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Histone Trimethylations and HDAC5 Regulate Spheroid Subpopulation and Differentiation Signaling of Human
Ming-Min Chang1,2, Yi-Kai Hong3,4, Chao-Kai Hsu3,4
1Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Abstract:
Human adipose-derived stem cells (ASCs) have shown immense potential for regenerative medicine. Our previous work demonstrated that chitosan nano-deposited surfaces induce spheroid formation and differentiation of ASCs for treating sciatic nerve injuries. However, the underlying cell fate and differentiation mechanisms of ASC-derived spheroids remain unknown. Here, we investigate the epigenetic regulation and signaling coordination of these therapeutic spheroids. During spheroid formation, we observed significant increases in histone 3 trimethylation at lysine 4 (H3K4me3), lysine 9 (H3K9me3), and lysine 27 (H3K27me3), accompanied by increased histone deacetylase (HDAC) activities and decreased histone acetyltransferase activities. Additionally, HDAC5 translocated from the cytoplasm to the nucleus, along with increased nuclear HDAC5 activities. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed the chitosan-induced ASC spheroids and discovered distinct cluster subpopulations, cell fate trajectories, differentiation traits, and signaling networks using the 10x Genomics platform, R studio/language, and the Ingenuity Pathway Analysis (IPA) tool. Specific subpopulations were identified within the spheroids that corresponded to a transient reprogramming state (Cluster 6) and the endpoint cell state (Cluster 3). H3K4me3 and H3K9me3 were discovered as key epigenetic regulators by IPA to initiate stem cell differentiation in Cluster 6 cells, and confirmed by qPCR and their respective histone methyltransferase inhibitors: SNDX-5613 (a KMT2A inhibitor for H3K4me3) and SUVi (an SUV39H1 inhibitor for H3K9me3). Moreover, H3K9me3 and HDAC5 were involved in regulating downstream signaling and neuronal markers during differentiation in Cluster 3 cells. These findings emphasize the critical role of epigenetic regulation, particularly H3K4me3, H3K9me3, and HDAC5, in shaping stem cell fate and directing lineage-specific differentiation.
Insights
Chitosan surfaces promote adipose-derived stem cell (ASCs) spheroid formation, revealing key epigenetic regulators like H3K4me3, H3K9me3, and HDAC5 that control cell fate and neuronal differentiation for regenerative medicine.
Area of Science:
- Stem cell biology
- Epigenetics
- Regenerative medicine
Background:
- Human adipose-derived stem cells (ASCs) show promise in regenerative medicine.
- Previous studies showed chitosan surfaces induce ASC spheroid formation for nerve repair.
- The mechanisms of ASC spheroid differentiation remain unclear.
Purpose of the Study:
- To investigate the epigenetic regulation and signaling pathways governing ASC spheroid formation and differentiation.
- To identify key molecular players in ASC cell fate determination induced by chitosan surfaces.
Main Methods:
- Chitosan nano-deposited surfaces were used to induce ASC spheroid formation.
- Histone modifications (H3K4me3, H3K9me3, H3K27me3) and histone deacetylase (HDAC) activities were analyzed.
- Single-cell RNA sequencing (scRNA-seq) was performed on ASC spheroids.
- Ingenuity Pathway Analysis (IPA), qPCR, and specific inhibitors (SNDX-5613, SUVi) were used to identify regulatory roles.
Main Results:
- Spheroid formation correlated with increased H3K4me3, H3K9me3, H3K27me3, and HDAC activity, with nuclear translocation of HDAC5.
- scRNA-seq identified distinct cell subpopulations, including transient reprogramming (Cluster 6) and differentiated (Cluster 3) states.
- IPA identified H3K4me3 and H3K9me3 as critical for initiating differentiation in Cluster 6.
- H3K9me3 and HDAC5 were found to regulate neuronal markers in Cluster 3.
Conclusions:
- Epigenetic modifications, specifically H3K4me3 and H3K9me3, are crucial for initiating stem cell differentiation in ASC spheroids.
- HDAC5 plays a significant role in regulating downstream signaling and neuronal differentiation.
- These findings elucidate the epigenetic mechanisms driving ASC spheroid differentiation, paving the way for optimized regenerative therapies.
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