Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×106±0.3×106 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.
Citation: Yanagimachi MD, Niwa A, Tanaka T, Honda-Ozaki F, Nishimoto S, Murata Y, et al. (2013) Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions. PLoS ONE 8(4): e59243. https://doi.org/10.1371/journal.pone.0059243
Editor: Katriina Aalto-Setala, University of Tampere, Finland
Received: August 14, 2012; Accepted: February 13, 2013; Published: April 3, 2013
Copyright: © 2013 Yanagimachi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by grants from the Ministry of Health, Labour and Welfare to TN, a grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to TN, grants from the Leading Project of MEXT to TN, a grant from Funding Program for World-Leading Innovative Research and Development on Science and Technology (FIRST Program) of Japan Society for the Promotion of Science (JSPS) to TN, grants from JSPS to TN and MKS, grants from the Takeda foundation, Mitsubishi Pharma Research Foundation and Suzuken memorial foundation to MKS and grants from Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to MDY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Monocytic lineage cells, such as monocytes, macrophages and dendritic cells (DCs), are central to immune responses and play key roles in various pathological conditions. – Monocytes are the myeloid progeny of hematopoietic stem/progenitor cells ; they are a type of mononuclear cell circulating in the bloodstream and act as gatekeepers in innate immunity. While they replenish macrophages and DCs, monocytes themselves respond to various inflammatory stimuli by migrating into inflamed tissues, phagocytosing pathological small particles and producing proinflammatory cytokines and chemokines. Therefore, monocytes not only contribute to host defense against pathogenic microorganisms, but are closely associated with the pathogenesis of chronic sterile inflammation.  Macrophages reside in tissues and robustly phagocytose microorganisms and cellular debris. One of the important hallmarks of monocytic lineage cells is their functional plasticity. In response to cytokines and microbial products, macrophages polarize into functionally distinct M1 and M2 cells.  Classically activated M1 macrophages are induced by interferon-γ (IFNγ), while alternatively activated M2 macrophages can be induced by IL-4 and IL-13. ,  M1 macrophages are generally characterized by high production of proinflammatory cytokines, while M2 are characterized by high production of anti-inflammatory cytokines. DCs are the most powerful antigen-presenting cells and have an indispensable role for the activation of T lymphocytes. Because of their ability to mediate communication between innate and acquired immunity, ex vivo expansion of DCs is expected to be a useful source of material for cancer immunotherapies, such as DC-based vaccines. – Moreover, recent reports of monocyte and/or DC deficiencies highlight the importance of understanding their development in humans.  However, there have been technical limitations for tracing the development of human monocytic cells, or for propagating them ex vivo.
Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are undifferentiated pluripotent cells that can be propagated indefinitely. – The development of monocytic cells from these pluripotent cells is of particular interest because it would provide an unlimited source of these cells for clinical applications and the examination of disease pathologies. Although the methods for hematopoietic differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established,  these methods usually depend on xenogeneic feeder cells and/or animal- or human-derived serum, and therefore have a relatively poor-reproducibility. For instance, batch-to-batch variability of serum or feeder cells can influence the characteristics of in vitro differentiated DCs.  Here, we describe a novel serum- and feeder cell-free method that robustly and repetitively produces monocytic lineage cells from human ESCs/iPSCs.
Materials and Methods
This study used human ESCs (cell line: KhES1) and iPSCs (cell lines: 201B7, 253G4, CIRA188Ai-W2, and CB-A11). , – 201B7, 253G4  and CIRA188Ai-W2  were previously described. A human ES cell line KhES1 was kindly provided by Dr. Norio Nakatsuji. Human iPS cell lines 201B7 and 253G4 were kindly provided by Dr. Shinya Yamanaka. CB-A11 was established from cord-blood mononuclear cells by using episomal vectors.  These ESCs/iPSCs were maintained on tissue culture dishes coated with growth factor-reduced Matrigel (Becton-Dickinson) in mTeSR1 serum-free medium (STEMCELL Technologies).
Monocytic Lineage Cell Differentiation Method
The monocytic lineage differentiation protocol was modified from a previously established hematopoietic differentiation protocol (Figure 1).  The protocol consists of 5 sequential steps by which mature MPs and DCs are differentiated from human pluripotent cells in a stepwise manner. In the first step, primitive streak cells were induced from undifferentiated ESCs/iPSCs, which were then differentiated into hemangioblast-like hematopoietic progenitors in the second step. In step 3, expanded hematopoietic progenitors were committed towards initial myeloid differentiation, and then differentiated into the monocytic lineage in step 4. Finally, CD14+ monocytes were differentiated into either MPs or DCs in step 5. The cytokines used in this study were purchased from R&D systems.
The protocol is composed of 5 steps. CD14-positive cells that are sorted between step-4 are differentiated into dendritic cells by step 5-1 or into macrophages by step 5-2. FL-3: Flt-3 ligand, TPO: Thrombopoietin.
Step 1: induction of primitive streak-like cells from undifferentiated human ES/iPS cells with BMP4.
BMP4 is an important molecule for the initial stage of mesodermal commitment of pluripotent stem cells in vitro.  Undifferentiated ESCs/iPSCs colonies were disseminated onto a 100 mm culture dish coated with growth factor-reduced Matrigel in mTeSR1 medium at a density of about 30 colonies per dish. Individual colonies were grown to a diameter of approximately 1 mm (Day 0), and BMP4 (80 ng/mL) was added to the mTeSR1 medium.
Step 2: generation of KDR+CD34+ hemangioblast-like cells with VEGF, basic FGF and SCF.
VEGF and SCF have been reported to be important cytokines for development of hemoangiogenic progenitors. – In this step, we also added basic FGF which enhances the development of mesodermal hematopoietic progenitors. ,  The mTeSR1 medium was replaced by StemPro-34 serum-free medium (Gibco) containing 2 mM glutamax (Invitrogen) on day 4, and then was supplemented with the step-2 cytokine cocktail composed of VEGF (80 ng/mL), basic FGF (25 ng/ml), and SCF (100 ng/mL).
Step 3: generation of hematopoietic cells with hematopoietic cytokines.
The cytokines in StemPro-34 medium were switched to the step-3 cytokine cocktail composed of SCF (50 ng/mL), IL-3 (50 ng/mL), TPO (Thrombopoietin) (5 ng/mL), M-CSF (50 ng/mL), and Flt-3 ligand (50 ng/mL), on day 6. Thereafter, the medium was changed on day 10.
Step 4: monocytic lineage-directed differentiation with Flt-3 ligand, GM-CSF and M-CSF.
The cytokines in StemPro-34 medium were switched to the step-4 cytokine cocktail composed of Flt-3 ligand (50 ng/mL), GM-CSF (25 ng/mL), and M-CSF (50 ng/mL) on day 13–15. The medium was changed every 3–4 days. The CD14+ monocytic lineage-directed cell fraction in supernatant was positively sorted by autoMACS pro (Miltenyi Biotec) with CD14 MicroBeads (Miltenyi Biotec) on days 15–28.
Step 5: differentiation into DCs (step 5-1) and MPs (step 5-2) from CD14+ monocytic lineage-cells.
CD14+ cells sorted by autoMACS pro (1.5×106 cells per well in a 6-well plate with Ultra-Low Attachment Surface (CORNING)) were cultured in StemPro-34 medium supplemented with GM-CSF (25 ng/mL) and IL-4 (40 ng/mL), with a medium change 4 days later, for differentiation into DCs (step 5-1). LPS (100 ng/mL, InvivoGen) and TNFα (0.2 ng/mL) were added for the last 2 days of the 7 day DC differentiation culture to promote maturation of DCs. CD14+ cells (1.5×106 cells per well in a 6-well tissue culture plate) were cultured in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS) and M-CSF (100 ng/mL) for 7 days with a medium change at day 4, for differentiation into macrophages (step 5-2). IFNγ (20 ng/ml) or IL-4 (20 ng/ml) was added for another day to promote differentiation into M1 or M2 macrophages, respectively.
Flow Cytometric Analysis
Flow cytometric analysis data were collected using the MACS Quant™ Analyzer (Miltenyi Biotec) and then analyzed utilizing the FlowJo software package (Treestar). The following antibodies were purchased from BD Biosciences: CD11b-FITC, CD11c-APC, CD34-PE, CD40-PE, CD43-FITC, CD80-PE, CD83-PE, CD86-FITC, CD205-Alexa fluor 647, CD206-FITC, CD209-PE, HLA-ABC-FITC and HLA-DR-FITC. CD14-APC and CD45-APC antibodies were purchased from Beckman Coulter. CD163-APC antibody was purchased from R&D systems. KDR (CD309)-Alexa fluor 647 and CX3CR1-PE antibodies were purchased from Biolegend.
May-Giemsa Staining and Esterase Staining
Cells were seeded onto glass slides by CYTOSPIN 4 (Thermo Scientific) and stained with May-Grunwald and Giemsa staining solution (MERCK) and Esterase staining solution (Muto pure chemicals) following the manufacturer’s instructions.
RNA Extraction and RT-PCR Analysis
RNA samples were prepared using the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Typically, 500 ng of total RNA were subjected to reverse transcription (RT) with a Sensiscript-RT kit (Qiagen). RT-PCR was performed for the evaluation of the expression of monocytic lineage