5-Aza-2'-Deoxycytidine and Valproic Acid in Combination with CHIR99021 and A83-01 Induce Pluripotency Genes Expression in Human Adult Somatic Cells.

A generation of induced pluripotent stem cells (iPSC) by ectopic expression of OCT4, SOX2, KLF4, and c-MYC has established promising opportunities for stem cell research, drug discovery, and disease modeling. While this forced genetic expression represents an advantage, there will always be an issue with genomic instability and transient pluripotency genes reactivation that might preclude their clinical application. During the reprogramming process, a somatic cell must undergo several epigenetic modifications to induce groups of genes capable of reactivating the endogenous pluripotency core. Here, looking to increase the reprograming efficiency in somatic cells, we evaluated the effect of epigenetic molecules 5-aza-2'-deoxycytidine (5AZ) and valproic acid (VPA) and two small molecules reported as reprogramming enhancers, CHIR99021 and A83-01, on the expression of pluripotency genes and the methylation profile of the OCT4 promoter in a human dermal fibroblasts cell strain. The addition of this cocktail to culture medium increased the expression of OCT4, SOX2, and KLF4 expression by 2.1-fold, 8.5-fold, and 2-fold, respectively, with respect to controls; concomitantly, a reduction in methylated CpG sites in OCT4 promoter region was observed. The epigenetic cocktail also induced the expression of the metastasis-associated gene S100A4. However, the epigenetic cocktail did not induce the morphological changes characteristic of the reprogramming process. In summary, 5AZ, VPA, CHIR99021, and A83-01 induced the expression of OCT4 and SOX2, two critical genes for iPSC. Future studies will allow us to precise the mechanisms by which these compounds exert their reprogramming effects.

Establishment of induced pluripotent stem cell lines derived from Parkinson's disease Mexican patients: A sporadic (UNAMi002-A) and a familial (UNAMi003-A) case carrying a mutation in PINK1.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in a prominent reduction of striatal dopamine levels leading to motor alterations. The mechanisms underlying neurodegeneration in PD remain unknown. Here, we generated an induced pluripotent stem cell line from dermal fibroblasts of a Mexican patient diagnosed with sporadic PD (UNAMi002-A) and another cell line from dermal fibroblasts of a patient carrying the point mutation c.1423delC in PINK1 (UNAMi003-A). These patient-derived iPS cell lines offer the possibility of modeling PD and understanding the mechanisms that contribute to dopamine neuron loss.

Human Embryonic Stem Cell-Derived Immature Midbrain Dopaminergic Neurons Transplanted in Parkinsonian Monkeys.

Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson's disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model.

Generation of a human induced pluripotent stem cell line (UNAMi001-A) from a Mexican patient with sporadic Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease caused by progressive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in motor alterations. The exact mechanisms underlying the dopaminergic neurodegeneration in PD are still unknown. Here, we generated a human induced pluripotent stem cell (iPSC) line from dermal fibroblasts of a Mexican patient diagnosed with sporadic PD. The generated iPS cell line (UNAMi001-A) express pluripotency markers, maintain a normal karyotype and display the ability to differentiate into all three germ layers. This is the first iPSC line from a Mexican patient and will be useful for PD modeling.