Genetic Engineering of Human Mesenchymal Stem Cell Lines to Produce Customized Extracellular Matrix Scaffolds for Tissue Regeneration

Core Concepts

CRISPR/Cas9 can be used to genetically edit human mesenchymal stem cell lines to produce engineered extracellular matrix (eECM) scaffolds with targeted composition and function, enabling the development of customized cell-free tissue engineering solutions.

Abstract

This study demonstrates the use of CRISPR/Cas9 gene editing to engineer human mesenchymal stem cell (hMSC) lines for the production of customized extracellular matrix (ECM) scaffolds. The key highlights are:

VEGF knockout in hMSC lines led to the generation of cartilage eECMs with reduced early-stage vascularization, but retained the capacity to undergo endochondral ossification upon in vivo implantation.

RUNX2 knockout in hMSC lines prevented cartilage hypertrophy in vitro, significantly delaying ectopic bone and bone marrow formation in vivo. This strategy resulted in superior cartilage regeneration in an osteochondral defect model.

The engineered eECMs can be produced as off-the-shelf, cell-free tissue engineering products, leveraging the standardization and design flexibility offered by the hMSC line platform.

This work provides a proof-of-concept for using CRISPR/Cas9 to edit the composition and function of eECMs, enabling the decoding of key factors driving effective tissue regeneration.

Stats

The VEGF protein content in the supernatant of MSOD-B control cells was 5000 pg/mL, while two VEGF-edited clones (MSOD-BΔV1 and MSOD-BΔV2) had undetectable levels of VEGF.
The bone/mineralized volume (BV/TV) ratio of the MSOD-B constructs was 16.46% at 2 weeks and 20.03% at 6 weeks, while the MSOD-BΔR1 constructs had a significantly lower BV/TV of 7.01% at 2 weeks and 4.25% at 6 weeks.
In the rat osteochondral defect model, the cartilage regeneration in the MSOD-BΔR1 group reached approximately 20.67% of the total healthy cartilage area, compared to only 1.05% in the MSOD-B group.

Quotes

"CRISPR/Cas9 can be harnessed to identify the necessary eECM factors driving endogenous repair."
"Our work paves the road towards the compositional eECMs editing and their exploitation in broad regenerative contexts."
"The exploitation of cell lines as an unlimited cell source for eECM generation offers unprecedented standardization in graft production and performance."

Key Insights Distilled From

Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

by Prithiviraj,... at www.biorxiv.org 07-28-2023

https://www.biorxiv.org/content/10.1101/2023.07.28.550935v2

Deeper Inquiries

What other key signaling pathways or structural components could be targeted using CRISPR/Cas9 to further optimize the regenerative capacity of the engineered eECMs?

In addition to VEGF and RUNX2, several other key signaling pathways and structural components could be targeted using CRISPR/Cas9 to enhance the regenerative capacity of engineered eECMs. One potential target could be the TGF-β pathway, which plays a crucial role in tissue repair and regeneration. By modulating the expression of TGF-β and its downstream effectors, such as SMAD proteins, it may be possible to fine-tune the regenerative potential of the eECMs. Additionally, targeting key growth factors like BMPs (Bone Morphogenetic Proteins) or FGFs (Fibroblast Growth Factors) could further enhance the osteogenic or chondrogenic differentiation capacity of the engineered tissues.
Furthermore, editing genes involved in ECM remodeling, such as MMPs (Matrix Metalloproteinases) or TIMPs (Tissue Inhibitors of Metalloproteinases), could help regulate the degradation and turnover of the ECM, leading to more efficient tissue regeneration. Targeting genes related to angiogenesis, such as Angiopoietins or PDGF (Platelet-Derived Growth Factor), could also promote vascularization and improve the integration of the engineered tissues with the host vasculature, enhancing their overall functionality and survival post-implantation.

How could the findings from this study be extended to other tissue engineering applications beyond skeletal repair, such as cardiac, neural, or skin regeneration?

The findings from this study on CRISPR/Cas9 editing of eECMs for skeletal repair could be extended to other tissue engineering applications by applying similar genetic editing strategies to target specific genes relevant to the regeneration of cardiac, neural, or skin tissues. For cardiac regeneration, genes involved in cardiomyocyte differentiation and maturation, such as GATA4 or NKX2.5, could be edited to enhance the cardiac repair capacity of engineered tissues. In neural regeneration, genes related to neural stem cell differentiation, axon guidance, or synaptogenesis could be targeted to promote nerve regeneration and functional recovery.
For skin regeneration, genes involved in keratinocyte proliferation, ECM synthesis, and wound healing processes could be edited to improve the regenerative potential of engineered skin substitutes. By customizing the genetic profile of the eECMs to match the specific requirements of each tissue type, it is possible to create tailored tissue engineering approaches that are optimized for cardiac, neural, or skin regeneration.

Given the potential for off-target effects with CRISPR/Cas9, what strategies could be employed to ensure the long-term safety and stability of the engineered eECM grafts in clinical applications?

To mitigate the potential off-target effects of CRISPR/Cas9 and ensure the long-term safety and stability of engineered eECM grafts in clinical applications, several strategies can be employed. Firstly, thorough validation and characterization of edited cell lines and eECMs should be conducted to identify and minimize off-target effects. This can include whole-genome sequencing, off-target prediction algorithms, and functional assays to assess the specificity of the editing.
Additionally, the use of inducible CRISPR/Cas9 systems, where the editing activity can be controlled or turned off after the desired modifications have been made, can help reduce the risk of off-target effects. Incorporating genetic safeguards, such as suicide genes or kill switches, can provide a fail-safe mechanism to eliminate edited cells with unintended mutations.
Regular monitoring and long-term follow-up studies should be implemented to track the stability and safety of the engineered eECM grafts post-implantation. This can involve imaging techniques, histological analysis, and functional assessments to evaluate the integration, functionality, and potential adverse effects of the engineered tissues over time. By implementing these strategies, the long-term safety and stability of engineered eECM grafts can be ensured in clinical applications.