TNF-NF-κB-p53 axis restricts in vivo survival of hPSC-derived dopamine neurons.

Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.

CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant.

Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ∼664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes (T2D) associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear enhancer gene regulatory activity during cell state transitions.

Comprehensive enhancer discovery is challenging because most enhancers, especially those contributing to complex diseases, have weak effects on gene expression. Our gene regulatory network modeling identified that nonlinear enhancer gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Using human embryonic stem cell definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set of enhancers for each of the core endoderm-specifying transcription factors. Many enhancers had strong effects mid-transition but weak effects post-transition, consistent with the nonlinear temporal responses to enhancer perturbation predicted by the modeling. Integrating three-dimensional genomic information, we were able to develop a CTCF-loop-constrained Interaction Activity model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and systematic enhancer discovery in both normal and pathological cell state transitions.