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Establishment of Human Brain Organoid Models of Tuberous Sclerosis


Tuberous Sclerosis (TSC) is a multisystem developmental disorder, which is associated with early-onset epilepsy, varying degrees of intellectual disability and high risk for psychiatric conditions including autism. It is defined by the presence of “cortical tubers”, which are focal cortical malformations composed of dysplastic neurons and astrocytes that can become seizure foci. TSC is caused by mutations in either TSC1 or TSC2, which encode for proteins forming a complex that is a negative regulator of mTORC1, a key cellular signaling node controlling cell growth and metabolism. Animal models have greatly increased our understanding of TSC; however they fail to recapitulate some of the key neuropathological features, namely the cortical tubers. This may be due to the contracted timeframe over which rodent cortical development occurs (days) compared to human cortical development (months) as well as the presence of human-specific progenitor cells. My research goal was to overcome these limitations by creating a human neuronal model of TSC that develops on a human timescale and recapitulates key features of early human cortical development.

I used CRISPR/Cas9 to create an isogenic panel of human embryonic stem cell lines harboring heterozygous or homozygous loss-of-function mutations in TSC1 or TSC2. I differentiated these cells into 3D cortical spheroids over five months in culture to model mid-gestational human fetal development. I found that while early forebrain development was largely normal, TSC1-/- or TSC2-/- cells in five-month-old spheroids were highly abnormal. Specifically, loss of function of TSC1/2 led to the formation of highly enlarged, dysplastic cells that strongly resembled the cortical tuber cells seen in patients. Furthermore, I revealed a significant differentiation bias of TSC1/2 mutant cells towards glial cell fates and away from neuronal fates, again reflecting what is seen in patient cortical tubers.

Cortical tubers are thought to arise due to a “second-hit” somatic mutation during progenitor cell proliferation that results in loss of heterozygosity. I further refined our human TSC model to mimic a somatic second-hit event, by generating a TSC2c/- stem cell line, in which all cells were heterozygous for TSC2 but would undergo biallelic inactivation in the presence of Cre recombinase. I also engineered a fluorescent Cre- reporter into these cells to enable lineage tracing of TSC2-/- cells over time. I found that cells with a second-hit mutation in TSC2 were large, dysplastic and primarily expressed glial markers, while the adjacent TSC2+/- cells showed normal morphology and development, mimicking a cortical tuber. Chronic treatment with rapamycin, a potent mTOR inhibitor, starting early in development rescued tuber cell morphology and caused marked shifts of differentiation towards a neuronal fate in both TSC2+/- and TSC2-/- cells, demonstrating mTORC1’s bidirectional control over human neuronal differentiation.

To elucidate the developmental programs of normal human corticogenesis and how these are altered by loss of TSC2, I have performed an in-depth single cell RNA- sequencing analysis across development in TSC2c/- spheroids. My analysis has confirmed the differentiation bias of TSC2-/- cells away from neuronal cell fates and revealed that this becomes pronounced primarily after two months of differentiation, before which TSC2+/- and TSC2-/- cells share similar identities. Furthermore, I discovered novel genetic markers of TSC2-/- cells: CLU, PTGDS, APOE, B2M that may contribute to their aberrant differentiation. Together, my thesis research has established novel, validated human brain spheroid models for TSC, which have generated new insights into the early molecular, cellular, and developmental mechanisms contributing to both normal and abnormal human brain development.

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