{"id":388631,"date":"2023-12-18T19:00:00","date_gmt":"2023-12-19T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/efficient-generation-of-a-self-organizing-neuromuscular-junction-model-from-human-pluripotent-stem-cells-nature-communications\/"},"modified":"2023-12-19T10:33:23","modified_gmt":"2023-12-19T15:33:23","slug":"efficient-generation-of-a-self-organizing-neuromuscular-junction-model-from-human-pluripotent-stem-cells-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/efficient-generation-of-a-self-organizing-neuromuscular-junction-model-from-human-pluripotent-stem-cells-nature-communications\/","title":{"rendered":"Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
We used five different hPSC lines (see methods section) to explore their potential to generate a self-organizing NMJ (soNMJ) model through an NMP state. We have previously used hPSC-derived NMP cells to generate NMOs in 3D under minimal culture media. However, the direct transfer of the protocol from 3D to 2D was insufficient to generate a functional NMJ model in adherent culture conditions. Analysis of the adherent cultures after applying the NMO conditions at days 20 and 50 revealed the presence of neurons expressing TUBB3 and a few muscle progenitors expressing DESMIN at day 20, which gave rise to only a few differentiated skeletal myofibers at day 50 (Supplementary Fig. 1<\/a>).<\/p>\n We reasoned that transposition of the 3D early NMO culture conditions in 2D may change the autocrine signal signature. BMP inhibition promotes the specification into paraxial mesoderm and skeletal muscles2<\/a>,18<\/a><\/sup> whereas TGF\u03b2 inhibition further enhances the efficiency of hPSCs differentiation into somites19<\/a><\/sup>. Thus, we employed both BMP and TGF\u03b2 inhibition using the dual SMAD inhibitor cocktail (2SMADi)19<\/a>,20<\/a><\/sup>. We first assessed the effect of 2SMADi at the NMP induction stage (day 0 \u2013 day 3) (Supplementary Fig. 2a<\/a>). We instructed the generation of NMPs using our previously established protocol21<\/a>,22<\/a><\/sup>. Initial exposure of hPSCs to WNT agonist (CHIR) and basic FGF (bFGF) signals for three days resulted in the efficient generation of NMP cells that co-expressed the nascent mesodermal marker BRACHYURY (TBXT) and the neural progenitor marker SOX2. Paraxial mesoderm TBX6+<\/sup> cells were also evident mainly in the periphery of the NMP colonies (Supplementary Fig. 2b, c<\/a>). We observed that simultaneous, early treatment of hPSCs with CHIR\/bFGF and 2SMADi resulted in the generation of cells that expressed high levels of SOX2 and low levels of TBXT in the absence of TBX6 (Supplementary Fig. 2b, c, d<\/a>). The low levels of TBXT agreed with the known role of SB431542 to mediate TBXT suppression23<\/a><\/sup>. Thus, the addition of 2SMADi changed the balance of TBXT and SOX2 towards a more SOX2-high fate (Supplementary Fig. 2e<\/a>).<\/p>\n To determine the effect of 2SMADi in rostro-caudal identity, we analyzed the expression of the posterior markers CDX2<\/i>, NKX1.2<\/i>, and HOXB1<\/i> and the anterior marker OTX2<\/i> in hPSCs treated during day 0 \u2013 day 3 with (i) 2SMADi, (ii) WNT\/bFGF\/2SMADi and (iii) WNT\/bFGF (Supplementary Fig. 2a<\/a>). Cells treated with 2SMADi alone acquired an anterior neural identity expressing the neural progenitor markers SOX2<\/i>, SOX1<\/i> and OTX2<\/i>, indicative of an anterior neural identity and in agreement with previously published studies20<\/a><\/sup> (Supplementary Fig. 2d<\/a>). The WNT\/bFGF condition alone resulted in the generation of NMPs that express the posterior markers CDX2<\/i> and NKX1.2<\/i> as previously described21<\/a>,22<\/a>,24<\/a><\/sup>. The simultaneous addition of 2SMADi and WNT\/bFGF resulted in the generation of posterior cells expressing CDX2<\/i> and HOXB1<\/i>. A striking difference between the WNT\/bFGF\/2SMADi and WNT\/bFGF treatments was the upregulation of NKX1.2<\/i> and SOX2<\/i> in the former (Supplementary Fig. 2d<\/a>); this upregulation was associated with a pre-neural tube identity25<\/a><\/sup>. We next examined the potential of the WNT\/bFGF\/2SMADi treated cells to differentiate into neural and mesodermal progenitors. To enhance the generation of both cell types, we continued the treatment from day 3 to day 6 with WNT\/bFGF\/2SMADi in the presence of retinoic acid (RA) and the SHH smoothened agonist (SAG) to induce a ventral identity (Fig. 1a<\/a>). Analysis of cells on day 6 revealed the exclusive differentiation into neural progenitor cells expressing SOX2<\/i>, SOX1<\/i>, PAX6<\/i>, and NKX1.2<\/i> (Fig. 1b, c<\/a>). Genes associated with a presomitic (PSM) identity, such as MEOX1<\/i>, FOXC1<\/i>, FOXC2<\/i>, and MYF5<\/i> were not expressed (Fig. 1b<\/a>). Thus, exposure of cells to the 2SMADi during the NMP induction stage precludes mesoderm formation. This was in agreement with the finding that 2SMADi promotes the preferential differentiation of hPSCs towards the neuroectodermal lineage20<\/a>,26<\/a><\/sup>.<\/p>\n