Table 2.
Overview of published hiPSC-derived microglia models. While all these protocols can be concluded to produce microglia-like phenotypes, co-culture models that provide cues associated with a CNS environment are the most promising.
| Article | Overview of protocol | Notable findings | Notable disadvantages |
|---|---|---|---|
| Almeida et al. (104) | Not described in publication | First to produce hiPSC-microglia | Transcriptomic profile not unlike immortalized microglia cell lines (BV-2) Generated through neuronal rather than myeloid pathway. |
| Muffat et al. (105) | Embryoid bodies were generated and resuspended in neuroglial differentiation media containing (supplement) with the addition of CSF-1/M-CSF and IL-34 | First published study with similar characteristics of fetal primary human and mouse microglia. | Appears to generate a mixed population of cells and is limited to monoculture experiments. |
| Abud et al. (106) | Microglia differentiation media utilizes neuronal base media DMEM/F-12 + +N2+B27 with small molecules M-CSF, IL-34, and TGFβ-1. An additional maturation media is utilized consisting of CD200 and CX3CL1, which is notably secreted by neurons for the final three days. | Successful transplantation of already ramified microglia within Alzheimer’s disease model mice. Subsequent in vivo evidence shows ability to interact with neurotoxic amyloid β | Requires an isolation step to begin differentiation part of haematopoiesis step, making it highly complex compared to pure single molecule methods. Not authentic YS ontogeny. |
| McQuade et al. (107) | Proprietary composition of initial hematopoietic differentiation media (STEMdiff hematopoietic kit) for an 11-day period followed by differentiation with IL-34, TGF-β1, and M-CSF/CSF-1. Includes the additional maturation step with CX3CL1 (fractalkine) and CD200 to induce ramification. | Successfully ramify following transplantation in mouse brain. Suggests IDE1 as a small molecule able to replace TGF-β in protocols utilizing this for differentiation. |
Describes itself as resembling developmental microglia but does not separate cited fetal vs adult datasets. Not authentic YS ontogeny |
| Takata et al. (108) | Generation of hematopoietic lineage macrophages terminally differentiated with SCF, IL-3 and CSF-1/M-CSF. Cells then co-cultured with mouse iPSC-derived neurons to further drive towards microglia phenotype | Described the requirement for tissue-dependent cues in order to make cells more microglia-like. Demonstrated potential of modelling infiltrating macrophages during adulthood. |
Primary characterization with mouse iPSCs. Not authentic YS ontogeny |
| Pandya et al. (109) | iPSCs were differentiated on OP9 feeder layers with OP9 differentiation medium (ODM) to myeloid progenitors. CD34+/CD43+ cells were sorted with MACS into myeloid progenitor media with GM-CSF and subsequently passaged and plated in astrocyte differentiation medium (ADM-IMDM base medium + GM-CSF, M-CSF and IL-3) then CD11+ cells were further isolated. Additionally, some experiments used CD39+ microglia sorted from a specific co-culture system with astrocytes. | Utilizes hematopoietic stem cells paired with astrocytes to obtain iPSC-derived microglia. Mouse iPSC-derived cells consistent with primary neonatal microglia profile. | Gene expression data primarily from mouse iPSC-derived microglia. The human microglia model requires an isolation step. Majority of characterization done in mouse model and the system does not utilize neuronal cells. Not authentic YS ontogeny |
| Ormel et al. (110) | This protocol was adapted from Lancaster and Knoblich (111), with the only change made in media composition being increasing the concentration of Heparin (0.1 ug/ml to 1 ug/ml) | Characterizes innate development of microglia in hiPSC-derived brain organoids, which exhibit some phagocytic function as synaptic material is present within the cells. | Replication of these findings is currently lacking in the literature regarding the spontaneous differentiation of microglia in the organoid. |
| Haenseler et al. (10) | Utilizes IL-3 and M-CSF to drive myelopoiesis yielding a pure macrophage precursor population. Microglia differentiation and ramification of these cells is successfully induced using a neuronal base media (DMEM/F-12+N2 as a base media) + small molecules IL-34 and GM-CSF compared to X-VIVO which is used in the cultivation of monocytes and macrophages. The protocol utilizes X-VIVO and M-CSF for the maturation to macrophages as comparison. | Once set up, fully matured microglia can be generated at 2-week intervals for a 5-month period. Functional validation completed in a co-culture system. Only protocol to demonstrate a myeloblastosias proto-oncogene transcription factor (MYB)-independent YS origin using a MYB knockout iPSC line in previous work (112), |
Requires a very sensitive 6–7-week period before microglia precursors can be collected. No assays showing functional integration into an animal model. |
The precise protocol used however is likely to be dependent on the experimental question under investigation. YS, Yolk Sac. Additional references not in main text (104).