Graham Gordon Walmsley

All Publications

• High-Throughput Screening of Surface Marker Expression on Undifferentiated and Differentiated Human Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Walmsley, G. G., Atashroo, D. A., Maan, Z. N., Hu, M. S., Zielins, E. R., Tsai, J. M., Duscher, D., Paik, K., Tevlin, R., Marecic, O., Wan, D. C., Gurtner, G. C., Longaker, M. T. 2015; 21 (15-16): 2281-2291

  Abstract

  Adipose tissue contains an abundant source of multipotent mesenchymal cells termed "adipose-derived stromal cells" (ASCs) that hold potential for regenerative medicine. However, the heterogeneity inherent to ASCs harvested using standard methodologies remains largely undefined, particularly in regards to differences across donors. Identifying the subpopulations of ASCs predisposed toward differentiation along distinct lineages holds value for improving graft survival, predictability, and efficiency. Human ASCs (hASCs) from three different donors were independently isolated by density-based centrifugation from adipose tissue and maintained in culture or differentiated along either adipogenic or osteogenic lineages using differentiation media. Undifferentiated and differentiated hASCs were then analyzed for the presence of 242 human surface markers by flow cytometry analysis. By comprehensively characterizing the surface marker profile of undifferentiated hASCs using flow cytometry, we gained novel insights into the heterogeneity underlying protein expression on the surface of cultured undifferentiated hASCs across different donors. Comparison of the surface marker profile of undifferentiated hASCs with hASCs that have undergone osteogenic or adipogenic differentiation allowed for the identification of surface markers that were upregulated and downregulated by osteogenic or adipogenic differentiation. Osteogenic differentiation induced upregulation of CD164 and downregulation of CD49a, CD49b, CD49c, CD49d, CD55, CD58, CD105, and CD166 while adipogenic differentiation induced upregulation of CD36, CD40, CD146, CD164, and CD271 and downregulation of CD49b, CD49c, CD49d, CD71, CD105, and CD166. These results lend support to the notion that hASCs isolated using standard methodologies represent a heterogeneous population and serve as a foundation for future studies seeking to maximize their regenerative potential through fluorescence-activated cell sorting-based selection before therapy.

  View details for DOI 10.1089/ten.tea.2015.0039

  View details for Web of Science ID 000359812700014

  View details for PubMedID 26020286

  View details for PubMedCentralID PMC4529076

• Identification and isolation of a dermal lineage with intrinsic fibrogenic potential SCIENCE Rinkevich, Y., Walmsley, G. G., Hu, M. S., Maan, Z. N., Newman, A. M., Drukker, M., Januszyk, M., Krampitz, G. W., Gurtner, G. C., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2015; 348 (6232): 302-?

  View details for DOI 10.1126/science.aaa2151

  View details for Web of Science ID 000352999000034

• Scarless wound healing: chasing the holy grail. Plastic and reconstructive surgery Walmsley, G. G., Maan, Z. N., Wong, V. W., Duscher, D., Hu, M. S., Zielins, E. R., Wearda, T., Muhonen, E., McArdle, A., Tevlin, R., Atashroo, D. A., Senarath-Yapa, K., Lorenz, H. P., Gurtner, G. C., Longaker, M. T. 2015; 135 (3): 907-917

  Abstract

  Over 100 million patients acquire scars in the industrialized world each year, primarily as a result of elective operations. Although undefined, the global incidence of scarring is even larger, extending to significant numbers of burn and other trauma-related wounds. Scars have the potential to exert a profound psychological and physical impact on the individual. Beyond aesthetic considerations and potential disfigurement, scarring can result in restriction of movement and reduced quality of life. The formation of a scar following skin injury is a consequence of wound healing occurring through reparative rather than regenerative mechanisms. In this article, the authors review the basic stages of wound healing; differences between adult and fetal wound healing; various mechanical, genetic, and pharmacologic strategies to reduce scarring; and the biology of skin stem/progenitor cells that may hold the key to scarless regeneration.

  View details for DOI 10.1097/PRS.0000000000000972

  View details for PubMedID 25719706

• Evolution of cranioplasty techniques in neurosurgery: historical review, pediatric considerations, and current trends. Journal of neurosurgery Feroze, A. H., Walmsley, G. G., Choudhri, O., Lorenz, H. P., Grant, G. A., Edwards, M. S. 2015; 123 (4): 1098-1107

  Abstract

  Cranial bone repair is one of the oldest neurosurgical practices. Reconstructing the natural contours of the skull has challenged the ingenuity of surgeons from antiquity to the present day. Given the continuous improvement of neurosurgical and emergency care over the past century, more patients survive such head injuries, thus necessitating more than ever before a simple, safe, and durable means of correcting skull defects. In response, numerous techniques and materials have been devised as the art of cranioplasty has progressed. Although the goals of cranioplasty remain the same, the evolution of techniques and diversity of materials used serves as testimony to the complexity of this task. This paper highlights the evolution of these materials and techniques, with a particular focus on the implications for managing pediatric calvarial repair and emerging trends within the field.

  View details for DOI 10.3171/2014.11.JNS14622

  View details for PubMedID 25699411

• Identification and characterization of an injury-induced skeletal progenitor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Marecic, O., Tevlin, R., McArdle, A., Seo, E. Y., Wearda, T., Duldulao, C., Walmsley, G. G., Nguyen, A., Weissman, I. L., Chan, C. K., Longaker, M. T. 2015; 112 (32): 9920-9925

  Abstract

  The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.

  View details for DOI 10.1073/pnas.1513066112

  View details for Web of Science ID 000359285100053

  View details for PubMedID 26216955

  View details for PubMedCentralID PMC4538608

• Assessing the Utility of Intraoperative Educational Time-Outs in the Promotion of Medical Student Knowledge. American journal of medical quality Feroze, A. H., Walmsley, G. G. 2015; 30 (4): 397-?

  View details for DOI 10.1177/1062860615588260

  View details for PubMedID 26001910

• Nanotechnology in bone tissue engineering NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE Walmsley, G. G., McArdle, A., Tevlin, R., Momeni, A., Atashroo, D., Hu, M. S., Feroze, A. H., Wong, V. W., Lorenz, P. H., Longaker, M. T., Wan, D. C. 2015; 11 (5): 1253-1263

  Abstract

  Nanotechnology represents a major frontier with potential to significantly advance the field of bone tissue engineering. Current limitations in regenerative strategies include impaired cellular proliferation and differentiation, insufficient mechanical strength of scaffolds, and inadequate production of extrinsic factors necessary for efficient osteogenesis. Here we review several major areas of research in nanotechnology with potential implications in bone regeneration: 1) nanoparticle-based methods for delivery of bioactive molecules, growth factors, and genetic material, 2) nanoparticle-mediated cell labeling and targeting, and 3) nano-based scaffold construction and modification to enhance physicochemical interactions, biocompatibility, mechanical stability, and cellular attachment/survival. As these technologies continue to evolve, ultimate translation to the clinical environment may allow for improved therapeutic outcomes in patients with large bone deficits and osteodegenerative diseases.Traditionally, the reconstruction of bony defects has relied on the use of bone grafts. With advances in nanotechnology, there has been significant development of synthetic biomaterials. In this article, the authors provided a comprehensive review on current research in nanoparticle-based therapies for bone tissue engineering, which should be useful reading for clinicians as well as researchers in this field.

  View details for DOI 10.1016/j.nano.2015.02.013

  View details for Web of Science ID 000363967100022

  View details for PubMedID 25791811

• Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent PLASTIC AND RECONSTRUCTIVE SURGERY Paik, K. J., Zielins, E. R., Atashroo, D. A., Maan, Z. N., Duscher, D., Luan, A., Walmsley, G. G., Momeni, A., Vistnes, S., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2015; 136 (1): 67-75

  Abstract

  Cell-assisted lipotransfer has shown much promise as a technique for improving fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown.Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 μl of fat. Fat graft volume retention was assessed by means of computed tomographic scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity.Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 μl of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, whereas fat grafts supplemented with 10 million cells showed significant lipodegeneration compared with fat alone: The authors' study demonstrates dose dependence in the number of stromal vascular fraction cells that can be added to a fat graft to enhance retention. Although cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the postgraft period.

  View details for DOI 10.1097/PRS.0000000000001367

  View details for Web of Science ID 000357096300002

  View details for PubMedID 25829158

• What Makes a Plastic Surgery Residency Program Attractive? An Applicant's Perspective PLASTIC AND RECONSTRUCTIVE SURGERY Atashroo, D. A., Luan, A., Vyas, K. S., Zielins, E. R., Maan, Z., Duscher, D., Walmsley, G. G., Lynch, M. P., Davenport, D. L., Wan, D. C., Longaker, M. T., Vasconez, H. C. 2015; 136 (1): 189-196

  Abstract

  Plastic surgery is among the most competitive specialties in medicine, but little is known about the attributes of programs that are most attractive to successful applicants. This study aimed to understand and provide insights regarding program characteristics that are most influential to students when ranking plastic surgery programs.An anonymous online survey was conducted with newly matched plastic surgery residents for the integrated and combined Match in 2012 and 2013. Subjects were queried regarding their demographics, qualifications, application experiences, and motivations for residency program selection.A total of 92 of 245 matched plastic surgery residents (38 percent) responded to the survey. The perception of resident happiness was the most positive factor influencing program ranking, followed by high operative volume, faculty mentorship, and strong research infrastructure. Perception of a program as "malignant" was the most negative attribute. Applicants with Step 1 scores greater than 245 received significantly more interviews (p =0.001) and considered resident benefits less important (p < 0.05), but geographic location more important (p =0.005). Applicants who published more than two articles also received more interviews (p =0.001) and ranked a strong research infrastructure and program reputation as significantly more important (p < 0.05). Forty-two percent of applicants completed an away rotation at the program with which they matched, and these applicants were more likely to match at their number one ranked program (p = 0.001).Plastic surgery applicants have differing preferences regarding the ideal training program, but some attributes resonate. These trends can guide programs for improvement in attracting the best applicants.

  View details for DOI 10.1097/PRS.0000000000001365

  View details for Web of Science ID 000357097900001

  View details for PubMedID 26111321

• Emerging drugs for the treatment of wound healing EXPERT OPINION ON EMERGING DRUGS Zielins, E. R., Brett, E. A., Luan, A., Hu, M. S., Walmsley, G. G., Paik, K., Senarath-Yapa, K., Atashroo, D. A., Wearda, T., Lorenz, H. P., Wan, D. C., Longaker, M. T. 2015; 20 (2): 235-246

  Abstract

  Wound healing can be characterized as underhealing, as in the setting of chronic wounds, or overhealing, occurring with hypertrophic scar formation after burn injury. Topical therapies targeting specific biochemical and molecular pathways represent a promising avenue for improving and, in some cases normalizing, the healing process.A brief overview of both normal and pathological wound healing has been provided, along with a review of the current clinical guidelines and treatment modalities for chronic wounds, burn wounds and scar formation. Next, the major avenues for wound healing drugs, along with drugs currently in development, are discussed. Finally, potential challenges to further drug development, and future research directions are discussed.The large body of research concerning wound healing pathophysiology has provided multiple targets for topical therapies. Growth factor therapies with the ability to be targeted for localized release in the wound microenvironment are most promising, particularly when they modulate processes in the proliferative phase of wound healing.

  View details for DOI 10.1517/14728214.2015.1018176

  View details for Web of Science ID 000356118400007

  View details for PubMedID 25704608

• Injuries to Appendage Extremities and Digit Tips: A Clinical and Cellular Update DEVELOPMENTAL DYNAMICS Rinkevich, Y., Maan, Z. N., Walmsley, G. G., Sen, S. K. 2015; 244 (5): 641-650

  Abstract

  The regrowth of amputated appendage extremities and the distal tips of digits represent models of tissue regeneration in multiple vertebrate taxa. In humans, digit tip injuries, including traumatic amputation and crush injuries, are among the most common type of injury to the human hand. Despite clinical reports demonstrating natural regeneration of appendages in lower vertebrates and human digits, current treatment options are suboptimal, and are complicated by the anatomical complexities and functions of the different tissues within the digits.In light of these challenges, we focus on recent advancements in understanding appendage regeneration from model organisms. We pay special attention to the cellular programs underlying appendage regeneration, where cumulative data from salamanders, fish, frogs, and mice indicate that regeneration occurs by the actions of lineage-restricted precursors. We focus on pathologic states and the interdependency that exists, in both humans and animal models, between the nail organ and the peripheral nerves for successful regeneration.The increased understanding of regeneration in animal models may open new opportunities for basic and translational research aimed at understanding the mechanisms that support limb regeneration, as well as amelioration of limb abnormalities and pathologies.

  View details for DOI 10.1002/dvdy.24265

  View details for Web of Science ID 000353953600002

  View details for PubMedID 25715837

• Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science Rinkevich, Y., Walmsley, G. G., Hu, M. S., Maan, Z. N., Newman, A. M., Drukker, M., Januszyk, M., Krampitz, G. W., Gurtner, G. C., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2015; 348 (6232)

  Abstract

  Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

  View details for DOI 10.1126/science.aaa2151

  View details for PubMedID 25883361

  View details for PubMedCentralID PMC5088503

• Live Fibroblast Harvest Reveals Surface Marker Shift In Vitro TISSUE ENGINEERING PART C-METHODS Walmsley, G. G., Rinkevich, Y., Hu, M. S., Montoro, D. T., Lo, D. D., McArdle, A., Maan, Z. N., Morrison, S. D., Duscher, D., Whittam, A. J., Wong, V. W., Weissman, I. L., Gurtner, G. C., Longaker, M. T. 2015; 21 (3): 314-321

  Abstract

  Current methods for the isolation of fibroblasts require extended ex vivo manipulation in cell culture. As a consequence, prior studies investigating fibroblast biology may fail to adequately represent cellular phenotypes in vivo. To overcome this problem, we describe a detailed protocol for the isolation of fibroblasts from the dorsal dermis of adult mice that bypasses the need for cell culture, thereby preserving the physiological, transcriptional, and proteomic profiles of each cell. Using the described protocol we characterized the transcriptional programs and the surface expression of 176 CD markers in cultured versus uncultured fibroblasts. The differential expression patterns we observed highlight the importance of a live harvest for investigations of fibroblast biology.

  View details for DOI 10.1089/ten.tec.2014.0118

  View details for Web of Science ID 000350043400009

  View details for PubMedID 25275778

• The role and regulation of osteoclasts in normal bone homeostasis and in response to injury. Plastic and reconstructive surgery McArdle, A., Marecic, O., Tevlin, R., Walmsley, G. G., Chan, C. K., Longaker, M. T., Wan, D. C. 2015; 135 (3): 808-816

  Abstract

  Bone is a dynamic tissue, with a range of diverse functions, including locomotion, protection of internal organs, and hematopoiesis. Optimum treatment of fractures and/or bone defects requires knowledge of the complex cellular interactions involved with bone healing and remodeling. Emerging data have underscored the importance of osteoclasts in this process, playing a key role both in normal bone turnover and in facilitating bone regeneration. In this review, the authors discuss the basic principles of osteoclast biology, including its cellular origins, its function, and key regulatory mechanisms, in addition to conditions that arise when osteoclast function is altered.

  View details for DOI 10.1097/PRS.0000000000000963

  View details for PubMedID 25719699

• Identification and specification of the mouse skeletal stem cell. Cell Chan, C. K., Seo, E. Y., Chen, J. Y., Lo, D., McArdle, A., Sinha, R., Tevlin, R., Seita, J., Vincent-Tompkins, J., Wearda, T., Lu, W., Senarath-Yapa, K., Chung, M. T., Marecic, O., Tran, M., Yan, K. S., Upton, R., Walmsley, G. G., Lee, A. S., Sahoo, D., Kuo, C. J., Weissman, I. L., Longaker, M. T. 2015; 160 (1-2): 285-298

  Abstract

  How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

  View details for DOI 10.1016/j.cell.2014.12.002

  View details for PubMedID 25594184

  View details for PubMedCentralID PMC4297645

• Transdermal deferoxamine prevents pressure-induced diabetic ulcers. Proceedings of the National Academy of Sciences of the United States of America Duscher, D., Neofytou, E., Wong, V. W., Maan, Z. N., Rennert, R. C., Inayathullah, M., Januszyk, M., Rodrigues, M., Malkovskiy, A. V., Whitmore, A. J., Walmsley, G. G., Galvez, M. G., Whittam, A. J., Brownlee, M., Rajadas, J., Gurtner, G. C. 2015; 112 (1): 94-99

  Abstract

  There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

  View details for DOI 10.1073/pnas.1413445112

  View details for PubMedID 25535360

• Assessment of viability of human fat injection into nude mice with micro-computed tomography. Journal of visualized experiments : JoVE Atashroo, D. A., Paik, K. J., Chung, M. T., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C. R., Walmsley, G. G., Wearda, T., Marecic, O., Longaker, M. T., Wan, D. C. 2015

  Abstract

  Lipotransfer is a vital tool in the surgeon's armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.(1) However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. (1-3) To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 µl of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes. Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

  View details for DOI 10.3791/52217

  View details for PubMedID 25590561

• Isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis. Journal of visualized experiments : JoVE Zielins, E. R., Tevlin, R., Hu, M. S., Chung, M. T., McArdle, A., Paik, K. J., Atashroo, D., Duldulao, C. R., Luan, A., Senarath-Yapa, K., Walmsley, G. G., Wearda, T., Longaker, M. T., Wan, D. C. 2015: 52181-?

  Abstract

  Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

  View details for DOI 10.3791/52181

  View details for PubMedID 25650785

• A mouse fetal skin model of scarless wound repair. Journal of visualized experiments : JoVE Walmsley, G. G., Hu, M. S., Hong, W. X., Maan, Z. N., Lorenz, H. P., Longaker, M. T. 2015: 52297-?

  Abstract

  Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

  View details for DOI 10.3791/52297

  View details for PubMedID 25650841

• Biomaterials for Craniofacial Bone Engineering JOURNAL OF DENTAL RESEARCH Tevlin, R., Mcardle, A., Atashroo, D., Walmsley, G. G., Senarath-Yapa, K., Zielins, E. R., Paik, K. J., Longaker, M. T., Wan, D. C. 2014; 93 (12): 1187-1195

  View details for DOI 10.1177/0022034514547271

  View details for Web of Science ID 000345340400002

• Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells SCIENTIFIC REPORTS Duscher, D., Rennert, R. C., Januszyk, M., Anghel, E., Maan, Z. N., Whittam, A. J., Perez, M. G., Kosaraju, R., Hu, M. S., Walmsley, G. G., Atashroo, D., Khong, S., Butte, A. J., Gurtner, G. C. 2014; 4

  View details for DOI 10.1038/srep07144

  View details for Web of Science ID 000346178900001

  View details for PubMedID 25413454

• Positive Selection for Bone Morphogenetic Protein Receptor Type-IB Promotes Differentiation and Specification of Human Adipose-Derived Stromal Cells Toward an Osteogenic Lineage TISSUE ENGINEERING PART A McArdle, A., Chung, M. T., Paik, K. J., Duldulao, C., Chan, C., Rennert, R., Walmsley, G. G., Senarath-Yapa, K., Hu, M., Seo, E., Lee, M., Wan, D. C., Longaker, M. T. 2014; 20 (21-22): 3031-3040

  Abstract

  Adipose tissue represents an abundant and easily accessible source of multipotent cells that may serve as an excellent building block for tissue engineering. However, adipose-derived stromal cells (ASCs) are a heterogeneous group and subpopulations may be identified with enhanced osteogenic potential.Human ASC subpopulations were prospectively isolated based on expression of bone morphogenetic protein receptor type-IB (BMPR-IB). Unsorted, BMPR-IB(+), and BMPR-IB(-) cells were analyzed for their osteogenic capacity through histological staining and gene expression. To evaluate their in vivo osteogenic potential, critical-sized calvarial defects were created in immunocompromised mice and treated with unsorted, BMPR-IB(+), or BMPR-IB(-) cells. Healing was assessed using microcomputed tomography and pentachrome staining of specimens at 8 weeks.Increased osteogenic differentiation was noted in the BMPR-IB(+) subpopulation, as demonstrated by alkaline phosphatase staining at day 7 and extracellular matrix mineralization with Alizarin red staining at day 14. This was also associated with increased expression for osteocalcin, a late marker of osteogenesis. Radiographic analysis demonstrated significantly enhanced healing of critical-sized calvarial defects treated with BMPR-IB(+) ASCs compared with unsorted or BMPR-IB(-) cells. This was confirmed through pentachrome staining, which revealed more robust bone regeneration in the BMPR-IB(+) group.BMPR-IB(+) human ASCs have an enhanced ability to form bone both in vitro and in vivo. These data suggest that positive selection for BMPR-IB(+) and manipulation of the BMP pathway in these cells may yield a highly osteogenic subpopulation of cells for bone tissue engineering.

  View details for DOI 10.1089/ten.tea.2014.0101

  View details for Web of Science ID 000344592600021

  View details for PubMedCentralID PMC4229710

• The role of stem cells in aesthetic surgery: fact or fiction? Plastic and reconstructive surgery McArdle, A., Senarath-Yapa, K., Walmsley, G. G., Hu, M., Atashroo, D. A., Tevlin, R., Zielins, E., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2014; 134 (2): 193-200

  Abstract

  Stem cells are attractive candidates for the development of novel therapies, targeting indications that involve functional restoration of defective tissue. Although most stem cell therapies are new and highly experimental, there are clinics around the world that exploit vulnerable patients with the hope of offering supposed stem cell therapies, many of which operate without credible scientific merit, oversight, or other patient protection.We review the potential, as well as drawbacks, for incorporation of stem cells in cosmetic procedures. A review of FDA-approved indications and ongoing clinical trials with adipose stem cells is provided. Furthermore, a "snapshot" analysis of websites using the search terms "stem cell therapy" or "stem cell treatment" or "stem cell facelift" was performed.Despite the protective net cast by regulatory agencies such as the FDA and professional societies such as the American Society of Plastic Surgeons, we are witnessing worrying advertisements for procedures such as stem cell facelifts, stem cell breast augmentations, and even stem cell vaginal rejuvenation. The marketing and promotion of stem cell procedures in aesthetic surgery is not adequately supported by clinical evidence in the majority of cases.Stem cells offer tremendous potential, but the marketplace is saturated with unsubstantiated and sometimes fraudulent claims that may place patients at risk. With plastic surgeons at the forefront of stem cell-based regenerative medicine, it is critically important that we provide an example of a rigorous approach to research, data collection, and advertising of stem cell therapies.

  View details for DOI 10.1097/PRS.0000000000000404

  View details for PubMedID 24732654

• Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

  View details for DOI 10.1073/pnas.1410097111

  View details for Web of Science ID 000338514800040

• Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration. Proceedings of the National Academy of Sciences of the United States of America Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

  Abstract

  The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

  View details for DOI 10.1073/pnas.1410097111

  View details for PubMedID 24958860

• Studies in fat grafting: Part I. Effects of injection technique on in vitro fat viability and in vivo volume retention. Plastic and reconstructive surgery Chung, M. T., Paik, K. J., Atashroo, D. A., Hyun, J. S., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Hu, M. S., Walmsley, G. G., Parisi-Amon, A., Momeni, A., Rimsa, J. R., Commons, G. W., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2014; 134 (1): 29-38

  Abstract

  Fat grafting has become increasingly popular for the correction of soft tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, we compare the biologic properties of fat following injection using two methods.Lipoaspiration samples were obtained from five female donors and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over twelve weeks following injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically.Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at 4, 6, 8, and 12 week time points. This corresponded with significantly greater histological scores for healthy fat and lower scores for injury following injection with the device.Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and our in vitro and in vivo data both support the use of the automated, low shear devices compared to the modified Coleman technique.

  View details for DOI 10.1097/PRS.0000000000000290

  View details for PubMedID 24622574

• Studies in fat grafting: Part II. Effects of injection mechanics on material properties of fat. Plastic and reconstructive surgery Atashroo, D., Raphel, J., Chung, M. T., Paik, K. J., Parisi-Amon, A., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Walmsley, G. G., Hu, M. S., Momeni, A., Domecus, B., Rimsa, J. R., Greenberg, L., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2014; 134 (1): 39-46

  Abstract

  Although fat grafting can address many soft-tissue deficits, results remain inconsistent. In this study, the authors compared physical properties of fat following injection using an automated, low-shear device or the modified Coleman technique.Lipoaspirate was obtained from nine patients and processed for injection using either a modified Coleman technique or an automated, low-shear device. Fat was passed through a 2-mm cannula and compared with minimally processed fat. A rheometer was used to measure the storage modulus and shear rate at which tissues began to lose their solid-like properties. Viscosity was also measured, and gross properties of treatment groups were evaluated qualitatively with a glass slide test.Fat injected through an automated, low-shear device closely matched physical properties of minimally processed fat. The storage modulus (G') of fat for the device group was greater than for the modified Coleman group, and the onset of breakdown was delayed. Similarly, viscosity measurement of fat from the automated device closely matched minimally processed fat and was greater than that of othe modified Coleman group.The physical properties of lipoaspirate processed using an automated, low-shear device with a 2-mm cannula preserved the intactness of fat more than the modified Coleman technique. The authors' rheologic data demonstrate less damage using an automated device compared with the modified Coleman technique and potentially support its use for improved fat graft integrity.

  View details for DOI 10.1097/PRS.0000000000000289

  View details for PubMedID 25028817

• Tissue engineering and regenerative repair in wound healing. Annals of biomedical engineering Hu, M. S., Maan, Z. N., Wu, J., Rennert, R. C., Hong, W. X., Lai, T. S., Cheung, A. T., Walmsley, G. G., Chung, M. T., McArdle, A., Longaker, M. T., Lorenz, H. P. 2014; 42 (7): 1494-1507

  Abstract

  Wound healing is a highly evolved defense mechanism against infection and further injury. It is a complex process involving multiple cell types and biological pathways. Mammalian adult cutaneous wound healing is mediated by a fibroproliferative response leading to scar formation. In contrast, early to mid-gestational fetal cutaneous wound healing is more akin to regeneration and occurs without scar formation. This early observation has led to extensive research seeking to unlock the mechanism underlying fetal scarless regenerative repair. Building upon recent advances in biomaterials and stem cell applications, tissue engineering approaches are working towards a recapitulation of this phenomenon. In this review, we describe the elements that distinguish fetal scarless and adult scarring wound healing, and discuss current trends in tissue engineering aimed at achieving scarless tissue regeneration.

  View details for DOI 10.1007/s10439-014-1010-z

  View details for PubMedID 24788648

• Studies in Fat Grafting: Part I. Effects of Injection Technique on In Vitro Fat Viability and In Vivo Volume Retention PLASTIC AND RECONSTRUCTIVE SURGERY Chung, M. T., Paik, K. J., Atashroo, D. A., Hyun, J. S., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Hu, M. S., Walmsley, G. G., Parisi-Amon, A., Momeni, A., Rimsa, J. R., Commons, G. W., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2014; 134 (1): 29-38

  Abstract

  Fat grafting has become increasingly popular for the correction of soft tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, we compare the biologic properties of fat following injection using two methods.Lipoaspiration samples were obtained from five female donors and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over twelve weeks following injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically.Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at 4, 6, 8, and 12 week time points. This corresponded with significantly greater histological scores for healthy fat and lower scores for injury following injection with the device.Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and our in vitro and in vivo data both support the use of the automated, low shear devices compared to the modified Coleman technique.

  View details for DOI 10.1097/PRS.0000000000000290

  View details for Web of Science ID 000338116400042

• Studies in Fat Grafting: Part II. Effects of Injection Mechanics on Material Properties of Fat PLASTIC AND RECONSTRUCTIVE SURGERY Atashroo, D., Raphel, J., Chung, M. T., Paik, K. J., Parisi-Amon, A., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Walmsley, G. G., Hu, M. S., Momeni, A., Domecus, B., Rimsa, J. R., Greenberg, L., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2014; 134 (1): 39-46

  Abstract

  Although fat grafting can address many soft-tissue deficits, results remain inconsistent. In this study, the authors compared physical properties of fat following injection using an automated, low-shear device or the modified Coleman technique.Lipoaspirate was obtained from nine patients and processed for injection using either a modified Coleman technique or an automated, low-shear device. Fat was passed through a 2-mm cannula and compared with minimally processed fat. A rheometer was used to measure the storage modulus and shear rate at which tissues began to lose their solid-like properties. Viscosity was also measured, and gross properties of treatment groups were evaluated qualitatively with a glass slide test.Fat injected through an automated, low-shear device closely matched physical properties of minimally processed fat. The storage modulus (G') of fat for the device group was greater than for the modified Coleman group, and the onset of breakdown was delayed. Similarly, viscosity measurement of fat from the automated device closely matched minimally processed fat and was greater than that of othe modified Coleman group.The physical properties of lipoaspirate processed using an automated, low-shear device with a 2-mm cannula preserved the intactness of fat more than the modified Coleman technique. The authors' rheologic data demonstrate less damage using an automated device compared with the modified Coleman technique and potentially support its use for improved fat graft integrity.

  View details for DOI 10.1097/PRS.0000000000000289

  View details for Web of Science ID 000338116400043

  View details for PubMedCentralID PMC4101917

• Gene expression in fetal murine keratinocytes and fibroblasts JOURNAL OF SURGICAL RESEARCH Hu, M. S., Januszyk, M., Hong, W. X., Walmsley, G. G., Zielins, E. R., Atashroo, D. A., Maan, Z. N., McArdle, A., Takanishi, D. M., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2014; 190 (1): 344-357

  Abstract

  Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis-the keratinocyte and fibroblast-during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration.Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate <2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways.By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts.Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

  View details for DOI 10.1016/j.jss.2014.02.030

  View details for Web of Science ID 000338444700051

• Induced pluripotent stem cells in regenerative medicine and disease modeling. Current stem cell research & therapy Walmsley, G. G., Hyun, J., McArdle, A., Senarath-Yapa, K., Hu, M. S., Chung, M. T., Wong, V. W., Longaker, M. T., Wan, D. C. 2014; 9 (2): 73-81

  Abstract

  In 2006, Dr. Yamanaka created the induced pluripotent stem cell (iPSC) by reprogramming adult fibroblasts back to an immature, pluripotent state. Effectively bypassing the ethical constraints of human embryonic stem cells, iPSCs have expanded the horizons of regenerative medicine by offering a means to derive autologous patient-matched cells and tissues for clinical transplantation. However, persisting safety concerns must be addressed prior to their widespread clinical application. In this review, we discuss the history of iPSCs, derivation strategies, and current research involving gene therapy and disease modeling. We review the potential of iPSCs for improving a range of cell-based therapies and obstacles to their clinical implementation.

  View details for PubMedID 24359141

• Abstract 135: improved engraftment of autologous skin grafts in diabetic mice with adipose-derived stem cells. Plastic and reconstructive surgery Hu, M., Hong, W. X., Senarath-Yapa, K., Zimmermann, A., Chung, M., Esquivel, M., McArdle, A., Walmsley, G., Maan, Z., Garza, R., Lorenz, H. P., Longaker, M. 2014; 133 (3): 151-?

  View details for DOI 10.1097/01.prs.0000444962.89785.8e

  View details for PubMedID 25942246

• Abstract 161: identification of cell-intrinsic mechanisms and differentially regulated genetic pathways responsible for the age-related functional decline in aged skeletal stem cells. Plastic and reconstructive surgery McArdle, A., Chan, C., Seita, J., Senarath-Yapa, K., Hu, M., Walmsley, G. G., Zielins, E., Atashroo, D., Tevlin, R., Weissman, I., Longaker, M. T. 2014; 133 (3): 178-?

  View details for DOI 10.1097/01.prs.0000444990.75431.f1

  View details for PubMedID 25942271

• Abstract 140: identification, characterization, and prospective isolation of a fibroblast lineage contributing to dermal development, cutaneous scarring, radiation fibrosis, and cancer stroma. Plastic and reconstructive surgery Walmsley, G. G., Rinkevich, Y., Hu, M. S., McArdle, A., Maan, Z. N., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2014; 133 (3): 157-?

  View details for DOI 10.1097/01.prs.0000444968.20280.4d

  View details for PubMedID 25942251

• Abstract 165: Enhanced Adipose-Derived Stromal Cell Osteogenesis through Surface Marker Enrichment and BMP Modulation using Magnet-assisted Transfection. Plastic and reconstructive surgery Chung, M. T., Morrison, S. D., Paik, K. J., McArdle, A., Walmsley, G., Senarath-Yapa, K., Hu, M. S., Tevlin, R., Zielins, E., Atashroo, D., Hong, W. X., Duldulao, C., Wearda, T., Garza, R. M., Momeni, A., Longaker, M. T., Wan, D. C. 2014; 133 (3): 181-182

  View details for DOI 10.1097/01.prs.0000444994.28797.34

  View details for PubMedID 25942275

• Abstract 151: short hairpin RNA interference therapy for diabetic murine wound closure and hindlimb ischemia. Plastic and reconstructive surgery Paik, K. J., Rennert, R., Chung, M. T., Sorkin, M., Duscher, D., Atashroo, D., Chen, H., Morrison, S. D., Zimmermann, A., Nauta, A., Ko, S., Tevlin, R., Zielins, E., Hu, M. S., McArdle, A., Walmsley, G., Senarath-Yapa, K., Hong, W. X., Garza, R. M., Duldulao, C., Wearda, T., Momeni, A., Wu, J. C., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2014; 133 (3): 167-168

  View details for DOI 10.1097/01.prs.0000444979.14443.08

  View details for PubMedID 25942261

• Epidermal or dermal specific knockout of PHD-2 enhances wound healing and minimizes ischemic injury. PloS one Zimmermann, A. S., Morrison, S. D., Hu, M. S., Li, S., Nauta, A., Sorkin, M., Meyer, N. P., Walmsley, G. G., Maan, Z. N., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2014; 9 (4)

  Abstract

  Hypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing.Epidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro.qRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-2 KO mice relative to control mice (*p<0.05). PHD-2 KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-2 tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05).PHD-2 plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-2 KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic factors such as VEGF.

  View details for DOI 10.1371/journal.pone.0093373

  View details for PubMedID 24695462

• Osteoclast derivation from mouse bone marrow. Journal of visualized experiments : JoVE Tevlin, R., McArdle, A., Chan, C. K., Pluvinage, J., Walmsley, G. G., Wearda, T., Marecic, O., Hu, M. S., Paik, K. J., Senarath-Yapa, K., Atashroo, D. A., Zielins, E. R., Wan, D. C., Weissman, I. L., Longaker, M. T. 2014

  View details for DOI 10.3791/52056

  View details for PubMedID 25407120

• Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells. Scientific reports Duscher, D., Rennert, R. C., Januszyk, M., Anghel, E., Maan, Z. N., Whittam, A. J., Perez, M. G., Kosaraju, R., Hu, M. S., Walmsley, G. G., Atashroo, D., Khong, S., Butte, A. J., Gurtner, G. C. 2014; 4: 7144-?

  Abstract

  Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.

  View details for DOI 10.1038/srep07144

  View details for PubMedID 25413454

  View details for PubMedCentralID PMC4239576

• Osteoclast derivation from mouse bone marrow. Journal of visualized experiments : JoVE Tevlin, R., McArdle, A., Chan, C. K., Pluvinage, J., Walmsley, G. G., Wearda, T., Marecic, O., Hu, M. S., Paik, K. J., Senarath-Yapa, K., Atashroo, D. A., Zielins, E. R., Wan, D. C., Weissman, I. L., Longaker, M. T. 2014

  Abstract

  Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

  View details for DOI 10.3791/52056

  View details for PubMedID 25407120

  View details for PubMedCentralID PMC4353410

• Wound healing: an update REGENERATIVE MEDICINE Zielins, E. R., Atashroo, D. A., Maan, Z. N., Duscher, D., Walmsley, G. G., Marecic, O., Hu, M., Senarath-Yapa, K., McArdle, A., Tevlin, R., Wearda, T., Paik, K. J., Duldulao, C., Hong, W. X., Gurtner, G. C., Longaker, M. T. 2014; 9 (6): 817-830

  Abstract

  Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

  View details for DOI 10.2217/RME.14.54

  View details for Web of Science ID 000345620600012

• Epidermal or Dermal Specific Knockout of PHD-2 Enhances Wound Healing and Minimizes Ischemic Injury. PloS one Zimmermann, A. S., Morrison, S. D., Hu, M. S., Li, S., Nauta, A., Sorkin, M., Meyer, N. P., Walmsley, G. G., Maan, Z. N., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2014; 9 (4)

  View details for DOI 10.1371/journal.pone.0093373

  View details for PubMedID 24695462

• Isolation of human adipose-derived stromal cells using laser-assisted liposuction and their therapeutic potential in regenerative medicine. Stem cells translational medicine Chung, M. T., Zimmermann, A. S., Paik, K. J., Morrison, S. D., Hyun, J. S., Lo, D. D., McArdle, A., Montoro, D. T., Walmsley, G. G., Senarath-Yapa, K., Sorkin, M., Rennert, R., Chen, H., Chung, A. S., Vistnes, D., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2013; 2 (10): 808-817

  Abstract

  Harvesting adipose-derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third-generation ultrasound-assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser-assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser-assisted lipoaspirate and suction-assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic nude mice. Although ASCs isolated from suction-assisted lipoaspirate and laser-assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34(+)CD31(-)CD45(-)) in the stromal vascular fraction were all significantly less with laser-assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro-computed tomography revealed significantly more healing with ASCs isolated from suction-assisted lipoaspirate relative to laser-assisted lipoaspirate at the 4-, 6-, and 8-week time points (p < .05). Therefore, as laser-assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction-assisted liposuction is preferable for tissue-engineering purposes.

  View details for DOI 10.5966/sctm.2012-0183

  View details for PubMedID 24018794

  View details for PubMedCentralID PMC3785265

• Enhancing in vivo survival of adipose-derived stromal cells through bcl-2 overexpression using a minicircle vector. Stem cells translational medicine Hyun, J., Grova, M., Nejadnik, H., Lo, D., Morrison, S., Montoro, D., Chung, M., Zimmermann, A., Walmsley, G. G., Lee, M., Daldrup-Link, H., Wan, D. C., Longaker, M. T. 2013; 2 (9): 690-702

  Abstract

  Tissue regeneration using progenitor cell-based therapy has the potential to aid in the healing of a diverse range of pathologies, ranging from short-gut syndrome to spinal cord lesions. However, there are numerous hurdles to be overcome prior to the widespread application of these cells in the clinical setting. One of the primary barriers to effective stem cell therapy is the hostile environment that progenitor cells encounter in the clinical injury wound setting. In order to promote cellular survival, stem cell differentiation, and participation in tissue regeneration, relevant cells and delivery scaffolds must be paired with strategies to prevent cell death to ensure that these cells can survive to form de novo tissue. The Bcl-2 protein is a prosurvival member of a family of proteins that regulate the mitochondrial pathway of apoptosis. Using several strategies to overexpress the Bcl-2 protein, we demonstrated a decrease in the mediators of apoptosis in vitro and in vivo. This was shown through the use of two different clinical tissue repair models. Cells overexpressing Bcl-2 not only survived within the wound environment at a statistically significantly higher rate than control cells, but also increased tissue regeneration. Finally, we used a nonintegrating minicircle technology to achieve this in a potentially clinically applicable strategy for stem cell therapy.

  View details for DOI 10.5966/sctm.2013-0035

  View details for PubMedID 23934910

• Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C. K., Lindau, P., Jiang, W., Chen, J. Y., Zhang, L. F., Chen, C., Seita, J., Sahoo, D., Kim, J., Lee, A., Park, S., Nag, D., Gong, Y., Kulkarni, S., Luppen, C. A., Theologis, A. A., Wan, D. C., DeBoer, A., Seo, E. Y., Vincent-Tompkins, J. D., Loh, K., Walmsley, G. G., Kraft, D. L., Wu, J. C., Longaker, M. T., Weissman, I. L. 2013; 110 (31): 12643-12648

  Abstract

  Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

  View details for DOI 10.1073/pnas.1310212110

  View details for Web of Science ID 000322441500042

  View details for PubMedID 23858471

  View details for PubMedCentralID PMC3732968

• Transcriptional Program Induced by Wnt Protein in Human Fibroblasts Suggests Mechanisms for Cell Cooperativity in Defining Tissue Microenvironments PLOS ONE Klapholz-Brown, Z., Walmsley, G. G., Nusse, Y. M., Nusse, R., Brown, P. O. 2007; 2 (9)

  Abstract

  The Wnt signaling system plays key roles in development, regulation of stem cell self-renewal and differentiation, cell polarity, morphogenesis and cancer. Given the multifaceted roles of Wnt signaling in these processes, its transcriptional effects on the stromal cells that make up the scaffold and infrastructure of epithelial tissues are of great interest.To begin to investigate these effects, we used DNA microarrays to identify transcriptional targets of the Wnt pathway in human lung fibroblasts. Cells were treated with active Wnt3a protein in culture, and RNA was harvested at 4 hours and 24 hours. Nuclear accumulation of ss-Catenin, as shown by immunofluorescence, and induction of AXIN2 demonstrate that fibroblasts are programmed to respond to extracellular Wnt signals. In addition to several known Wnt targets, we found many new Wnt induced genes, including many transcripts encoding regulatory proteins. Transcription factors with important developmental roles, including HOX genes, dominated the early transcriptional response. Furthermore, we found differential expression of several genes that play direct roles in the Wnt signaling pathway, as well as genes involved in other cell signaling pathways including fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling. The gene most highly induced by Wnt3a was GREMLIN2, which encodes a secreted BMP antagonist.Elevated expression of GREMLIN2 suggests a new role for Wnt signals in the maintenance of stem cell niches, whereby Wnt signals induce nearby fibroblasts to produce a BMP antagonist, inhibiting differentiation and promoting expansion of stem cells in their microenvironment. We suggest that Wnt-induced changes in the gene expression program of local stromal cells may play an important role in the establishment of specialized niches hospitable to the self-renewal of normal or malignant epithelial stem cells in vivo.

  View details for DOI 10.1371/journal.pone.0000945

  View details for Web of Science ID 000207455800024

  View details for PubMedID 17895986