This integrated molecular, single-nucleus, and electrophysiological characterization defines the cellular heterogeneity of DPSC-to-neuron reprogramming and provides a framework for protocol refinement and future patient-specific disease modeling.
Abstract
Direct neuronal reprogramming offers an alternative to induced pluripotent stem cell-based differentiation by converting somatic cells directly into neurons without passage through pluripotency. However, commonly used fibroblast-based protocols are often slow and inefficient. Here, we evaluated human dental pulp stem cells (DPSCs), which originate from the cranial neural crest and possess intrinsic neurogenic potential, as a developmentally relevant source of induced neurons (iNs). Using an all-in-one lentiviral vector, we converted DPSCs into iNs within 17 days, compared with 28 days for fibroblasts reprogrammed with the same vector, and achieved significantly higher neuronal purity under the respective established protocols. Multi-omic profiling revealed coordinated suppression of mesenchymal and cell-cycle programs and induction of neuronal, synaptic, and metabolic pathways. Single-nucleus RNA sequencing resolved fibroblast-like, transitional, maturing neuronal, GABAergic-like, and alternative fates, while trajectory inference suggested divergent neuronal and non-neuronal conversion paths. Whole-cell recordings showed that a subset of DPSC-iNs developed early neuronal excitability and voltage-gated inward and outward currents. Together, our findings establish DPSCs as an accessible and developmentally relevant source for rapid direct neuronal conversion. This integrated molecular, single-nucleus, and electrophysiological characterization defines the cellular heterogeneity of DPSC-to-neuron reprogramming and provides a framework for protocol refinement and future patient-specific disease modeling.
Direct reprogramming of somatic cells into induced dopaminergic neurons (iDANs) is a promising strategy for replacing degenerated dopamine neurons in Parkinson’s disease. One current bottleneck for translation of direct neuronal reprogramming is the low reprogramming efficiency and difficulty to acquire enough tyrosine hydroxylase (TH) positive iDANs. Here we systematically refine the culture conditions used for dopaminergic reprogramming of human stem cell-derived glial progenitor cells (GPCs) and adult dermal fibroblasts (DFs). Starting from a standard reprogramming medium containing base medium, growth factors and small molecules, we systematically compared three base media and removed individual small molecules to determine their specific contribution to reprogramming outcome. The GPCs and DFs were reprogrammed using established transcription factor-based strategies combined with REST inhibition. We found that the two starting cell types benefited from different culture conditions. For GPCs, B27/Neurobasal medium without small molecules yielded the highest neuronal and TH reprogramming efficiency, whereas DFs yielded the highest efficiencies in N2/DMEM/Neurobasal containing small molecules. Further characterization of the GPC-derived iDANs showed that these cells were functional, expressed key dopaminergic markers, and released dopamine upon stimulation. Together, these findings demonstrate that media composition is a key parameter influencing dopaminergic reprogramming efficiency across starting cell types in vitro.
Kerstin Laurin, Janko Kajtez, J. Wickham et al.· Scientific Reports· 0 citations
Recognising the central role of VSELs/progenitors and their niche in maintaining tissue homeostasis in vivo could resolve existing roadblocks and guide more effective endogenous regenerative therapies for diseased tissues and age-related dysfunctions.
D. Bhartiya, N. Sharma, Anish Tripathi et al.· Stem Cell Reviews and Report...· 0 citations
Neurological diseases, often caused by irreversible loss of terminally differentiated neurons, present considerable challenges to treatment due to the limited regenerative capacity of these neurons. Although induced pluripotent stem cells hold promise for neuronal regeneration, their clinical application is constrained by risks, including tumorigenicity, incomplete neuronal maturation, and immune rejection. Recent advancements in direct neuronal reprogramming, which bypasses the intermediate pluripotent stage by directly converting non-neuronal cells into functional neurons, offer a compelling alternative for in situ neuronal replacement in neurodegenerative diseases. Key transcription factors, such as NeuroD1, Ascl1, Sox2, as well as CRISPR activation (CRISPRa) of NGN2 and ISL1, have been explored to convert glial cells into neurons. However, several challenges remain. This review discusses the current applications of direct neuronal reprogramming technology in several neurological diseases. We further highlight the potential contamination issues in adeno-associated virus (AAV) delivery systems and propose a code of conduct to avoid artifacts and pitfalls. Finally, we point out future directions for expanding direct reprogramming targets, integrating organoid-based disease modeling, and advancing reprogramming regulation techniques.
Yu Chen, Zhe Zheng, P. Zhao et al.· Frontiers in Neuroscience· 0 citations
Oligodendrocytes (OLs) are essential for central nervous system (CNS) function through their role in axon myelination, and their dysfunction is implicated in a range of neurological disorders. Despite this, in vitro modeling of human OL biology remains limited by the scarcity of primary human OLs. Furthermore, while induced pluripotent stem cells (iPSCs) offer a promising source of human OLs, current differentiation protocols remain complex, inefficient, and time-consuming. Here, we present an optimized protocol to generate OLs from iPSCs (iOLs) using defined soluble factors supplemented in the culture media. Within 28 days, cultures yielded an average of 85% O4+ iOLs, and by Day 38, approximately 70% expressed MBP, a key marker of mature OLs. Bulk RNA-seq analysis confirmed a stepwise transcriptional progression consistent with OL lineage identity, with upregulation of key OL-specific transcripts and gene expression profiles. Comparative transcriptomic analyses further revealed increased expression of gene networks associated with myelination, extracellular matrix remodeling, and gliogenesis when iOLs were cultured on aligned nanofiber scaffolds. Importantly, iOLs formed compact myelin sheaths around axons and showed enhanced maturation in a three-dimensional (3D) environment, highlighting the importance of spatial and matrix-derived cues in OL development. This study establishes a cost- and time-efficient approach for generating functional OLs from iPSCs, with broad applicability for disease modeling, drug screening, and the development of regenerative therapies.
Unknown authors· Journal of Neuroscience Rese...· 0 citations
Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.
Summary Direct reprogramming of human fibroblasts into hematopoietic stem cells (HSCs) offers a promising strategy for generating autologous cells to treat blood and immune disorders. Current protocols are limited by low efficiency and insufficient tools for evaluating reprogramming outcomes. Although functional assays are the standard for confirming cell identity, they require fully reprogrammed cells, limiting their utility during protocol development. To address this, we assembled a single-cell transcriptomic reference atlas of hematopoietic reprogramming and tested an algorithmically predicted transcription factor recipe for HSC induction. Long-read single-cell RNA sequencing of CD34+ reprogrammed cells revealed progressive loss of fibroblast identity alongside induction of early hematopoietic and endothelial programs, with reference-atlas benchmarking placing reprogrammed cells in an intermediate transcriptomic state between fibroblasts, endothelial cells, and HSCs. Isoform-level analysis further revealed transcriptional remodeling not captured by gene-level analyses. This experimental-computational framework offers a generalizable strategy for characterizing partially reprogrammed states and guiding optimization of reprogramming protocols.
Unknown authors· iScience· 0 citations
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