A Standardized Nomenclature Design for Systematic Referencing and Identification of Animal Cellular Material.

The documentation, preservation and rescue of biological diversity increasingly uses living biological samples. Persistent associations between species, biosamples, such as tissues and cell lines, and the accompanying data are indispensable for using, exchanging and benefiting from these valuable materials. Explicit authentication of such biosamples by assigning unique and robust identifiers is therefore required to allow for unambiguous referencing, avoid identification conflicts and maintain reproducibility in research. A predefined nomenclature based on uniform rules would facilitate this process. However, such a nomenclature is currently lacking for animal biological material. We here present a first, standardized, human-readable nomenclature design, which is sufficient to generate unique and stable identifying names for animal cellular material with a focus on wildlife species. A species-specific human- and machine-readable syntax is included in the proposed standard naming scheme, allowing for the traceability of donated material and cultured cells, as well as data FAIRification. Only when it is consistently applied in the public domain, as publications and inter-institutional samples and data are exchanged, distributed and stored centrally, can the risks of misidentification and loss of traceability be mitigated. This innovative globally applicable identification system provides a standard for a sustainable structure for the long-term storage of animal bio-samples in cryobanks and hence facilitates current as well as future species conservation and biomedical research.

mtDNA analysis using Mitopore.

Mitochondrial DNA (mtDNA) analysis is crucial for the diagnosis of mitochondrial disorders, forensic investigations, and basic research. Existing pipelines are complex, expensive, and require specialized personnel. In many cases, including the diagnosis of detrimental single nucleotide variants (SNVs), mtDNA analysis is still carried out using Sanger sequencing. Here, we developed a simple workflow and a publicly available webserver named Mitopore that allows the detection of mtDNA SNVs, indels, and haplogroups. To simplify mtDNA analysis, we tailored our workflow to process noisy long-read sequencing data for mtDNA analysis, focusing on sequence alignment and parameter optimization. We implemented Mitopore with eliBQ (eliminate bad quality reads), an innovative quality enhancement that permits the increase of per-base quality of over 20% for low-quality data. The whole Mitopore workflow and webserver were validated using patient-derived and induced pluripotent stem cells harboring mtDNA mutations. Mitopore streamlines mtDNA analysis as an easy-to-use fast, reliable, and cost-effective analysis method for both long- and short-read sequencing data. This significantly enhances the accessibility of mtDNA analysis and reduces the cost per sample, contributing to the progress of mtDNA-related research and diagnosis.

Balancing neuronal activity to fight neurodevelopmental disorders.

In a recent study, Rylaarsdam and colleagues revealed that mutant PACS1 gene, which causes a rare neurodevelopmental syndrome, affects the firing ability of human neurons without dysregulating the cellular architecture of brain organoids. These findings suggest aberrant neuronal electrophysiology as a possible interventional target for pediatric diseases impairing brain development.

Cortical brain organoid slices (cBOS) for the study of human neural cells in minimal networks.

Brain organoids derived from human pluripotent stem cells are a promising tool for studying human neurodevelopment and related disorders. Here, we generated long-term cultures of cortical brain organoid slices (cBOS) grown at the air-liquid interphase from regionalized cortical organoids. We show that cBOS host mature neurons and astrocytes organized in complex architecture. Whole-cell patch-clamp demonstrated subthreshold synaptic inputs and action potential firing of neurons. Spontaneous intracellular calcium signals turned into synchronous large-scale oscillations upon combined disinhibition of NMDA receptors and blocking of GABAA receptors. Brief metabolic inhibition to mimic transient energy restriction in the ischemic brain induced reversible intracellular calcium loading of cBOS. Moreover, metabolic inhibition induced a reversible decline in neuronal ATP as revealed by ATeam1.03YEMK. Overall, cBOS provide a powerful platform to assess morphological and functional aspects of human neural cells in intact minimal networks and to address the pathways that drive cellular damage during brain ischemia.

Mitochondrial DNA integrity and metabolome profile are preserved in the human induced pluripotent stem cell reference line KOLF2.1J.

Quality control of human induced pluripotent stem cells (iPSCs) is critical to ensure reproducibility of research. Recently, KOLF2.1J was characterized and published as a male iPSC reference line to study neurological disorders. Emerging evidence suggests potential negative effects of mtDNA mutations, but its integrity was not analyzed in the original publication. To assess mtDNA integrity, we conducted a targeted mtDNA analysis followed by untargeted metabolomics analysis. We found that KOLF2.1J mtDNA integrity was intact at the time of publication and is still preserved in the commercially distributed cell line. In addition, the basal KOLF2.1J metabolome profile was similar to that of the two commercially available iPSC lines IMR90 and iPSC12, but clearly distinct from an in-house-generated ERCC6R683X/R683X iPSC line modeling Cockayne syndrome. Conclusively, we validate KOLF2.1J as a reference iPSC line, and encourage scientists to conduct mtDNA analysis and unbiased metabolomics whenever feasible.

Modeling mitochondrial DNA diseases: from base editing to pluripotent stem-cell-derived organoids.

Mitochondrial DNA (mtDNA) diseases are multi-systemic disorders caused by mutations affecting a fraction or the entirety of mtDNA copies. Currently, there are no approved therapies for the majority of mtDNA diseases. Challenges associated with engineering mtDNA have in fact hindered the study of mtDNA defects. Despite these difficulties, it has been possible to develop valuable cellular and animal models of mtDNA diseases. Here, we describe recent advances in base editing of mtDNA and the generation of three-dimensional organoids from patient-derived human-induced pluripotent stem cells (iPSCs). Together with already available modeling tools, the combination of these novel technologies could allow determining the impact of specific mtDNA mutations in distinct human cell types and might help uncover how mtDNA mutation load segregates during tissue organization. iPSC-derived organoids could also represent a platform for the identification of treatment strategies and for probing the in vitro effectiveness of mtDNA gene therapies. These studies have the potential to increase our mechanistic understanding of mtDNA diseases and may open the way to highly needed and personalized therapeutic interventions.

Generation of an induced pluripotent stem cell line from a Huntington's disease patient with a long HTT-PolyQ sequence.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal length of CAG repeats in the gene HTT, leading to an elongated poly-glutamine (poly-Q) sequence in huntingtin (HTT). We used non-integrative Sendai virus to reprogram fibroblasts from a patient with juvenile onset HD to induced pluripotent stem cells (iPSCs). Reprogrammed iPSCs expressed pluripotency-associated markers, exhibited a normal karyotype, and following directed differentiation generated cell types belonging to the three germ layers. PCR analysis and sequencing confirmed the HD patient-derived iPSC line had one normal HTT allele and one with elongated CAG repeats, equivalent to ≥180Q.

Teriflunomide as a therapeutic means for myelin repair.

BACKGROUND: Promotion of myelin repair in the context of demyelinating diseases such as multiple sclerosis (MS) still represents a clinical unmet need, given that this disease is not only characterized by autoimmune activities but also by impaired regeneration processes. Hence, this relates to replacement of lost oligodendrocytes and myelin sheaths-the primary targets of autoimmune attacks. Endogenous remyelination is mainly mediated via activation and differentiation of resident oligodendroglial precursor cells (OPCs), whereas its efficiency remains limited and declines with disease progression and aging. Teriflunomide has been approved as a first-line treatment for relapsing remitting MS. Beyond its role in acting via inhibition of de novo pyrimidine synthesis leading to a cytostatic effect on proliferating lymphocyte subsets, this study aims to uncover its potential to foster myelin repair. METHODS: Within the cuprizone mediated de-/remyelination model teriflunomide dependent effects on oligodendroglial homeostasis and maturation, related to cellular processes important for myelin repair were analyzed in vivo. Teriflunomide administration was performed either as pulse or continuously and markers specific for oligodendroglial maturation and mitochondrial integrity were examined by means of gene expression and immunohistochemical analyses. In addition, axon myelination was determined using electron microscopy. RESULTS: Both pulse and constant teriflunomide treatment efficiently boosted myelin repair activities in this model, leading to accelerated generation of oligodendrocytes and restoration of myelin sheaths. Moreover, teriflunomide restored mitochondrial integrity within oligodendroglial cells. CONCLUSIONS: The link between de novo pyrimidine synthesis inhibition, oligodendroglial rescue, and maintenance of mitochondrial homeostasis appears as a key for successful myelin repair and hence for protection of axons from degeneration.

Generation of two mother-child pairs of iPSCs from maternally inherited Leigh syndrome patients with m.8993 T > G and m.9176 T > G MT-ATP6 mutations.

We generated two pairs of mother-child iPSCs lines for Maternally Inherited Leigh Syndrome (MILS) carrying the m.8993 T > G and m.9176 T > G mutations in the MT-ATP6 gene. We delivered reprogramming factors OCT4, SOX2, KLF4, and c-MYC via Sendai virus. All iPSCs lines had a normal karyotype, expressed pluripotency markers, and differentiated into the three germ layers. Both patient-iPSCs retained the same degrees of heteroplasmy as their source fibroblasts (>97.0 %). In maternal iPSCs, the heteroplasmy remained 0.0 % in the case of the m.8993 T > G mutation and dropped from 55.0 % to 1.0 % in the case of m.9176 T > G mutation.

Generation of induced pluripotent stem cells from three individuals with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal glutamine (Q) expansion in the huntingtin protein due to elongated CAG repeats in the gene HTT. We used non-integrative episomal plasmids to generate induced pluripotent stem cells (iPSCs) from three individuals affected by HD: CH1 (58Q), and two twin brothers CH3 (44Q) and CH4 (44Q). The iPSC lines exhibited one healthy HTT allele and one with elongated CAG repeats, as confirmed by PCR and sequencing. All iPSC lines expressed pluripotency markers, exhibited a normal karyotype, and generated cells of the three germ layers in vitro.

A Novel Gene Controls a New Structure: PiggyBac Transposable Element-Derived 1, Unique to Mammals, Controls Mammal-Specific Neuronal Paraspeckles.

Although new genes can arrive from modes other than duplication, few examples are well characterized. Given high expression in some human brain subregions and a putative link to psychological disorders [e.g., schizophrenia (SCZ)], suggestive of brain functionality, here we characterize piggyBac transposable element-derived 1 (PGBD1). PGBD1 is nonmonotreme mammal-specific and under purifying selection, consistent with functionality. The gene body of human PGBD1 retains much of the original DNA transposon but has additionally captured SCAN and KRAB domains. Despite gene body retention, PGBD1 has lost transposition abilities, thus transposase functionality is absent. PGBD1 no longer recognizes piggyBac transposon-like inverted repeats, nonetheless PGBD1 has DNA binding activity. Genome scale analysis identifies enrichment of binding sites in and around genes involved in neuronal development, with association with both histone activating and repressing marks. We focus on one of the repressed genes, the long noncoding RNA NEAT1, also dysregulated in SCZ, the core structural RNA of paraspeckles. DNA binding assays confirm specific binding of PGBD1 both in the NEAT1 promoter and in the gene body. Depletion of PGBD1 in neuronal progenitor cells (NPCs) results in increased NEAT1/paraspeckles and differentiation. We conclude that PGBD1 has evolved core regulatory functionality for the maintenance of NPCs. As paraspeckles are a mammal-specific structure, the results presented here show a rare example of the evolution of a novel gene coupled to the evolution of a contemporaneous new structure.

RNA-based generation of iPSCs from a boy carrying the mutation m.9185 T>C in the mitochondrial gene MT-ATP6 and from his healthy mother.

We used a non-integrative self-replicating RNA vector to establish four iPSC lines: two iPSC lines from a young male carrying the mutation m.9185 T>C in the mitochondrial gene MT-ATP6 (present at virtual homoplasmic level), and two iPSC lines from his healthy mother (carrying the mutation in only about 4 % of mtDNA copies). All iPSC lines exhibited pluripotency characteristics, were capable to give rise to cells of the three germ layers in vitro, and presented a normal karyotype. The derived iPSC lines retained the MT-ATP6 mutation at levels similar to those observed in the parental fibroblasts.

High-content screening of mitochondrial polarization in neural cells derived from human pluripotent stem cells.

We present a high-content screening (HCS) protocol for quantifying mitochondrial activity in live neural cells from human induced pluripotent stem cells (iPSCs). The assessment is based on mitochondrial membrane potential, which is influenced by the efficiency of mitochondrial bioenergetics. We describe how to perform the analysis using both an HCS platform and the open-source software CellProfiler. The protocol can identify the mitochondrial fitness of human neurons and may be used to carry out high-throughput compound screenings in patient-derived neural cells. For complete details on the use and execution of this protocol, please refer to Lorenz et al. (2017) and Zink et al. (2020).

High-content analysis of neuronal morphology in human iPSC-derived neurons.

We present a high-content analysis (HCA) protocol for monitoring the outgrowth capacity of human neurons derived from induced pluripotent stem cells (iPSCs). We describe steps to perform HCA imaging, followed by quantifying the morphology of dendrites and axons within a high-throughput system to evaluate neurons obtained through various differentiation approaches. This protocol can be used to screen for modulators of neuronal morphogenesis or neurotoxicity. The approach can be applied to patient-derived iPSCs to identify patient-specific defects and possible therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Zink et al. (2020) and Inak et al. (2021). The protocol can be used in combination with Zink et al. (2022).

A call for consensus guidelines on monitoring the integrity of nuclear and mitochondrial genomes in human pluripotent stem cells.

Despite the widespread use of human pluripotent stem cells (hPSCs), there is no general consensus with respect to monitoring the integrity of nuclear and mitochondrial genome of hPSCs. We argue that clear guidelines should be in place, and we have identified frequently asked questions that can be used to develop a practical guide for hPSCs users.

Generation of four iPSC lines from four patients with Leigh syndrome carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6.

We report the generation of four human iPSC lines (8993-A12, 8993-B12, 8993-C11, and 8993-D7) from fibroblasts of four patients affected by maternally inherited Leigh syndrome (MILS) carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6. We used Sendai viruses to deliver reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The established iPSC lines expressed pluripotency markers, exhibited a normal karyotype, were capable to form cells of the three germ layers in vitro, and retained the MT-ATP6 mutations at the same homoplasmic level of the parental fibroblasts.

Homozygous mutation in MCM7 causes autosomal recessive primary microcephaly and intellectual disability.

BACKGROUND: Minichromosomal maintenance (MCM) complex components 2, 4, 5 and 6 have been linked to human disease with phenotypes including microcephaly and intellectual disability. The MCM complex has DNA helicase activity and is thereby important for the initiation and elongation of the replication fork and highly expressed in proliferating neural stem cells. METHODS: Whole-exome sequencing was applied to identify the genetic cause underlying the neurodevelopmental disease of the index family. The expression pattern of Mcm7 was characterised by performing quantitative real-time PCR, in situ hybridisation and immunostaining. To prove the disease-causative nature of identified MCM7, a proof-of-principle experiment was performed. RESULTS: We reported that the homozygous missense variant c.793G>A/p.A265T (g.7:99695841C>T, NM_005916.4) in MCM7 was associated with autosomal recessive primary microcephaly (MCPH), severe intellectual disability and behavioural abnormalities in a consanguineous pedigree with three affected individuals. We found concordance between the spatiotemporal expression pattern of Mcm7 in mice and a proliferative state: Mcm7 expression was higher in early mouse developmental stages and in proliferative zones of the brain. Accordingly, Mcm7/MCM7 levels were detectable particularly in undifferentiated mouse embryonal stem cells and human induced pluripotent stem cells compared with differentiated neurons. We further demonstrate that the downregulation of Mcm7 in mouse neuroblastoma cells reduces cell viability and proliferation, and, as a proof-of-concept, that this is counterbalanced by the overexpression of wild-type but not mutant MCM7. CONCLUSION: We report mutations of MCM7 as a novel cause of autosomal recessive MCPH and intellectual disability and highlight the crucial function of MCM7 in nervous system development.

Activated microglia and neuroinflammation as a pathogenic mechanism in Leigh syndrome.

Neuroinflammation is one of the main mechanisms leading to neuronal death and dysfunction in neurodegenerative diseases. The role of microglia as primary mediators of inflammation is unclear in Leigh syndrome (LS) patients. This study aims to elucidate the role of microglia in LS progression by a detailed multipronged analysis of LS neuropathology, mouse and human induced pluripotent stem cells models of Leigh syndrome. We described brain pathology in three cases of Leigh syndrome and performed immunohistochemical staining of autopsy brain of LS patients. We used mouse model of LS (Ndufs4-/-) to study the effect of microglial partial ablation using pharmacologic approach. Genetically modified human induced pluripotent stem cell (iPS) derived neurons and brain organoid with Ndufs4 mutation were used to investigate the neuroinflammation in LS. We reported a novel observation of marked increased in Iba1+ cells with features of activated microglia, in various parts of brain in postmortem neuropathological examinations of three Leigh syndrome patients. Using an Ndufs4-/- mouse model for Leigh syndrome, we showed that partial ablation of microglia by Pexidartinib initiated at the symptom onset improved neurological function and significantly extended lifespan. Ndufs4 mutant LS brain organoid had elevated NLRP3 and IL6 pro-inflammatory pathways. Ndufs4-mutant LS iPSC neurons were more susceptible to glutamate excitotoxicity, which was further potentiated by IL-6. Our findings of LS human brain pathology, Ndufs4-deficient mouse and iPSC models of LS suggest a critical role of activated microglia in the progression of LS encephalopathy. This study suggests a potential clinical application of microglial ablation and immunosuppression during the active phase of Leigh syndrome.

TLR4 Associated Signaling Disrupters as a New Means to Overcome HERV-W Envelope-Mediated Myelination Deficits.

Myelin repair in the adult central nervous system (CNS) is driven by successful differentiation of resident oligodendroglial precursor cells (OPCs) and thus constitutes a neurodegenerative process capable to compensate for functional deficits upon loss of oligodendrocytes and myelin sheaths as it is observed in multiple sclerosis (MS). The human endogenous retrovirus type W (HERV-W) represents an MS-specific pathogenic entity, and its envelope (ENV) protein was previously identified as a negative regulator of OPC maturation-hence, it is of relevance in the context of diminished myelin repair. We here focused on the activity of the ENV protein and investigated how it can be neutralized for improved remyelination. ENV-mediated activation of toll like receptor 4 (TLR4) increases inducible nitric oxide synthase (iNOS) expression, prompts nitrosative stress, and results in myelin-associated deficits, such as decreased levels of oligodendroglial maturation marker expression and morphological alterations. The intervention of TLR4 surface expression represents a potential means to rescue such ENV-dependent deficits. To this end, the rescue capacity of specific substances, either modulating V-ATPase activity or myeloid differentiation 2 (MD2)-mediated TLR4 glycosylation status, such as compound 20 (C20), L48H437, or folimycin, was analyzed, as these processes were demonstrated to be relevant for TLR4 surface expression. We found that pharmacological treatment can rescue the maturation arrest of oligodendroglial cells and their myelination capacity and can prevent iNOS induction in the presence of the ENV protein. In addition, downregulation of TLR4 surface expression was observed. Furthermore, mitochondrial integrity crucial for oligodendroglial cell differentiation was affected in the presence of ENV and ameliorated upon pharmacological treatment. Our study, therefore, provides novel insights into possible means to overcome myelination deficits associated with HERV-W ENV-mediated myelin deficits.