Biology subjects

Tristan, C. A.

Publications and source records attributed to Tristan, C. A..

9 recordsLinked to original sources

bioRxiv2025

Modeling Friedreich's ataxia with Bergmann glia-enriched human cerebellar organoids

The human cerebellum is a specialized brain region that is involved in various neurological and psychiatric diseases but has been challenging to study in vitro due its complex neurodevelopment and cellular diversity. Despite the progress in generating neural tissues from human induced pluripotent stem cells (iPSCs), an organoid model that recapitulates the key features of cerebellar development has not been widely established. Here, we report the generation of a 60-day method for human cerebellar organoids (hCBOs) that is characterized by induction of rhombomere 1 (R1) cellular identity followed by derivation of neuronal and glial cell types of the cerebellum. In contrast to forebrain organoids with multiple neural rosettes and inside-out neuronal migration, hCBOs develop a SOX2+ cerebellar plate on the outermost surface of organoids with outside-in neuronal migration, which is a characteristic hallmark of cerebellar histogenesis. These hCBOs produced various other cell types including granule neurons, Purkinje cells, Golgi neurons, and deep cerebellar nuclei. By using a glial induction strategy, we generate Bergmann glial cells (BGCs) within the hCBOs that not only serve as scaffolds for granule cells migration but also enhance electrophysiological response of the hCBOs. Furthermore, by generating hCBOs from patients with Friedreichs ataxia (FRDA), we revealed abnormal disease-specific phenotypes that could be reversed by histone deacetylase (HDAC) inhibitors and gene editing by CRISPR-Cas9. Taken together, our advanced hCBO model provides new opportunities to investigate the molecular and cellular mechanisms of cerebellar ontogenesis and utilize patient-derived iPSCs for translational research.

developmental biology↗

bioRxiv2024

Scalable Hypothalamic Arcuate Neuron Differentiation from Human Pluripotent Stem Cells Suitable for Modeling Metabolic and Reproductive Disorders

The hypothalamus, composed of several nuclei, is essential for maintaining our bodys homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

developmental biology↗

bioRxiv2022

A Comprehensive Roadmap of Human Placental Development in vitro

Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However, lineage specification into trophectoderm (TE) remains poorly understood and access to well-characterized placental cells for biomedical research is limited, largely depending on fetal tissues or cancer cell lines. Here, we developed novel strategies enabling highly efficient TE specification that generates cytotrophoblast (CTB) and multinucleated primary syncytiotrophoblast (STB) followed by establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise induction of lineage- and cell-type-specific genes and substantiated typical features of placental cells using morphological, biochemical, integrated multi-omics, and single-cell analyses. Our data provide conclusive evidence that conventional hPSCs can be directly and exclusively converted into TE, thereby providing an unlimited source of diverse placental cell types suitable for a broad range of biomedical applications.

developmental biology↗

bioRxiv2022

Scalable Generation of Pseudo-Unipolar Sensory Neurons from Human Pluripotent Stem Cells

Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types amenable for translational research. Here, we developed a highly efficient and reproducible method that differentiates hPSCs into peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest cells, followed by differentiation into sensory neurons with an in vivo-like pseudo-unipolar morphology. Detailed characterization confirmed that the hPSC-derived nociceptors displayed molecular and cellular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors, including critical pain-relevant drug targets (e.g., TRPV1, TAC1, CALCA, NAV1.7, NAV1.8). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.

developmental biology↗

bioRxiv2022

Enhancing the Fitness of Embryoid Bodies and Organoids by Chemical Cytoprotection

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, and kidney) can be optimized by using the CEPT small molecule cocktail, a polypharmacology approach that ensures cytoprotection and cell survival. Application of CEPT (chroman 1, emricasan, polyamines, trans-ISRIB) for just 24 hours during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, and cellular differentiation. Various qualification methods confirmed that CEPT treatment consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

developmental biology↗

bioRxiv2022

SEQUIN: rapid and reproducible analysis of RNA-seq data in R/Shiny

SEQUIN is a new web application (app) that allows fast and intuitive RNA-sequencing data analysis for organisms, tissues, and single cells. Integrated app functions enable uploading datasets, quality control, gene set enrichment, data visualization, and differential gene expression analysis. We also present the iPSC Profiler, a practical tool that helps to measure pluripotency and cell differentiation. Freely available to the public, SEQUIN empowers scientists to investigate transcriptome data firsthand with cutting edge statistical methods.

bioinformatics↗

bioRxiv2021

Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient chemically defined astrocyte differentiation strategy that overcomes current limitations. This approach largely bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like radial glia signatures. Activation of NOTCH and JAK/STAT pathways in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to-astrocyte differentiation and absence of neurogenesis. The morphological and functional identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g. electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic platform and used to model Alexander disease. In conclusion, our findings uncover remarkable plasticity in neural lineage progression that can be exploited to manufacture large numbers of human hPSC-derived astrocytes for drug development and regenerative medicine.

developmental biology↗

bioRxiv2020

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).

bioengineering↗

bioRxiv2019

A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term growth and functional differentiation. Pluripotent cells are capable of extensive self-renewal, yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. Here, we deployed innovative high-throughput screening strategies to identify a small molecule cocktail that dramatically improves viability of hPSCs and their differentiated progeny. We discovered that the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. In proof-of-principle experiments we then demonstrated the strong improvements that CEPT provided for several key applications in stem cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body and organoid formation, single-cell cloning, genome editing, and new iPSC line generation. Thus, CEPT represents a unique polypharmacology strategy for comprehensive cytoprotection, providing a new rationale for efficient and safe utilization of hPSCs. Conferring cell fitness by multi-target drug combinations may become a common approach in cryobiology, drug development, and regenerative medicine.

cell biology↗