ORIGINAL RESEARCH article
Front. Immunol.
Sec. Alloimmunity and Transplantation
This article is part of the Research TopicThe Significance of Induced Pluripotent Stem Cells in Translational MedicineView all 5 articles
Robust, scalable and xeno-free protocol for differentiating human induced pluripotent stem cells into functional macrophages
Provisionally accepted
Miquel De Homdedeu1,2Rubén Escribá1Kenia Rodríguez-González1
Sergio Querol3Jesús Fernández-Sojo1,2
Belen Alvarez-Palomo1,2*- 1Banc de Sang i Teixits, Barcelona, Spain
- 2Vall d'Hebron Institut de Recerca, Barcelona, Spain
- 3Fundacion Josep Carreras contra la Leucemia, Barcelona, Spain
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Human induced pluripotent stem cells (hiPSCs)-derived macrophages (iMacs) exhibit key macrophage phenotypic and functional properties, positioning them as promising candidates for allogenic cell immunotherapies. However, an efficient, scalable and good manufacturing practices (GMP)-compatible differentiation protocol is noticeably lacking. To meet this need, we aimed to develop a robust protocol for differentiating clinical-grade hiPSC lines into functional iMacs, designed for scalability and immediate GMP translation. We tested different media compositions, cytokine concentrations, seeding densities, culture regimens (2D vs. 3D) and coatings across key developmental stages. With the optimized protocol, we differentiated three different clinical-grade hiPSC lines towards mesoderm through embryoid body (EB) formation by agitation in 3D. Hematopoietic progenitor-producing EBs were induced to produce myeloid progenitors in agitation for 7 days. Then, myeloid progenitors were harvested and transferred to a G-Rex production platform to differentiate and further polarize into M1 or M2 iMacs. Cellular differentiation was assessed using flow cytometry panels through all developmental stages. At the final differentiated stage, iMacs were functionally characterized using the pHrodo phagocytosis assay. Pro-inflammatory cytokine secretion was analyzed by ELISA, and cell morphology was assessed by May-Grünwald Giemsa staining in Cytospin preparations. The full protocol was performed using feeder-free, scalable and either GMP or GMP-translatable reagents. This protocol enabled the production of iMacs with a mean purity of 98% viable cells, and a mean differentiation fold of 250X from a single hiPSC.
Keywords: Allogenic cell therapy, Gas-permeable Rapid Expansion system (G- Rex), Good manufacturing practice (GMP), induced pluripotent stem cells (iPSCs), Macrophage differentiation, Phagocytosis
Received: 06 Oct 2025; Accepted: 17 Dec 2025.
Copyright: © 2025 De Homdedeu, Escribá, Rodríguez-González, Querol, Fernández-Sojo and Alvarez-Palomo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Belen Alvarez-Palomo
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