Authors :
Presenting Author: M. Carmen Varela, BSc Ba – University of Michigan
Miranda Walker, BSc – University of Michigan
Jack Parent, MD – University of Michigan
Jeyoon Bok, BS – University of Michigan. Ann Arbor
Emmanuel Crespo, PhD – University of Michigan. Ann Arbor
Tyler Thenstedt, BS – University of Michigan. Ann Arbor
Leah Goldstein, Undergraduate – University of Michigan. Ann Arbor
Andrew Tidball, PhD – University of Michigan. Ann Arbor
Yukun Yuan, MD, PhD – University of Michigan
Lori Isom, PhD – University of Michigan.
Jianping Fu, PhD – University of Michigan. Ann Arbor
Michael Uhler, PhD – University of Michigan. Ann Arbor
Rationale:
Developmental and epileptic encephalopathies (DEEs) and other neurological disorders are increasingly linked to dysfunction of medial ganglionic eminence (MGE)-derived GABAergic cortical interneurons (cINs), particularly parvalbumin (PV)-expressing cIN subtypes. The fast-spiking properties of PV cells are crucial for regulating network dynamics and cortical excitability. Rodent DEE models often do not fully recapitulate human phenotypes, and access to human brain tissue during development is extremely limited. Current human in vitro models fail produce mature PV cINs, limiting their use for disease modeling and therapeutic screening. This work addresses a long-standing gap in the brain organoid field by establishing an MGE-specified human organoid model that efficiently generates MGE cell types including PV cINs.Methods:
We hypothesized that precise modulation of developmental signaling pathways could guide human brain organoids toward an MGE-like identity. We varied the timing and concentration of WNT inhibition and Sonic Hedgehog (SHH) activation in a single-rosette brain organoid system to promote ventral forebrain patterning and achieve an MGE identity. To ensure reproducibility, MGE organoids (MGEOs) were generated from three human pluripotent stem cell lines, both male and female sources, including embryonic stem cells, and reprogrammed fibroblasts and peripheral blood cells. We characterized IN development and specification in our MGEOs at various timepoints ranging from 18-250 days in vitro using scRNAseq, RT-qPCR, immunostaining, and functional assays including multi-electrode array (MEA) and whole-cell patch-clamp recordings. MGEOs were also fused with cortical organoids to assess interneuron migration and network integration.Results:
Our model produced organoids with strong MGE identity, marked by expression of NKX2.1, LHX6, LHX8, and SOX6. MGEOs generated key MGE-derived cell types including astrocytes, oligodendrocytes, and cIN subtypes including somatostatin, calretinin, and notably high levels of PV. Major PV subtypes including basket and axoaxonic cell were represented in our MGEOs. Upon fusion with cortical-patterned organoids, MGEO cINs rapidly migrated and integrated into the cortical tissue, mimicking in vivo tangential migration. MEA recordings of long-term assembloids revealed complex synchronized network-level activity and cells with fast-spiking properties. This activity was sensitive to bicuculline, confirming functional integration of MGEO INs into the cortical organoid network.Conclusions:
We present a novel MGE-specified organoid model that robustly generates mature, fast-spiking neurons and PV interneurons, including basket and axoaxonic subtypes. This model overcomes previous barriers to ventral forebrain specification and provides a powerful tool to study interneuron development, migration, circuit integration, and neuron-glial interactions. Its capacity to model human MGE development and function offers valuable opportunities for research into epilepsy, neurodevelopmental disorders, and cIN-related neuropsychiatric conditions.
Funding:
This work was supported by NIH (NINDS) U54NS117170 (JMP), and the NSF Graduate Research Fellowship Program (MCV)