Biology subjects

Connor, B.

Publications and source records attributed to Connor, B..

4 recordsLinked to original sources

bioRxiv2024

Reprogrammed Human Lateral Ganglionic Eminence Precursors Generate Striatal Neurons and Restore Motor Function in a Rat Model of Huntington's Disease.

BackgroundHuntingtons disease (HD) is a genetic neurological disorder predominantly characterised by the progressive loss of GABAergic medium spiny neurons in the striatum resulting in motor dysfunction. One potential strategy for the treatment of HD is the development of cell replacement therapies to restore neuronal circuitry and function by the replacement of lost neurons. We propose the generation of lineage-specific human lateral ganglionic eminence precursors (hiLGEP) using direct reprogramming technology provides a novel and clinically viable cell source for cell replacement therapy for HD. MethodshiLGEPs were derived by direct reprogramming of adult human dermal fibroblasts (aHDFs) using chemically modified mRNA (cmRNA) and a defined reprogramming medium. hiLGEPs were differentiated in vitro using an optimised striatal differentiation medium. Acquisition of a striatal precursor and neural cell fate was assessed through gene expression and immunocytochemical analysis of key markers. hiLGEP-derived striatal neuron functionality in vitro was demonstrated by calcium imaging using Cal-520. To investigate the ability for hiLGEP to survive, differentiate and functionally integrate in vivo, we transplanted hiLGEPs into the striatum of quinolinic acid (QA)-lesioned rats and performed behavioural assessment using the cylinder test over the course of 14 weeks. Survival and differentiation of hiLGEPs was assessed at 8 and 14-weeks post-transplant by immunohistochemical analysis. ResultsWe demonstrate the capability to generate hiLGEPs from aHDFs using cmRNA encoding the pro-neural genes SOX2 and PAX6, combined with a reprogramming medium containing Go6983, Y-27632, N-2 and Activin A. hiLGEPs generated functional DARPP32+ neurons following 14 days of culture in BrainPhys media supplemented with dorsomorphin and Activin A. We investigated the ability for hiLGEPs to survive transplantation, differentiate to medium spiny-like striatal neurons and improve motor function in the QA lesion rat model of HD. Fourteen weeks after transplantation, we observed STEM121+ neurons co-expressing MAP2, DARPP32, GAD65/67, or GABA. Rats transplanted with hiLGEPs also demonstrated reduction in motor function impairment as determined by spontaneous exploratory forelimb use when compared to saline transplanted animals. ConclusionThis study provides proof-of-concept and demonstrates for the first time that aHDFs can be directly reprogrammed to hiLGEPs which survive transplantation, undergo neuronal differentiation to generate medium spiny-like striatal neurons, and reduce functional impairment in the QA lesion rat model of HD. Significance statementThe present study reports for the first time that human lateral ganglionic eminence precursor (hiLGEP) cells directly reprogrammed from adult human fibroblasts using chemically modified mRNA can survive transplantation into the quinolinic acid-lesioned rat striatum and generate medium spiny striatal neurons. Most importantly, the authors show that transplantation of directly reprogrammed hiLGEPs restores motor function impairment by 14 weeks post-transplantation. This work provides proof of concept and demonstrates that directly reprogrammed hiLGEPs offer an effective and clinically viable cell source for cell replacement therapy to treat Huntingtons disease.

neuroscience↗

bioRxiv2024

Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes

BackgroundiPSC-derived cells are increasingly used to model complex diseases in vitro because they can be patient derived and can differentiate into any cell in the adult human body. Recent studies have demonstrated the generation of brain pericytes using a neural crest-based differentiation protocol. However, the inflammatory response of these iPSC-derived brain pericytes has not been investigated. We aimed to investigate the response of iPSC-derived brain pericytes to common inflammatory stimuli, thereby assessing the suitability of these cells to study inflammatory disease. MethodsBrain pericytes were differentiated from iPSCs for 42 days. The expression of brain pericyte markers was assessed by RT-qPCR and immunofluorescent staining at days 0, 15, 21, and 42 of differentiation to validate the brain pericyte-like phenotype. Nuclear localisation of NF{kappa}B and STAT1 was assessed by immunofluorescence following IL-1{beta}- and TNF-treatment in day 21 and day 42 iPSC-derived pericytes, and primary human pericytes. Cytometric bead array assessed the concentration of secreted inflammatory factors in the cell medium and phagocytosis was investigated using fluorescent carboxylated beads and flow cytometry. ResultsAt day 42 of differentiation, but not at day 21, cells expressed brain pericyte markers. Generally, iPSC-derived pericytes lacked consistent responses to inflammatory treatment compared to primary human pericytes. Day 21 and 42 iPSC-derived pericytes exhibited a NF{kappa}B response to IL-1{beta} treatment comparable to primary human pericytes. Day 21 iPSC-derived pericytes exhibited a STAT1 response with IL-1{beta} treatment which was absent in day 42 cells, but present in a subset of primary human pericytes. TNF treatment presented similar NF{kappa}B responses between day 21 and 42 iPSC-derived and primary human pericytes, but a STAT1 response was again present in a subset of primary human pericytes which was absent in both day 21 and day 42 iPSC-derived pericytes. Numerous differences were observed in the secretion of cytokines and chemokines following treatment of iPSC-derived and primary human pericytes with IL-1{beta} and TNF. iPSC-derived pericytes exhibited greater rates of phagocytosis than primary human pericytes. ConclusionsWith the increase in iPSC-derived cells in research, labs should undertake validation of lineage specificity when adapting an iPSC-derived differentiation protocol. In our hands, the inflammatory response of iPSC-derived pericytes was different to that of primary human pericytes, raising concern regarding the use of iPSC-derived pericytes to study neuroinflammatory disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/613375v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e3612eorg.highwire.dtl.DTLVardef@1014537org.highwire.dtl.DTLVardef@6729adorg.highwire.dtl.DTLVardef@e14825_HPS_FORMAT_FIGEXP M_FIG C_FIG Brain pericytes can be generated from iPSCs. The work presented here shows the generation of phenotypically distinct pericytes from the original protocol, demonstrating the significant variability present within some iPSC differentiation protocols. Furthermore, functional differences are demonstrated between iPSC-derived brain pericytes and primary brain pericytes, revealing limitations in the use of iPSC-derived brain pericytes to model brain pericyte biology. Key PointsO_ST_ABSWhat is already known about this topic?C_ST_ABSBrain pericyte-like cells can be generated from induced pluripotent stem cells, however their responses to inflammatory stimuli has not been assessed. What does this study add?iPSC-derived brain pericytes exhibit different inflammatory responses compared to primary brain pericytes, showing that some iPSC-derived cell models are not appropriate for modelling all aspects of a cells biology. Furthermore, the iPSC-derived pericytes generated here were markedly different to those generated from the original article. It is therefore important for each lab to optimise the generation of iPSC-derived cell in their own hands to account for potential inter-lab variability.

cell biology↗

bioRxiv2024

Screening activity of brain cancer-derived factors on primary human brain pericytes

Brain cancers offer poor prognoses to patients accompanied by symptoms that drastically impact the patient and their family. Brain tumours recruit local non-transformed cells to provide trophic support and immunosuppression within the tumour microenvironment, supporting tumour progression. Given the localization and supportive role of pericytes at the brain vasculature, we explored the potential for brain pericytes to contribute to the brain cancer microenvironment. To investigate this, primary brain pericytes were treated with factors commonly upregulated in brain cancers. Changes to brain pericyte cell signalling, inflammatory secretion, and phagocytosis were investigated. The TGF{beta} superfamily cytokines TGF{beta} and GDF-15 activated SMAD2/3 and inhibited C/EBP-{delta}, revealing a potential mechanism behind the pleiotropic action of TGF{beta} on brain pericytes. IL-17 induced secretion of IL-6 without activating NF{kappa}B, STAT1, SMAD2/3, or C/EBP-{delta} signalling pathways. IL-27 and IFN{gamma} induced STAT1 signalling and significantly reduced pericyte phagocytosis. The remaining brain cancer-derived factors did not induce a measured response, indicating that these factors may act on other cell types or require co-stimulation with other factors to produce significant effects. Together, these findings show potential mechanisms by which brain pericytes contribute to aspects of inflammation and starts to uncover the supportive role brain pericytes may play in brain cancers.

cancer biology↗

bioRxiv2022

Transcytosis-mediated anterograde transport of TrkA receptors is necessary for sympathetic neuron development and function

In neurons, many membrane proteins, synthesized in cell bodies, must be efficiently delivered to axons to influence neuronal connectivity, synaptic communication, and repair. Previously, we found that axonal targeting of TrkA neurotrophin receptors in sympathetic neurons occurs via an atypical transport mechanism called transcytosis, which relies on TrkA interactions with PTP1B, a protein tyrosine phosphatase. Here, we generated TrkAR685A mice, where TrkA receptor signaling is preserved, but its PTP1B-dependent transcytosis is disrupted, to show that this mode of axonal transport is essential for sympathetic neuron development and autonomic function. TrkAR685A mice have decreased axonal TrkA levels in vivo, developmental loss of sympathetic neurons, and reduced innervation of targets. Postnatal TrkAR685A mice exhibit reduced pupil size and eyelid ptosis, indicative of sympathetic dysfunction. These findings establish the necessity of transcytosis in supplying TrkA receptors to sympathetic axons and highlight the physiological relevance of this axon targeting mechanism in the nervous system.

neuroscience↗