Generation and characterization of integration-free induced pluripotent stem cells from patients with autoimmune disease

Autoimmune diseases (AIDs), a heterogeneous group of immune-mediated disorders, are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs, the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However, stem cell transplantation therapy has limitations, including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology, which allows the generation of patient-specific pluripotent stem cells, could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS), Sjögren''s syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally, all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS, SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs, including AS, SS and SLE.     

Patient-Derived Induced Pluripotent Stem Cell Models for Phenotypic Screening in the Neuronal Ceroid Lipofuscinoses

Batten disease or neuronal ceroid lipofuscinosis (NCL) is a group of rare, fatal, inherited neurodegenerative lysosomal storage disorders. Numerous genes (CLN1–CLN8, CLN10–CLN14) were identified in which mutations can lead to NCL; however, the underlying pathophysiology remains elusive. Despite this, the NCLs share some of the same features and symptoms but vary in respect to severity and onset of symptoms by age. Some common symptoms include the progressive loss of vision, mental and motor deterioration, epileptic seizures, premature death, and in the rare adult-onset, dementia. Currently, all forms of NCL are fatal, and no curative treatments are available. Induced pluripotent stem cells (iPSCs) can differentiate into any cell type of the human body. Cells reprogrammed from a patient have the advantage of acquiring disease pathogenesis along with recapitulation of disease-associated phenotypes. They serve as practical model systems to shed new light on disease mechanisms and provide a phenotypic screening platform to enable drug discovery. Herein, we provide an overview of available iPSC models for a number of different NCLs. More specifically, we highlight findings in these models that may spur target identification and drug development.     

Dopamine promotes formation and secretion of non-fibrillar alpha-synuclein oligomers

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) and by abnormal aggregation of α-synuclein. Previous studies have suggested that DA can interact with α-synuclein, thus modulating the aggregation process of this protein; this interaction may account for the selective vulnerability of DA neurons in patients with PD. However, the relationship between DA and α-synuclein, and the role in progressive degeneration of DA neurons remains elusive. We have shown that in the presence of DA, recombinant human α-synuclein produces non-fibrillar, SDS-resistant oligomers, while β-sheet-rich fibril formation is inhibited. Pharmacologic elevation of the cytoplasmic DA level increased the formation of SDS-resistant oligomers in DA-producing neuronal cells. DA promoted α-synuclein oligomerization in intracellular vesicles, but not in the cytosol. Furthermore, elevation of DA levels increased secretion of α-synuclein oligomers to the extracellular space, but the secretion of monomers was not changed. DA-induced secretion of α-synuclein oligomers may contribute to the progressive loss of the dopaminergic neuronal population and the pronounced neuroinflammation observed in the SNpc in patients with PD.     

Stiffness-Tunable Hydrogel-Sandwich Culture Modulates the YAP-Mediated Mechanoresponse in Induced-Pluripotent Stem Cell Embryoid Bodies and Augments Cardiomyocyte Differentiation

Microenvironmental factors, including substrate stiffness, regulate stem cell behavior and differentiation. However, the effects of substrate stiffness on the behavior of induced pluripotent stem cell (iPSC)- derived embryoid bodies (EB) remain unclear. To investigate the effects of mechanical cues on iPSC-EB differentiation, a 3D hydrogel-sandwich culture (HGSC) system is developed that controls the microenvironment surrounding iPSC-EBs using a stiffness-tunable polyacrylamide hydrogel assembly. Mouse iPSC-EBs are seeded between upper and lower polyacrylamide hydrogels of differing stiffness (Young's modulus [E’] = 54.3 ± 7.1 kPa [hard], 28.1 ± 2.3 kPa [moderate], and 5.1 ± 0.1 kPa [soft]) and cultured for 2 days. HGSC induces stiffness-dependent activation of the yes-associated protein (YAP) mechanotransducer and actin cytoskeleton rearrangement in the iPSC-EBs. Moreover, moderate-stiffness HGSC specifically upregulates the mRNA and protein expression of ectoderm and mesoderm lineage differentiation markers in iPSC-EBs via YAP-mediated mechanotransduction. Pretreatment of mouse iPSC-EBs with moderate-stiffness HGSC promotes cardiomyocyte (CM) differentiation and structural maturation of myofibrils. The proposed HGSC system provides a viable platform for investigating the role of mechanical cues on the pluripotency and differentiation of iPSCs that can be beneficial for research into tissue regeneration and engineering.     

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.     

Self-organizing map of gene regulatory networks for cell phenotypes during reprogramming

The induced pluripotent cells (iPSCs) are derived from somatic cells by reprogramming their genetic profiles. Such a process requires coordinated dynamic expression of hundreds of genes and proteins. As both deterministic and stochastic elements control the reprogramming process, it is not easy to have a way to reflect the status of gene regulatory network in those reprogramming cells. In this study, we applied self-organizing maps (SOMs) on those complex gene expression data from different pluripotent cells, including partially reprogrammed and fully reprogrammed induced pluripotent cells (iPSCs), embryonic stem cells (ESCs), and adult stem cells came from different tissues. We showed that our SOMs have good correlation with the previously reported PluriNet of stem cells and they are pictorial diagrams which can reflect the intrinsic status of cells.     

PEP-1-GLRX1 Reduces Dopaminergic Neuronal Cell Loss by Modulating MAPK and Apoptosis Signaling in Parkinson’s Disease

Parkinson’s disease (PD) is characterized mainly by the loss of dopaminergic neurons in the substantia nigra (SN) mediated via oxidative stress. Although glutaredoxin-1 (GLRX1) is known as one of the antioxidants involved in cell survival, the effects of GLRX1 on PD are still unclear. In this study, we investigated whether cell-permeable PEP-1-GLRX1 inhibits dopaminergic neuronal cell death induced by 1-methyl-4-phenylpyridinium (MPP⁺) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We showed that PEP-1-GLRX1 protects cell death and DNA damage in MPP⁺-exposed SH-SY5Y cells via the inhibition of MAPK, Akt, and NF-κB activation and the regulation of apoptosis-related protein expression. Furthermore, we found that PEP-1-GLRX1 was delivered to the SN via the blood–brain barrier (BBB) and reduced the loss of dopaminergic neurons in the MPTP-induced PD model. These results indicate that PEP-1-GLRX1 markedly inhibited the loss of dopaminergic neurons in MPP⁺- and MPTP-induced cytotoxicity, suggesting that this fusion protein may represent a novel therapeutic agent against PD.     

MicroRNA‐2861 and nanofibrous scaffold synergistically promote human induced pluripotent stem cells osteogenic differentiation

Tissue engineering using new strategies has become a growing and promising method for treating large tissue lesions in the body. On the other hand, microRNAs (miRNAs), which are small non‐coding regulatory RNAs, are a new class of genetic materials that can have effective pharmacological roles. The combination of these two themes has created promising prospects for the treatment of diseases. Herein, human induced pluripotent stem cells (iPSCs) were transduced with miRNA‐2861 and then the osteogenic differentiation potential of transduced iPSCs and non‐transduced iPSCs was investigated while cultured on the electrospun poly lactic‐co‐glycolic acid (PLGA) nanofibrous scaffold and culture plate. MiR‐2861‐transduced iPSCs showed a significantly higher viability, mineralization, alkaline phosphatase (ALP) activity, calcium content, and bone‐related gene expression in comparison with those iPSCs that non‐transduced. The results also indicated that this increase is improved when miR‐2861 transduced iPSCs are cultured on the PLGA nanofibrous scaffold synergistically. This synergy was also confirmed by the results obtained from of Western blot analysis. It can be concluded that, miR‐2861, by negative regulation of those proteins that decrease/inhibit osteogenic differentiation and PLGA nanofibrous scaffold by preparation of a suitable artificial extracellular matrix, have a great positive impact in improving iPSCs osteogenic differentiation potential and this blend can be proposed to use in bone tissue engineering application.     

Harpagide,a natural product,promotes synaptic vesicle release as measured by nanoelectrode amperometry

Parkinson''s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DAergic) neurons and low level of dopamine (DA) in the midbrain. Recent studies suggested that some natural products can protect neurons against injury, but their role on neurotransmitter release and the underlying mechanisms remained unknown. In this work, nanoelectrode electrochemistry was used for the first time to quantify DA release inside single DAergic synapses. Our results unambiguously demonstrated that harpagide, a natural product, effectively enhances synaptic DA release and restores DA release at normal levels from injured neurons in PD model. These important protective and curative effects are shown to result from the fact that harpagide efficiently inhibits the phosphorylation and aggregation of α-synuclein by alleviating the intracellular reactive oxygen level, being beneficial for vesicle loading and recycling. This establishes that harpagide offers promising avenues for preventive or therapeutic interventions against PD and other neurodegenerative disorders.

Nanoelectrode amperometry was used to monitor DA release inside single DAergic synapses, and demonstrated that harpagide effectively enhances synaptic DA release by reducing intracellular ROS generation and inhibiting α-Syn phosphorylation.     

Concise Synthesis and X-Ray Crystal Structure of N-Benzyl-2-(pyrimidin-4′-ylamino)-thiazole-4-carboxamide (Thiazovivin), a Small-Molecule Tool for Stem Cell Research

Stem cell research is one of the most promising fields of modern biomedical research and regenerative medicine. Limited availability and ethical concerns suggest the renouncement of embryonic stem cells (ESCs), thus raising the need for more efficient procedures for the generation of stem cells, ideally through reprogramming of mammalian cells. The small molecule N-benzyl-2-(pyrimidin-4′-ylamino)-thiazole-4-carboxamide (thiazovivin) is known to improve the generation of human induced pluripotent stem cells (iPSCs) from human fibroblasts. We herein describe a highly efficient procedure for the synthesis of thiazovivin over just five steps, which should be suitable for a large-scale application, and the first x-ray crystal structure of the target compound.

[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource: Full experimental and spectral details.]     

Neuroinflammation Modulation via α7 Nicotinic Acetylcholine Receptor and Its Chaperone,RIC-3

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in or on various cell types and have diverse functions. In immune cells nAChRs regulate proliferation, differentiation and cytokine release. Specifically, activation of the α7 nAChR reduces inflammation as part of the cholinergic anti-inflammatory pathway. Here we review numerous effects of α7 nAChR activation on immune cell function and differentiation. Further, we also describe evidence implicating this receptor and its chaperone RIC-3 in diseases of the central nervous system and in neuroinflammation, focusing on multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Deregulated neuroinflammation due to dysfunction of α7 nAChR provides one explanation for involvement of this receptor and of RIC-3 in neurodegenerative diseases. In this review, we also provide evidence implicating α7 nAChRs and RIC-3 in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) involving neuroinflammation. Besides, we will describe the therapeutic implications of activating the cholinergic anti-inflammatory pathway for diseases involving neuroinflammation.     

A Long‐Living Bioengineered Neural Tissue Platform to Study Neurodegeneration

The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long‐term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk‐based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic‐like indicators of increased pathological β‐amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long‐term stability of the platform is suited to study chronic brain disease including neurodegeneration.     

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson’s disease (PD), multiple system atrophy, Alzheimer’s disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.     

Electrochemical Oxidations of Central Nervous System Indoleamines,Catecholamines and Alkaloids: Potential Significance into Neurodegenerative Diseases

The serotonergic neurotransmitter 5-hydroxytryptamine (5-HT), the catecholaminergic neurotransmittcr dopaminc (DA) and various tetrahydroisoquinoline (TIQ) and tetrahydro-β-carboline (THβC) alkaloids are all easily oxidized compounds. Aberrant oxidative transformations of 5-HT and other central indoles might be involved in neurodegenerative Alzheimer's Disease (AD). Changes in the oxidation chemistry of DA appear to be fundamental in substantia nigra neurons in Parkinson's Disease (PD). Various TIQ and THβC alkaloids are elevated in the brain as a result of ethanol drinking. Recent studies into the electrochemical oxidation chemistry of 5-HT, DA and various TIQ and THβC alkaloids have been reviewed. The potential roles of the oxidation chemistry of these compounds in neurodegenerative AD, PD and alcoholism are discussed.     

Proteomics of Caenorhabditis elegans over-expressing human alpha-synuclein analyzed by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry: identification of actin and several ribosomal proteins as negative markers at early Parkinson's disease stages

It has been known that the over-expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease (PD), leads to neurodegeneration in PD models. In this study, the changes in protein expression between the transgenic over-expressing human alpha-synuclein wild type (alpha-synWT) and the control Caenorhabditis elegans were elucidated by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) proteome analysis, which is a highly selective, sensitive, repeatable and quantitative method for protein identification. Because the alpha-synuclein wild-type worms showed moderate levels of dopamine loss without overt behavioral abnormalities, it was suggested that the changes in proteins in the alpha-synWT are related in the sequence of the formation of Lewy bodies. Among more than 400 protein peaks detected, actin and several ribosomal proteins were identified for the first time as negative markers at early PD stages. Actin was suggested to be one of the important targets in the elucidation of the etiology of neuronal diseases such as PD or other synucleinopathies.     

Regenerative chemical biology: current challenges and future potential

The enthusiasm surrounding the clinical potential of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is tempered by the fact that key issues regarding their safety, efficacy, and long-term benefits have thus far been suboptimal. Small molecules can potentially relieve these problems at major junctions of stem cell biology and regenerative therapy. In this review we will introduce recent advances in these important areas and the first generation of small molecules used in the regenerative context. Current chemical biology studies will provide the archetype for future interdisciplinary collaborations and improve clinical benefits of cell-based therapies.     

Exogenous Addition of 25-Hydroxycholesterol Reduces Level of Very Long-Chain Fatty Acids in X-Linked Adrenoleukodystrophy

X-Linked adrenoleukodystrophy (X-ALD) is a severe metabolic disorder characterized by the accumulation of very long-chain fatty acids (VLCFAs). Recently, we demonstrated that levels of 25-hydroxycholesterol (25-HC) and cholesterol 25-hydroxylase (CH25H) were found to be elevated in X-ALD. Herein, we report that the exogenous addition of 25-HC significantly reduces C26:0 levels in X-ALD patient-derived fibroblasts and oligodendrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from X-ALD patients. Moreover, 25-HC treatment was found to down-regulate the expression of ELOVL1, a key enzyme for the synthesis of C26. In addition, activation of liver X receptor (LXR), a molecular target of endogenous 25-HC, also reduced C26:0 level. The reduction of C26:0 levels by 25-HC treatment might result, at least partially, from the decrease of ELOVL1 expression as well as the activation of LXR. Our findings could provide a better understanding of the role of 25-HC in X-ALD and useful information to find therapeutic agents to treat X-ALD.     

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80 min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells.