Direct Generation of Human Cortical Organoids from Primary Cells - PubMed (original) (raw)

Direct Generation of Human Cortical Organoids from Primary Cells

Monique Schukking et al. Stem Cells Dev. 2018.

Abstract

The study of variations in human neurodevelopment and cognition is limited by the availability of experimental models. While animal models only partially recapitulate the human brain development, genetics, and heterogeneity, human-induced pluripotent stem cells can provide an attractive experimental alternative. However, cellular reprogramming and further differentiation techniques are costly and time-consuming and therefore, studies using this approach are often limited to a small number of samples. In this study, we describe a rapid and cost-effective method to reprogram somatic cells and the direct generation of cortical organoids in a 96-well format. Our data are a proof-of-principle that a large cohort of samples can be generated for experimental assessment of the human neural development.

Keywords: high-throughput; induced pluripotent stem cell; neural development; organoids.

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Conflict of interest statement

Dr. Muotri is a cofounder and has an equity interest in TISMOO, a company dedicated to genetic analysis focusing on therapeutic applications customized for autism spectrum disorder and other neurological disorders with genetic origins. The terms of this arrangement have been reviewed and approved by the University of California San Diego following its conflict of interest policies. The other authors declare no conflicts of interest.

Figures

<b>FIG. 1.</b>

**FIG. 1.

MEFs facilitate the reprogramming process. (A) Left is human skin fibroblasts on top of MEFs (F-on-M) in a 10 cm disk and right is MEFs on top of human skin fibroblasts (M-on-F) in a six-well plate. Images made using EVOS microscope at 4 × . Scale bar, 1,000 μm. (Day 2) Pictures were taken 2 days after transduction. F-on-M pictures just taken after reseeding fibroblasts on top of MEFs. M-on-F pictures just taken after seeding MEFs on top of fibroblasts. (Day 15) Pictures were taken 15 days after transduction. iPSC colonies started to show. (Day 17) Pictures were taken 17 days after transduction. (B) Relative number of primary colonies formed per 25,000 fibroblasts counted on reprogramming day 17. Results are presented as mean ± SEM, n = 2. The SEM for F-on-M is zero. EVOS, EVOS Cell Imaging Systems (ThermoFisher); iPSC, induced pluripotent stem cell; MEFs, mouse embryonic fibroblasts; SEM, standard error of the mean.

<b>FIG. 2.</b>

**FIG. 2.

Cortical organoids formed from nonpassaged iPSCs. The cortical organoid protocol was started in a 24-well plate using nonpassaged iPSCs at day 24 and 32 of the reprogramming protocol. To control for extensive cellular turnover, iPSCs of passage 45 were included. (A) Images of the organoids formed at protocol days 3, 17, and 24. Images made using EVOS at magnification 4 × . Scale bar, 1,000 μm. (B) Graphical diagram of the protocol used to generate cortical organoids. (C) Cortical organoids stained for MAP2, a marker for neural differentiation, NESTIN, marker for NPCs and nuclei marker DAPI. Images made using EVOS at magnification 20 × . Scale bar, 50 μm. NPCs, neural progenitor cells.

<b>FIG. 3.</b>

**FIG. 3.

Skin fibroblasts reprogram in 96-well plate using 1.0 μL Sendai virus per well. Human primary skin fibroblasts at passage 6 were seeded at four different concentrations. On the following day, a serial dilution of 1.0, 2.8, 5.7, and 11.3 μL of Sendai virus was added. RT-qPCR were shown of gene expression levels of the pluripotency markers KLF4, LIN28, MYC, NANOG, OCT4, and SOX2. Expression levels were depicted as relative to HPRT. Significance was represented as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (A) Visualization of the amount of iPSC colonies per well at reprogramming day 19 following a color scaling system: white, no colonies; black, 1 colony; gray, 1–9 colonies; dots, 10–19 colonies; stripes, 20 or more colonies. (B) Relative mRNA expression of pluripotency markers in the unpassaged iPSCs formed in the wells D7 to D11 of the 96-well plate shown in Fig. 3A. (C) Relative mRNA expression of pluripotency markers in preestablished iPSCs. (D) Gene expression levels of unpassaged iPSCs compared with gene expression levels of preestablished iPSCs depicted as fold change. The expression levels of each gene were quantified, normalized to B2M (reference gene), and the results are presented as mean ± SEM. Significance was calculated with unpaired Student's _t_-test with Welch's correction if variances were not equal. HPRT, hypoxanthine guanine phosphoribosyl transferase; RT-qPCR, quantitative reverse transcription–polymerase chain reaction.

<b>FIG. 4.</b>

**FIG. 4.

Direct generation of cortical organoids from reprogrammed fibroblasts in a 96-well plate. Direct differentiation protocol in a 96-well plate using primary iPSC colonies that were formed in a 96-well plate. (A) One well of reprogrammed skin fibroblasts from the experiment of Fig. 3 was seeded to two wells of an ultralow attachment 96-well plate. The iPSCs were at reprogramming day 23. The cortical organoids protocol was followed as described in the Materials and Methods section. (B) Top: Unsuccessful organoid forming of the primary iPSC colonies. Bottom: Successful organoid forming of primary colonies showed in organoid formation starting between days 2 and 3. Scale bar, 1,000 μm. (C) Relative amount of primary iPSC colonies in one well that successfully differentiated into two organoids. Data are shown as mean ± SEM, n = 2. (D) Developing cortical organoids followed until day 8. Images were made using the EVOS microscope at magnification 4 × . Scale bar, 1,000 μm.

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