Carrie Harrison

Stimulation of the follicular bulge LGR5+ and LGR6+ stem cells with the gut-derived human alpha defensin 5 results in decreased bacterial presence, enhanced wound healing, and hair growth from tissues devoid of adnexal structures.

Background: Discovery of leucine-rich repeat-containing G-protein–coupled receptors 5 and 6 (LGR5 and LGR6) as markers of adult epithelial stem cells of the skin and intestine permits researchers to draw on the intrinsic cellular fundamentals of wound healing and proliferation dynamics of epithelial surfaces. In this study, the authors use the intestine-derived human alpha defensin 5 to stimulate epithelial proliferation, bacterial reduction, and hair production in burn wound beds to provide the field with initial insight on augmenting wound healing in tissues devoid of adnexal stem cells. Methods: Murine third-degree burn wound beds were treated with (1) intestine-derived human alpha defensin 5, (2) skin-derived human beta defensin 1, and (3) sulfadiazine to determine their roles in wound healing, bacterial reduction, and hair growth. Results: The human alpha defensin 5 peptide significantly enhanced wound healing and reduced basal bacterial load compared with human beta defensin 1 and sulfadiazine. Human alpha defensin 5 was the only therapy to induce LGR stem cell migration into the wound bed. In addition, gene heat mapping showed significant mRNA up-regulation of key wound healing and Wnt pathway transcripts such as Wnt1 and Wisp1. Ex vivo studies showed enhanced cell migration in human alpha defensin 5–treated wounds compared with controls. Conclusions: Application of human alpha defensin 5 increases LGR stem cell migration into wound beds, leading to enhanced healing, bacterial reduction, and hair production through the augmentation of key Wnt and wound healing transcripts. These findings can be used to derive gut protein–based therapeutics in wound healing.

Transplantation of the LGR6+ epithelial stem cell into full-thickness cutaneous wounds results in enhanced healing, nascent hair follicle development, and augmentation of angiogenic analytes.

Background: The recently discovered leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6+) epithelial stem cell located within the follicular bulge of the adnexal compartment is capable of producing all cellular lineages of the skin. In this study, the authors sought to determine whether these cells can be transplanted for use as a type of cellular therapy for the repair of full-thickness wounds in which the native stem cell niche has been obliterated. Methods: Full-thickness murine skin was harvested and LGR6+GFP epithelial stem cells were isolated using fluorescence-activated cell sorting. This enriched epithelial stem cell population was then transplanted by means of local injection into wound beds on the dorsum of nude mice. Viability, migration, healing, the development of nascent hair follicles, and gene and proteomic expression studies were performed to determine whether the engraftment of LGR6+GFP epithelial stem cells enhanced healing when compared with controls. Results: Wound beds receiving LGR6+GFP epithelial stem cells showed enhanced healing; nascent follicle growth; and augmentation of the Wnt, vascular endothelial growth factor, epidermal growth factor, and platelet-derived growth factor pathways when compared with controls. Conclusions: The LGR6+ epithelial stem cells appear to hold great promise for the development of a clinically useful stem cell–based therapy for the repair of full-thickness wounds and hair regeneration. These results indicate that transplantation of LGR6+ epithelial stem cells promotes epithelialization, hair growth, and angiogenesis in tissues destined for scar formation.

Adipose-derived stem cell to epithelial stem cell transdifferentiation: a mechanism to potentially improve understanding of fat grafting's impact on skin rejuvenation.

BACKGROUND Recent evidence suggests that lipofilling improves overlying skin composition and appearance. Adipose-derived stem cells (ADSC) have been implicated. OBJECTIVE The authors identify ADSC transdifferentiation into epithelial stem cells through coexpression of GFP+ (green fluorescent protein positive) ADSC with the epithelial stem cell marker p63 in an in vivo fat grafting model. METHODS Six male, GFP+ mice served as adipose tissue donors. Twelve nude mice served as recipients. Recipients were subdivided into 2 arms (6 mice/each arm) and received either whole-fat specimen (group 1) or isolated and purified ADSC + peptide hydrogel carrier (group 2) engrafted into a 1-cm(2) left parascapular subdermal plane. The right parascapular subdermal plane served as control. Skin flaps were harvested at 8 weeks and subjected to (1) confocal fluorescent microscopy and (2) reverse transcriptase polymerase chain reaction (RT-PCR) for p63 mRNA expression levels. RESULTS Gross examination of skin flaps demonstrated subjectively increased dermal vessel presence surrounding whole-fat and ADSC specimens. The GFP+ cells were seen within overlying dermal architecture after engraftment and were found to coexpress p63. Significantly increased levels of p63 expression were found in the ADSC + hydrogel skin flaps. CONCLUSIONS We offer suggestive evidence that GFP+ ADSC are found within the dermis 8 weeks after engraftment and coexpress the epithelial stem cell marker p63, indicating that ADSC may transdifferentiate into epithelial stem cells after fat grafting. These findings complement current understanding of how fat grafts may rejuvenate overlying skin.

Transplantation of an LGR6+ Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin.

Background: Recent literature has shown that full-thickness wounds, devoid of the stem cell niche, can subsequently be reconstructed with functional skin elements following migration of the LGR6+ epithelial stem cell into the wound bed. In this study, the authors use a variety of LGR6+ epithelial stem cell–seeded scaffolds to determine therapeutic utility and regenerative potential in the immediate reconstruction of full-thickness wounds. Methods: Isolated LGR6+ epithelial stem cells were seeded onto a spectrum of acellular matrices and monitored in both in vitro and in vivo settings to determine their relative capacity to regenerate tissues and heal wounds. Results: Wound beds containing LGR6+ stem cell–seeded scaffolds showed significantly augmented rates of healing, epithelialization, and hair growth compared with controls. Gene and proteomic expression studies indicate that LGR6+ stem cell–seeded constructs up-regulate WNT, epidermal growth factor, and angiogenesis pathways. Finally, the addition of stromal vascular fraction to LGR6+ stem cell–seeded constructs induces polarized tissue formation, nascent hair growth, and angiogenesis within wounds. Conclusions: LGR6+ stem cells are able to undergo proliferation, differentiation, and migration following seeding onto a variety of collagen-based scaffolding. In addition, deployment of these constructs induces epithelialization, hair growth, and angiogenesis within wound beds. The addition of stromal vascular fraction to LGR6+ stem cell–containing scaffolds initiated an early form of tissue polarization, providing for the first time a clinically applicable stem cell–based construct that is capable of the repair of full-thickness wounds and hair regeneration. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

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.

Abstract 154: the anti-neoplastic effect of aminosterol squalamine on melanoma.

PurPose: Squalamine, a recently discovered aminosterol, has notably been shown to be effective as both an antibiotic and an inhibitor of angiogenesis. This intrinsic anti-angiogenic effect of squalamine has been studied in the literature for its clinical capacity to reduce cancer progression. Here, we suggest an additional role of squalamine in inducing melanoma destruction via induction of apoptosis and or necrosis of melanotic cells through angiogenic inhibition.

What's your diagnosis? Dyspnea and Porphyria in Rats

cages with hardwood bedding. Based on the surgical history of these rats, what could cause clinical and necropsy signs such as these? Our veterinary staff received a male 375-g Wistar rat in severe respiratory distress. On the previous day the rat had undergone surgery. An experienced research technologist, who had completed the identical procedure successfully in the past, did the surgery. He had anesthetized the rat with pentobarbital, 42 mg/kg intraperitoneally (i.p.) and did not require a second dose for adequate maintenance of anesthesia. Completion of the surgical procedure required approximately 60 minutes. Using aseptic technique, the research technologist placed a silicone implant subcutaneously in the right inguinal region and sutured it to the abdominal wall to prevent migration. After the surgery, he gave the rat a subcutaneous (s.c.) injection of buprenorphine (0.08 mg/kg) and returned him to his cage. When checked the next morning, the rat was salivating and had some bedding stuck to the area around his mouth. Approximately 36 hours after surgery, the rat was dyspneic and appeared to be choking. He was breathing heavily through his mouth and was salivating excessively, causing bedding to become matted around his mouth (Fig. 1). Porphyrin staining was present around the rat’s eyes and on his front feet (Fig. 1). Consequently, we decided to euthanize the rat. The research technician who euthanized the rat and removed the implant noted no obvious complications associated with the surgery. Veterinary staff did a necropsy immediately after euthanasia and found the esophagus to be grossly distended and strawlike in color (Fig. 2). The distension and discoloration extended from the cardiac orifice of the stomach to the cranial aspect of the thorax. The stomach was full and appeared larger than normal (Fig. 2), and the intestines were distended from gas with a small amount of fecal matter present. Esophageal and gastric contents were strawcolored, fibrous, compacted, dry, and crumbly upon manipulation (Fig. 3). Almost no food was present in the stomach. We did not see lesions in any other organs: the lungs were pink, the trachea was clear of any apparent obstruction or damage, the heart was unremarkable, and the thoracic cavity was free of fluid. We tested serum inhouse for Sendai virus, mycoplasma, and coronavirus using the ImmunoComb system (Charles River Laboratories, Wilmington, MA), with negative results. Several days later, another rat presented with history and clinical signs similar to those of the first rat. We also euthanized and necropsied this rat, and found similar lesions. We had maintained both rats on ad libitum food and water before and after Thomas M. Donnelly, DVM, Column Editor