{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T03:38:20Z","timestamp":1781062700711,"version":"3.54.1"},"reference-count":56,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,30]],"date-time":"2025-12-30T00:00:00Z","timestamp":1767052800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"iPSC Neurodegenerative Disease Initiative","award":["NIH 75N95020Q00265"],"award-info":[{"award-number":["NIH 75N95020Q00265"]}]},{"name":"JAX Scientific Service Innovation Funding"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>We present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Building upon an established transcription factor-driven approach, our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in brain tumors and disease modeling, neuroinflammation research, and therapeutic screening.<\/jats:p>","DOI":"10.3390\/bioengineering13010045","type":"journal-article","created":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T16:08:00Z","timestamp":1767197280000},"page":"45","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Xeno-Free Protocol for Rapid Differentiation of Human iPSC-Derived Microglia from the KOLF2.1J Reference Line"],"prefix":"10.3390","volume":"13","author":[{"given":"N\u00e9lio A. J.","family":"Oliveira","sequence":"first","affiliation":[{"name":"The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-7498-7023","authenticated-orcid":false,"given":"Katherine R.","family":"Lewkowicz","sequence":"additional","affiliation":[{"name":"The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3669-7826","authenticated-orcid":false,"given":"Patricia A.","family":"Clow","sequence":"additional","affiliation":[{"name":"The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5296-8051","authenticated-orcid":false,"given":"Michael E.","family":"Ward","sequence":"additional","affiliation":[{"name":"National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1058-3831","authenticated-orcid":false,"given":"Mark R.","family":"Cookson","sequence":"additional","affiliation":[{"name":"Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8334-3329","authenticated-orcid":false,"given":"William C.","family":"Skarnes","sequence":"additional","affiliation":[{"name":"The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1283-5972","authenticated-orcid":false,"given":"Justin A.","family":"McDonough","sequence":"additional","affiliation":[{"name":"The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT 06032, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1149","DOI":"10.1038\/s41593-022-01131-4","article-title":"A CRISPRi\/a platform in human iPSC-derived microglia uncovers regulators of disease states","volume":"25","author":"Drager","year":"2022","journal-title":"Nat. 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