Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population that is capable of nearly indefinite expansion and subsequent differentiation into the various neuronal and supporting cell types that comprise the CNS. However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS. In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation. Furthermore, exogenous manipulation of WNT signaling during neural differentiation, through either activation or inhibition, reduces this heterogeneity in NPC cultures, thereby promoting the formation of regionally homogeneous NPC and neuronal cultures. The ability to manipulate WNT signaling to generate regionally specific NPCs and neurons will be useful for studying human neural development and will greatly enhance the translational potential of hPSCs for neural-related therapies.

The evolutionary conserved WNT signaling pathway orchestrates numerous complex biological processes during development and is critical to the maintenance of tissue integrity and homeostasis in the adult. As it relates to the central nervous system, WNT signaling plays several roles as it relates to neurogenesis, synaptic formation, memory, and learning. Thus, dysfunction of this pathway is associated with multiple diseases and disorders, including several neurodegenerative disorders. Alzheimer’s disease (AD) is characterized by several pathologies, synaptic dysfunction, and cognitive decline. In this review, we will discuss the various epidemiological, clinical, and animal studies that demonstrate a precise link between aberrant WNT signaling and AD-associated pathologies. In turn, we will discuss the manner in which WNT signaling influences multiple molecular, biochemical, and cellular pathways upstream of these end-point pathologies. Finally, we will discuss how merging tools and technologies can be used to generate next generation cellular models to dissect the relationship between WNT signaling and AD.

The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.

Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.

CRISPR-based technologies are paramount in genome engineering and synthetic biology. Prime editing (PE) is a technology capable of installing genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. Prime editing guide RNAs (pegRNAs) simultaneously encode both guide and edit template sequences. They are more design intensive than CRISPR single guide RNAs (sgRNAs). As such, application of PE technology is hindered by the limited throughput of manual pegRNA design. To that end, we designed a software tool, Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE), that enables high-throughput automated design of pegRNAs and prime editing strategies. PINE-CONE translates edit coordinates and sequences into pegRNA designs, accessory guides, and oligonucleotides for facile cloning workflows. To demonstrate PINE-CONE’s utility in studying disease-relevant genotypes, we rapidly design a library of pegRNAs targeting Alzheimer’s Disease single nucleotide polymorphisms (SNPs). Overall, PINE-CONE will accelerate the application of PEs in synthetic biology and biomedical research.