Biology subjects

Bocchi, V. D.

Publications and source records attributed to Bocchi, V. D..

6 recordsLinked to original sources

bioRxiv2026

Timed binary WNT activity is sufficient to model the early human neural axis

The vertebrate nervous system develops into functionally distinct domains along the body axis[1]. Developmental studies implicate multiple signaling pathways in coordinating this process, but the order of specification events, sufficiency of individual signals, and molecular basis of competency remain unclear [2,3], leaving the field without a unified model of neuraxis patterning. Enteric nervous system (ENS) and midbrain dopaminergic (mDA) progenitors have reached clinical and preclinical testing [4-11], yet neither is produced at full purity, and yields vary markedly across human pluripotent stem cell (hPSC) lines, reflecting incomplete control of axial identity. Here we use single-cell genetic fate mapping to demonstrate that hPSCs can commit to anterior or posterior cranial fate within the first 24 hours of differentiation, before neural induction. Building on this finding, we show that the duration of a single saturating WNT pulse is sufficient to specify three 'first-order' domains: anterior cranial (0 h), posterior cranial (24 h) and trunk (48 h), each pre-neural and competent for CNS, neural crest and epidermal lineages. Nested 'second-order' domains are specified within first-order domains in response to WNT, FGF, and retinoic acid. Mechanistically, an anterior gene regulatory network repressing WNT targets is irreversibly shut down during the transition to posterior identities. Applying this binary WNT patterning logic, we generate mDA neurons and ENS progenitors robustly across hPSC lines and establish an assembloid model combining first-order progenitors to produce all major regions of the nervous system.

developmental biology↗

bioRxiv2026

Developmental rewiring of EHMT1 function during human cortical neurogenesis

Ubiquitously expressed chromatin regulators orchestrate cell-type-specific gene expression programs, yet how they acquire distinct regulatory functions during development remains fundamentally unresolved. Using temporally controlled protein depletion during human cortical neurogenesis in vitro, we show that the autism-associated histone methyltransferase EHMT1 is developmentally repurposed from epiblast to cortical neurons. Broadly expressed zinc finger proteins recruit EHMT1 to distinct cis-regulatory elements within changing chromatin landscapes associated with lineage-specific transcription factor activity, with extensive remodeling of EHMT1 enhancer occupancy during neural induction dependent on ZNF462. This genomic rewiring is accompanied by a fundamental shift from restraining premature developmental gene expression in pluripotent cells to supporting neuronal programs, with distinct transcriptional consequences of EHMT1 loss across neural progenitors and excitatory and inhibitory neuronal subtypes. Timed depletion and recovery experiments identify the neural progenitor stage as the onset of a developmental period during which EHMT1 is required for faithful neuronal maturation. Remarkably, EHMT1 can re-engage stage-specific targets after prolonged deficiency, allowing extensive transcriptional recovery with only limited molecular scarring. These findings reveal how ubiquitous epigenetic machinery acquires developmental specificity while demonstrating the resilience of developmental gene regulatory programs to prolonged disruption, with implications for neurodevelopmental disorders.

developmental biology↗

bioRxiv2026

Decoding subtype development and function in human pluripotent stem cell-derived midbrain dopaminergic neurons

Midbrain dopaminergic (mDA) neurons comprise molecularly and functionally distinct subtypes with differential vulnerability in neurodegenerative and psychiatric disorders. However, the mechanisms specifying subtype identity remain poorly understood, and protocols for the selective derivation of human mDA subtypes are lacking. Here we establish a strategy to derive substantia nigra (A9) and ventral tegmental area (A10) mDA neurons from human pluripotent stem cells (hPSCs). A9 identity is specified by dual-SMAD activation through Activin A and BMP7 at the midbrain floor-plate progenitor stage, whereas A10 identity is promoted by BMP inhibition. A9 mDA neurons are purified based on ALDH1A1 expression, and subtype identity is maintained by continued TGF-{beta} modulation and ESRRB activation in vitro and upon transplantation in vivo. Single-cell RNA sequencing and biochemical analyses demonstrate that hPSC-derived A9 neurons exhibit increased oxidative phosphorylation, neuromelanin-like pigmentation, elevated dopamine synthesis and release, and electrophysiological properties characteristic of A9 mDA neurons in vivo. Integration with human fetal midbrain datasets confirms strong transcriptional concordance between in vitro-derived and in vivo mDA subtypes. Neuromelanin-like structures produced by hPSC-derived A9 neurons trigger pro-inflammatory cytokine secretion from hPSC-derived microglia, and A9 neurons are primed to upregulate MHC-I genes in response to interferon-{gamma}, features which may contribute to the selective vulnerability of A9 neurons. Together, these results establish a robust in vitro platform to interrogate human mDA subtype development, function, and selective vulnerability, enabling mechanistic studies relevant to Parkinson's disease and the development of cell-based therapies.

developmental biology↗

bioRxiv2026

Rapid and efficient oligodendrocyte differentiation from human pluripotent stem cells via dual inhibition of BMP and Notch signaling

The protracted timing required for oligodendrocyte differentiation from human pluripotent stem cells (hPSCs) has limited their use in disease modeling, drug screening, and cell therapy. In particular, the signals that drive oligodendrocyte specification and maturation after neural induction and ventral patterning remain poorly understood. Here, we present a protocol to derive human oligodendrocytes from hPSCs that is based solely on extrinsic cues, and we identify dual inhibition of BMP and Notch signaling as critical drivers of oligodendrocyte commitment and maturation. By day 42 of differentiation, up to 70% of the cells are positive for the oligodendrocyte marker O4, with minimal astrocyte contamination, and show robust expression of mature myelin markers including MBP, MOG, and MAG. These hPSC-derived oligodendrocytes closely match the molecular identity of primary fetal human oligodendrocytes as assessed by single-cell RNA sequencing and are functional as shown by in vitro myelination assays. In addition to the rapid generation of myelinating oligodendrocytes, the new protocol can be modularly adapted for the efficient production of PDGFR+ oligodendrocyte precursors or mixed glial populations containing AQP4+ astrocytes, thereby providing a cellular toolbox for the study of human glial lineages in translational applications.

developmental biology↗

bioRxiv2025

Enhanced yield and subtype identity of hPSC-derived midbrain dopamine neuron by modulation of WNT and FGF18 signaling

While clinical trials are ongoing using human pluripotent stem cell-derived midbrain dopamine (mDA) neuron precursor grafts in Parkinsons disease (PD), current protocols to derive mDA neurons remain suboptimal. In particular, the yield of TH+ mDA neurons after in vivo grafting and the expression of some mDA neuron and subtype-specific markers can be further improved. For example, characterization of mDA grafts by single cell transcriptomics has yielded only a small proportion of mDA neurons and a considerable fraction of contaminating cell populations. Here we present an optimized mDA neuron differentiation strategy that builds on our clinical grade ("Boost") protocol but includes the addition of FGF18 and IWP2 treatment ("Boost+") at the mDA neurogenesis stage. We demonstrate that Boost+ mDA neurons show higher expression of EN1, PITX3 and ALDH1A1. Improvements in both mDA neurons yield and transcriptional similarity to primary mDA neurons is observed both in vitro and in grafts. Furthermore, grafts are enriched in authentic A9 mDA neurons by single nucSeq. Functional studies in vitro demonstrate increased dopamine production and release and improved electrophysiological properties. In vivo analyses show increased percentages of TH+ mDA neurons resulting in efficient rescue of amphetamine induced rotation behavior in the 6-OHDA rat model and rescue of some motor deficits in non-drug induced assays, including the ladder rung assay that is not improved by Boost mDA neurons. The Boost+ conditions present an optimized protocol with advantages for disease modeling and mDA neuron grafting paradigms.

cell biology↗

bioRxiv2020

Dose-dependent and disease-modifying effects of striatal infusion of cholesterol in Huntington's disease

A variety of pathophysiological mechanisms are implicated in Huntingtons disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.

neuroscience↗