Regulation of ADSC Osteoinductive Potential Using Notch Pathway Inhibition and Gene Rescue: A Potential On/Off Switch for Clinical Applications in Bone Formation and Reconstructive Efforts.
BACKGROUND
Although there has been tremendous research in the ability of mesenchymal-derived adipose derived stem cells (ADSCs) to form bone, less is known regarding the molecular mechanisms that regulate the osteogenic potential of ADSCs. Notch, which consists of a key family of regulatory ligands involved in bone formation, is expressed in the bone marrow-derived mesenchymal stem cell niche and is critical for proliferation, migration, and ultimately osseous differentiation. The authors investigate how Notch impacts ADSC proliferation and osteogenic differentiation to determine a translatable application of these cells in bone regeneration.


METHODS
Enriched ADSC populations were isolated from tissue and examined for their ability to respond to Notch pathway signaling events. Proliferation, viability, extracellular matrix deposition, and osteoinduction were assessed following Notch activation and inhibition. Notch pathway rescue was conducted using a lentiviral vector encoding a downstream Notch-1 intracellular domain (NICD).


RESULTS
Proliferation, osteogenic induction, and the ability to form bone elements were reduced following Notch inhibition (p < 0.05). However, ADSCs, while in the presence of the Notch inhibition, were able to be rescued following lentiviral transduction with NICD, restoring osteogenic potential at both the molecular and cellular functional levels (p < 0.05).


CONCLUSIONS
These data suggest a potential translatable "on/off switch," using endogenous Notch signaling to regulate the proliferation, differentiation, and osteogenic potential of ADSCs. Although Notch inhibition reduced ADSC proliferation and down-regulated osteoinduction, targeted gene therapy and the delivery of the downstream NICD peptide restored bone formation, suggesting pragmatic clinical utility of ADSCs for bone regeneration.
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Denver M
DM
Lough
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Christopher
C
Chambers
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Gunter
G
Germann
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Ruben
R
Bueno
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Joel
J
Reichensperger
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Edward
E
Swanson
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Mary
M
Dyer
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Lisa
L
Cox
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Carrie
C
Harrison
Springfield, Ill.; and Baltimore, Md.|From the Department of Surgery, Institute for Plastic Surgery, Laboratory for Regenerative Medicine and Applied Sciences, Southern Illinois University; and the Department of Plastic and Reconstructive Surgery, The Johns Hopkins University Hospital.
Michael W
MW
Neumeister
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D004151:Dipeptides / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015316:Genetic Therapy / Q000379:methods*; D048091:Guided Tissue Regeneration / Q000379:methods*; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D008822:Mice, Transgenic; D010012:Osteogenesis / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D051880:Receptors, Notch / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D050151:Subcutaneous Fat / Q000166:cytology; D023822:Tissue Engineering / Q000379:methods*
D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D051880:Receptors, Notch
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