Synergistic CRISPRa-Regulated Chondrogenic Extracellular Matrix Deposition Without Exogenous Growth Factors

Abstract

Stem cell therapies have shown promise for regenerative treatment for musculoskeletal conditions, but their success is mixed. To enhance regenerative effects, growth factors are utilized to induce differentiation into native cell types, but uncontrollable in vivo conditions inhibit differentiation, and precise control of expressed matrix proteins is difficult to achieve. To address these issues, we investigated a novel method of enhancing regenerative phenotype through direct upregulation of major cartilaginous tissue proteins, aggrecan (ACAN), and collagen II (COL2A1) using dCas9-VPR CRISPR gene activation systems. We demonstrated increased expression and deposition of targeted proteins independent of exogenous growth factors in pellet culture. Singular upregulation of COL2A1/ACAN interestingly indicates that COL2A1 upregulation mediates the highest sulfated glycosaminoglycan (sGAG) deposition, in addition to collagen II deposition. Through RNA-seq analysis, this was shown to occur by COL2A1 upregulation mediating broader chondrogenic gene expression changes. Multiplex upregulation of COL2A1 and ACAN together resulted in the highest sGAG, and collagen II deposition, with levels comparable to those in chondrogenic growth factor-differentiated pellets. Overall, this work indicates dCas9-VPR systems can robustly upregulate COL2A1 and ACAN deposition without growth factors, to provide a novel, precise method of controlling stem cell phenotype for cartilage and intervertebral disc cell therapies and tissue engineering. Impact statement Stem cell therapies have come about as a potential regenerative treatment for musculoskeletal disease, but clinically, they have mixed results. To improve stem cell therapies, growth factors are used to aid a regenerative cell phenotype, but their effects are inhibited by in vivo musculoskeletal disease environments. This article describes CRISPR gene activation-based cell engineering methods that provide a growth factor-free method of inducing chondrogenic extracellular matrix deposition. This method is demonstrated to be as/more potent as growth factors in inducing a chondrogenic phenotype in pellet culture, indicating potential utility as a method of enhancing stem cell therapies for musculoskeletal disease.

Keywords: CRISPRa; cartilage; cell engineering; intervertebral disc; stem cell.

FIG. 1.

Design and verification of CRISPRa vectors. (A) Map of lentiviral vectors for dCas9-VPR expression cassette and gRNA expression cassette. (B) Sequence of gRNAs designed to target each gene and their respective PAM sequence. (C) Gene expression changes of ACAN in cells transduced with ACAN targeting gRNAs relative to NTC cells (n = 3, *p < 0.05 relative to NTC group). (D) Gene expression changes of COL2A1 in cells transduced with COL2A1 targeting gRNAs relative to NTC cells (n = 3, *p < 0.05 relative to NTC group). (E) Difference in normalized gene expression of ACAN or COL2A1 in NTC cells or ACAN or COL2A1 upregulated cells relative to COL2A1 cells with gRNA inducing highest gene upregulation (n = 3, *p < 0.05 relative to COL2A1 group). CRISPRa, CRISPR gene activation; NTC, nontarget control.

FIG. 2.

Qualitative analysis of pellet cultures with singular gene upregulation. (A) Macroscopic images of cell pellets demonstrating their morphological similarities and differences (scale bar is 2 mm). (B) Alcian blue staining of cell pellets (scale bar is 40 μm). (C) Collagen II IHC of cell pellets (scale bar is 40 μm). IHC, immunohistochemistry.

FIG. 3.

Biochemical assays quantifying ECM content in pellet cultures. (A) Total sGAG produced by pellet cultures. (B) Total collagen produced by pellet cultures (n = 5–10, *p < 0.05 relative to NTC, dotted line represents amounts produced by NTC + GF cell pellets). sGAG, sulfated glycosaminoglycan.

FIG. 4.

RNA-seq analysis of cells cultured in pellet culture for 1 week. (A) Heat map of differentially expressed genes in all cell groups (B, C) Differential gene expression analysis of ACAN (B) or COL2A1 (C) upregulated cells versus NTC cells. (D) Top five biological processes associated with genes differentially expressed after COL2A1 upregulation. (E) Fold change and significance of gene expression changes in proteoglycans and enzymes associated with proteoglycans and chondroitin sulfate synthesis after COL2A1 upregulation.

FIG. 5.

Verification of multiplex gene upregulation by CRISPRa vectors. (A) Map of lentiviral vectors for dCas9-VPR expression cassette and gRNA expression cassettes expressed in hADSCs for multiplex gene upregulation. (B) Sequence of gRNAs targeting each gene and their respective PAM sequence. (C) Gene expression changes of ACAN in cells with multiplex ACAN & COL2A1 upregulation relative to NTC cells (n = 3, *p < 0.05 relative to NTC group). (D) Gene expression changes of COL2A1 in cells with multiplex ACAN & COL2A1 upregulation relative to NTC cells (n = 3, *p < 0.05 relative to NTC group).

FIG. 6.

Qualitative analysis of pellet cultures. (A) Macroscopic images of cell pellets demonstrating their morphological similarities and differences (scale bar is 2 mm). (B) Alcian blue staining of cell pellets (scale bar is 40 μm). (C) Collagen II IHC of cell pellets (scale bar is 40 μm).

FIG. 7.

Biochemical assays quantifying ECM content in pellet cultures. (A) Total sGAG produced by pellet cultures. (B) Total collagen produced by pellet cultures (n = 5–6, *p < 0.05 relative to NTC, dotted line represents amounts produced by NTC + GF cell pellets).