CRISPRi-Driven Osteogenesis in Adipose-Derived Stem Cells for Bone Healing and Tissue Engineering

Abstract

Engineered bone tissue synthesized from mesenchymal stem cell progenitors has numerous applications throughout the fields of regenerative medicine and tissue engineering. However, these multipotent cells offer little tissue-building assistance without differentiation direction from environmental cues such as bone morphogenetic proteins (BMPs). Unfortunately, BMP dosing and environmental cues can be difficult to control both in vitro and after in vivo delivery. Several BMP antagonists are expressed by cells in response to BMP dosing that bind extracellular BMPs and reduce their effective concentration. Here, we use CRISPR-guided gene-modulation technology to downregulate the expression of three BMP antagonists, noggin, gremlin-1, and gremlin-2, in adipose-derived stem cells (ASCs). We show that regulating noggin using this method results in ASC osteogenesis without the need for exogenous growth factors. To demonstrate the versatility and the precision capabilities of these engineered cells, we employ them with CRISPRa multiplex-engineered chondrogenic cells as a proof-of-concept tissue engineering application by creating a tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the tendon/ligament enthesis or intervertebral disc attachment. In doing so, we show that multiple CRISPR multiplex-engineered cell types can be utilized in concert to provide a high degree of tissue developmental control without the use of exogenous growth factors.