���ϳԹ�

UNLABELLED Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue.

The human iPSC cell line, RP2-FiPS4F1 (RCPFi001-A), derived from dermal fibroblasts from the patient with retinitis pigmentosa caused by the mutation of the gene PRPF8, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors.

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterized patient with autism spectrum disorder (ASD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The pluripotency of transgene-free iPSCs was verified by immunocytochemistry for pluripotency markers and by spontaneous in vitro differentiation towards the 3 germ layers. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of ASD, also for drug testing, early biomarker discovery and gene therapy studies.

Peripheral blood was collected from a clinically diagnosed 64-year old male multiple schwannoma patient. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers, and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma.

Peripheral blood was collected from a clinically diagnosed 79-year old male sporadic Parkinson's disease patient. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers, and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model can be used to study the mechanism of sporadic Parkinson's disease and to test new drugs. Resource Table.

Peripheral blood was collected from a clinically diagnosed 72-year old male patient with later onset Alzheimer's disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers, and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for studying the pathological mechanism of Alzheimer's disease.

Peripheral blood was collected from a clinically diagnosed 60-year old female patient with multiple schwannoma. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers, and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma.

The heart wall tissue, or the myocardium, is one of the main targets in cardiovascular disease prevention and treatment. Animal models have not been sufficient in mimicking the human myocardium as evident by the very low clinical translation rates of cardiovascular drugs. Additionally, current in vitro models of the human myocardium possess several shortcomings such as lack of physiologically relevant co-culture of myocardial cells, lack of a 3D biomimetic environment, and the use of non-human cells. In this study, we address these shortcomings through the design and manufacture of a myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem cell (hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated among iCMs as well as in capillary-like side channels, to better mimic the microvasculature seen in native myocardium. We first fully characterized iCMs using immunostaining, genetic, and electrochemical analysis and iECs through immunostaining and alignment analysis to ensure their functionality, and then seeded these cells sequentially into the MOC device. We showed that iECs could be cultured within the microfluidic device without losing their phenotypic lineage commitment, and align with the flow upon physiological level shear stresses. We were able to incorporate iCMs within the device in a spatially controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be viable and functional within the device up to 7 days, and were integrated with the iECs. The iCMs and iECs in this study were derived from the same hiPSC cell line, essentially mimicking the myocardium of an individual human patient. Such devices are essential for personalized medicine studies where the individual drug response of patients with different genetic backgrounds can be tested in a physiologically relevant manner.

A patient specific point mutation (c.1288GtextgreaterT) of Men1 gene was introduced into wide type iPSC line with CRISPR/Cas9 and single-stranded donor oligonucleotides carrying the mutation. The mutated iPSC line has a heterozygous c.1288GtextgreaterT mutation on exon-9 of Men1 that was confirmed by sequencing analysis. The karyotype of this line was normal and the pluripotency was demonstrated by its ability to differentiate into three germ layers. These artificially created Men1 mutation in wild type iPSC line will help to dissect out the molecular basis of two patients carried the same mutation from one family who were differentially represented hypoglycemia.

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with S1647T variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model will be useful for further function studies and therapeutic screening.

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with N551K variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model can complement in vivo PD models for pathophysiological studies and drug screening.