Inflammatory Response and Exosome Biogenesis of Choroid Plexus Organoids Derived from Human Pluripotent Stem Cells

Abstract

The choroid plexus (ChP) is a complex structure in the human brain that is responsible for the secretion of cerebrospinal fluid (CSF) and forming the blood-CSF barrier (B-CSF-B). Human-induced pluripotent stem cells (hiPSCs) have shown promising results in the formation of brain organoids in vitro; however, very few studies to date have generated ChP organoids. In particular, no study has assessed the inflammatory response and the extracellular vesicle (EV) biogenesis of hiPSC-derived ChP organoids. In this study, the impacts of Wnt signaling on the inflammatory response and EV biogenesis of ChP organoids derived from hiPSCs was investigated. During days 10-15, bone morphogenetic protein 4 was added along with (+/-) CHIR99021 (CHIR, a small molecule GSK-3β inhibitor that acts as a Wnt agonist). At day 30, the ChP organoids were characterized by immunocytochemistry and flow cytometry for TTR (~72%) and CLIC6 (~20%) expression. Compared to the -CHIR group, the +CHIR group showed an upregulation of 6 out of 10 tested ChP genes, including CLIC6 (2-fold), PLEC (4-fold), PLTP (2-4-fold), DCN (~7-fold), DLK1 (2-4-fold), and AQP1 (1.4-fold), and a downregulation of TTR (0.1-fold), IGFBP7 (0.8-fold), MSX1 (0.4-fold), and LUM (0.2-0.4-fold). When exposed to amyloid beta 42 oligomers, the +CHIR group had a more sensitive response as evidenced by the upregulation of inflammation-related genes such as TNFα, IL-6, and MMP2/9 when compared to the -CHIR group. Developmentally, the EV biogenesis markers of ChP organoids showed an increase over time from day 19 to day 38. This study is significant in that it provides a model of the human B-CSF-B and ChP tissue for the purpose of drug screening and designing drug delivery systems to treat neurological disorders such as Alzheimer's disease and ischemic stroke.

Keywords: Wnt signaling; choroid plexus organoids; extracellular vesicles; human pluripotent stem cells; inflammatory response.

Figure 1

ChP organoid differentiation from hiPSCs. (A) Illustration of ChP differentiation; (B) the ChP organoid morphology for CHIR+/− conditions during the differentiation. Scale bar: 200 µm. BMP: bone morphogenetic protein; EBs: embryoid bodies. EV: extracellular vesicles.

Figure 2

Expression of ChP markers for CHIR+/− conditions. (A) Immunostaining of ChP markers at day 32. Scale bar: 50 µm. (B) Flow cytometry histograms of ChP markers for CHIR+/− conditions. The assays were performed at day 30. Black line: negative control. Red line: marker of interest. TTR (transthyretin) and CLIC6 (chloride intracellular channel 6) are ChP markers. CHIR(+/−) indicates treatment with CHIR99021.

Figure 3

mRNA expression of ChP markers at days 18 and 30 for CHIR+/− conditions. mRNA expression was determined by RT-qPCR. (A) Day 18; (B) day 30 of differentiation. N = 3. * indicates p < 0.05. CHIR(+/−) indicates treatment with CHIR99021.

Figure 4

Inflammatory response to Aβ42 oligomer stimulation for ChP organoids of CHIR+/− conditions. mRNA expression determined by RT-qPCR for (A) proinflammatory markers (day 33); (B) anti-inflammatory markers (day 33); these markers were relative to the CHIR+ condition. (C) MMP2, MMP3, and MMP9 expression affected by Aβ42 oligomers (day 33); (D) MMP2, MMP3, and MMP9 expression affected by CHIR at days 19 and 38. N = 3. * indicates p < 0.05. The MMP2, MMP3, and MMP9 expression was relative to the CHIR−D19 condition. CHIR(+/−) indicates treatment with CHIR99021, D19 and D38 indicates days 19 and 38, AB(+/−) indicates treatment with amyloid β 42 oligomers. The CHIR+ group is more responsive than the CHIR− group.

Figure 5

Extracellular vesicle biogenesis of ChP organoids with CHIR+/− conditions. (A) mRNA expression determined by RT-qPCR for (i) ESCRT-dependent EV biogenesis markers; (ii) ESCRT-independent EV biogenesis markers; N = 3. * indicates p < 0.05 for the compared conditions, and # indicates p < 0.05 compared to the CHIR− condition at the same time point. CHIR(+/−) indicates treatment with CHIR99021 (3 µM), D19 and D38 indicates days 19 and 38 during differentiation. (B) Nanoparticle tracking analysis (NTA) for the isolated ChP organoid-EVs; (i) particle size distribution and zeta potential by ZetaView; (ii) particle size distribution by NanoSight. (C) Transmission electron microscopy images showing exosome morphology; scale bar: 100 nm.