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Although several in vitro studies have investigated how motor neurons and endothelial cells interact with skeletal myocytes independently, there is limited knowledge about the combined effect of neural and vascular cells on muscle maturation and development.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Here, we report a triculture system comprising human-induced pluripotent stem cell (iPSC)-derived skeletal myocytes, human iPSC-derived motor neurons, and primary human endothelial cells maintained under controlled media conditions. Briefly, iPSCs were differentiated to generate skeletal muscle progenitor cells (SMPCs). These SMPCs were seeded at a density of 5\u2009\u00d7\u200910\n                      <jats:sup>4<\/jats:sup>\n                      cells\/well in 12-well plates and allowed to differentiate for 7\u00a0days before adding iPSC-derived motor neurons at\u00a0a concentration of 0.5\u2009\u00d7\u200910\n                      <jats:sup>4<\/jats:sup>\n                      cells\/well. The neuromuscular coculture was maintained for another 7\u00a0days in coculture media before addition of primary human umbilical vein endothelial cells (HUVEC) also at 0.5\u2009\u00d7\u200910\n                      <jats:sup>4<\/jats:sup>\n                      cells\/well. The triculture was maintained for another 7\u00a0days in triculture media comprising equal portions of muscle differentiation media, coculture media, and vascular media. Extensive morphological, genetic, and molecular characterization was performed to understand the combined and individual effects of neural and vascular cells on skeletal muscle maturation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      We observed that motor neurons independently promoted myofiber fusion, upregulated neuromuscular junction genes, and maintained a molecular niche supportive of muscle maturation. Endothelial cells independently did not support myofiber fusion and downregulated expression of\n                      <jats:italic>LRP4<\/jats:italic>\n                      but did promote expression of type II specific myosin isoforms. However, neurovascular cells in combination exhibited additive increases in myofiber fusion and length, enhanced production of Agrin, along with upregulation of several key genes like\n                      <jats:italic>MUSK<\/jats:italic>\n                      ,\n                      <jats:italic>RAPSYN<\/jats:italic>\n                      ,\n                      <jats:italic>DOK-7<\/jats:italic>\n                      , and\n                      <jats:italic>SLC2A4<\/jats:italic>\n                      . Interestingly, more divergent effects were observed in expression of genes like\n                      <jats:italic>MYH8<\/jats:italic>\n                      ,\n                      <jats:italic>MYH1<\/jats:italic>\n                      ,\n                      <jats:italic>MYH2<\/jats:italic>\n                      ,\n                      <jats:italic>MYH4<\/jats:italic>\n                      , and\n                      <jats:italic>LRP4<\/jats:italic>\n                      and secretion of key molecular factors like amphiregulin and IGFBP-4.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Neurovascular cells when cultured in combination with skeletal myocytes promoted myocyte fusion with concomitant increase in expression of various neuromuscular genes. This triculture system may be used to gain a deeper understanding of the effects of the neurovascular niche on skeletal muscle biology and pathophysiology.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13395-024-00336-4","type":"journal-article","created":{"date-parts":[[2024,3,7]],"date-time":"2024-03-07T09:02:52Z","timestamp":1709802172000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Motor neurons and endothelial cells additively promote development and fusion of human iPSC-derived skeletal myocytes"],"prefix":"10.1186","volume":"14","author":[{"given":"Suradip","family":"Das","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melanie C.","family":"Hilman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feikun","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Foteini","family":"Mourkioti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenli","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"D. 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K. C. is a co-founder of Axonova Medical, LLC, and Innervace, Inc., which are University of Pennsylvania spin-out companies focused on translation of advanced regenerative therapies to treat nervous system disorders. US Patent Application 17\/291,869 and PCT International Patent Application PCT\/US2019\/060585 titled \u201cEngineering of Innervated Tissue and Modulation of Peripheral Organ Activity\u201d (D. K. C, SD) are related to the techniques described in this paper. The other authors declare that they have no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"5"}}