Reenacting Neuroectodermal Exposure of Hematopoietic Progenitors Enables Scalable Production of Cryopreservable
Mona Mathews1,2, Jannis Wißfeld3, Lea Jessica Flitsch2
1LIFE & BRAIN GmbH, Venusberg-Campus 1, 53127, Bonn, Germany.
Abstract:
Human microglia, as innate immune cells of the central nervous system (CNS), play a central role in the pathogenesis of a large number of neurological and psychiatric disorders. However, experimental access to primary human microglia for biomedical applications such as disease modeling is extremely limited. While induced pluripotent stem cells (iPSCs) could provide an alternative source of microglia, the reenactment of their complex ontogenesis with a yolk sac origin and subsequent priming upon CNS invasion has remained a challenge. Here, we report a developmentally informed in vitro differentiation method for large-scale production and cryopreservation of iPSC-derived microglia (iPSdMiG). Specifically, iPSCs were propagated in conditions yielding both yolk sac hematopoietic derivatives and early neuroepithelial cells. To enable large-scale production, we implemented 3D bioreactor-based dynamic culture conditions and the use of novel mesh macrocarriers. Under these conditions, microglia could be harvested across a time period of at least 6 weeks, with 1 × 106 iPSCs giving rise to up to 45 × 106 iPSdMiG. The transcriptomic profile of iPSdMiG showed high similarity to adult human microglia, and harvested cells were immunopositive for typical microglial markers. In addition, iPSdMiG were able to secrete pro-inflammatory cytokines, engaged in phagocytotic activity, produced reactive oxygen species and lent themselves to co-culture studies in neural 2D and 3D systems. Importantly, iPSdMiG were efficiently cryopreserved, enabling the establishment of donor-specific microglia cell banks for disease modeling, drug discovery and eventually cell therapy. Main points. Scalable generation of iPSC-derived multi-lineage embryoid bodies on macrocarriers, reproducibly releasing microglia exhibiting characteristic markers and function. Cells are transcriptomically similar to primary human microglia and cryopreservable.
Insights
This study presents a new method to generate large quantities of human microglia from induced pluripotent stem cells (iPSCs). These iPSC-derived microglia (iPSdMiG) mimic primary cells and are suitable for disease modeling and drug discovery.
Area of Science:
- Neuroscience
- Immunology
- Stem Cell Biology
Background:
- Primary human microglia are crucial for understanding neurological disorders but are difficult to obtain for research.
- Induced pluripotent stem cells (iPSCs) offer a potential source, but replicating microglial development has been challenging.
Purpose of the Study:
- To develop a scalable and developmentally informed in vitro method for producing human microglia from iPSCs.
- To characterize the function and viability of these iPSC-derived microglia (iPSdMiG) for biomedical applications.
Main Methods:
- Utilized a 3D bioreactor system with novel mesh macrocarriers for large-scale iPSC culture.
- Differentiated iPSCs to generate yolk sac hematopoietic derivatives and early neuroepithelial cells, facilitating microglial development.
- Harvested iPSdMiG over a 6-week period, yielding substantial cell numbers.
Main Results:
- Generated up to 45 million iPSdMiG from 1 million iPSCs.
- iPSdMiG exhibited transcriptomic profiles highly similar to adult human microglia and expressed key microglial markers.
- Demonstrated functional characteristics including cytokine secretion, phagocytosis, and reactive oxygen species production.
Conclusions:
- The developed method enables scalable, reproducible production of functional iPSC-derived microglia.
- These iPSdMiG are suitable for disease modeling, drug discovery, and potential cell therapy.
- Efficient cryopreservation allows for the creation of donor-specific microglia cell banks.
