While we cannot exclude the

While we cannot exclude the possibility that some MYB-dependent hematopoietic progenitor ras inhibitor (HPCs)/HSCs are generated during normal WT iPSC differentiation, we would expect a reduction in monocyte/macrophage production if the majority of monocytes/macrophages were derived from MYB-dependent progenitors. In contrast, we observed an increase in production of monocytes/macrophages in the MYB−/− iPSCs, without any major change in phenotype or function of the cells. Furthermore, using a similar EB-based human embryonic stem cell differentiation protocol, Vanhee et al. (2015) observed that multipotent HPCs expressing high levels of MYB were not generated in their cultures and that macrophages were generated from precursors not expressing detectable MYB. Combined with our data, this strongly suggests that in WT iPSC differentiation, most, if not all, monocytes/macrophages are produced in an MYB-independent fashion, and the contribution of MYB-dependent multilineage HPC/HSC-derived hematopoiesis in our EB-based monocyte differentiation protocol is negligible. Interestingly, in addition to the increased monocyte/macrophage production, we observed an increased number of CD34+ CD45+ HPCs within MYB−/− EBs. A very similar phenotype has been observed in mouse Myb−/− ESC differentiation by Clarke et al. (2000). First, they observed that Myb−/− ESCs were capable of macrophage and primitive erythrocyte colony formation, but the kinetics of formation was different between the control line and the Myb−/− ESCs. The number of CFU-E was lower in Myb−/− while the generation of CFU-M was increased at day 7 when compared with the WT control. Second, no BFU-E were generated by Myb−/− ESCs, indicating a block to definitive erythrocyte production. Third, they observed a higher number of CD34+Sca-1+ HPCs within the Myb−/− EBs, but these progenitors were unable to progress further in differentiation. This could be due to an increased hematopoietic commitment, progenitor proliferation, or an accumulation of committed erythrocyte/granulocyte progenitors that cannot progress further through differentiation due to the lack of Myb. It will be interesting to understand the precise mechanism of action underlying this increase in precursor cells and monocytes/macrophages. With the mounting data suggesting that tissue-resident macrophages and BM monocyte-derived macrophages can play different roles in diseases such as cancer (Lahmar et al., 2015) and parasite infection (Rückerl and Allen, 2014), having access to authentic embryonic-derived monocytes and macrophages in vitro will be of considerable scientific value. Patient-derived tissue-resident macrophages are very difficult to obtain, are inherently genetically variable, and are notoriously difficult to genetically modify, making their study laborious and unreliable. On the other hand, iPSCs can be generated from a patient with a specific genetic background and can be modified by multiple mechanisms, such as lentiviral transduction or CRISPR-Cas9 gene editing. The demonstration that MYB-independent monocytes/macrophages can be generated in our differentiation protocol lays the foundation for their use in the development of reliable protocols for generating the tissue-specific subtypes of macrophages for the in vitro study of their role in pathology and homeostasis. Moreover, iPSC differentiation is a potential source of tissue-resident macrophages for cell therapy, as has recently been shown in the mouse with the use of murine pluripotent stem cell-derived Myb−/− alveolar-like macrophages as a cell source for treating a mouse model of adenosine deaminase deficiency (ADA−/−) (Litvack et al., 2016).
Experimental Procedures
Author Contributions
Acknowledgments J.B. was funded by the RCUK–Medical Research Council and the Heatley Merck Sharpe and Dohme studentship. The Oxford Martin School (LC0910-004) and the Wellcome Trust (WTISSF121302) provide core support to the James Martin Stem Cell Facility within the Sir William Dunn School of Pathology. Samples and associated clinical data were supplied by the Oxford Parkinson\'s Disease Centre study, funded by the Monument Trust Discovery Award from Parkinson\'s UK, a charity registered in England and Wales (2581970) and in Scotland (SC037554), with the support of the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre based at Oxford University Hospitals NHS Trust and University of Oxford, and the NIHR Comprehensive Local Research Network. The High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (generation of Illumina genotyping) is funded by Wellcome Trust Grant Reference 090532/Z/09/Z.