miR-218 Promotes Dopaminergic Differentiation and Controls Neuron Excitability and Neurotransmitter Release through the Regulation of a Synaptic-Related Genes Network.

In the brain, microRNAs (miRNAs) are believed to play a role in orchestrating synaptic plasticity at a higher level by acting as an additional mechanism of translational regulation, alongside the mRNA/polysome system. Despite extensive research, our understanding of the specific contribution of individual miRNA to the function of dopaminergic neurons (DAn) remains limited. By performing a dopaminergic-specific miRNA screening, we have identified miR-218 as a critical regulator of DAn activity in male and female mice. We have found that miR-218 is specifically expressed in mesencephalic DAn and is able to promote dopaminergic differentiation of embryonic stem cells and functional maturation of transdifferentiated induced DA neurons. Midbrain-specific deletion of both genes encoding for miR-218 (referred to as miR-218-1 and mir218-2) affects the expression of a cluster of synaptic-related mRNAs and alters the intrinsic excitability of DAn, as it increases instantaneous frequencies of evoked action potentials, reduces rheobase current, affects the ionic current underlying the action potential after hyperpolarization phase, and reduces dopamine efflux in response to a single electrical stimulus. Our findings provide a comprehensive understanding of the involvement of miR-218 in the dopaminergic system and highlight its role as a modulator of dopaminergic transmission.SIGNIFICANCE STATEMENT In the past decade, several miRNAs have emerged as potential regulators of synapse activity through the modulation of specific gene expression. Among these, we have identified a dopaminergic-specific miRNA, miR-218, which is able to promote dopaminergic differentiation and regulates the translation of an entire cluster of synapse related mRNAs. Deletion of miR-218 has notable effects on dopamine release and alters the intrinsic excitability of dopaminergic neurons, indicating a direct control of dopaminergic activity by miR-218.

Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells.

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies.

Levodopa-loaded nanoparticles for the treatment of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) resulting in dopamine (DA) deficiency, which manifests itself in motor symptoms including tremors, rigidity and bradykinesia. Current PD treatments aim at symptom reduction through oral delivery of levodopa (L-DOPA), a precursor of DA. However, L-DOPA delivery to the brain is inefficient and increased dosages are required as the disease progresses, resulting in serious side effects like dyskinesias. To improve PD treatment efficacy and to reduce side effects, recent research focuses on the encapsulation of L-DOPA into polymeric- and lipid-based nanoparticles (NPs). These formulations can protect L-DOPA from systemic decarboxylation into DA and improve L-DOPA delivery to the central nervous system. Additionally, NPs can be modified with proteins, peptides and antibodies specifically targeting the blood-brain barrier (BBB), thereby reducing required dosages and free systemic DA. Alternative delivery approaches for NP-encapsulated L-DOPA include intravenous (IV) administration, transdermal delivery using adhesive patches and direct intranasal administration, facilitating increased therapeutic DA concentrations in the brain. This review provides an overview of the recent advances for NP-mediated L-DOPA delivery to the brain, and debates challenges and future perspectives on the field.

Shaping Synthetic Multicellular and Complex Multimaterial Tissues via Embedded Extrusion-Volumetric Printing of Microgels.

In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck toward creating physiologically-relevant models. Addressing this limitation, a novel technique is introduced, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing spatially pattern multiple inks/cell types. Light-responsive microgels are developed for the first time as bioresins (µResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. µResins can be sculpted within seconds with tomographic light projections into centimeter-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP is applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models.

Direct Cell Conversion of Somatic Cells into Dopamine Neurons: Achievements and Perspectives.

In the last decade, direct reprogramming has emerged as a novel strategy to obtain mature and functional dopamine neurons from somatic cells. This approach could overcome issues linked to the use of human pluripotent stem cells such as ethical concerns and safety problems that can arise from the overgrowth of undifferentiated cells after transplantation. Several conversion methodologies have been developed to obtain induced DA neurons (iDANs) or induced DA neuron progenitors (iDPs). iDANs have also proved to successfully integrate in mice striatum, alleviating Parkinson's disease (PD) motor symptoms. In the next decade, human iDANs and/or iDPs could be translated to clinic to achieve a patient-tailored therapy, but current critical issues hinder this goal, such as the low conversion rate of adult human fibroblasts and the risks associated with lentiviral delivery of conversion factors. In this study, we summarize the strategies and recent improvements developed for the generation of mouse and human iDANs/iDPs. Furthermore, we discuss the more recent application of in vivo direct conversion, which may enable clinical therapies for PD by means of brain in situ delivery of dopaminergic reprogramming transcription factors.

Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation.

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression.

MicroRNA Roles in Cell Reprogramming Mechanisms.

Cell reprogramming is a groundbreaking technology that, in few decades, generated a new paradigm in biomedical science. To date we can use cell reprogramming to potentially generate every cell type by converting somatic cells and suitably modulating the expression of key transcription factors. This approach can be used to convert skin fibroblasts into pluripotent stem cells as well as into a variety of differentiated and medically relevant cell types, including cardiomyocytes and neural cells. The molecular mechanisms underlying such striking cell phenotypes are still largely unknown, but in the last decade it has been proven that cell reprogramming approaches are significantly influenced by non-coding RNAs. Specifically, this review will focus on the role of microRNAs in the reprogramming processes that lead to the generation of pluripotent stem cells, neurons, and cardiomyocytes. As highlighted here, non-coding RNA-forced expression can be sufficient to support some cell reprogramming processes, and, therefore, we will also discuss how these molecular determinants could be used in the future for biomedical purposes.

Induced pluripotent stem cell-derived and directly reprogrammed neurons to study neurodegenerative diseases: The impact of aging signatures.

Many diseases of the central nervous system are age-associated and do not directly result from genetic mutations. These include late-onset neurodegenerative diseases (NDDs), which represent a challenge for biomedical research and drug development due to the impossibility to access to viable human brain specimens. Advancements in reprogramming technologies have allowed to obtain neurons from induced pluripotent stem cells (iPSCs) or directly from somatic cells (iNs), leading to the generation of better models to understand the molecular mechanisms and design of new drugs. Nevertheless, iPSC technology faces some limitations due to reprogramming-associated cellular rejuvenation which resets the aging hallmarks of donor cells. Given the prominent role of aging for the development and manifestation of late-onset NDDs, this suggests that this approach is not the most suitable to accurately model age-related diseases. Direct neuronal reprogramming, by which a neuron is formed via direct conversion from a somatic cell without going through a pluripotent intermediate stage, allows the possibility to generate patient-derived neurons that maintain aging and epigenetic signatures of the donor. This aspect may be advantageous for investigating the role of aging in neurodegeneration and for finely dissecting underlying pathological mechanisms. Here, we will compare iPSC and iN models as regards the aging status and explore how this difference is reported to affect the phenotype of NDD in vitro models.

Liver Organoids: Updates on Disease Modeling and Biomedical Applications.

Liver organoids are stem cell-derived 3D structures that are generated by liver differentiation signals in the presence of a supporting extracellular matrix. Liver organoids overcome low complexity grade of bidimensional culture and high costs of in vivo models thus representing a turning point for studying liver disease modeling. Liver organoids can be established from different sources as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hepatoblasts and tissue-derived cells. This novel in vitro system represents an innovative tool to deeper understand the physiology and pathological mechanisms affecting the liver. In this review, we discuss the current advances in the field focusing on their application in modeling diseases, regenerative medicine and drug discovery.

Brain Organoids: Filling the Need for a Human Model of Neurological Disorder.

Neurological disorders are among the leading causes of death worldwide, accounting for almost all onsets of dementia in the elderly, and are known to negatively affect motor ability, mental and cognitive performance, as well as overall wellbeing and happiness. Currently, most neurological disorders go untreated due to a lack of viable treatment options. The reason for this lack of options is s poor understanding of the disorders, primarily due to research models that do not translate well into the human in vivo system. Current models for researching neurological disorders, neurodevelopment, and drug interactions in the central nervous system include in vitro monolayer cell cultures, and in vivo animal models. These models have shortcomings when it comes to translating research about disorder pathology, development, and treatment to humans. Brain organoids are three-dimensional (3D) cultures of stem cell-derived neural cells that mimic the development of the in vivo human brain with high degrees of accuracy. Researchers have started developing these miniature brains to model neurodevelopment, and neuropathology. Brain organoids have been used to model a wide range of neurological disorders, including the complex and poorly understood neurodevelopmental and neurodegenerative disorders. In this review, we discuss the brain organoid technology, placing special focus on the different brain organoid models that have been developed, discussing their strengths, weaknesses, and uses in neurological disease modeling.

Direct Cell Reprogramming of Mouse Fibroblasts into Functional Astrocytes Using Lentiviral Overexpression of the Transcription Factors NFIA, NFIB, and SOX9.

Astrocytes play an important role in maintaining brain homeostasis and their dysfunction is involved in a number of neurological disorders. An accessible source of astrocytes is essential to model neurological diseases and potential cell therapy approaches. Cell reprogramming techniques offer possibilities to reprogram terminally differentiated cells into other cell types. By overexpressing the three astrocytic transcription factors NFIA, NFIB, and SOX9, we showed that it is possible to directly transdifferentiate fibroblasts into functional astrocytes. These induced astrocytes (iAstrocytes) express glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100B), as well as other astrocytic markers. Moreover, electrophysiological properties indicate that iAstrocytes are functionally comparable to native brain astrocytes. Here we describe an optimized protocol to generate iAstrocytes starting from skin fibroblasts and this approach can be adapted for a wide range of somatic cell types.

Noncoding RNAs and Midbrain DA Neurons: Novel Molecular Mechanisms and Therapeutic Targets in Health and Disease.

Midbrain dopamine neurons have crucial functions in motor and emotional control and their degeneration leads to several neurological dysfunctions such as Parkinson's disease, addiction, depression, schizophrenia, and others. Despite advances in the understanding of specific altered proteins and coding genes, little is known about cumulative changes in the transcriptional landscape of noncoding genes in midbrain dopamine neurons. Noncoding RNAs-specifically microRNAs and long noncoding RNAs-are emerging as crucial post-transcriptional regulators of gene expression in the brain. The identification of noncoding RNA networks underlying all stages of dopamine neuron development and plasticity is an essential step to deeply understand their physiological role and also their involvement in the etiology of dopaminergic diseases. Here, we provide an update about noncoding RNAs involved in dopaminergic development and metabolism, and the related evidence of these biomolecules for applications in potential treatments for dopaminergic neurodegeneration.

CD33 and SIGLECL1 Immunoglobulin Superfamily Involved in Dementia.

Sialic acid-binding immunoglobulin-type lectins, which are predominantly expressed in immune cells, represent a family of immunomodulatory receptors with inhibitory and activating signals, in both healthy and disease states. Genetic factors are important in all forms of dementia, especially in early onset dementia. CD33 was recently recognized as a genetic risk factor for Alzheimer disease (AD). Here, we present a 2-generation family with 4 members, the father and the 3 siblings, characterized by an early form of unusual dementia exhibiting a behavioral variant close to behavioral variant frontotemporal dementia phenotype and severe forms of memory loss suggestive of AD. We analyzed the CD33 gene in this family and identified 10 single nucleotide polymorphisms (SNPs) in a linkage disequilibrium block associated with the disease. We also identified a tag SNP, rs2455069-A>G, in CD33 exon 2 that could be involved with dementia risk. Additionally, we excluded the presence of C9orf72 expansion mutations and other mutations previously associated with sporadic FTD and AD. The tag SNP association was also analyzed in selected sporadic AD patients from the same Southern Italy region. We demonstrate that CD33 and SIGLECL1 have a significantly increased level of expression in these patients.

Transdifferentiation of Mouse Embryonic Fibroblasts into Dopaminergic Neurons Reactivates LINE-1 Repetitive Elements.

In mammals, LINE-1 (L1) retrotransposons constitute between 15% and 20% of the genome. Although only a few copies have retained the ability to retrotranspose, evidence in brain and differentiating pluripotent cells indicates that L1 retrotransposition occurs and creates mosaics in normal somatic tissues. The function of de novo insertions remains to be understood. The transdifferentiation of mouse embryonic fibroblasts to dopaminergic neuronal fate provides a suitable model for studying L1 dynamics in a defined genomic and unaltered epigenomic background. We found that L1 elements are specifically re-expressed and mobilized during the initial stages of reprogramming and that their insertions into specific acceptor loci coincides with higher chromatin accessibility and creation of new transcribed units. Those events accompany the maturation of neuronal committed cells. We conclude that L1 retrotransposition is a non-random process correlating with chromatin opening and lncRNA production that accompanies direct somatic cell reprogramming.

Bioprinting Neural Systems to Model Central Nervous System Diseases.

To date, pharmaceutical progresses in central nervous system (CNS) diseases are clearly hampered by the lack of suitable disease models. Indeed, animal models do not faithfully represent human neurodegenerative processes and human in vitro 2D cell culture systems cannot recapitulate the in vivo complexity of neural systems. The search for valuable models of neurodegenerative diseases has recently been revived by the addition of 3D culture that allows to re-create the in vivo microenvironment including the interactions among different neural cell types and the surrounding extracellular matrix (ECM) components. In this review, the new challenges in the field of CNS diseases in vitro 3D modeling are discussed, focusing on the implementation of bioprinting approaches enabling positional control on the generation of the 3D microenvironments. The focus is specifically on the choice of the optimal materials to simulate the ECM brain compartment and the biofabrication technologies needed to shape the cellular components within a microenvironment that significantly represents brain biochemical and biophysical parameters.

A Chemically Defined Hydrogel for Human Liver Organoid Culture.

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is poorly defined, suffers from high batch-to-batch variability and is of xenogeneic origin, which limits the clinical application of organoids. Here, a novel hydrogel based on polyisocyanopeptides (PIC) and laminin-111 is described for human liver organoid cultures. PIC is a synthetic polymer that can form a hydrogel with thermosensitive properties, making it easy to handle and very attractive for clinical applications. Organoids in an optimized PIC hydrogel proliferate at rates comparable to those observed with Matrigel; proliferation rates are stiffness-dependent, with lower stiffnesses being optimal for organoid proliferation. Moreover, organoids can be efficiently differentiated toward a hepatocyte-like phenotype with key liver functions. This proliferation and differentiation potential maintain over at least 14 passages. The results indicate that PIC is very promising for human liver organoid culture and has the potential to be used in a variety of clinical applications including cell therapy and tissue engineering.

Allele-specific silencing as treatment for gene duplication disorders: proof-of-principle in autosomal dominant leukodystrophy.

Allele-specific silencing by RNA interference (ASP-siRNA) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. This approach has been effectively used to target autosomal dominant mutations and single nucleotide polymorphisms linked with aberrantly expanded trinucleotide repeats. Here, we propose ASP-siRNA as a preferable choice to target duplicated disease genes, avoiding potentially harmful excessive downregulation. As a proof-of-concept, we studied autosomal dominant adult-onset demyelinating leukodystrophy (ADLD) due to lamin B1 (LMNB1) duplication, a hereditary, progressive and fatal disorder affecting myelin in the CNS. Using a reporter system, we screened the most efficient ASP-siRNAs preferentially targeting one of the alleles at rs1051644 (average minor allele frequency: 0.45) located in the 3' untranslated region of the gene. We identified four siRNAs with a high efficacy and allele-specificity, which were tested in ADLD patient-derived fibroblasts. Three of the small interfering RNAs were highly selective for the target allele and restored both LMNB1 mRNA and protein levels close to control levels. Furthermore, small interfering RNA treatment abrogates the ADLD-specific phenotypes in fibroblasts and in two disease-relevant cellular models: murine oligodendrocytes overexpressing human LMNB1, and neurons directly reprogrammed from patients' fibroblasts. In conclusion, we demonstrated that ASP-silencing by RNA interference is a suitable and promising therapeutic option for ADLD. Moreover, our results have a broad translational value extending to several pathological conditions linked to gene-gain in copy number variations.

KMT2B Is Selectively Required for Neuronal Transdifferentiation, and Its Loss Exposes Dystonia Candidate Genes.

Transdifferentiation of fibroblasts into induced neuronal cells (iNs) by the neuron-specific transcription factors Brn2, Myt1l, and Ascl1 is a paradigmatic example of inter-lineage conversion across epigenetically distant cells. Despite tremendous progress regarding the transcriptional hierarchy underlying transdifferentiation, the enablers of the concomitant epigenome resetting remain to be elucidated. Here, we investigated the role of KMT2A and KMT2B, two histone H3 lysine 4 methylases with cardinal roles in development, through individual and combined inactivation. We found that Kmt2b, whose human homolog's mutations cause dystonia, is selectively required for iN conversion through suppression of the alternative myocyte program and induction of neuronal maturation genes. The identification of KMT2B-vulnerable targets allowed us, in turn, to expose, in a cohort of 225 patients, 45 unique variants in 39 KMT2B targets, which represent promising candidates to dissect the molecular bases of dystonia.

Cell reprogramming approaches in gene- and cell-based therapies for Parkinson's disease.

Degeneration of dopamine (DA) neurons in the substantia nigra pars compacta is the pathological hallmark of Parkinson's disease (PD). In PD multiple pathogenic mechanisms initiate and drive this neurodegenerative process, making the development of effective treatments challenging. To date, PD patients are primarily treated with dopaminergic drugs able to temporarily enhance DA levels, therefore relieving motor symptoms. However, the drawbacks of these therapies including the inability to alter disease progression are constantly supporting the search for alternative treatment approaches. Over the past years efforts have been put into the development of new therapeutic strategies based on the delivery of therapeutic genes using viral vectors or transplantation of DA neurons for cell-based DA replacement. Here, past achievements and recent advances in gene- and cell-based therapies for PD are outlined. We discuss how current gene and cell therapy strategies hold great promise for the treatment of PD and how the use of stem cells and recent developments in cellular reprogramming could contribute to open a new avenue in PD therapy.

miR-34b/c Regulates Wnt1 and Enhances Mesencephalic Dopaminergic Neuron Differentiation.

The differentiation of dopaminergic neurons requires concerted action of morphogens and transcription factors acting in a precise and well-defined time window. Very little is known about the potential role of microRNA in these events. By performing a microRNA-mRNA paired microarray screening, we identified miR-34b/c among the most upregulated microRNAs during dopaminergic differentiation. Interestingly, miR-34b/c modulates Wnt1 expression, promotes cell cycle exit, and induces dopaminergic differentiation. When combined with transcription factors ASCL1 and NURR1, miR-34b/c doubled the yield of transdifferentiated fibroblasts into dopaminergic neurons. Induced dopaminergic (iDA) cells synthesize dopamine and show spontaneous electrical activity, reversibly blocked by tetrodotoxin, consistent with the electrophysiological properties featured by brain dopaminergic neurons. Our findings point to a role for miR-34b/c in neuronal commitment and highlight the potential of exploiting its synergy with key transcription factors in enhancing in vitro generation of dopaminergic neurons.