Modeling PTEN overexpression-induced microcephaly in human brain organoids

The phosphatase and tensin homolog (PTEN) protein, encoded by the PTEN gene on chromosome 10, is a negative regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway. Loss of PTEN has been linked to an array of human diseases, including neurodevelopmental disorders such as macrocephaly and autism. However, it remains unknown whether increased dosage of PTEN can lead to human disease. A recent human genetics study identifies chromosome 10 microduplication encompassing PTEN in patients with microcephaly. Here we generated a human brain organoid model of increased PTEN dosage. We showed that mild PTEN overexpression led to reduced neural precursor proliferation, premature neuronal differentiation, and the formation of significantly smaller brain organoids. PTEN overexpression resulted in decreased AKT activation, and treatment of wild-type organoids with an AKT inhibitor recapitulated the reduced brain organoid growth phenotypes. Together, our findings provide functional evidence that PTEN is a dosage-sensitive gene that regulates human neurodevelopment, and that increased PTEN dosage in brain organoids results in microcephaly-like phenotypes. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00841-3.


Neural precursor culture
Differentiation of human embryonic stem cells to neural precursors in 2D adherent culture was performed as previously described (2,3). Briefly, hPSCs were passaged onto Matrigel-coated dishes using PBS without Ca 2+ /Mg 2+ , filtered through a 40um cell strainer (Falcon, 352340) to remove MEFs, and cultured directly in NGD 0.5X plus 2.5uM dorsomorphin (Stemgent, 04-0024), 10ng/mL bFGF (Thermo, PHG0263) and human insulin (additional 20ug/mL, Sigma, 9278) until super-confluent. bFGF and additional insulin were removed after a week, and NGD 0.5X plus dorsomorphin was replaced every day for 10 days. Cells were subsequently passaged with Accutase (Thermo, A1110501) when rosette lawns were observed throughout the culture. ROCK inhibitor Y27632 (Stemgent, 04-0012, 10uM) was added to the medium during the first 3 passages. After the first passage, neural precursors were expanded and maintained in NGD 0.5X plus 10ng/ml bFGF and 20ug/mL human insulin. For AKT inhibitor experiments, passage 9 to for 7 days.
hPSCs were plated onto Matrigel-coated dishes and fed mTeSR medium (Stem Cell Technologies, 85875) plus 10uM ROCK inhibitor Y27632 (Stemgent, 04-0012), and lentiviral particles. hPSCs were subsequently passaged as single cells at clonal density to MEFs, and single clones were picked and expanded. Expression of lentiviral-transgene was evaluated using GFP fluorescence, quantitative RT-PCR and immuno-blotting.

Histology and imaging
Cells and tissues were fixed with 4% (w/v) paraformaldehyde (Sigma, 158127) in PBS.

RNA extraction, reverse transcription and quantitative PCR
Cells and organoids were homogenized and total RNA extracted using the RNeasy kit (Qiagen, 74106) following manufacturer's instructions. Total RNA concentrations were measured using NanoDrop ND-1000 or DeNovix DS-11+ spectrophotometer. RNA was reverse transcribed into cDNA using Superscript IV reverse transcriptase (Thermo, 18090200) with random hexamer primers, or a mix of oligo d(T)20 primers and random hexamer primers, according to manufacturer's instructions. Transcript representation was determined by quantitative PCR using PowerUp SYBR Green PCR mix (Applied Biosystems, A25778), with primer pairs against PTEN, SOX2, TBR2, DCX, CTIP2 and GAPDH. Cellular RNA raw Ct values were normalized to GAPDH. Detailed primer information is described in Table S2. For the purpose of quantitative analysis, transcripts not detected were assigned a Ct value of 40.

Protein purification and immuno-blotting
Total protein was extracted from cells and tissues using RIPA lysis buffer containing  Table S1. Membranes blotted for phospho-proteins were stripped and re-probed with antibodies against total proteins. Membranes blotted for PTEN were stripped and re-probed for Actin. Values for phospho-proteins were normalized to total proteins, and PTEN was normalized to Actin.

Statistics
All data values were presented as mean +/-SEM. Student's t tests were applied to data with two groups. ANOVA analyses were used for comparisons of data with greater than two groups. Post hoc group comparisons were performed with Turkey test. A value of p<0.05 was considered significant.  Results are mean +/-SEM. *p<0.05, **p<0.01.