Cell Cycle Inhibitor p21 Expression Decrease In PPMS Cerebral Organoids Leads To Exhaustion Of The Progenitor Pool Independently Of Apoptosis Pathway

Multiple sclerosis (MS) is an auto-immune neurological disorder characterized by inflammation, demyelination, and neural degeneration. The origin and evolution of MS are still poorly understood because of lack of animal models and relative inaccessibility to human brain tissue. Cerebral organoids represent an interesting tool to study neurological disorders as they recapitulate early human neurodevelopment, including the generation, proliferation and differentiation of neural progenitors into glial cell and neurons.

The purpose of this work was to use patient with MS induced pluripotent stem cells derived cerebral organoids to study the genetic components of MS. This model was used to analyze the human neural stem cell population in control and MS organoids after 42 days in vitro.

The cell cycle inhibitor p21 is essential for the maintenance of stem cell quiescence and self-renewal. Immunostainings showed a high expression of p21 in the ventricular zone of control organoids. This expression was highly reduced in PPMS organoids compared to control and other subtypes of MS. Further analysis of the stem cells revealed that this decrease of p21 was associated with a significant reduction of the expression of the stem cell marker SOX2 and of the proliferation marker Ki67 in PPMS organoids compared to control, indicating a smaller stem cell pool and a reduced proliferation rate respectively.

To explore if p21 expression in organoids is involved in the DNA damage and apoptosis pathway, immunostainings for γH2AX, p53, and CC3 were performed.  Low to no γH2AX-positive cells were found in control and PPMS organoids, indicating low DNA damage. No difference was observed between the two conditions. p53 was detected in the ventricular zone/subventricular zone in both control and PPMS organoids, but there was no difference in p53 expression between the two conditions. A few CC3+ cells were detected in organoids, particularly localized in the upper cortical layers, with low to no positive cells in the stem cell population. No difference in CC3 expression was detected in PPMS compared to control organoids, indicating that there were no or low involvement of the apoptosis pathway in MS samples.

Our data indicates that the reduced p21 expression in PPMS is independent of the p53/apoptosis pathway and is related to cell cycle only, leading to an exhaustion of the progenitor pool. Patient derived cerebral organoids may be a viable human model to study MS pathogenesis.