Microglia-sufficient brain organoids
A technology of microglia and organoids, applied in the field of cell culture, can solve problems such as failure to fully reproduce the human fetal brain
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Embodiment 1
[0135] Example 1: Primitive-like macrophages (iMac) colonize brain organoids and differentiate into functionally active microglia-like cells (iMicro)
[0136] use Figure 1A Protocol shown to generate brain organoids and primitive-like macrophages (iMacs) from the same human iPSCs. Microglia have been observed in the human fetal brain as early as 4.5 weeks after conception. The iMacs were co-cultured with relatively young brain organoids (day 26) mimicking fetal brains in the first trimester. Co-culture was performed using organoid growth medium supplemented with 100 ng / mL CSF-1, with half of the medium replaced every three days. Additionally, the ultra-low-attachment 24-well plate used for co-cultivation minimizes adsorption of the iMac to the culture plate, thereby maximizing its physical interaction with the organoid. Additionally, one organoid is grown per well, which ensures sufficient space and nutrients for its survival and growth.
[0137] After 15 days of co-cultu...
Embodiment 2
[0140] Example 2: Addition of iMac reduces organoid size and number of neural progenitor cells (NPCs)
[0141] To investigate the effect of iMac on organoid development, organoid growth throughout the co-culture was examined. Organoids showed continuous growth in the absence of iMacs. However, when co-cultured with iMacs, organoid growth was significantly reduced and they became more spherical in shape ( Figure 2A , Figure 8 ). To understand whether there were any changes in the composition of the cells in the organoids after co-cultivation with iMacs, the organoids were enzymatically digested and single cell suspensions were stained with antibodies to identify macrophages, NPCs by flow cytometry , neuronal and glial progenitor cells. Importantly, when organoids were co-cultured with iMacs, the number and proportion of NPCs in the organoids decreased ( Figure 2B ). This is further supported by the observation that iMac-rich regions in organoids often lack NPCs.
Embodiment 3
[0142] Example 3: Addition of iMac promotes axonal development in organoids
[0143] To understand how iMicro affects brain organoid growth and NPC numbers, a single-cell RNA-seq experiment was set up that allows determination of all transcriptomic changes in organoid cells in the presence or absence of macrophages. The 10X Genomics Chromium System for rapid droplet-based encapsulation of single cells using an emulsion method with gel beads was used for organoids obtained from three co-cultures (co-culture), in the absence of iMac Single-cell suspensions of three organoids (organoids only) cultured in the absence of organoids ( Figure 3A ).
[0144] Cluster analysis was then performed on the transcriptomics, which allowed the identification of four separate clusters of cells ( Figure 3B ). By comparing with neuronal markers (MYT1L, DCX, NRXN1, MAP2, TUBB), NPC markers (VIM, PAX6, SOX2, HES1), mesenchymal markers (COL1A2, COL5A1, DCN, LUM) and macrophage The expression of...
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