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1.
Front Neurosci ; 17: 1251906, 2023.
Article in English | MEDLINE | ID: mdl-37781243

ABSTRACT

Spinal cord injury can attenuate both motor and sensory function with minimal potential for full recovery. Research utilizing human induced pluripotent stem cell (hiPSC) -derived spinal cell types for in vivo remodeling and neuromodulation after spinal cord injury has grown substantially in recent years. However, the majority of protocols for the differentiation of spinal neurons are lengthy, lack the appropriate dorsoventral or rostrocaudal specification, and are not typically replicated in more than one cell line. Furthermore, most researchers currently utilize hiPSC-derived motor neurons for cell transplantation after injury, with very little exploration of spinal sensory neuron transplantation. The lack of studies that utilize sensory populations may be due in part to the relative scarcity of dorsal horn differentiation protocols. Building upon our previously published work that demonstrated the rapid establishment of a primitive ectoderm population from hiPSCs, we describe here the production of a diverse population of both ventral spinal and dorsal horn progenitor cells. Our work creates a novel system allowing dorsal and ventral spinal neurons to be differentiated from the same intermediate ectoderm population, making it possible to construct the dorsal and ventral domains of the spinal cord while decreasing variability. This technology can be used in tandem with biomaterials and pharmacology to improve cell transplantation for spinal cord injury, increasing the potential for neuroregeneration.

2.
J Cell Mol Med ; 26(7): 1932-1942, 2022 04.
Article in English | MEDLINE | ID: mdl-35257489

ABSTRACT

Numerous interventions have been explored in animal models using cells differentiated from human induced pluripotent stem cells (iPSCs) in the context of neural injury with some success. Our work seeks to transplant cells that are generated from hiPSCs into regionally specific spinal neural progenitor cells (sNPCs) utilizing a novel accelerated differentiation protocol designed for clinical translation. We chose a xenotransplantation model because our laboratory is focused on the behaviour of human cells in order to bring this potential therapy to translation. Cells were transplanted into adult immunodeficient rats after moderate contusion spinal cord injury (SCI). Twelve weeks later, cells derived from the transplanted sNPCs survived and differentiated into neurons and glia that filled the lesion cavity and produced a thoracic spinal cord transcriptional program in vivo. Furthermore, neurogenesis and ionic channel expression were promoted within the adjacent host spinal cord tissue. Transplanted cells displayed robust integration properties including synapse formation and myelination by host oligodendrocytes. Axons from transplanted hiPSC sNPC-derived cells extended both rostrally and caudally from the SCI transplant site, rostrally approximately 6 cm into supraspinal structures. Thus, iPSC-derived sNPCs may provide a patient-specific cell source for patients with SCI that could provide a relay system across the site of injury.


Subject(s)
Induced Pluripotent Stem Cells , Neural Stem Cells , Spinal Cord Injuries , Animals , Axons/pathology , Cell Differentiation/physiology , Humans , Induced Pluripotent Stem Cells/metabolism , Neural Stem Cells/metabolism , Rats , Recovery of Function , Spinal Cord/pathology , Spinal Cord Injuries/pathology , Synapses/pathology
3.
J Biol Chem ; 294(40): 14674-14685, 2019 10 04.
Article in English | MEDLINE | ID: mdl-31395656

ABSTRACT

Human Schlafen 11 (SLFN11) is an interferon-stimulated gene (ISG) that we previously have demonstrated to ablate translation of HIV proteins based on the virus's distinct codon preference. Additionally, lack of SLFN11 expression has been linked to the resistance of cancer cells to DNA-damaging agents (DDAs). We recently resolved the underlying mechanism, finding that it involves SLFN11-mediated cleavage of select tRNAs predominantly employed in the translation of the ATR and ATM Ser/Thr kinases, thereby establishing SLFN11 as a novel tRNA endonuclease. Even though SLFN11 is thus involved in two of the most prominent diseases of our time, cancer and HIV infection, its regulation remained thus far unresolved. Using MS and bioinformatics-based approaches combined with site-directed mutagenesis, we show here that SLFN11 is phosphorylated at three different sites, which requires dephosphorylation for SLFN11 to become fully functionally active. Furthermore, we identified protein phosphatase 1 catalytic subunit γ (PPP1CC) as the upstream enzyme whose activity is required for SLFN11 to cleave tRNAs and thereby act as a selective translational inhibitor. In summary, our work has identified both the mechanism of SLFN11 activation and PPP1CC as the enzyme responsible for its activation. Our findings open up future studies of the PPP1CC subunit(s) involved in SLFN11 activation and the putative kinase(s) that inactivates SLFN11.


Subject(s)
DNA Damage/genetics , Nuclear Proteins/genetics , Protein Biosynthesis , Protein Phosphatase 1/genetics , Ataxia Telangiectasia Mutated Proteins/genetics , Computational Biology , HIV Infections/genetics , HIV Infections/pathology , Humans , Interferons/genetics , Mutagenesis, Site-Directed , Mutation , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation/genetics , RNA, Transfer/genetics
4.
Nat Struct Mol Biol ; 25(11): 1047-1058, 2018 11.
Article in English | MEDLINE | ID: mdl-30374083

ABSTRACT

Transcriptome analysis reveals a strong positive correlation between human Schlafen family member 11 (SLFN11) expression and the sensitivity of tumor cells to DNA-damaging agents (DDAs). Here, we show that SLFN11 preferentially inhibits translation of the serine/threonine kinases ATR and ATM upon DDA treatment based on distinct codon usage without disrupting early DNA damage response signaling. Type II transfer RNAs (tRNAs), which include all serine and leucine tRNAs, are cleaved in a SLFN11-dependent manner in response to DDAs. Messenger RNAs encoded by genes with high TTA (Leu) codon usage, such as ATR, display utmost susceptibility to translational suppression by SLFN11. Specific attenuation of tRNA-Leu-TAA sufficed to ablate ATR protein expression and restore the DDA sensitivity of SLFN11-deficient cells. Our study uncovered a novel mechanism of codon-specific translational inhibition via SLFN11-dependent tRNA cleavage in the DNA damage response and supports the notion that SLFN11-deficient tumor cells can be resensitized to DDAs by targeting ATR or tRNA-Leu-TAA.


Subject(s)
Cell Death/physiology , DNA Damage , Nuclear Proteins/metabolism , RNA, Transfer/metabolism , Ataxia Telangiectasia Mutated Proteins/biosynthesis , Ataxia Telangiectasia Mutated Proteins/genetics , Camptothecin/pharmacology , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Codon/genetics , HEK293 Cells , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Protein Biosynthesis/drug effects , RNA, Small Interfering/genetics , RNA, Transfer/classification , RNA, Transfer/genetics , RNA, Transfer, Leu/genetics , RNA, Transfer, Leu/metabolism , Topoisomerase I Inhibitors/pharmacology
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