Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

Abstract

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.

Functional Analysis and Transcriptomic Profiling of iPSC-Derived Macrophages and Their Application in Modeling Mendelian Disease

“…Using human iPSCs derived by our Sendai virus reprogramming protocol, 7 We present 1) a relatively simple protocol that uses fewer cytokines for rapid, high-throughput generation of IPSDM of high purity and functional homogeneity. 27, 28 2) comparable morphological, functional and transcriptome profiles in IPSDM and HMDM, 3) remarkably similar in vitro functional plasticity and polarization in phagocytosis and cholesterol efflux capacity and inflammatory cytokine secretion in IPSDM and HMDM, 4) almost identical hallmark cholesterol-metabolism phenotypes in IPSDM and HMDM of TD patients, and 5) novel insights via IPSDM into macrophage inflammatory response in human ABCA1 deficiency. Thus, our IPSDM system paves the way towards large-scale applications in functional, disease and therapeutic modeling of macrophages in innate immunity and cellular cholesterol homeostasis in human.…”

Pluripotent Cell Models of Fanconi Anemia Identify the Early Pathological Defect in Human Hemoangiogenic Progenitors

“…Abbreviations: a-MEM, alpha Minimum Essential Medium; bFGF, basic fibroblast growth factor; EPO, erythropoietin; IL-3, interleukin 3; NS, not significant; SCF, stem cell factor; TPO, thyroid peroxidase; VEGF, vascular endothelial growth factor. the FA-iPSC lines into hematopoietic progenitor cells through the previously reported serum-and feeder-free monolayer culture system [10,11]. Under this protocol, the progenitor cells committed to the hematopoietic lineage are obtained as KDR 1 CD34…”

“…Plasmids were kindly provided by Dr. Keisuke Okita (Kyoto University). For hematopoietic differentiation, a two-dimensional hematopoietic differentiation system was used, as described previously [10,11].…”

Human Induced Pluripotent Stem Cell–Derived Macrophages for Unraveling Human Macrophage Biology

“…9, 10 In 2013, modified from a protocol published by Karlsson et al for human embryonic stem cell (hESC) to macrophage differentiation, 11 van Wilgenburg et al 12 established an embryoid bodies (EBs)-based protocol for IPSDM differentiation in which hematopoiesis was induced under serum-free condition followed by directed differentiation to macrophages with M-CSF either in serum-free media or in media with serum. Masakatsu et al 13 developed a monolayer-based protocol by culturing iPSC on a layer of extracellular matrix component under serum-free condition and differentiated floating “monocyte-like” cells to macrophage in media with serum and M-CSF. In 2015, our group 8 published an EB-based differentiation protocol and for the first time performed transcriptomic characterization using deep RNA-sequencing (RNA-seq) in HMDM and IPSDM lines derived from the same subjects.…”