Biology subjects

Balmas, E.

Publications and source records attributed to Balmas, E..

5 recordsLinked to original sources

bioRxiv2026

Genetic control couples TGFβ-independent pluripotent stem cell renewal to deterministic forward programming across germ layers

Reproducible and scalable manufacture of human induced pluripotent stem cell (hiPSC) derivatives requires control over expansion and differentiation. Transcription factor-mediated forward programming can place hiPSC differentiation under genetic control, but expansion still relies on extracellular growth-factor supplementation, increasing process complexity and operator dependence. Here we introduce GERALT (Genetically Enhanced Renewal and Autonomous Lineage Transdifferentiation), a push-pull genetic circuit assembled through targeted safe-harbour integration that couples NANOG-supported, TGF{beta}-independent renewal to doxycycline-inducible lineage programming and simultaneous NANOG silencing. GERALT maintained pluripotency during extended TGF{beta} withdrawal in both adherent and suspension culture, yet remained programmable towards myogenic, neuronal, and hepatic fates. Compared with the established OPTi-OX platform in myogenesis, GERALT enabled differentiation in simplified medium while canalising and accelerating commitment. GERALT also supported direct myogenic programming in suspension and enabled direct casting of hiPSCs into hydrogel followed by in-tissue programming, yielding electrically responsive engineered muscle tissues. These results extend genetic control upstream into stem cell renewal, facilitating deployment of new approach methodologies and other biomanufacturing applications.

synthetic biology↗

bioRxiv2026

Combinatorial and Inducible CRISPRa/i Enables Canalized hiPSC Forward Programming and Iterative Refinement via Single-Cell Genomics

Synthetic gene-regulation logic is established in immortalized cell lines but remains largely aspirational in human induced pluripotent stem cells (hiPSCs) and derivatives. This gap constrains both mechanistic discovery and translational engineering in physiologically relevant models. We developed CIRI (Combinatorial Inducible CRISPR in IPSCs), an isogenic, safe-harbor-engineered platform in which tetracycline-responsive single guide RNAs (sgR-NAs) carry modular RNA aptamers that recruit RNA-binding proteins and effector domains. This design enables multimodal regulation from a single catalytically inactive Cas9 (dCas9), exemplified by orthogonal CRISPR activation and interference (CRISPRa/i). After optimizing sgRNA-aptamer architectures, we achieved robust CRISPRa and CRISPRi in hiPSCs and hiPSC-derived cardiac organoids. CIRI rapidly channels hiPSC forward programming into skeletal myocytes by activating MYOD1 while repressing NANOG, POU5F1/OCT4, and SOX2. Combinatorial pooled dual-guide single-cell RNA sequencing screens identify ID3 as a road-block and KDM6B and SMARCD3 as synergistic enhancers of myogenic maturation. Together, CIRI establishes a programmable synthetic biology framework in human stem cell models. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/729073v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@18ce445org.highwire.dtl.DTLVardef@de8d94org.highwire.dtl.DTLVardef@1212763org.highwire.dtl.DTLVardef@1a0d83d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Opposing CTCF and GATA4 activities set the pace of chromatin topology remodeling during cardiomyogenesis

Reorganization of the three-dimensional chromatin structure is a critical feature of human embryonic development. Yet, the mechanisms regulating integrative remodelling of local structures (e.g., loops) and global architecture (e.g., A/B compartmentalization) remain un-clear. Here, we investigate this aspect in the context of cardiomyogenesis, characterized by pronounced B-to-A remodelling of several cardiac-specific genes such as TTN. We focus on the roles of the pioneer transcription factor GATA4 and the architectural protein CTCF. Using an inducible knockdown system during human induced pluripotent stem cell differ-entiation, we show that GATA4 is essential for timely topological activation of key cardiac genes, while partial depletion of CTCF, anticipating physiological downregulation during de-velopment, enhances this process. Deletion of a single CTCF binding site on TTN leads to modest gene decompaction and transcriptional activation. Bulk and single-cell RNA se-quencing of chamber-specific cardiac organoids reveals that loss of GATA4 delays differ-entiation and sustains proliferation of early cardiomyocytes, whereas premature CTCF de-pletion accelerates yet alters cardiomyocyte maturation. These findings suggest that CTCF and GATA4 have antagonistic roles on chromatin dynamics during cardiomyogenesis, form-ing a rheostat that maintains accurate developmental tempo. Disruption of this mecha-nism may contribute to congenital heart defects caused by mutations in these factors.

molecular biology↗

bioRxiv2024

CALIPERS: Cell cycle-aware live imaging for phenotyping experiments and regeneration studies

Cell cycle progression, migration, and proliferation shape development and regeneration, but simultaneous live-cell imaging remains challenging as conventional fluorescent cell cycle indicators (FUCCI) monopolize the green and red channels used by most structural and functional biosensors. To overcome this, we integrated a spectrally re-engineered FUCCI variant, open-source analysis software, and four-color human stem cell reporter lines into CALIPERS: a method for Cell-cycle-Aware Live-cell Imaging in Phenotyping and Regeneration Studies.

bioengineering↗

bioRxiv2018

Maternal group 2 innate lymphoid cells control fetal growth and protect from endotoxin-induced abortion in mice

Group 2 innate lymphoid cells (ILC2s) adapt to tissue physiology and contribute to immunity, inflammatory pathology and metabolism. We show that mouse uterine ILC2s have a heightened type-2 gene signature and expand during pregnancy. Indeed, maternal ILC2s promote fetal growth and protect against fetal mortality upon systemic endotoxin challenge. Absence of ILC2s leads to utero-placental abnormalities, including poor vascular remodelling, increased Il1b and decreased Il4, Il5, and Il13 gene expression, and reduced alternative activation of dendritic cells (DCs) and macrophages. Placentas exhibit signs of adaptation to stress, including larger maternal blood spaces and increased expression of nutrient transporter genes. Endotoxin induces the expansion of IL-1{beta}-producing uterine DCs and, in response, more uterine ILC2s produce IL-4, IL-5 and IL-13. In a protective feedback mechanism, these cytokines suppress IL-1{beta}-producing DCs, in line with a protective role of uILC2s against endotoxin-induced abortion. Uterine ILC2s emerge as pivotal for both normal and complicated pregnancies.

immunology↗