPF31 with dysfunction and degeneration of the retinal pigment epithelium (RPE). Thanks to the ability to obtain and differentiate induced pluripotent stem cells (iPSC), retinal models can be generated to study the disease mechanism and to evaluate new therapies. This work is based on a personalized cellular model obtained by differentiating RPE from iPSCs of a patient with PRPF31 c.165G mutation, which will be used to study the mechanism of the disease. Methods: iPSCs and previously differentiated RPE cells have been cultured and imaged. The characterization of the RNA level expression of specific genes of both cell types has been performed by RT-PCR. Expression at the protein level has been analyzed by Western blot. At the physiological level, the ability of the cellular model to establish an epithelial barrier has been evaluated by transepithelial electrical resistance (TER). Results: Phase contrast images showed a characteristic and distintive morphology of iPSC and RPE cells. RT-PCR showed the silencing of pluripotency genes such as NANOG in RPE cells, as well as the exclusive expression of specific genes such as CRALBP and RPE65 in RPE. In the comparative study of the cellular models of patient and healthy control, it was observed a variation in the expression levels of the PRPF31 and RPE65 genes. In Western blot, the PRPF31 protein detected in the patient's RPE showed a different band pattern compared with healthy control and iPSCs. Finally, TER showed a similar maturation of the two cell models compared, indicating that PRPF31 c.165G mutation does not affect the cells adhesions. Conclusions: The cellular model of RPE with PRPF31 c.165G mutation has been correctly differentiated, allowing the study of the consequences at the cellular level of this genetic defect. The decrease found in RPE65 gene expression suggests that this could be the mechanism by wich PRPF31 c.165G mutation causes RP, because RPE65 insufficiency is a known cause of blindness

Optimization and application of encapsulation technology for cell therapy in diabetes.

189 p.El eje central de esta tesis doctoral consiste en la búsqueda del conjunto de técnicas más adecuadas para la obtención de células productoras de insulina diferenciadas a partir de células mesenquimales en el interior de microcápsulas, cuya composición se habrá optimizado para recrear el ambiente de estos tipos celulares con la mayor fidelidad posible. Para ello, se han llevado a cabo los siguientes estudios: (I) Obtención de un biomaterial compuesto de alginato y ácido hialurónico apto para microencapsulación con similares propiedades físicas y químicas a las microcápsulas de alginato.(II) Estudios in vitro con células encapsuladas para comprobar que el biomaterial recrea el ambiente in vivo de las células mesenquimales.(III) Comparativa del comportamiento biológico de células productoras de insulina encapsuladas en matrices de alginato y alginato-hialurónico.(IV) Diferenciación de células mesenquimales procedentes de distintos tejidos encapsuladas en mezclas de alginato y alginato-hialurónico para la obtención de células ß productoras de insulina

Review of Advanced Hydrogel-Based Cell Encapsulation Systems for Insulin Delivery in Type 1 Diabetes Mellitus

Type 1 Diabetes Mellitus (T1DM) is characterized by the autoimmune destruction of beta-cells in the pancreatic islets. In this regard, islet transplantation aims for the replacement of the damaged beta-cells through minimally invasive surgical procedures, thereby being the most suitable strategy to cure T1DM. Unfortunately, this procedure still has limitations for its widespread clinical application, including the need for long-term immunosuppression, the lack of pancreas donors and the loss of a large percentage of islets after transplantation. To overcome the aforementioned issues, islets can be encapsulated within hydrogel-like biomaterials to diminish the loss of islets, to protect the islets resulting in a reduction or elimination of immunosuppression and to enable the use of other insulin-producing cell sources. This review aims to provide an update on the different hydrogel-based encapsulation strategies of insulin-producing cells, highlighting the advantages and drawbacks for a successful clinical application.Authors thank the support to research on cell microencapsulation from the University of the Basque Country UPV/EHU (EHUa16/06 to L.SB) and the Basque Country Government (Grupos Consolidados, ref. no.: IT907-16 to J.L. P). Authors also thank ICTS "NANBIOSIS", specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) at the University of Basque Country UPV/EHU in Vitoria-Gasteiz

Generation of the human iPSC line ESi082-A from a patient with macular dystrophy associated to mutations in the CRB1 gene

Retinal dystrophies associated to mutations in the CRB1 gene comprise a wide array of clinical presentations. A blood sample from a patient with a family history of CRB1-retinal dystrophy was used to prepare the iPSC line ESi082-A. The genotype of the donor, affected of a perifoveal-bilateral macular dystrophy includes one frameshift deletion and one hypomorphic allele. ESi082-A cell line has been characterized for pluripotency and will be used to prepare retinal cellular models to study the dysfunction leading to the disease.This work was supported by Andalusian Ministry of Health, Equality and Social Policies (PI-0099-2018).Ye

Generation of the human iPSC line ESi082-A from a patient with macular dystrophy associated to mutations in the CRB1 gene

Retinal dystrophies associated to mutations in the CRB1 gene comprise a wide array of clinical presentations. A blood sample from a patient with a family history of CRB1-retinal dystrophy was used to prepare the iPSC line ESi082-A. The genotype of the donor, affected of a perifoveal-bilateral macular dystrophy includes one frameshift deletion and one hypomorphic allele. ESi082-A cell line has been characterized for pluripotency and will be used to prepare retinal cellular models to study the dysfunction leading to the disease.This work was supported by Andalusian Ministry of Health, Equality and Social Policies (PI-0099-2018)

Type 1 Diabetes Mellitus reversal via implantation of magnetically purified microencapsulated pseudoislets

Microencapsulation of pancreatic islets for the treatment of Type I Diabetes Mellitus (T1DM) generates a high quantity of empty microcapsules, resulting in high therapeutic graft volumes that can enhance the host’s immune response. We report a 3D printed microfluidic magnetic sorting device for microcapsules purification with the objective to reduce the number of empty microcapsules prior transplantation. In this study, INS1E pseudoislets were microencapsulated within alginate (A) and alginate-poly-L-lysine-alginate (APA) microcapsules and purified through the microfluidic device. APA microcapsules demonstrated higher mechanical integrity and stability than A microcapsules, showing better pseudoislets viability and biological function. Importantly, we obtained a reduction of the graft volume of 77.5% for A microcapsules and 78.6% for APA microcapsules. After subcutaneous implantation of induced diabetic Wistar rats with magnetically purified APA microencapsulated pseudoislets, blood glucose levels were restored into normoglycemia (< 200 mg/dL) for almost 17 weeks. In conclusion, our described microfluidic magnetic sorting device represents a great alternative approach for the graft volume reduction of microencapsulated pseudoislets and its application in T1DM disease.University of the Basque Country UPV/EHU (Spain) (EHUa16/06 to L.SB, and ESPPOC 16/65), the Basque Country Government (Spain) (Grupos Consolidados with Grant N° IT907-16 to JL. P, Elkartek with Grant N°KK-2017/ 0000088 and RIS3 with Grant N°307616FKA4) and the Spanish Government (Spain) (RYC-2012-10796). Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country UPV/EHU, Prof. Maechler from the University of Geneva Medical Center for providing the INS1E cell line, Adhesive Research for providing PSA sheets and Prof. Martínez de Pancorbo for her laboratory facilities at University of the Basque Country UPV/EHU