Current State-of-the-Art and Unresolved Problems in Using Human Induced Pluripotent Stem Cell-Derived Dopamine Neurons for Parkinson's Disease Drug Development.

Human induced pluripotent stem (iPS) cells have the potential to give rise to a new era in Parkinson's disease (PD) research. As a unique source of midbrain dopaminergic (DA) neurons, iPS cells provide unparalleled capabilities for investigating the pathogenesis of PD, the development of novel anti-parkinsonian drugs, and personalized therapy design. Significant progress in developmental biology of midbrain DA neurons laid the foundation for their efficient derivation from iPS cells. The introduction of 3D culture methods to mimic the brain microenvironment further expanded the vast opportunities of iPS cell-based research of the neurodegenerative diseases. However, while the benefits for basic and applied studies provided by iPS cells receive widespread coverage in the current literature, the drawbacks of this model in its current state, and in particular, the aspects of differentiation protocols requiring further refinement are commonly overlooked. This review summarizes the recent data on general and subtype-specific features of midbrain DA neurons and their development. Here, we review the current protocols for derivation of DA neurons from human iPS cells and outline their general weak spots. The associated gaps in the contemporary knowledge are considered and the possible directions for future research that may assist in improving the differentiation conditions and increase the efficiency of using iPS cell-derived neurons for PD drug development are discussed.

Generation of induced pluripotent stem cell line RCPCMi004-A derived from patient with Parkinson's disease with deletion of the exon 2 in PARK2 gene.

IPSC line RCPCMi004-A was generated from skin fibroblasts collected from a male patient with early onset Parkinson's disease. The patient carries a heterozygous deletion of the exon 2 of PARK2 gene. The reprogramming of fibroblasts was performed with Sendai viruses containing Oct-4, Sox-2, Klf-4 and c-Myc. Pluripotency was confirmed by immunofluorescence, RT-PCR, and formation of embryoid bodies. The RCPCMi004-A cell line carries the same deletion in PARK2 gene. The RCPCMi004-A cell line can be used to model Parkinson's disease in vitro.

Parkinson's Disease-Associated Changes in the Expression of Neurotrophic Factors and their Receptors upon Neuronal Differentiation of Human Induced Pluripotent Stem Cells.

Parkinson's disease (PD) is a neurodegenerative pathology resulting from the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Neurotrophic factors (NTFs) and their receptors are key regulators of the survival, differentiation, and development of neurons. However, the role of these factors in the pathogenesis of PD is still unclear. Here, we analyzed the expression of NTFs and their receptors in human induced pluripotent stem cells (iPSCs) derived from the fibroblasts of patients with PD and healthy donors (HDs). Four PD-derived iPSC lines with different mutations and three cell lines from HDs at different stages of neuronal differentiation were used for RT-qPCR analysis and ELISA. We found that the mRNA levels of most analyzed genes were altered in PD-derived cells compared with those in HD-derived cells at all stages. Importantly, irrespective of PD-associated mutations, the mRNA levels of the BDNF and GDNF genes were mostly increased or unchanged in predominantly DA terminally differentiated neurons (TDNs) compared with those in HD-derived cells. Strikingly, in contrast to BDNF and GDNF mRNA levels, BDNF and GDNF protein levels were lower in almost all PD-derived TDNs than in HD-derived cells, thus indicating the dysregulation of NTF expression at the post-transcriptional level. We suggest that this dysregulation is one of the important signs of PD development.

The Use of Human Induced Pluripotent Stem Cells for Testing Neuroprotective Activity of Pharmacological Compounds.

Development of therapeutic preparations involves several steps, starting with the synthesis of chemical compounds and testing them in different models for selecting the most effective and safest ones to clinical trials and introduction into medical practice. Cultured animal cells (both primary and transformed) are commonly used as models for compound screening. However, cell models display a number of disadvantages, including insufficient standardization (primary cells) and disruption of cell genotypes (transformed cells). Generation of human induced pluripotent stem cells (IPSCs) offers new possibilities for the development of high-throughput test systems for screening potential therapeutic preparations with different activity spectra. Due to the capacity to differentiate into all cell types of an adult organism, IPSCs are a unique model that allows examining the activity and potential toxicity of tested compounds during the entire differentiation process in vitro. In this work, we demonstrated the efficiency of IPSCs and their neuronal derivatives for selecting substances with the neuroprotective activity using two classes of compounds - melanocortin family peptides and endocannabinoids. None of the tested compounds displayed cyto- or embryotoxicity. Both melanocortin peptides and endocannabinoids exerted neuroprotective effect in the neuronal precursors and IPSC-derived neurons subjected to hydrogen peroxide. The endocannabinoid N-docosahexaenoyl dopamine exhibited the highest neuroprotective effect (~70%) in the differentiated cultures enriched with dopaminergic neurons; the effect of melanocortin Semax was ~40%. The possibility of using other IPSC derivatives for selecting compounds with the neuroprotective activity is discussed.

Expression and Functional Properties of NMDA and GABAA Receptors during Differentiation of Human Induced Pluripotent Stem Cells into Ventral Mesencephalic Neurons.

Ionotropic glutamate and GABA receptors regulate the differentiation and determine the functional properties of mature neurons. Both insufficient and excessive activity of these neurotransmission systems are associated with various nervous system diseases. Our knowledge regarding the expression profiles of these receptors and the mechanisms of their regulation during the differentiation of specialized human neuron subtypes is limited. Here the expression profiles of the NMDA and GABAA receptor subunits were explored during in vitro differentiation of human induced pluripotent stem cells (iPSCs) into ventral mesencephalic neurons. The correlation between the neuronal maturation and the expression dynamics of these genes was investigated, and the functional activity of these receptors was assessed by calcium imaging. The role of NMDA and GABAA receptors in neurite outgrowth and the development of spontaneous activity was analyzed using the viral transduction of neural progenitors with the reporter genes TagGFP and TagRFP. The data indicate that agonists of the investigated receptors can be employed for optimization of existing protocols for neural differentiation of iPSCs, in particular for acceleration of neuronal maturation.

Genome Editing and the Problem of Tetraploidy in Cell Modeling of the Genetic Form of Parkinsonism.

The prevalent form of familial parkinsonism is caused by mutations in the LRRK2 gene encoding for the mitochondrial protein kinase. In the review, we discuss possible causes of appearance of tetraploid cells in neuronal precursors obtained from induced pluripotent stem cells from patients with the LRRK2-associated form of parkinsonism after genome editing procedure. As LRRK2 protein participates in cell proliferation and maintenance of the nuclear envelope, spindle fibers, and cytoskeleton, mutations in the LRRK2 gene can affect protein functions and lead, via various mechanisms, to the mitotic machinery disintegration and chromosomal aberration. These abnormalities can appear at different stages of fibroblast reprogramming; therefore, editing of the LRRK2 nucleotide sequence should be done during or before the reprogramming stage.

Cytogenetic Analysis of the Results of Genome Editing on the Cell Model of Parkinson's Disease.

We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson's disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.

Neuroprotective Properties of Endocannabinoids N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine Examined in Neuronal Precursors Derived from Human Pluripotent Stem Cells.

Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1-10 µM range. However, both agents at 10 µM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.

Expression of Type I Cannabinoid Receptors at Different Stages of Neuronal Differentiation of Human Fibroblasts.

Differential expression of type 1 cannabinoid receptors (CR1) was evaluated at different stages of human skin fibroblast transformation into terminally differentiated neurons. Immunocytochemical staining detected no CR1 on fibroblasts, but their transformation into induced pluripotent stem cells was accompanied by marked stimulation of CR1 expression. In neuronal precursors, the receptors were located mainly on cell bodies and at the base of their processes. This distribution was retained at the terminal stage of differentiation of induced pluripotent stem cells into neurons.

The Transcriptional Changes of trim Genes Associated with Parkinson's Disease on a Model of Human Induced Pluripotent Stem Cells.

Over the last few years, in vitro models, based on patient-derived induced pluripotent stem cells (iPSCs), have received considerable attention for modeling different neurodegenerative disorders. Using this model, we analyzed transcription of 15 tripartite motif (trim) genes in iPSCs, derived from the different groups: Parkinson's disease (PD) patients bearing mutations in different genes, patient with the sporadic form of PD, and the healthy individuals. The transcription was observed during neuronal differentiation of the cells in vitro into neuronal stem cells and terminally differentiated neurons. The transcription of over 50 % of these genes, belonging to different sub-groups of the TRIM family, varied between PD patients and healthy individuals during the reprogramming of fibroblasts into iPSCs and the following neuronal differentiation. Moreover, the transcription of the trim6 and trim24 genes was different between cells, derived from PD patients, and control cells at all stages. The transcription of the four trim genes (trim5α, 26, 27, 31) remained unchanged during almost all investigated stages, compared with the controls. We suppose that the revealed changes in the transcription of several trim genes reflect their possible role in neurodegenerative processes at the early stages of PD. These genes may act as a gear unit between the PD progression and the deregulation of the immune system.

Investigation of the effects of GABA receptor agonists in the differentiation of human induced pluripotent stem cells into dopaminergic neurons.

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca2+ level ([Ca2+] i ) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus, the presence of active GABA-A receptors, associated with phenotype determination via Ca2+-signalling was demonstrated in differentiating human DA neurons.

Fibroblast-like cells as an effective feeder for the cultivation and derivation of new lines of human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) can be a highly informative model of hereditary and sporadic human diseases. In the future, such cells can be used in substitution and regenerative therapy of a wide range of diseases and for the treatment of injuries and burns. The ability of iPSCs derived from patients with Parkinson's disease to differentiate into fibroblast-like cells (derivatives) was studied. It was found that these cells can serve as an effective feeder layer not only to maintain the pluripotency of allogenic and autologous iPSCs but also to derive new iPSC lines.

Investigation of the effect of α-melanocyte-stimulating hormone on proliferation and early stages of differentiation of human induced pluripotent stem cells.

We have studied the influence of α-melanocyte-stimulating hormone (α-MSH) on proliferation and early stages of differentiation of human induced pluripotent stem cells (iPSc). We have demonstrated that α-MSH receptor genes are expressed in undifferentiated iPSc. The expression levels of MCR1, MCR2, and MCR3 increased at the embryoid body (EB) formation stage. The formation of neural progenitors was accompanied by elevation of MCR2, MCR3, and MCR4 expression. α-MSH had no effect on EB generation and iPSc proliferation at concentrations ranging from 1 nM to 10 µM. At the same time, α-MSH increased the generation of neural rosettes in human iPSc cultures more than twice.

Phenotypical Differences in Neuronal Cultures Derived via Reprogramming the Fibroblasts from Patients Carrying Mutations in Parkinsonian Genes LRRK2 and PARK2.

Fibroblasts isolated from skin biopsy specimens from patients with genetic forms of Parkinson's disease, carriers of mutations in LRRK2 and PARK2 genes, and from a healthy volunteer were reprogrammed using lentiviral vectors into induced pluripotent stem cells (iPSC). iPSC were differentiated into neuron-like cells using a cocktail of differentiation factors (N2, B27, and Noggin). The iPSC lines derived from patients with different mutations and from a healthy volunteer cultured under the same conditions were characterized by different proportion of neuronal precursors and differentiated neurons. Control Po2 line contained 56% precursors, while B15 line with LRRK2 gene mutation (G2019S) contained 35% precursor cells. Similar regularities were characteristic of Tr5 culture carrying compound heterozygous mutations in PARK2 gene (del202-203AG and IVS1+1G/A) and containing 4% neuronal precursors. Further comparative studies of iPSC carrying various mutations and comparison with normal human cells will help to understand the molecular pathogenesis of some genetic variants of Parkinson's disease.

Induced pluripotent stem cells: from derivation to application in biochemical and biomedical research.

This review considers different methods for obtaining induced pluripotent stem (iPS) cells and their use in biochemical and biomedical research. Some viral and nonviral methods for obtaining iPS cells are described. Basic factors involved in reprogramming are considered. It is also demonstrated that the most suitable source of iPS cells are skin fibroblasts. Properties of iPS cells and embryonic stem cells are compared, and some advantages of iPS cells for biological and biomedical investigations are emphasized. The possibilities for application of iPS cells in the development of cell models of some neurodegenerative diseases, drug screening, and cell therapy are also considered.

The Production and Characteristics of a Mouse's Embryonic Stem Cell Lineage, Transfected by the Glia Neurotrophic Factor and Gene Fused with the Green Fluorescent Protein Gene.

The influence that the expression of the human (glial-derived neurotrophic factor (GDNF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (SC) of a mouse with R1 lineage, as well as their ability to form embroid bodies (EB), has been studied. Before that, using a PCR (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic SC, the expression of the receptors' genes is being fulfilled for the neurotropfic RET and GFRα1 glia factor. The mouse's embryonic SC lineage has been obtained, transfected by the human GDNF gene, and has been fused with the "green" fluorescent protein (GFP) gene. The presence of the expression of the human GDNF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic SC transfected by the GDNF gene has been shown. No significant influence of the expression of the GDNF gene on the morphology and the proliferative activity of the transfected embryonic SCs has been found when compared with the control ones.

Influence of pub Gene Expression on Differentiation of Mouse Embryonic Stem Cells into Derivatives of Ecto-, Meso-, and Endoderm in vitro.

The influence of low and high pub gene expression on the initial stages of the differentiation of mouse embryonic stem cells into derivatives of ecto-, meso-, and endoderm in vitro was investigated. As follows from the results of a RT -PCR analysis, the expression of the vimentin, somatostatin, GATA 4, and GATA 6 genes, being the markers of endodermal differentiation, does not vary in both the cells with high pub gene expression and the cells with low pub gene expression, as well as in the corresponding control lines. The cells with high pub gene expression are characterized by an increase in the expression of mesodermal differentiation gene-markers (trI card, trI skel, c-kit, and IL-7), whereas the cells with low pub gene expression are specified by a decrease in their expression. According to the analyses carried out, the reverse is characteristic of the expression of ectodermal differentiation gene-markers (nestin, ≤-III tubulin, gfap, and th). Expression of these genes decreases in cell lines with high pub gene expression, whereas their expression increases with the decrease in pub gene expression. Hence, it is suggested that the variations in the pub gene expression in the embryonic stem cells influence significantly the mesodermal and ectodermal differentiation of these cells.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

Effects of human presenilin 1 isoforms on proliferation and survival of rat pheochromocytoma cell line PC12.

Missense mutations in human presenilin 1 gene (hPS1) cause an autosomal dominant, early onset form of Alzheimer's disease (AD). To study effects of mutant presenilin on processes of cell growth, differentiation, and susceptibility to apoptotic signals, we produced a series of rat pheochromocytoma PC12 poly- and monoclonal cell lines stably expressing wild type hPS1 and hPS1 with mutations in amino (N-) and carboxyl (C-) terminal regions of the PS1 protein. Employing a heterologous rat PC12 cell system, we demonstrated that: 1) AD mutations inhibit, in part, processing of hPS1 holoprotein; 2) negative selection against highly expressed hPS1 may occur in polyclonal cell cultures; 3) expression of N-terminus mutant (M146V) hPS1 increases susceptibility to apoptosis in differentiated neuronal PC12 cells under deprivation conditions; 4) monoclones with hPS1 C-terminal AD mutation (C410Y) have lower proliferation rates than monoclones expressing wild type hPS1 under deprivation conditions and during NGF-induced neuronal differentiation. The data demonstrate deleterious effect of PS1 AD mutations. The effect depends on the level of expression of the hPS1 isoforms, the number of passages, and trophic and differentiation conditions used for growing PC12 cells.