Rigor and reproducibility in human brain organoid research: Where we are and where we need to go.

Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.

Mass spectrometry imaging as an emerging tool for studying metabolism in human brain organoids.

Human brain organoids are emerging models to study human brain development and pathology as they recapitulate the development and characteristics of major neural cell types, and enable manipulation through an in vitro system. Over the past decade, with the advent of spatial technologies, mass spectrometry imaging (MSI) has become a prominent tool for metabolic microscopy, providing label-free, non-targeted molecular and spatial distribution information of the metabolites within tissue, including lipids. This technology has never been used for studies of brain organoids and here, we set out to develop a standardized protocol for preparation and mass spectrometry imaging of human brain organoids. We present an optimized and validated sample preparation protocol, including sample fixation, optimal embedding solution, homogenous deposition of matrices, data acquisition and processing to maximize the molecular information derived from mass spectrometry imaging. We focus on lipids in organoids, as they play critical roles during cellular and brain development. Using high spatial and mass resolution in positive- and negative-ion modes, we detected 260 lipids in the organoids. Seven of them were uniquely localized within the neurogenic niches or rosettes as confirmed by histology, suggesting their importance for neuroprogenitor proliferation. We observed a particularly striking distribution of ceramide-phosphoethanolamine CerPE 36:1; O2 which was restricted within rosettes and of phosphatidyl-ethanolamine PE 38:3, which was distributed throughout the organoid tissue but not in rosettes. This suggests that ceramide in this particular lipid species might be important for neuroprogenitor biology, while its removal may be important for terminal differentiation of their progeny. Overall, our study establishes the first optimized experimental pipeline and data processing strategy for mass spectrometry imaging of human brain organoids, allowing direct comparison of lipid signal intensities and distributions in these tissues. Further, our data shed new light on the complex processes that govern brain development by identifying specific lipid signatures that may play a role in cell fate trajectories. Mass spectrometry imaging thus has great potential in advancing our understanding of early brain development as well as disease modeling and drug discovery.

Mutations in the transcriptional regulator MeCP2 severely impact key cellular and molecular signatures of human astrocytes during maturation.

Mutations in the MECP2 gene underlie a spectrum of neurodevelopmental disorders, most commonly Rett syndrome (RTT). We ask whether MECP2 mutations interfere with human astrocyte developmental maturation, thereby affecting their ability to support neurons. Using human-based models, we show that RTT-causing MECP2 mutations greatly impact the key role of astrocytes in regulating overall brain bioenergetics and that these metabolic aberrations are likely mediated by dysfunctional mitochondria. During post-natal maturation, astrocytes rely on neurons to induce their complex stellate morphology and transcriptional changes. While MECP2 mutations cause cell-intrinsic aberrations in the astrocyte transcriptional landscape, surprisingly, they do not affect the neuron-induced astrocyte gene expression. Notably, however, astrocytes are unable to develop complex mature morphology due to cell- and non-cell-autonomous aberrations caused by MECP2 mutations. Thus, MECP2 mutations critically impact key cellular and molecular features of human astrocytes and, hence, their ability to interact and support the structural and functional maturation of neurons.

Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

BASP1 labels neural stem cells in the neurogenic niches of mammalian brain.

The mechanisms responsible for determining neural stem cell fate are numerous and complex. To begin to identify the specific components involved in these processes, we generated several mouse neural stem cell (NSC) antibodies against cultured mouse embryonic neurospheres. Our immunohistochemical data showed that the NSC-6 antibody recognized NSCs in the developing and postnatal murine brains as well as in human brain organoids. Mass spectrometry revealed the identity of the NSC-6 epitope as brain abundant, membrane-attached signal protein 1 (BASP1), a signaling protein that plays a key role in neurite outgrowth and plasticity. Western blot analysis using the NSC-6 antibody demonstrated multiple BASP1 isoforms with varying degrees of expression and correlating with distinct developmental stages. Herein, we describe the expression of BASP1 in NSCs in the developing and postnatal mammalian brains and human brain organoids, and demonstrate that the NSC-6 antibody may be a useful marker of these cells.

Activity-dependent aberrations in gene expression and alternative splicing in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that affects about 1 in 10,000 female live births. The underlying cause of RTT is mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2); however, the molecular mechanism by which these mutations mediate the RTT neuropathology remains enigmatic. Specifically, although MeCP2 is known to act as a transcriptional repressor, analyses of the RTT brain at steady-state conditions detected numerous differentially expressed genes, while the changes in transcript levels were mostly subtle. Here we reveal an aberrant global pattern of gene expression, characterized predominantly by higher levels of expression of activity-dependent genes, and anomalous alternative splicing events, specifically in response to neuronal activity in a mouse model for RTT. Notably, the specific splicing modalities of intron retention and exon skipping displayed a significant bias toward increased retained introns and skipped exons, respectively, in the RTT brain compared with the WT brain. Furthermore, these aberrations occur in conjunction with higher seizure susceptibility in response to neuronal activity in RTT mice. Our findings advance the concept that normal MeCP2 functioning is required for fine-tuning the robust and immediate changes in gene transcription and for proper regulation of alternative splicing induced in response to neuronal stimulation.

ADAR1 is involved in the regulation of reprogramming human fibroblasts to induced pluripotent stem cells.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional, site-specific modification process that is catalyzed by Adenosine Deaminase Acting on RNA (ADAR) gene family members. Since ADARs act on double-stranded RNA, most A-to-I editing occurs within repetitive elements, particularly Alu elements, as the result of the inherent property of these sequences to fold and form double strands. ADAR1-mediated A-to-I RNA editing was recently implicated in the regulation of human embryonic stem cells (hESCs). Spontaneous and neuronal differentiation of hESC was shown to result in a decrease in A-to-I editing levels. Knockdown of ADAR1 in hESCs results in an elevation of the expression of differentiation-related genes. In addition, we found that hESCs over-expressing ADAR1 could not be generated. The current study shows that the editing levels of induced pluripotent stem cells (iPSCs) change throughout reprogramming, from a source cell level to a level similar to that of hESCs. Up- or down-regulation of the ADAR1 level in human foreskin fibroblast (HFF) cells before induction of reprogramming results in varied reprogramming efficiencies. Furthermore, HFF-iPSC early clones derived from source cells in which the ADAR1 level was down-regulated lose their iPSC properties shortly after iPSC colony formation and instead exhibit characteristics of cancer cells. Taken together, our results imply a role for ADAR1 in the regulation of pluripotency induction as well as in the maintenance of early iPSC properties.

Altered A-to-I RNA editing in human embryogenesis.

Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.

Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.

An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.

Consistent levels of A-to-I RNA editing across individuals in coding sequences and non-conserved Alu repeats.

BACKGROUND: Adenosine to inosine (A-to-I) RNA-editing is an essential post-transcriptional mechanism that occurs in numerous sites in the human transcriptome, mainly within Alu repeats. It has been shown to have consistent levels of editing across individuals in a few targets in the human brain and altered in several human pathologies. However, the variability across human individuals of editing levels in other tissues has not been studied so far. RESULTS: Here, we analyzed 32 skin samples, looking at A-to-I editing level in three genes within coding sequences and in the Alu repeats of six different genes. We observed highly consistent editing levels across different individuals as well as across tissues, not only in coding targets but, surprisingly, also in the non evolutionary conserved Alu repeats. CONCLUSIONS: Our findings suggest that A-to-I RNA-editing of Alu elements is a tightly regulated process and, as such, might have been recruited in the course of primate evolution for post-transcriptional regulatory mechanisms.

Alu sequences in undifferentiated human embryonic stem cells display high levels of A-to-I RNA editing.

Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3'UTRs, 5'UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.

Widespread cleavage of A-to-I hyperediting substrates.

A-to-I RNA editing is the conversion of adenosine to inosine in double-stranded cellular and viral RNAs. Recently, abundant hyperediting of human transcripts, affecting thousands of genes, has been reported. Most of these editing sites are confined to intramolecular hairpin double-stranded RNA (dsRNA) structures formed by pairing of neighboring, reversely oriented, primate-specific Alu repeats. The biological implication of this extensive modification is still a mystery. A number of studies have shown that heavily edited transcripts are often retained in the nucleus. A recent study found that the edited region in transcripts of the mouse Slc7a2 gene is post-transcriptionally cleaved upon stress, enabling the release of the mRNA to the cytoplasm, followed by its translation. Here, we aim to test whether this scenario might be relevant for many other hyperedited Alu targets. Bioinformatics analysis of publicly available mRNA and expressed sequence tag data provides evidence showing that neighboring, reversely oriented, Alu elements are often cleaved at both ends of the region harboring the inverted repeats followed by rejoining of the two parts of the transcript on both sides of the inverted repeats, resulting in almost inosine-free mRNA products. Deleted segments vary among transcripts of the same gene and are not flanked by the canonical splicing signal sequences. The tissue distribution of these events seems to correlate with known A-to-I editing patterns, suggesting that it depends on the dsRNA structure being edited. Results are experimentally verified by polymerase chain reaction and cloning data. A database of 566 human and 107 mouse putative cleavage loci is supplied.

Establishing a dynamic process for the formation, propagation, and differentiation of human embryoid bodies.

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe here a robust and well-defined scale up platform for human embryoid body (EB) formation, propagation, and differentiation. The efficacy of the dynamic process as compared to the static cultivation in Petri dishes was analyzed. Our optimized conditions include specific bioreactor and impeller type, seeding and propagation parameters, and scale up. Quantitative analyses of viable cell concentrations, apoptosis percentages, and EB yield revealed 6.7-fold enhancement in the generation of hESC-derived cells after 10 cultivation days. Other metabolic indices such as glucose consumption, lactic acid production and pH all pointed to efficient cell expansion in the dynamic cultures. The hydrodynamic conditions during seeding and cultivation were found to be crucial for the EB formation and propagation. The EBs' prearrangement in the static system and EB cultivation in the Glass Ball Impeller spinner flask resulted in high EB yield, a round homogenous shape, and the fastest growth rate. The appearance of representative genes of the three germ layers as well as primitive neuronal tube organization and blood vessel formation indicated that the initial developmental events in the human EBs are not interfered by the dynamic system. Furthermore, well developed endothelial networks and contracting EBs with functional cardiac muscle were also obtained after two cultivation weeks. Collectively, our study defines the technological platform for the controlled large-scale generation of hESC-derived cells for clinical and industrial applications.

Design principle of gene expression used by human stem cells: implication for pluripotency.

Human embryonic stem cells (ESC) are undifferentiated and are endowed with the capacities of self-renewal and pluripotential differentiation. Adult stem cells renew their own tissue, but whether they can transdifferentiate to other tissues is still controversial. To understand the genetic program that underlies the pluripotency of stem cells, we compared the transcription profile of ESC with that of progenitor/stem cells of human hematopoietic and keratinocytic origins, along with their mature cells to be viewed as snapshots along tissue differentiation. ESC gene profiles show higher complexity with significantly more highly expressed genes than adult cells. We hypothesize that ESC use a strategy of expressing genes that represent various differentiation pathways and selection of only a few for continuous expression upon differentiation to a particular target. Such a strategy may be necessary for the pluripotency of ESC. The progenitors of either hematopoietic or keratinocytic cells also follow the same design principle. Using advanced clustering, we show that many of the ESC expressed genes are turned off in the progenitors/stem cells followed by a further down-regulation in adult tissues. Concomitantly, genes specific to the target tissue are up-regulated toward mature cells of skin or blood.

Vascular gene expression and phenotypic correlation during differentiation of human embryonic stem cells.

The study of the cascade of events of induction and sequential gene activation that takes place during human embryonic development is hindered by the unavailability of postimplantation embryos at different stages of development. Spontaneous differentiation of human embryonic stem cells (hESCs) can occur by means of the formation of embryoid bodies (EBs), which resemble certain aspects of early embryos to some extent. Embryonic vascular formation, vasculogenesis, is a sequential process that involves complex regulatory cascades. In this study, changes of gene expression along the development of human EBs for 4 weeks were studied by large-scale gene screening. Two main clusters were identified-one of down-regulated genes such as POU5, NANOG, TDGF1/Cripto (TDGF, teratocarcinoma-derived growth factor-1), LIN28, CD24, TERF1 (telomeric repeat binding factor-1), LEFTB (left-right determination, factor B), and a second of up-regulated genes such as TWIST, WNT5A, WT1, AFP, ALB, NCAM1. Focusing on the vascular system development, genes known to be involved in vasculogenesis and angiogenesis were explored. Up-regulated genes include vasculogenic growth factors such as VEGFA, VEGFC, FIGF (VEGFD), ANG1, ANG2, TGFbeta3, and PDGFB, as well as the related receptors FLT1, FLT4, PDGFRB, TGFbetaR2, and TGFbetaR3, other markers such as CD34, VCAM1, PECAM1, VE-CAD, and transcription factors TAL1, GATA2, and GATA3. The reproducibility of the array data was verified independently and illustrated that many genes known to be involved in vascular development are activated during the differentiation of hESCs in culture. Hence, the analysis of the vascular system can be extended to other differentiation pathways, allocating human EBs as an in vitro model to study early human development.

Vascular development in early human embryos and in teratomas derived from human embryonic stem cells.

During early human embryonic development, blood vessels are stimulated to grow, branch, and invade developing tissues and organs. Pluripotent human embryonic stem cells (hESCs) are endowed with the capacity to differentiate into cells of blood and lymphatic vessels. The present study aimed to follow vasculogenesis during the early stages of developing human vasculature and to examine whether human neovasculogenesis within teratomas generated in SCID mice from hESCs follows a similar course and can be used as a model for the development of human vasculature. Markers and gene profiling of smooth muscle cells and endothelial cells of blood and lymphatic vessels were used to follow neovasculogenesis and lymphangiogenesis in early developing human embryos (4-8 weeks) and in teratomas generated from hESCs. The involvement of vascular smooth muscle cells in the early stages of developing human embryonic blood vessels is demonstrated, as well as the remodeling kinetics of the developing human embryonic blood and lymphatic vasculature. In teratomas, human vascular cells were demonstrated to be associated with developing blood vessels. Processes of intensive remodeling of blood vessels during the early stages of human development are indicated by the upregulation of angiogenic factors and specific structural proteins. At the same time, evidence for lymphatic sprouting and moderate activation of lymphangiogenesis is demonstrated during these developmental stages. In the teratomas induced by hESCs, human angiogenesis and lymphangiogenesis are relatively insignificant. The main source of blood vessels developing within the teratomas is provided by the murine host. We conclude that the teratoma model has only limited value as a model to study human neovasculogenesis and that other in vitro methods for spontaneous and guided differentiation of hESCs may prove more useful.