Biology subjects

Oro, A. E.

Publications and source records attributed to Oro, A. E..

8 recordsLinked to original sources

bioRxiv2023

A Spatiotemporal and Machine-Learning Platform Accelerates the Manufacturing of hPSC-derived Esophageal Mucosa

Human pluripotent stem cell-derived tissue engineering offers great promise in designer cell-based personalized therapeutics. To harness such potential, a broader approach requires a deeper understanding of tissue-level interactions. We previously developed a manufacturing system for the ectoderm-derived skin epithelium for cell replacement therapy. However, it remains challenging to manufacture the endoderm-derived esophageal epithelium, despite both possessing similar stratified structure. Here we employ single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for human esophageal development. We illuminate the cellular diversity, dynamics and signal communications for the developing esophageal epithelium and stroma. Using the machine-learning based Manatee, we prioritize the combinations of candidate human developmental signals for in vitro derivation of esophageal basal cells. Functional validation of the Manatee predictions leads to a clinically-compatible system for manufacturing human esophageal mucosa. Our approach creates a versatile platform to accelerate human tissue manufacturing for future cell replacement therapies to treat human genetic defects and wounds.

developmental biology↗

bioRxiv2023

Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy

While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Upregulation of the major p63 isoform {Delta}Np63, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression.

cell biology↗

bioRxiv2023

Acquisition of drug resistance in basal cell nevus syndrome tumors through basal to squamous cell carcinoma transition

While basal cell carcinomas (BCCs) arise from ectopic hedgehog pathway activation and can be treated with pathway inhibitors, sporadic BCCs display high resistance rates while tumors arising in Gorlin syndrome patients with germline Patched (PTCH1) mutations are uniformly suppressed by inhibitor therapy. In rare cases, Gorlin syndrome patients on long-term inhibitor therapy will develop individual resistant tumor clones that rapidly progress, but the basis of this resistance remains unstudied. Here we report a case of an SMOi-resistant tumor arising in a Gorlin patient on suppressive SMOi for nearly a decade. Using a combination of multi-omics and spatial transcriptomics, we define the tumor populations at the cellular and tissue level to conclude that Gorlin tumors can develop resistance to SMOi through the previously described basal to squamous cell carcinoma transition (BST). Intriguingly, through spatial whole exome genomic analysis, we nominate PCYT2, ETNK1, and the phosphatidylethanolamine biosynthetic pathway as novel genetic suppressors of BST resistance. These observations provide a general framework for studying tumor evolution and provide important clinical insight into mechanisms of resistance to SMOi for not only Gorlin syndrome but sporadic BCCs as well.

cancer biology↗

bioRxiv2023

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa

BackgroundGene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. MethodsWe developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar- free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. FindingsiPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT- treated mice. InterpretationDEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

bioengineering↗

bioRxiv2022

Integrated single-cell chromatin and transcriptomic analyses of human scalp reveal etiological insights into genetic risk for hair and skin disease

Genome-wide association studies (GWAS) of hair and skin disease have identified many genetic variants associated with disease phenotypes, but identifying causal variants and interpreting their function requires deciphering gene-regulatory networks in disease-relevant cell types. To this end, we generated matched scRNA- and scATAC-seq profiles of human scalp biopsies, identifying diverse cell types of the hair follicle niche. By interrogating the integrated datasets across multiple levels of cellular resolution, we infer 50-100% more enhancer-gene links than prior approaches, and show that the aggregate accessibility at linked enhancers for highly-regulated genes predicts expression. We use these gene-regulatory maps to prioritize cell types, genes, and causal variants implicated in the pathobiology of androgenetic alopecia (AGA), eczema, and other complex traits. AGA GWAS signals are strongly and specifically enriched in dermal papilla cell open chromatin regions, supporting the role of these cells as key drivers of AGA pathogenesis. Finally, we trained machine-learning models to nominate SNPs that affect gene expression through disruption of specific transcription factor binding motifs, identifying candidate functional SNPs linked to expression of WNT10A in AGA and IL18RAP in eczema. Together, this work reveals principles of gene regulation and identifies gene regulatory consequences of natural genetic variation in complex skin and hair diseases.

genomics↗

bioRxiv2018

Feedback Regulation between Initiation and Maturation Networks Orchestrates the Chromatin Dynamics of Epidermal Lineage Commitment

Tissue development results from lineage-specific transcription factors (TF) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we interrogate the epigenomic landscape during epidermal differentiation and create an inference network that ranks the coordinate effects of TF-accessible regulatory element-target gene expression triplets on lineage commitment. We discover two critical transition periods: surface ectoderm initiation and keratinocyte maturation, and identify TFAP2C and p63 as lineage initiation and maturation factors, respectively. Surprisingly, we find that TFAP2C, and not p63, is sufficient to initiate surface ectoderm differentiation, with TFAP2C-initiated progenitor cells capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively auto-regulate its expression and close a subset of the TFAP2C-initiated early program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.

cell biology↗

bioRxiv2018

Morphogen-Lineage Selector Interactions During Surface Epithelial Commitment

Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into tissues such as keratinocytes requires a better understanding of epigenetic interactions between the inductive morphogens retinoic acid (RA) and bone morphogenetic protein 4 (BMP), and the master regulator p634,5. Here we develop a robust, defined, keratinocyte differentiation system, and use a multi-dimensional genomics approach to interrogate the contributions of the morphogens and lineage selector to chromatin dynamics during early surface ectoderm commitment. In stark contrast to other master regulators6-9, we find using p63 gain and loss of function hESC lines, that p63 effects major transcriptional changes only after morphogenetic action. Morphogens alter chromatin accessibility and histone modifications, establishing an epigenetic landscape for p63 to modify. In turn, p63 closes chromatin accessibility and promotes the accumulation of repressive H3K27me3 histone modifications at sites distal to where it binds. Surprisingly, cohesin HiChIP10 visualization of genome-wide chromosome conformation reveals that both p63 and the morphogens contribute to dynamic long-range genomic interactions that increase the probability of negative transcriptional regulation at p63 target loci. p63-regulated accessibility, not H3K27me3 deposition, appears to drive early transcriptional changes. We illustrate morphogen-selector interactions by studying p63 negative feedback regulation of TFAP2Ci11, whereby disruption of the single p63 binding site results in a loss of p63-mediated transcriptional control and dramatic increases in TFAP2C and p63 expression. Our study reveals the unexpected dependency of p63 on morphogenetic signaling to control long-range chromatin interactions during tissue specification and provides novel insights into how master regulators specify diverse morphological outcomes.

genetics↗

bioRxiv2018

MTSS1/Src family kinase Dysregulation Underlies Multiple Inherited Ataxias

The genetically heterogeneous Spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of non-receptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-Metastasis (MTSS1) is a novel ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically-approved Src inhibitor corrects Purkinje basal firing, and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.\n\nSignificance StatementThe Src family of non-receptor tyrosine kinases (SFK) are essential for nervous system function, and may contribute to neurodegeneration. Spinocerebellar ataxias (SCAs) are neurodegenerative diseases where Purkinje neurons fire irregularly and degenerate leading to motor problems. We show that the SFK suppressor Missing-in-Metastasis (MTSS1) is a novel ataxia gene that links multiple SCAs. MTSS1 loss results in increased SFK activity, degenerating Purkinje neurons with low basal firing rates, and cell death. Surprisingly, mouse models for three different SCAs show elevated SFK activity, with SCA1 and SCA2 models displaying dramatically reduced MTSS1 protein levels. Treatment of each SCA model with SFK inhibitor corrects Purkinje basal firing, and delays ataxia progression in MTSS1 mutants. Our results identify a common link among disparate neurodegenerative diseases.

neuroscience↗