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

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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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Crossref · 10.3389/fimmu.2025.1719452

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Figures

Figure 1 Differentiation of hiPSC into M0, M1 and M2 polarized iMacs. (A) Scheme of differentiation of hiPSC into M0, M1 and M2 polarized iMacs. (B) Representative brightfield images showing the different cellular stages: hiPSCs (adhered, compact colonies, with a high nucleus-to-cytoplasm ratio, and no signs of spontaneous differentiation), EBs (at day 3 they measure >300µm, have spherical morphology and show clear boundaries), HSCs (small and very bright round cells), MPs (slightly bigger and very bright round cells), M0 iMacs (large, foamy, and cells with a vacuolated cytoplasm), and M1(Elongated, spindle-shaped cells)- and M2(large, round, and flat-shaped cells with an amoeboid appearance)-polarized iMacs. Images were acquired at x40-x200 magnifications, scale bar represents 100 µm. EB, embryoid Body; HSC, hematopoietic stem cell; MP, myeloid progenitor; iMacs, hiPSCs-derived macrophages; d, day.
Figure 1 Differentiation of hiPSC into M0, M1 and M2 polarized iMacs. (A) Scheme of differentiation of hiPSC into M0, M1 and M2 polarized iMacs. (B) Representative brightfield images showing the different cellular stages: hiPSCs (adhered, compact colonies, with a high nucleus-to-cytoplasm ratio, and no signs of spontaneous differentiation), EBs (at day 3 they measure >300µm, have spherical morphology and show clear boundaries), HSCs (small and very bright round cells), MPs (slightly bigger and very bright round cells), M0 iMacs (large, foamy, and cells with a vacuolated cytoplasm), and M1(Elongated, spindle-shaped cells)- and M2(large, round, and flat-shaped cells with an amoeboid appearance)-polarized iMacs. Images were acquired at x40-x200 magnifications, scale bar represents 100 µm. EB, embryoid Body; HSC, hematopoietic stem cell; MP, myeloid progenitor; iMacs, hiPSCs-derived macrophages; d, day.CC BY · PubMed Central
Figure 2 Characterization of secreted hematopoietic stem cells. Representative plots of the gating strategy used during flow cytometry analysis: exclusion of cellular debris using Forward Scatter (FSC-A) and Side Scatter (SSC-A); selection of single cells using FSC-A and FSC-H; identification of live cells using SSC-A and ViaKrome 405; scatter dot plot showing the expression of CD34 and CD43; and scatter dot plot showing the expression of CD34 and CD45.
Figure 2 Characterization of secreted hematopoietic stem cells. Representative plots of the gating strategy used during flow cytometry analysis: exclusion of cellular debris using Forward Scatter (FSC-A) and Side Scatter (SSC-A); selection of single cells using FSC-A and FSC-H; identification of live cells using SSC-A and ViaKrome 405; scatter dot plot showing the expression of CD34 and CD43; and scatter dot plot showing the expression of CD34 and CD45.CC BY · PubMed Central
Figure 3 Optimization of differentiation of hematopoietic stem cells into myeloid progenitors. Bar plots comparing different strategies for optimizing the HSC-to-MP differentiation stage. (A) Fold change of CD34+ to MPs relative to 24mw Coating. (B) Fold change of CD34+ to MPs relative to cell culture regimen. (C) Density of differentiated MPs generated by seeding a single EB with different amounts of CD34+ HSCs. (D) Density of differentiated MPs generated by seeding different amount of EBs with the same amount of CD34+ HSCs. (E) Fold change of CD34+ to MPs relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (F) Expression of CD14 in the differentiated MP population relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (G) Fold change of CD34+ to MPs relative to partial replacement of M-CSF by GM-CSF in 6mw plates. (H) Expression of CD14 in the differentiated MP population relative to partial replacement of M-CSF by GM-CSF in 6mw plates. (n=4-13; *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney and Kruskal-Wallis test with Dunn’s post-test; mean ± SD).
Figure 3 Optimization of differentiation of hematopoietic stem cells into myeloid progenitors. Bar plots comparing different strategies for optimizing the HSC-to-MP differentiation stage. (A) Fold change of CD34+ to MPs relative to 24mw Coating. (B) Fold change of CD34+ to MPs relative to cell culture regimen. (C) Density of differentiated MPs generated by seeding a single EB with different amounts of CD34+ HSCs. (D) Density of differentiated MPs generated by seeding different amount of EBs with the same amount of CD34+ HSCs. (E) Fold change of CD34+ to MPs relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (F) Expression of CD14 in the differentiated MP population relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (G) Fold change of CD34+ to MPs relative to partial replacement of M-CSF by GM-CSF in 6mw plates. (H) Expression of CD14 in the differentiated MP population relative to partial replacement of M-CSF by GM-CSF in 6mw plates. (n=4-13; *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney and Kruskal-Wallis test with Dunn’s post-test; mean ± SD).CC BY · PubMed Central
Figure 4 Optimization of differentiation of myeloid progenitors into macrophages. Bar plots and fold-change curves comparing different strategies for optimizing the MP-to-iMacs differentiation stage. (A) Fold change of MPs to iMacs relative to 24mw Coating. (B) Fold change of MPs to iMacs relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (C) Expression of CD14 in the differentiated iMacs population relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (D) Fold change of MPs to iMacs relative to cell culture regimen. (E) Density of differentiated iMacs obtained by seeding MPs at varying densities in 24mw plates. The colored range indicates the optimal seeding window. of MPs. (F) Density of differentiated iMacs obtained by seeding MPs at varying densities in 24mw G-Rex. The colored range indicates the optimal seeding window. of MPs. (G) Fold-change curves of MP-to-iMac differentiation as a function of MP seeding density in 6mw, 24mw, and 24mw G-Rex plates. The colored range indicates the optimal seeding window. of MPs. (H) Fold change of MPs to iMacs relative to number of M-CSF spiking in 24mw G-Rex. (n=4-31; *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney and Kruskal-Wallis test with Dunn’s post-test; mean ± SD).
Figure 4 Optimization of differentiation of myeloid progenitors into macrophages. Bar plots and fold-change curves comparing different strategies for optimizing the MP-to-iMacs differentiation stage. (A) Fold change of MPs to iMacs relative to 24mw Coating. (B) Fold change of MPs to iMacs relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (C) Expression of CD14 in the differentiated iMacs population relative to partial or full replacement of M-CSF by GM-CSF in 24mw plates. (D) Fold change of MPs to iMacs relative to cell culture regimen. (E) Density of differentiated iMacs obtained by seeding MPs at varying densities in 24mw plates. The colored range indicates the optimal seeding window. of MPs. (F) Density of differentiated iMacs obtained by seeding MPs at varying densities in 24mw G-Rex. The colored range indicates the optimal seeding window. of MPs. (G) Fold-change curves of MP-to-iMac differentiation as a function of MP seeding density in 6mw, 24mw, and 24mw G-Rex plates. The colored range indicates the optimal seeding window. of MPs. (H) Fold change of MPs to iMacs relative to number of M-CSF spiking in 24mw G-Rex. (n=4-31; *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney and Kruskal-Wallis test with Dunn’s post-test; mean ± SD).CC BY · PubMed Central
Figure 5 Phenotypic and functional characterization of M0 iMacs. (A) Representative plots of the gating strategy used during flow cytometry analysis: exclusion of cellular debris using FSC-A and SSC-A; selection of single cells using FSC-A and FSC-H; identification of live cells using SSC-A and ViaKrome 405; identification of iMacs using SSC-A and CD14; and scatter dot plot showing the expression of CD86 and CD206. (B) Representative brightfield Cytospin staining for M0 iMacs. (C) Comparison of Live Cells, and CD14, CD86, and CD206 expression between macrophages derived from primary peripheral blood monocytes and iPSC-derived macrophages. (PB-Macs: n=1 used as reference; iMacs: n=3; data shown as mean ± SD.) (D) Cytokine levels produced by iMacs: GM-CSF, Granzyme-B, IL-6, IL-10, IL-17A, and TNF. Images were acquired tat ×200 magnification, scale bar represents 100 µm. (n=2; mean ± SD).
Figure 5 Phenotypic and functional characterization of M0 iMacs. (A) Representative plots of the gating strategy used during flow cytometry analysis: exclusion of cellular debris using FSC-A and SSC-A; selection of single cells using FSC-A and FSC-H; identification of live cells using SSC-A and ViaKrome 405; identification of iMacs using SSC-A and CD14; and scatter dot plot showing the expression of CD86 and CD206. (B) Representative brightfield Cytospin staining for M0 iMacs. (C) Comparison of Live Cells, and CD14, CD86, and CD206 expression between macrophages derived from primary peripheral blood monocytes and iPSC-derived macrophages. (PB-Macs: n=1 used as reference; iMacs: n=3; data shown as mean ± SD.) (D) Cytokine levels produced by iMacs: GM-CSF, Granzyme-B, IL-6, IL-10, IL-17A, and TNF. Images were acquired tat ×200 magnification, scale bar represents 100 µm. (n=2; mean ± SD).CC BY · PubMed Central
Figure 6 Phenotypic and functional characterization of M0, M1 and M2 iMacs. (A) Overlay of MFI histograms comparing HLA-DR and CD206 expression between M0, M1 and M2 iMacs. (B) Overlay of MFI histograms comparing pHrodo expression in M1 iMacs after incubation with Deep Red E. coli BioParticles for 30min or 2h 30min at 37°C. (C) Fluorescence microscopy images of M0, M1, and M2 iMacs after internalization of pHrodo E. coli BioParticles for 2h 30min at 37°C. Images were acquired tat ×200 magnification, scale bar represents 100 µm.
Figure 6 Phenotypic and functional characterization of M0, M1 and M2 iMacs. (A) Overlay of MFI histograms comparing HLA-DR and CD206 expression between M0, M1 and M2 iMacs. (B) Overlay of MFI histograms comparing pHrodo expression in M1 iMacs after incubation with Deep Red E. coli BioParticles for 30min or 2h 30min at 37°C. (C) Fluorescence microscopy images of M0, M1, and M2 iMacs after internalization of pHrodo E. coli BioParticles for 2h 30min at 37°C. Images were acquired tat ×200 magnification, scale bar represents 100 µm.CC BY · PubMed Central

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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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