Induced pluripotent stem cells as a new approach in therapy for stroke

• Autores: Esteban López Arias
• Directores de la Tesis: José Antonio Castillo Sánchez (dir. tes.), Tomas Sobrino Moreiras (codir. tes.), Francisco Campos Pérez (codir. tes.)
• Lectura: En la Universidade de Santiago de Compostela ( España ) en 2020
• Idioma: inglés
• Tribunal Calificador de la Tesis: Lino Ferreira (presid.), Manuel Collado Rodríguez (secret.), Antonia María Gutiérrez Fernández (voc.)
• Programa de doctorado: Programa de Doctorado en Medicina Molecular por la Universidad de Santiago de Compostela
• Enlaces
  • Tesis en acceso abierto en: MINERVA
• Resumen

  • Ischemic and haemorrhagic stroke remain as diseases without treatment for recovery nor restauration of brain tissue, despite all the efforts of biomedical community to find out some neuroprotective or neurorestorative drug. Being the first cause of disability in Europe, stroke patients cost thousands of million euros per years to public health European cares; and not only as medical expenses but also direct and indirect social costs.

    Many of efforts aimed to heal stroke focus on cell therapy, both in preclinical and clinical phases. Induced pluripotent stem cells (iPSCs) appeared in 2006 as a revolution in the way of getting stem cells. iPSCs are dedifferentiated as embryonic stem cells but can be obtained from somatic, adult and differentiated tissues. This open big opportunities in autologous grafts.

    That is why we hypothesized that the systemic administration of iPSCs may improve the progress in stroke animal model, ischemic and haemorrhagic. As far as we know, we injected iPSCs intravenously in stroke animal models for the first time.

    The first section presents the results of obtaining of iPSCs derived from primary mouse embryonic fibroblasts carrying a polycistronic cassette for the inducible expression of four reprogramming factors, and characterization of these cells by the standard methods.

    In the second section of this study, we carried out a protocol to evaluate the potential therapeutic effect of intravenous administration of iPSCs in an ischemic stroke model in rats. The ischemic stroke was inducted by transient middle cerebral artery occlusion (tMCAO). The day after induction of cerebral ischemia, magnetic resonance imaging (MRI) and functional tests were performed. After this first follow-up, 3 million of iPSCs in 1 ml PBS were administrated in jugular vein in treated group (iv iPSCs) and 1 ml of PBS in control group. MRI and functional tests were repeated at 7, 14, 28 and 56 days. At 56 days we also acquired positron emission tomography (PET) images of whole body to check tumour formation. And finally, rats were perfused and their brains extracted for histological analysis of neurogenesis and angiogenesis.

    There were no differences in progression of lesion volumes between groups. iPSCs-treated group obtained better results in neurological tests, but not in motor test. Also, we found a greater neurogenesis in iv iPSCs group.

    The third section is about the potential therapeutic effect of iPSCs administration in a haemorrhagic stroke model. The established protocol was similar to in the second section. The haemorrhage was inducted by injection of 5 μl collagenase in striatum. The first follow-ups consisted in MRI and functional tests same as in the previous section at 1, 7, 14, 28 and 56 days after model induction The treatments were administrated after first follow-up: 3 million of iPSCs in 1 ml PBS in jugular and 500000 iPSCs in 5 μl PBS in perihematoma (ic iPSCs). Control groups with injection of vehicle were also performed. PET images of whole body were acquired, and brains were extracted for histological study.

    The hematoma volume progression did not show any differences between groups. ic iPSCs group presented worse punctuation in motor and neurological tests in comparison to its respective control, meanwhile iv iPSCs group obtained less deficit than its control in motor function. We observed an increased neuroblast positive area in ic iPSCs group compared to its control and enhanced activated neuroblasts in iv iPSCs group.

    No differences were found in angiogenesis in any group, and PET studies did not reveal any tumour formation in both models.

    In fourth section, cell tracking study was carried out to find where iPSCs travelled after systemic administration. iPSCs were labelled by 10 and 18 nm gold nanoparticles (AuNPs), apoptosis and pluripotency maintaining of labelled cells were evaluated to analyse the effect of AuNPs integration in cell metabolism. 18 nm-AuNPs-labelled cells were injected in ischemic rats in jugular vein. Organs were extracted and analysed by inductively coupled plasma mass spectrometry to measure quantity of gold. Most gold was found in liver (near to 80%) and to a lesser extent in spleen and lungs.