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Cited by 3Pluripotent Stem Cells ResearchMesenchymal stem cell researchHematopoietic Stem Cell Transplantation

The DOI matches its Crossref record; 31 of the 31 references it cites were found in a registry or an index.

Robust, scalable and xeno-free protocol for differentiating human induced pluripotent stem cells into functional macrophages

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De Homdedeu, Miquel, Escribá, Rubén, Rodríguez-González, Kenia, Querol, Sergi, Fernandez-Sojo, Jesus, Alvarez-Palomo, Belén·2026·Frontiers in Immunology
DOI 10.3389/fimmu.2025.1719452PMC PMC12833622
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Abstract

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.

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Metadata

Authors
De Homdedeu, M., Escribá, R., Rodríguez-González, K., Querol, S., Fernandez-Sojo, J., & Alvarez-Palomo, B.
Affiliations
  • Vall d'Hebron Institut de Recerca (ES)
  • Banc de Sang i Teixits (ES)
  • Fundación Josep Carreras Contra la Leucemia (ES)
ORCID
Venue
Frontiers in Immunology
Publisher
Frontiers Media SA
Published
2026-01-12
Volume
16
Pages
1719452-1719452
ISSN
1664-3224 (ISSN-L)
DOI
10.3389/fimmu.2025.1719452
PMC
PMC12833622
OpenAlex
W7122704894
Cited by
3
References
31
Citations by year
2026: 3
FWCI
6.20
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Immunology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Ministerio de Ciencia e Innovación
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
Keywords
human induced pluripotent stem cells, macrophage differentiation, IMACS, xeno-free culture, hematopoietic progenitor cells, myeloid progenitor cells, macrophage polarization, phagocytosis, scalable cell production
MeSH terms
Cell Differentiation, Cell Line, Cells, Cultured, Humans, Macrophages, Phagocytosis, Cytokines, Cell Culture Techniques, Induced Pluripotent Stem Cells

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