Optimizing the Method for Differentiation of Macrophages from Human Induced Pluripotent Stem Cells

Abstract

Macrophage is a very promising cell type for cancer immunotherapy, yet it is difficult to obtain enough functional macrophages for clinical cell therapy. Herein, we descibe a reliable method to produce functional macrophages through the differentiation of human induced pluripotent stem cells (hiPSCs). By optimizing the size control of embryoid bodies (EBs), we accelerated the differentiation process of macrophages and increased the production of macrophages without attenuating macrophage functions. Our final yield of macrophages was close to 50-fold of starting iPSCs. The macrophages showed phagocytic capacity in vitro and a xenograft tumor model. M0 macrophages could be further polarized into M1 and M2 subtypes, and M1 cells exhibited typical proinflammatory characteristics. Moreover, we found that hematopoietic differentiation originated from the outside of EB and matured inward gradually. Taken together, our protocol provides an effective method for the generation of macrophages comparable to blood-derived macrophages, which provides potential value for cell therapy and gene editing studies.

Germline Neurofibromin 1 mutation enhances the anti‐tumour immune response and decreases juvenile myelomonocytic leukaemia tumourigenicity

“…In the CTR group, the expression of CD80 and CD86 was upregulated in M1 cells and the expression of CD163 and CD206 was higher in M2 (Figure 3E), which is consistent with the previous study. 26 In the group of NF1-KO, however, the expression of CD80, CD86, CD163 and CD206 in the M1 and M2 subtypes was not upregulated compared with the M0 subtype (Figure 3E). These results suggest that NF1 deletion mutations inhibit the polarization of M0 macrophages, particularly toward the M2 subtypes.…”

“…Differentiating iPSCs into macrophages (iMacs) was performed as previously described 26 . In brief, 8000 cells/well were seeded into an untreated round‐bottom 96‐well (Costar 3788) plate in 100 μL medium per well (day 0).…”

“…As previously described, 26 at least 2 × 10 5 macrophages were mixed with 1‐fold luoresbrite™ Polychromatic Red 1.0 μm Latex beads (Polyscience Inc., 18660) and incubated for 24 h in RPMI1640 medium containing 10% FBS (Gibco). After 24 h, macrophages were washed, and the samples were run on a FACS Calibur system (BD Canto Plus) and analysed using FlowJo v10 (FlowJo, LLC).…”

“…To verify the effects of NF1 deletion or mutations on macrophage function, we performed a monocyte-macrophage differentiation as previously described. 26 Our study showed that NF1-KO, NF1-G (Q83P) and NF1-G (Q83P) S (C42fs) hiPSC colonies generated significantly more total cell numbers on day 8, day 14, day 18, day 22 and day 28 (Figure 3A,B) along with the population numbers of CD45 + /CD11b + and CD11b + /CD14 + (Figure 3C,D and Figure S4A,B) on day 22 compared with non-mutated iPSC colonies with dose-dependent effects on the NF1 expression. Moreover, we also characterized the cells generated on day 22 of differentiation by Wright-Giemsa staining.…”

“…Differentiating iPSCs into macrophages (iMacs) was performed as previously described. 26 In brief, 8000 cells/well were seeded into an untreated round-bottom 96-well (Costar 3788) plate in 100 μL medium per well (day 0). Fresh APEL medium (100 μL) with recombinant human bFGF (10 ng/mL), BMP4 (20 ng/mL), VEGF (20 ng/mL) and SCF (40 ng/mL) was exchanged every 2~3 days (days 1-7).…”