Direct Reprogramming to Human Induced Neuronal Progenitors from Fibroblasts of Familial and Sporadic Parkinson’s Disease Patients

In Parkinson’s disease (PD) research, human neuroblastoma and immortalized neural cell lines have been widely used as in vitro models. The advancement in the field of reprogramming technology has provided tools for generating patient-specific induced pluripotent stem cells (hiPSCs) as well as human induced neuronal progenitor cells (hiNPCs). These cells have revolutionized the field of disease modeling, especially in neural diseases. Although the direct reprogramming to hiNPCs has several advantages over differentiation after hiPSC reprogramming, such as the time required and the simple procedure, relatively few studies have utilized hiNPCs. Here, we optimized the protocol for hiNPC reprogramming using pluripotency factors and Sendai virus. In addition, we generated hiNPCs of two healthy donors, a sporadic PD patient, and a familial patient with the LRRK2 G2019S mutation (L2GS). The four hiNPC cell lines are highly proliferative, expressed NPC markers, maintained the normal karyotype, and have the differentiation potential of dopaminergic neurons. Importantly, the patient hiNPCs show different apoptotic marker expression. Thus, these hiNPCs, in addition to hiPSCs, are a favorable option to study PD pathology.

Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC

Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1, encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it, we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However, the mALK2 leads to inhibitory pluripotency maintenance, or impaired clonogenic potential after single-cell dissociation as an inevitable step, which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus, current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome, and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors, CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617G>A. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern, as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time, labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies, which is hampered by inhibitory pluripotency-maintenance requirements, or vulnerability of single-cell-dissociated iPSCs.