Engineering myelinating human brain organoids for personalized therapy of multiple sclerosis

Introduction: Multiple sclerosis (MS) is an autoimmune demyelinating disease mediated by the complex interplay between innate and adaptive immune systems in the brain microenvironment. In MS, autoreactive T cells and activated macrophages are often found in the active demyelinating edges of the patient’s white matter. However, direct phenotyping of their counterparts in patients’ peripheral blood has remained largely uninformative as the immune cell phenotype profile overlaps significantly with healthy donors due to low autoreactive T cell frequencies in circulation and lack of brain microenvironment stimulations to properly observe their phenotypes related to MS. Here, we engineer myelinating human brain organoids for modeling the demyelination and neuroinflammation in MS and testing the treatment response for personalized therapy of multiple sclerosis.

Materials and

Methods: Culture of human cortical organoids from stem cells. Human cortical organoids were established as our well-established protocol. Human embryonic stem cells (WA09) were obtained from WiCell under a material transfer agreement approved by both WiCell Institute and Indiana University.

Engineering myelinating organoids using nanofibre devices. The 3D printed devices integrated with electrospun nanofibers were coated with fibronectin for >4 hours. The mature (>1.5 months old) human cortical organoids were cultured onto nanofibre devices and cocultured with OPCs for forming myelinating neurites from the organoids.

Characterization of demyelination and neuroinflammation on-chip: Immune cells from MS patients were labeled with different fluorescence dyes, and cultured with engineered cortical organoids. The demyelination and neuroinflammation induced by MS patient immune cells were imaged using an inverted Olympus spinning disk microscope and analyzed.

Statistical analysis. All data were extracted and analyzed using Prism 7 (GraphPad Software). P-values between 2 samples were analyzed by student’s t-tests. P-values among 3 or more samples were analyzed by one-way ANOVA followed by Tukey's honestly significant difference (HSD) post hoc test. P-values were denoted as following: * < 0.05; ** < 0.01; *** < 0.005; **** < 0.001.

Results, Conclusions, and Discussions: by introducing T cells and macrophages isolated from MS patients’ blood into our on-chip myelinating human brain organoids, we could observe T cell-induced activation of macrophages, and macrophages’ phagocytosis of myelin in real-time, as well as evaluate their responses to various drug treatments. By incorporating time-lapse confocal imaging and multi-color flow cytometry analysis, we simultaneously interrogate the phenotypes of T cells and macrophages, their cytokine secretion profiles, and on-chip demyelination processes using as few as 10,000 PBMC cells per sample. Additionally, our functional phenotyping assays could distinguish MS patients from healthy donors’ immunocytes as well as evaluate their responses to various standard-of-care MS treatments through imaging-based parameters such as on-chip demyelination and T-monocyte co-localization. Moreover, we identified an expansion of memory CD4 T (mCD4 T) cells, Th1 CD4 T cells, and differentiation and polarization of monocytes towards the M2 macrophage phenotypes using flow-cytometry-based parameters. Our findings indicate that the peripheral immune cells including T cells and monocytes of MS patients are functionally distinct from those of healthy donors. Characterization of them using our functional phenotyping assays could reveal underlying mechanisms of MS disease development and potentially serve as a tool to discover novel therapies as well as guide treatment selections for personalized therapy.

Acknowledgements (Optional): F.G. acknowledges Indiana University departmental start-up funds, and the National Institute of Health Awards (DP2AI160242, and U01DA056242). The authors also thank the Indiana University Imaging Center (NIH1S10OD024988-01) for using their instruments.