Epigenetic remodeling during monolayer cell expansion reduces therapeutic potential

Abstract

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies require two-dimensional (2D) cell expansion processes to achieve large cell populations critical for the repair of damaged (e.g. connective and musculoskeletal) tissues. However, the influence of mechanical memory on cell fate following expansion is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we show that the organization of histone H3 trimethylated at lysine 9 (H3K9me3) and expression of tissue-identifying genes in primary cartilage cells (chondrocytes) transferred to three-dimensional (3D) hydrogels depends on the number of previous population doublings on tissue culture plastic during 2D cell expansion. Decreased levels of H3K9me3 occupying promoters of dedifferentiation genes after the 2D culture were also retained in 3D culture. Suppression of H3K9me3 during expansion of cells isolated from a murine model similarly resulted in the loss of the chondrocyte phenotype and global remodeling of nuclear architecture. In contrast, increasing levels of H3K9me3 through inhibiting H3K9 demethylases partially rescued the chondrogenic nuclear architecture and gene expression, which has important implications for tissue repair therapies, where expansion of large numbers of phenotypically-suitable cells is required. Overall, our findings indicate mechanical memory in primary cells is encoded in the chromatin architecture, which impacts cell fate and the phenotype of expanded cells.SIGNIFICANCE STATEMENTTissue regeneration procedures, such as cartilage defect repair (e.g. Matrix-induced Autologous Chondrocyte Implantation) often require cell expansion processes to achieve sufficient cells to transplant into an in vivo environment. However, the chondrocyte cell expansion on 2D stiff substrates induces epigenetic changes that persist even when the chondrocytes are transferred to a different (e.g. 3D) or in vivo environment. Treatments to alter epigenetic gene regulation may be a viable strategy to improve existing cartilage defect repair procedures and other tissue engineering procedures that involve cell expansion.

Epigenetic Priming Enhances Chondrogenic Potential of Expanded Chondrocytes for Cartilage Repair

“…Inhibiting the demethylase of H3K9me3, surprisingly, did not alter the peripheral enrichment of H3K9me3. We have previously found that ML324 applied at higher concentrations (10µM) to chondrocytes during the 2D expansion process causes an increase in dense regions of H3K9me3 toward the nuclear envelop and an increase in chondrogenic gene expression 9 . Therefore, we anticipated that treating the chondrocytes with ML324 prior to encapsulation would increase the chondrogenic phenotype in 3D culture.…”

“…Chondrocytes were encapsulated at a concentration of 10×10 6 cells/mL after isolation (PD0), 8 population doublings (PD8) and 16 population doublings (PD16) in 3D hyaluronan-poly(ethylene) glycol diacrylate (HA-PEGDA) hydrogels (20 mg/1mL of HA and 8.6 mg/mL PEGDA) and cultured for 1, 5, and 10 days as previously described 9 .…”

“…Increasing evidence links spatial organization of the genome to gene function and cellular memory. Recent literature, along with work from our lab, reveals that chromatin architecture maintains mechanical memories after exposure to previous non-native or stiff environments 9,13 . Specifically, we have shown that a structural change of trimethylated H3K9 (H3K9me3) marked chromatin, an increase in H3K9me3 foci, occurs with dedifferentiation of chondrocytes and retains a memory of the dedifferentiated state, preventing redifferentiation 9 .…”

“…When the dedifferentiated chondrocytes are transferred to a 3D scaffold , it is well-known that the chondrocyte hyaline morphology returns 8 . However, our previous work demonstrates that the dedifferentiated fibrotic phenotype is remembered in 3D culture and gene expression of hyaline cartilage does not return 9 . Thus, chondrocytes are influenced by the previous 2D mechanical environment when transferred to the 3D environment, demonstrating evidence of mechanical memory 4,10,11 .…”

“…culture and gene expression of hyaline cartilage does not return 9 . Thus, chondrocytes are influenced by the previous 2D mechanical environment when transferred to the 3D environment, demonstrating evidence of mechanical memory 4,10,11 .…”