Control of Adipogenic Differentiation in Mesenchymal Stem Cells via Endogenous Gene Activation Using CRISPR-Cas9

Abstract

Mesenchymal stem cells (MSCs) are of interest in regenerative medicine owing to their multilineage differentiation and self-renewal properties. Understanding the in vivo differentiation process is necessary for clinical applications including cell therapy and transplantation. This remains challenging owing to the lack of induction methods that imitate the natural programming process. Endogenous gene regulation of tissue-specific transcription factors is therefore desirable. In the present study, we demonstrated endogenous activation of adipogenic genes through the dCas9-based transcription system and achieved efficient induction of different types of adipocyte-like cells from MSCs. Interestingly, the MSCs converted via single-gene activation exhibited morphological and molecular properties of white adipocytes, while beige adipocyte-like cells were induced via multiplex gene activation of three specific transcription factors. These results reveal that the fate of MSCs can be effectively manipulated by direct activation of specific endogenous gene expression using a dCas9-based activator with reduced exogenous additives.

CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration

“…To date, CRISPRa has been harnessed to induce neuronal differentiation of embryonic stem cells (15), mouse embryonic fibroblasts (MEF) (21) or induced pluripotent stem cells (iPSC) (12,36), reprogram MEF (23) or fibroblast (37) to iPSC, as well as induce MSC differentiation into adipogenic lineage (38). CRISPRi has also been exploited for gene knockdown in pluripotent stem cells to regulate cell fate (22) or induce spatiotemporal mosaic self-patterning (22).…”

“…Besides, CRISPRa/CRISPRi systems are commonly delivered using plasmids (13,43), lentivirus (10,38,44) or adeno-associated virus (AAV) (39). For instance, CRISPRa is delivered using lentivirus for activation of endogenous adipogenic genes and induction of BMSC differentiation into adipocyte-like cells (38). CRISPRa is also carried using AAV to activate utrophin to ameliorate muscular dystrophy symptoms in mouse models (39).…”

“…dCas9, MPH and Com-KRAB) in a single vector. Consequently, the dCas9 regulator, effector and sgRNA are often split into separate viral vectors (21,38,39) and concurrent delivery of all CRISPRa/CRISPRi components to the same cells is necessary, and challenging (46). Furthermore, lentivirus integrates the gene cassettes into chromosome, which is undesired from the perspective of regenerative medicine.…”

CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration

“…CRISPRa technology has been exploited for programmable gene activation for genome-wide genetic screening 27, identification of long non-coding RNA involved in drug resistance 28 and in vitro cell reprogramming. With regard to cell fate manipulation in vitro , CRISPRa has been harnessed to convert mouse embryonic fibroblasts to neuronal cells 29, reprogram mouse embryonic 30 or human skin 31 fibroblasts to induced pluripotent stem cells (iPSC), accelerate the differentiation of human iPSC into neurons and astrocytes 32, as well as induce mesenchymal stem cell differentiation into adipocyte-like cells 33. Moreover, CRISPRa has been employed to treat muscular dystrophy symptoms in mouse models 34.…”

“…Here we exploited the SAM system 22 to simultaneously active 3 neurotrophic genes in ASC sheets in order to promote nerve regeneration. We chose the SAM system because it was proven to be superior or at least comparable to other CRISPRa systems with regard to activation magnitude 37, and SAM system was already used to treat muscular dystrophy 34 and cell reprogramming 33, 38. In these studies, the SAM-based CRISPRa systems were delivered by plasmids 22 or viral vectors such as adeno-associated virus (AAV) or lentivirus 33, 38.…”