Abstract
Monocytes and macrophages are essential for immune defense and tissue hemostasis. They are also the underlying trigger of many diseases. The availability of robust and short protocols to induce monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) will benefit many applications of immune cells in biomedical research. Here, we describe a protocol to derive and functionally characterize these cells. Large numbers of hiPSC‐derived monocytes (hiPSC‐mono) could be generated in just 15 days. These monocytes were fully functional after cryopreservation and could be polarized to M1 and M2 macrophage subtypes. hiPSC‐derived macrophages (iPSDMs) showed high phagocytotic uptake of bacteria, apoptotic cells, and tumor cells. The protocol was effective across multiple hiPSC lines. In summary, we developed a robust protocol to generate hiPSC‐mono and iPSDMs which showed phenotypic features of macrophages and functional maturity in different bioassays. © 2020 The Authors. Basic Protocol 1: Differentiation of hiPSCs toward monocytes Support Protocol 1: Isolation and cryopreservation of monocytes Support Protocol 2: Characterization of monocytes Basic Protocol 2: Differentiation of different subtypes of macrophages Support Protocol 3: Characterization of hiPSC‐derived macrophages (iPSDMs) Support Protocol 4: Functional characterization of different subtypes of macrophages
Highlights
Monocytes and macrophages play crucial roles in protective immunity and tissue hemostasis and trigger or exacerbate many pathological conditions, including diabetes, atherosclerosis, fibrosis, and cancer (Wynn, Chawla, & Pollard, 2013)
Previous studies have shown that human induced pluripotent stem cells can be induced to form hemogenic endothelium (HE), identified as CD144+CD34+ and CD73, which could be further differentiated into CD43+ hematopoietic progenitors (HPCs) and to erythro-myeloid progenitors (EMPs); these are reminiscent of EMPs in the yolk sac during embryonic hematopoiesis
All of these studies indicate that iPSDMs can be a unique source of patient-specific, tissue-resident macrophages given that they can be produced in unlimited cell numbers from a renewable donor source of choice and can adopt a tissue resident macrophage-like identity
Summary
Monocytes and macrophages play crucial roles in protective immunity and tissue hemostasis and trigger or exacerbate many pathological conditions, including diabetes, atherosclerosis, fibrosis, and cancer (Wynn, Chawla, & Pollard, 2013). CD14+ monocytes can be harvested and isolated either on day 14 or day 15 depending on differentiation efficiency and/or on the hiPSC line, and optimal time can be determined either by FACS or by observing a small number of cells that start to adhere to the plate and differentiate towards macrophages. HPCs derived from this protocol on day 9 show multilineage differentiation potential in a colony forming unit (CFU) assay, developing to erythroid, myeloid (granulocytes, monocytes, macrophages), and megakaryocyte lineages (Cao et al, 2019). Uenishi and colleagues showed that T lymphoid cells can be generated from hiPSC-derived HPCs differentiated using a comparable method, even though Tenascin C was used as an extracellular matrix protein to promote lymphoid differentiation (Uenishi et al, 2014) These results suggest that HPCs derived using this protocol can be used for the induction of other hematopoietic lineages, including granulocytes, erythrocytes, megakaryocytes, and T lymphocytes. Always prewarm medium to room temperature and add growth factors to the medium right before refreshing
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