Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives

Abstract

Summary A renewable source of human monocytes and macrophages would be a valuable alternative to primary cells from peripheral blood (PB) in biomedical research. We developed an efficient protocol to derive monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) and performed a functional comparison with PB-derived cells. hiPSC-derived monocytes were functional after cryopreservation and exhibited gene expression profiles comparable with PB-derived monocytes. Notably, hiPSC-derived monocytes were more activated with greater adhesion to endothelial cells under physiological flow. hiPSC-derived monocytes were successfully polarized to M1 and M2 macrophage subtypes, which showed similar pan- and subtype-specific gene and surface protein expression and cytokine secretion to PB-derived macrophages. hiPSC-derived macrophages exhibited higher endocytosis and efferocytosis and similar bacterial and tumor cell phagocytosis to PB-derived macrophages. In summary, we developed a robust protocol to generate hiPSC monocytes and macrophages from independent hiPSC lines that showed aspects of functional maturity comparable with those from PB.

Generation and Functional Characterization of Monocytes and Macrophages Derived from Human Induced Pluripotent Stem Cells

“…Previously we described a detailed protocol to study adhesion of monocytes to hiPSC‐ECs in a microfluidic chip (Halaidych et al., 2018b). The protocol was applied to study adhesion of human monocytic cell line (THP1; Halaidych et al., 2018a), blood‐mono, and hiPSC‐mono (Cao et al., 2019). We have previously shown that hiPSC‐ECs when compared to primary HUVECs exhibit lower induction of leukocyte pro‐adhesive receptors, such as E‐selectin and ICAM‐1, and lack upregulation of VCAM‐1 upon treatments with various pro‐inflammatory cytokines (TNF‐α, LPS, and IL‐1β; Halaidych et al., 2018a).…”

“…We have previously shown that hiPSC‐ECs when compared to primary HUVECs exhibit lower induction of leukocyte pro‐adhesive receptors, such as E‐selectin and ICAM‐1, and lack upregulation of VCAM‐1 upon treatments with various pro‐inflammatory cytokines (TNF‐α, LPS, and IL‐1β; Halaidych et al., 2018a). Pre‐conditioning of hiPSC‐ECs with bone morphogenetic protein 9 (BMP9) enhances inflammatory responses and results in an increase in VCAM‐1 expression (Cao et al., 2019), which is an important receptor for very late antigen 4 (VLA‐4, α4β1, CD49d, and CD29) integrin on leukocytes. VLA‐4 is highly expressed on T cells and neutrophils and is absent on blood‐mono.…”

“…This article describes two basic protocols: The efficient monolayer differentiation of monocytes from hiPSCs (Basic Protocol 1), and then induction of their differentiation to different macrophage subtypes from cryopreserved monocytes (Basic Protocol 2); this is an adaption of our previous publication (Cao et al., 2019). Using our protocol, functional monocytes and polarized macrophage subtypes can be efficiently derived from hiPSCs within 2 and 3 weeks, respectively.…”

Human‐induced pluripotent stem cell‐derived blood products: state of the art and future directions

“…Most iPSC differentiations have the capability to produce MФs for up to 4-5 months [89]. Recently, Cao et al [93] introduced a 2D differentiation method that resulted in a total monocyte yield of~1.5-2 9 10 7 (~39 monocyte/input iPSC) after 15 days of differentiation, which could be further differentiated to MФs in 6 days. Ackermann et al [92] introduced the first iPSC-MФ bioreactor utilization.…”

“…[89,93]. In addition, they have the ability to phagocytose and to produce multiple cytokines (IL-6, IL-1b, tumor necrosus factor a etc.)…”