Microglia constitute approximately 10% of the cells within the central nervous system, where they serve multifaceted roles from clearing infectious agents and cellular debris to sculpting neural circuits during brain development. Dysregulation of microglial function is increasingly recognized as a driver of neuroinflammation, which precedes and exacerbates hallmark protein aggregation in devastating disorders such as Alzheimer’s, Parkinson’s, Huntington’s diseases, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. Understanding microglial biology and manipulating their activity therapeutically has long been hampered by the scarcity of human cells and significant interspecies differences that limit rodent models’ translational reliability.

The core breakthrough rests on the use of TFome™—an innovative synthetic biology platform that systematically screens and applies combinations of human transcription factors (TFs) to steer iPSC fate decisions with remarkable precision and speed. Transcription factors are proteins that act as master regulators driving entire gene expression networks, thereby orchestrating cellular identity and function. Prior to this study, attempts to cultivate microglia-like cells from stem cells were inefficient, protracted, and often yielded immature or functionally limited cells. Employing iterative rounds of TF screening and single-cell RNA sequencing (scRNA-seq), the Wyss-HMS team distilled a potent sextet of microglia-specifying TFs that unlock rapid differentiation and maturation in only four days.

The researchers initiated the process by curating a smartly selected panel of 40 candidate TFs, informed by developmental biology and disease-specific expression profiles characteristic of primary human microglia. By randomly expressing combinations of five to seven TFs in single iPSCs and assessing their genetic profiles through scRNA-seq, the team identified a triumvirate of TFs—SPI1, CEBPA, and FLI1—that induced partial microglial programming. Recognizing that this initial combination was insufficient for full functional maturation, the group supplemented the cocktail with three additional TFs—MEF2C, CEBPB, and IRF8—elevating the cellular phenotype to closely match native microglia both transcriptionally and morphologically.

Crucially, these engineered microglia-like cells exhibited hallmark responses to neuroinflammatory stimuli, a fundamental functional test. Exposure to interferon gamma (IFNγ), a cytokine elevated during brain infections and neurodegenerative states, provoked activation of microglia-specific gene expression programs. Remarkably, the aggregates of TDP-43 protein—a pathological feature in ALS—similarly elicited microglial gene expression changes, underscoring the physiological relevance of these stem cell-derived microglia surrogates.

The implications of this technology extend far beyond mere cell culture convenience. By expediting the derivation of highly functional human microglia, the TFome™ platform empowers researchers to faithfully model neuroinflammatory processes implicated in myriad neurological diseases. It paves the way for high-throughput drug screening, mechanistic exploration, and personalized medicine interventions with patient-specific iPSC lines. Moreover, the modularity and adaptability of TFome™ technology portend its application to other elusive cell types, potentially revolutionizing regenerative medicine and cell therapy product development.

This advance builds upon prior work in which the Wyss Institute team developed a comprehensive library of 1,732 human transcription factors and variants, laying the foundation for precision control of stem cell fates. Notably, the founders also established GC Therapeutics, a biotechnology startup aiming to translate transcription factor-based cell engineering into commercially viable cell therapies. The current study signifies a major refinement of their platform, showcasing iterative design and data-driven optimization harnessed by single-cell transcriptomics to achieve rapid and target-specific cellular identity.

Underlying this cellular engineering feat is an integrated interdisciplinary effort combining synthetic biology, computational genomics, bioinformatics, and neural cell biology. Collaborators included experts in statistics and single-cell data analysis who developed algorithms to rank TF combinations by their effectiveness in recapitulating authentic microglial gene expression signatures. Such iterative screening—cycling through design, experimental testing, and computational validation—proved essential to identifying the optimal transcriptional code for human microglia induction.

The team’s focus on microglia originated from a longstanding interest in creating complex brain organoids—three-dimensional miniaturized tissue models—which aim to recapitulate cellular diversity and functional intercellular interactions present in the human brain. While TFome™ technology had allowed generation of neuronal, oligodendrocyte, stromal, and vascular components of brain organoids, microglia presented a tougher challenge due to their unique developmental origins and transcriptional programs. Addressing this gap enhances the physiological relevance of brain organoids, expanding their utility in modeling neurodevelopment, neurodegeneration, and neuroinflammation.

Looking forward, the researchers envision fine-tuning TF expression dynamics—varying timing, dosage, and sequence—to engineer microglia subtypes with specialized activities. This precision could unravel cell-type-specific contributions to brain pathologies and enable targeted interventions that modulate particular microglial functions. Their approach exemplifies a synthetic biology paradigm in which modular genetic parts enable custom design of complex cellular phenotypes within unprecedented timeframes.

In sum, this iterative transcription factor screening method exemplifies a leap forward in stem cell biology and neuroimmunology. By successfully producing microglia-like cells that combine rapid generation with mature, functionally relevant profiles, investigators have unlocked a promising new avenue for studying brain immune cells and their roles in health and disease. As neurodegenerative disorders continue to exact a growing global toll, innovative tools such as this offer fresh hope for decoding disease mechanisms and discovering effective therapeutics.

Subject of Research: Cells

Article Title: Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC

News Publication Date: 10-Jun-2025

Web References:

• Wyss Institute at Harvard University: https://wyss.harvard.edu/
• Harvard Medical School: https://hms.harvard.edu/
• GC Therapeutics: https://www.gc-tx.com/
• TFome™ technology Nature publication: https://www.nature.com/articles/s41587-020-0742-6
• CircaVent drug discovery platform: https://wyss.harvard.edu/technology/circavent-a-drug-discovery-platform-for-mental-health-conditions/

References:
Liu, S., Zhang, F., Li, L., et al. Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC. Nature Communications. 2025 Jun 10.

Image Credits: Wyss Institute at Harvard University

The team, led by Fu, Okawa, and Vinaikosol, focused on the effects of shaking culture, a prevalent mechanical stimulation technique, on the circadian clock machinery inherent in iPSCs undergoing osteogenic differentiation. Circadian rhythms, the approximately 24-hour cycles governing physiological processes, have previously been shown to influence cellular functions ranging from metabolism to cell division. However, their role and modulation during stem cell fate determination have remained enigmatic and underexplored.

Central to the study is the TEAD-Fbxl3-CRY axis, a molecular cascade that integrates mechanical cues with circadian regulation. TEAD transcription factors, known for their pivotal role in controlling gene expression linked to cell proliferation and differentiation, appear to orchestrate the downstream activity of Fbxl3, an F-box protein that targets circadian repressors such as CRY for ubiquitination and proteasomal degradation. This axis acts as an essential intermediary transforming mechanical forces from the shaking culture environment into tangible alterations in the molecular circadian clock.

Experimental evidence from carefully controlled shaking culture systems demonstrated a marked attenuation of circadian amplitude in iPSCs progressing toward osteoblast-like phenotypes. This attenuation manifests as dampened oscillations in core clock genes, particularly cryptochromes (CRYs), which are fundamental repressors within the circadian feedback loop. The dampened rhythms suggest that continuous mechanical stimulation disrupts the normal temporal signaling required for finely tuned gene expression during differentiation.

The importance of this finding lies in the intricate interplay between circadian biology and stem cell fate decisions. Osteogenic differentiation is a tightly regulated process involving sequential activation and repression of lineage-specific genes. Disruption of circadian rhythms may lead to aberrant timing in gene expression, potentially influencing the functionality and quality of differentiated bone cells. This concept challenges existing paradigms that often overlook temporal regulation in the differentiation milieu.

Moreover, the identification of the TEAD-Fbxl3-CRY axis as a key mediator provides a molecular handle to manipulate circadian dynamics experimentally. By modulating TEAD activity or Fbxl3 expression, it may be possible to rescue or fine-tune circadian oscillations even under mechanical stress. Such interventions could enhance the robustness and predictability of stem cell-derived osteogenic therapies, which are essential for skeletal repair and regeneration.

Mechanotransduction, the process through which cells convert mechanical stimuli into biochemical signals, has been increasingly recognized as a critical regulator in stem cell biology. Shaking culture represents a form of dynamic mechanical stimulation that mimics physiological movements inherent to bone tissue. Insights from this study suggest that mechanotransduction not only influences cytoskeletal organization and gene expression but extends its regulatory reach to the cellular timekeeping mechanisms.

Interestingly, this attenuation of circadian rhythms under shaking conditions contrasts with previous observations in static cultures, highlighting the complexity of in vitro differentiation systems. These findings underscore the necessity to consider culture conditions as active variables that profoundly affect cellular phenotypes and molecular states. It also raises questions about how in vivo mechanical forces influence circadian biology during bone development and remodeling.

The researchers employed sophisticated techniques such as real-time bioluminescence reporting of circadian gene activity, quantitative PCR, and protein interaction assays to delineate the molecular underpinnings of this phenomenon. The convergence of these methodologies enabled a comprehensive characterization of the TEAD-Fbxl3-CRY pathway’s involvement and established a direct causative link between mechanical cues and circadian modulation.

Looking ahead, this work paves the way for designing biomimetic culture systems that could harness or circumvent mechanical influences on circadian regulation, tailoring differentiation protocols with temporal precision. Such approaches could optimize the generation of bone tissue constructs with enhanced functionality and integration potential upon transplantation.

Beyond osteogenesis, the broader implications of this research hint at an overarching principle wherein mechanical environments serve as temporal “zeitgebers” (time-givers), synchronizing or disrupting circadian clocks across diverse stem cell types and tissues. Future studies may explore whether similar mechanisms operate in other lineage pathways and how these dynamics affect aging, disease susceptibility, and tissue homeostasis.

This revelation of shaking culture’s influence on circadian rhythms also resonates with an evolving appreciation of chronobiology in regenerative medicine. By aligning cell therapy strategies with circadian principles, clinicians and researchers may improve therapeutic efficacy, minimize adverse effects, and promote long-term tissue health.

In conclusion, the identification of the TEAD-Fbxl3-CRY axis as a bridge between mechanical stimuli and circadian attenuation in iPSC-derived osteogenic cells represents a significant advance in stem cell biology. It challenges us to rethink the temporal dimension within differentiation paradigms, emphasizing that the “when” can be just as critical as the “what” and “how” in guiding cell fate and function. As this line of research progresses, it promises to unlock new strategies for better harnessing the potential of stem cells in medicine, engineering, and understanding fundamental biological rhythms.

Subject of Research:
Article Title:
Article References: Fu, Y., Okawa, H., Vinaikosol, N. et al. Shaking culture attenuates circadian rhythms in induced pluripotent stem cells during osteogenic differentiation through the TEAD-Fbxl3-CRY axis. Cell Death Discov. 11, 252 (2025). https://doi.org/10.1038/s41420-025-02533-6
Image Credits: AI Generated
DOI: https://doi.org/10.1038/s41420-025-02533-6