mRNA Reprogramming of T8993G Leigh's Syndrome Fibroblast Cells to Create Induced Pluripotent Stem Cell Models for Mitochondrial Disorders.

Early molecular and developmental events impacting many incurable mitochondrial disorders are not fully understood and require generation of relevant patient- and disease-specific stem cell models. In this study, we focus on the ability of a nonviral and integration-free reprogramming method for deriving clinical-grade induced pluripotent stem cells (iPSCs) specific to Leigh's syndrome (LS), a fatal neurodegenerative mitochondrial disorder of infants. The cause of fatality could be due to the presence of high abundance of mutant mitochondrial DNA (mtDNA) or decline in respiration levels, thus affecting early molecular and developmental events in energy-intensive tissues. LS patient fibroblasts (designated LS1 in this study), carrying a high percentage of mutant T8993G mtDNA, were reprogrammed using a combined mRNA-miRNA nonviral approach to generate human iPSCs (hiPSCs). The LS1-hiPSCs were evaluated for their self-renewal, embryoid body (EB) formation, and differentiation potential, using immunocytochemistry and gene expression profiling methods. Sanger sequencing and next-generation sequencing approaches were used to detect the mutation and quantify the percentage of mutant mtDNA in the LS1-hiPSCs and differentiated derivatives. Reprogrammed LS-hiPSCs expressed pluripotent stem cell markers including transcription factors OCT4, NANOG, and SOX2 and cell surface markers SSEA4, TRA-1-60, and TRA-1-81 at the RNA and protein level. LS1-hiPSCs also demonstrated the capacity for self-renewal and multilineage differentiation into all three embryonic germ layers. EB analysis demonstrated impaired differentiation potential in cells carrying high percentage of mutant mtDNA. Next-generation sequencing analysis confirmed the presence of high abundance of T8993G mutant mtDNA in the patient fibroblasts and their reprogrammed and differentiated derivatives. These results represent for the first time the derivation and characterization of a stable nonviral hiPSC line reprogrammed from a LS patient fibroblast carrying a high abundance of mutant mtDNA. These outcomes are important steps toward understanding disease origins and developing personalized therapies for patients suffering from mitochondrial diseases.

Human iNPC therapy leads to improvement in functional neurologic outcomes in a pig ischemic stroke model.

Introduction: Stroke is the leading cause of disability in the United States but current therapies are limited with no regenerative potential. Previous translational failures have highlighted the need for large animal models of ischemic stroke and for improved assessments of functional outcomes. The aims of this study were first, to create a post-stroke functional outcome assessment scale in a porcine model of middle cerebral artery occlusion (MCAO) and second, to use this scale to determine the effect of human-induced-pluripotent-cell-derived neural progenitor cells (iNPCs) on functional outcome in this large animal stroke model. Materials and Methods: Eight 6-month-old Landrace mix pigs underwent permanent MCAO. Five days following MCAO, pigs received intraparenchymal injections of either iNPCs or PBS. A post-stroke assessment scale was developed to measure functional outcome. Evaluations were performed at least 1-3 days prior to MCAO and repeated 1 day, 3 days, and 5 days post-stroke as well as 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, 9 weeks, and 12 weeks post-injection. Comparisons of scores between animals receiving iNPCs or PBS only were compared using a two-way ANOVA and a Tukey's post-hoc t test. Results: The developed scale was able to consistently determine differences between healthy and stroked pigs at all time points. iNPC-treated pigs showed a significantly faster recovery in their overall scores relative to PBS-only treated pigs with the parameters of appetite and body posture exhibiting the most improvement in the iNPC-treated group. Conclusions: We developed a robust and repeatable functional assessment tool that can reliably detect stroke and recovery, while also showing for the first time that iNPC therapy leads to functional recovery in a translational pig ischemic stroke model. These promising results suggest that iNPCs may 1 day serve as a first in class cell therapeutic for ischemic stroke.

Induced Pluripotent Stem Cell-Derived Neural Stem Cell Therapy Enhances Recovery in an Ischemic Stroke Pig Model.

Induced pluripotent stem cell-derived neural stem cells (iNSCs) have significant potential as an autologous, multifunctional cell therapy for stroke, which is the primary cause of long term disability in the United States and the second leading cause of death worldwide. Here we show that iNSC transplantation improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in a novel ischemic pig stroke model. Longitudinal multiparametric magnetic resonance imaging (MRI) following iNSC therapy demonstrated reduced changes in white matter integrity, cerebral blood perfusion, and brain metabolism in the infarcted tissue. The observed tissue level recovery strongly correlated with decreased immune response, enhanced neuronal protection, and increased neurogenesis. iNSCs differentiated into neurons and oligodendrocytes with indication of long term integration. The robust recovery response to iNSC therapy in a translational pig stroke model with increased predictive potential strongly supports that iNSCs may be the critically needed therapeutic for human stroke patients.

Rapamycin up-regulation of autophagy reduces infarct size and improves outcomes in both permanent MCAL, and embolic MCAO, murine models of stroke.

BACKGROUND AND PURPOSE: The role of autophagy in response to ischemic stroke has been confusing with reports that both enhancement and inhibition of autophagy decrease infarct size and improve post-stroke outcomes. We sought to clarify this by comparing pharmacologic modulation of autophagy in two clinically relevant murine models of stroke. METHODS: We used rapamycin to induce autophagy, and chloroquine to block completion of autophagy, by treating mice immediately after stroke and at 24 hours post-stroke in two different models; permanent Middle Cerebral Artery Ligation (MCAL), which does not allow for reperfusion of distal trunk of middle cerebral artery, and Embolic Clot Middle Cerebral Artery Occlusion (eMCAO) which allows for a slow reperfusion similar to that seen in most human stroke patients. Outcome measures at 48 hours post-stroke included infarct size analysis, behavioral assessment using Bederson neurological scoring, and survival. RESULTS: Chloroquine treatment reduced the lesion size by approximately 30% and was significant only in the eMCAO model, where it also improved the neurological score, but did not increase survival. Rapamycin reduced lesion size by 44% and 50% in the MCAL and eMCAO models, respectively. Rapamycin also improved the neurological score to a greater degree than chloroquine and improved survival. CONCLUSIONS: While both inhibition and enhancement of autophagy by pharmacological intervention decreased lesion size and improved neurological scores, the enhancement with rapamycin showed a greater degree of improvement in outcomes as well as in survival. The protective action seen with chloroquine may be in part due to off-target effects on apoptosis separate from blocking lysosomal activity in autophagy. We conclude pharmacologic induction of autophagy is more advantageous than its blockade in physiologically-relevant permanent and slow reperfusion stroke models.