Biology subjects

Wells, M. F.

Publications and source records attributed to Wells, M. F..

5 recordsLinked to original sources

bioRxiv2025

Cell villages and Dirichlet modeling map human cell fitness genetics

The capacity of cells to proliferate and survive is central to development and disease. Assays that measure cell fitness are therefore a cornerstone of biology, but traditional techniques lack donor diversity and have high technical variability that impedes scale and reproducibility. To overcome these barriers, we designed and validated a "cell village"-based fitness screening approach using pooled cultures of 12-39 genetically distinct human neural progenitor cell (NPC) lines. We also developed Townlet to establish a foundational statistical framework based on Dirichlet regression for analyzing proportional data from cell villages. Applying these systems, we identified hyperproliferation in NPCs harboring the autism risk factor chromosome 16p11.2 deletion, mapped common genetic variants near ZFHX3 associated with NPC proliferation rate, and discovered genetic modifiers of lead (Pb) sensitivity implicating ARNT2. Together, these experimental and analytical tools advance a scalable, genetically diverse in vitro platform for dissecting human variation in cell fitness and gene-environment interactions.

genetics↗

bioRxiv2023

Reprogramming method does not impact the neuronal differentiation potential of 16p11.2 deletion patient iPSCs

A major impediment to the actualization of the induced pluripotent stem cell (iPSC)-based personalized medicine revolution is the lack of widely accepted standard operating procedures (SOPs) across different groups and institutions. The various methods employed can include choice of starting materials, reprogramming agents, and culture conditions, with each of these factors hypothesized to influence the reprogramming efficiency and transcriptional identity of iPSCs. As such, we systematically compared iPSC reprogramming procedures using cells derived from the somatic cells of three patients with 16p11.2 deletion syndrome (16p11.2del) and found remarkable similarity among the different methods. FACS analysis revealed that regardless of somatic cell type (fibroblast, lymphocyte, erythroblast), route of reprogramming factor introduction (mRNA, Sendai virus, episome), donor sex, or facility (Rutgers, NYSCF), 16p11.2del patient iPSCs were viable as high purity cultures expressing pluripotency marker proteins. This observation was supported at the transcript level by qPCR analysis, which demonstrated the ability for the iPSCs to differentiate into all three embryonic germ cell lineages after 12 days in culture as embryoid bodies. NGN2-mediated differentiation of these iPSCs produced functional neurons that formed active synaptic networks as revealed by multi-electrode array (MEA) recordings. Importantly, no group-wise comparisons among the reprogramming methods yielded consistent statistically significant differences, indicating that these procedures are equally capable of producing pluripotent stem cells that can efficiently differentiate into mature, functional neurons. This work highlights the utility of these reprogramming methods and supports the use of differentially reprogrammed iPSCs for direct comparative studies of human neurodevelopment.

developmental biology↗

bioRxiv2021

Natural variation in gene expression and Zika virus susceptibility revealed by villages of neural progenitor cells

Variation in the human genome contributes to abundant diversity in human traits and vulnerabilities, but the underlying molecular and cellular mechanisms are not yet known, and will need scalable approaches to accelerate their recognition. Here, we advanced and applied an experimental platform that analyzes genetic, molecular, and phenotypic heterogeneity across cells from very many human donors cultured in a single, shared in vitro environment, with algorithms (Dropulation and Census-seq) for assigning phenotypes to individual donors. We used natural genetic variation and synthetic (CRISPR-Cas9) genetic perturbations to analyze the vulnerability of neural progenitor cells to infection with Zika virus. These analyses identified a common variant in the antiviral IFITM3 gene that regulated IFITM3 expression and explained most inter-individual variation in NPCs susceptibility to Zika virus infectivity. These and other approaches could provide scalable ways to recognize the impact of genes and genetic variation on cellular phenotypes. HIGHLIGHTSO_LIMeasuring cellular phenotypes in iPSCs and hPSC-derived NPCs from many donors C_LIO_LIEffects of donor sex, cell source, genetic and other variables on hPSC RNA expression C_LIO_LINatural genetic variation and synthetic perturbation screens both identify IFITM3 in NPC susceptibility to Zika virus C_LIO_LIA common genetic variant in IFITM3 explains most inter-individual variation in NPC susceptibility to Zika virus C_LI

genomics↗

bioRxiv2021

Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

neuroscience↗

bioRxiv2018

Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

Neural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe human Stem cell-derived NGN2-accelerated Progenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum gene PTEN.

neuroscience↗