An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

Autor:	Dorion MF; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Casas D; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Shlaifer I; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Yaqubi M; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Fleming P; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Karpilovsky N; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; McGill Parkinson Program and Neurodegenerative Disorders Research Group, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Chen CX; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Nicouleau M; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Piscopo VEC; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., MacDougall EJ; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; McGill Parkinson Program and Neurodegenerative Disorders Research Group, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Alluli A; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Goldsmith TM; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Schneider A; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Dorion S; Faculty of Arts and Sciences, Université de Montréal, Montreal, H3T 1NB, Canada., Aprahamian N; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., MacDonald A; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Thomas RA; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; McGill Parkinson Program and Neurodegenerative Disorders Research Group, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Dudley RWR; Department of Pediatric Surgery, Division of Neurosurgery, Montreal Children's Hospital, McGill University Health Centers, Montreal, H4A 3J1, Canada., Hall JA; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Fon EA; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; McGill Parkinson Program and Neurodegenerative Disorders Research Group, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Antel JP; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Stratton JA; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., Durcan TM; Early Drug Discovery Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada., La Piana R; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada. roberta.lapiana@mcgill.ca., Healy LM; Neuroimmunology Unit, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada. luke.healy@mcgill.ca.; Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, H3A 2B4, Canada. luke.healy@mcgill.ca.
Jazyk:	angličtina
Zdroj:	Molecular neurodegeneration [Mol Neurodegener] 2024 Apr 05; Vol. 19 (1), pp. 31. Date of Electronic Publication: 2024 Apr 05.
DOI:	10.1186/s13024-024-00723-x
Abstrakt:	Background: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. Methods: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. Results: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam 3 CSK 4 . Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1R WT/KO and CSF1R WT/E633K iMGL compared to their respective isogenic controls. Conclusions: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities. (© 2024. The Author(s).)
Databáze:	MEDLINE
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