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Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion [re-analysis]

ABSTRACT: Direct conversion generates patient-specific, disease-relevant cell types such as neurons that are rare, limited, or difficult to isolate from common and easily accessible cells such as skin cells. However, low rates of direct conversion and complex protocols limit scalability and thus the potential of cell-fate conversion for biomedical applications. Systems-level optimization of conversion protocols across molecular and cellular scales can support scalable cell manufacturing for diverse application. We use insights from our work on how proliferation and transcription factor levels act together to drive direct conversion to optimize the conversion protocol by examining process parameters including transcript design, delivery via AAV, retrovirus and lentivirus, cell seeding density, and the impact of media conditions. Thus, we report a compact, portable conversion process that boosts proliferation and increases direct conversion of mouse fibroblasts to induced motor neurons to achieve high conversion rates of above 1,000%, corresponding to more than 10 motor neurons yielded per cell seeded which we achieve through expansion. Our optimized, direct conversion process generates functional motor neurons at scales relevant for cell therapies (>10^7 cells) that graft with the mouse central nervous system. High-efficiency, compact, direct conversion systems will support scaling to patient-specific, neural cell therapies.

ORGANISM(S): Mus musculus

PROVIDER: GSE287882 | GEO | 2025/02/03

REPOSITORIES: GEO

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Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion

Project description:Direct conversion generates patient-specific, disease-relevant cell types such as neurons that are rare, limited, or difficult to isolate from common and easily accessible cells such as skin cells. However, low rates of direct conversion and complex protocols limit scalability and thus the potential of cell-fate conversion for biomedical applications. Systems-level optimization of conversion protocols across molecular and cellular scales can support scalable cell manufacturing for diverse application. We use insights from our work on how proliferation and transcription factor levels act together to drive direct conversion to optimize the conversion protocol by examining process parameters including transcript design, delivery via AAV, retrovirus and lentivirus, cell seeding density, and the impact of media conditions. Thus, we report a compact, portable conversion process that boosts proliferation and increases direct conversion of mouse fibroblasts to induced motor neurons to achieve high conversion rates of above 1,000%, corresponding to more than 10 motor neurons yielded per cell seeded which we achieve through expansion. Our optimized, direct conversion process generates functional motor neurons at scales relevant for cell therapies (>107 cells) that graft with the mouse central nervous system. High-efficiency, compact, direct conversion systems will support scaling to patient-specific, neural cell therapies.

2025-02-03 | GSE287783 | GEO

Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion

Project description:Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion

| PRJNA1214691 | ENA

Generation of induced motor neurons (iMNs) from human fibroblasts facilitates locomotor recovery after spinal cord injury

Project description:Generation of autologous motor neurons holds a great promise for cell replacement therapy to treat spinal cord injury (SCI). Direct conversion allows the generation of target cell types from somatic cells, however, current protocols are not practicable for therapeutic purposes since converted cells are post-mitotic populations that are not readily scalable. Therefore, therapeutic effects of directly converted neurons have not been elucidated yet. Here, we show that human fibroblasts can be converted into induced motor neurons (iMNs) by sequential induction of OCT4 and LHX3. Our strategy enables large production of pure iMNs through self-renewing iMN-intermediate cell stage which is distinct from neural progenitors. iMNs exhibited the hallmarks of spinal motor neurons including cellular morphology, marker expression, electrophysiological property, synaptic activity, and formation of neuromuscular junctions. Remarkably, transplantation of iMNs showed therapeutic effects, promoting locomotor functional recovery in SCI model. Taken together, our advanced strategy will provide tools to acquire sufficient iMNs that may represent a promising cell source for personalized cell therapy.

2020-06-29 | GSE149664 | GEO

Accelerated high-yield generation of limb-innervating motor neurons from human stem cells

Project description:Human pluripotent stem cells are a promising source of diverse cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons, is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that induce up to 50% motor neurons within 3 weeks from human pluripotent stem cells with defined subtype identities that are relevant to neurodegenerative diseases. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3-). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays. We analyze 3 samples including 2 positive samples and 1 negative sample. Descriptions are as follow: a) Positive Sample 1: SHH-derived, day 21 GFP-high FACS purified motor neurons.b) Positive Sample 2: S+P-derived, day 21 GFP-high FACS purified motor neurons. c) Negative: S+P condition, day 21 no GFP FACS purified motor neurons

2013-02-06 | E-GEOD-41795 | biostudies-arrayexpress

Mouse embryonic stem cells can differentiate via multiple paths to the same state [RNA-seq]

Project description:In embryonic development, cells must differentiate through stereotypical sequences of intermediate states to generate mature states of a particular fate. By contrast, direct programming can generate similar fates through alternative routes, by directly expressing terminal transcription factors. Yet the cell state transitions defining these new routes are unclear. We applied single-cell RNA sequencing to compare two mouse motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both undergo similar early neural commitment. Then, rather than transitioning through spinal intermediates like the standard protocol, the direct programming path diverges into a novel transitional state. This state has specific and abnormal gene expression. It opens a 'loop' or 'worm hole' in gene expression that converges separately onto the final motor neuron state of the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons from embryos.

2017-10-17 | GSE97390 | GEO

Mouse embryonic stem cells can differentiate via multiple paths to the same state [array]

2017-10-17 | GSE97360 | GEO

Accelerated high-yield generation of limb-innervating motor neurons from human stem cells

2013-02-06 | GSE41795 | GEO

Genome-wide analysis of transcription in in vitro- and in vivo-derived motor neurons

Project description:RNA sequencing analysis of Hb9::GFP mouse embryonic fibroblasts, Hb9::GFP+ primary mouse embryonic motor neurons at day E13.5, Hb9::GFP+ mouse embryonic stem cell-derived motor neurons, Hb9::GFP+ mouse induced pluripotent stem cell derived motor neurons, and Hb9::GFP+ mouse induced motor neurons generated using transcription factor overexpression. The goal of this project is to evaluate the ability of directed differentiation and lineage conversion techniques to generate a bona fide neuronal subtype.

2016-11-08 | GSE89619 | GEO

Rapid Generation of Regionally Specified CNS Neurons by Sequential Patterning and Conversion of Human Induced Pluripotent Stem Cells

Project description:The differentiation of patient-specific induced pluripotent stem cells (iPSCs) into specific neuronal subtypes has been exploited as an approach to modeling a variety of neurological disorders. However, achieving a highly pure population of neurons is challenging when using directed differentiation methods, especially for neuronal subtypes generated by complex and protracted protocols. In this study, we efficiently produced highly pure populations of regionally specified CNS neurons by using a modified NGN2-Puromycin direct conversion protocol. The protocol is amenable across a range of iPSC lines, with an efficiency above 97% by day 21, and above 95% of neurons positive for MAP2. This NGN2-Puromycin conversion resulted in a significant number of peripheral neurons, but by incorporating a short CNS patterning step, we eliminated these peripheral neurons. Furthermore, we used the patterning step to control the rostral-caudal identity. This approach to sequential patterning and NGN2-Puromycin conversion, when patterned with SMAD inhibitors, produced pure populations of forebrain neurons; when SMAD inhibitors and WNT agonists were applied, the approach produced anterior hindbrain excitatory neurons, resulting in a neuronal population containing VSX2/SHOX2 V2a interneurons. Overall, this sequential patterning and conversion protocol can be used for the production of a variety of CNS excitatory neurons from patient-derived iPSCs, which is a highly versatile system for investigating early disease events for a range of neurological disorders including Alzheimer's disease, motor neurons disease and spinal cord injury.

2020-06-24 | GSE149864 | GEO

Gene expresssion in human iPSC-derived neurons from normal and SPG3A donors

Project description:We performed RNA-seq experiments on two samples (cortical neurons and spinal motor neurons) from normal induced pluripotent stem cells (iPSCs), and another two samples (cortical neurons and spinal motor neurons) derived from SPG3A (an early onset form of hereditary spastic paraplegia) iPSCs. This initial experiment is to test the system and set up a baseline for future studies. Cortical projection neurons and spinal motor neurons were differentiated from same batch of iPSCs in parallel to minimize variations. The differentiation of cortical neurons and spinal motor neurons are based on protocols well-established in our group.

2019-03-25 | E-MTAB-7770 | biostudies-arrayexpress

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