Gibbin mesodermal regulation patterns epithelial development.

Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm1-6. However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin, encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene7-9, as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions.

A scalable and cGMP-compatible autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

We present Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a scalable platform producing autologous organotypic iPS cell-derived induced skin composite (iSC) grafts for definitive treatment. Clinical-grade manufacturing integrates CRISPR-mediated genetic correction with reprogramming into one step, accelerating derivation of COL7A1-edited iPS cells from patients. Differentiation into epidermal, dermal and melanocyte progenitors is followed by CD49f-enrichment, minimizing maturation heterogeneity. Mouse xenografting of iSCs from four patients with different mutations demonstrates disease modifying activity at 1 month. Next-generation sequencing, biodistribution and tumorigenicity assays establish a favorable safety profile at 1-9 months. Single cell transcriptomics reveals that iSCs are composed of the major skin cell lineages and include prominent holoclone stem cell-like signatures of keratinocytes, and the recently described Gibbin-dependent signature of fibroblasts. The latter correlates with enhanced graftability of iSCs. In conclusion, DEBCT overcomes manufacturing and safety roadblocks and establishes a reproducible, safe, and cGMP-compatible therapeutic approach to heal lesions of DEB patients.

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.

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

Background: Gene 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. Methods: We 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. Findings: iPS 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. Interpretation: DEBCT 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.

TFAP2C- and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment.

Tissue development results from lineage-specific transcription factors (TFs) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we define epigenomic landscape during epidermal differentiation of human pluripotent stem cells (PSCs) and create inference networks that integrate gene expression, chromatin accessibility, and TF binding to define regulatory mechanisms during keratinocyte specification. We found two critical chromatin networks during surface ectoderm initiation and keratinocyte maturation, which are driven by TFAP2C and p63, respectively. Consistently, TFAP2C, but not p63, is sufficient to initiate surface ectoderm differentiation, and TFAP2C-initiated progenitor cells are 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 autoregulate its own expression and close a subset of the TFAP2C-initiated surface ectoderm program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.

Retinoic acid and BMP4 cooperate with p63 to alter chromatin dynamics during surface epithelial commitment.

Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.

The Rickettsia Endosymbiont of Ixodes pacificus Contains All the Genes of De Novo Folate Biosynthesis.

Ticks and other arthropods often are hosts to nutrient providing bacterial endosymbionts, which contribute to their host's fitness by supplying nutrients such as vitamins and amino acids. It has been detected, in our lab, that Ixodes pacificus is host to Rickettsia species phylotype G021. This endosymbiont is predominantly present, and 100% maternally transmitted in I. pacificus. To study roles of phylotype G021 in I. pacificus, bioinformatic and molecular approaches were carried out. MUMmer genome alignments of whole genome sequence of I. scapularis, a close relative to I. pacificus, against completely sequenced genomes of R. bellii OSU85-389, R. conorii, and R. felis, identified 8,190 unique sequences that are homologous to Rickettsia sequences in the NCBI Trace Archive. MetaCyc metabolic reconstructions revealed that all folate gene orthologues (folA, folC, folE, folKP, ptpS) required for de novo folate biosynthesis are present in the genome of Rickettsia buchneri in I. scapularis. To examine the metabolic capability of phylotype G021 in I. pacificus, genes of the folate biosynthesis pathway of the bacterium were PCR amplified using degenerate primers. BLAST searches identified that nucleotide sequences of the folA, folC, folE, folKP, and ptpS genes possess 98.6%, 98.8%, 98.9%, 98.5% and 99.0% identity respectively to the corresponding genes of Rickettsia buchneri. Phylogenetic tree constructions show that the folate genes of phylotype G021 and homologous genes from various Rickettsia species are monophyletic. This study has shown that all folate genes exist in the genome of Rickettsia species phylotype G021 and that this bacterium has the genetic capability for de novo folate synthesis.