Kshemendra Senarath-Yapa

Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation

In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13, and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs), as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age, with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

Biomaterials for Craniofacial Bone Engineering

Conditions such as congenital anomalies, cancers, and trauma can all result in devastating deficits of bone in the craniofacial skeleton. This can lead to significant alteration in function and appearance that may have significant implications for patients. In addition, large bone defects in this area can pose serious clinical dilemmas, which prove difficult to remedy, even with current gold standard surgical treatments. The craniofacial skeleton is complex and serves important functional demands. The necessity to develop new approaches for craniofacial reconstruction arises from the fact that traditional therapeutic modalities, such as autologous bone grafting, present myriad limitations and carry with them the potential for significant complications. While the optimal bone construct for tissue regeneration remains to be elucidated, much progress has been made in the past decade. Advances in tissue engineering have led to innovative scaffold design, complemented by progress in the understanding of stem cell–based therapy and growth factor enhancement of the healing cascade. This review focuses on the role of biomaterials for craniofacial bone engineering, highlighting key advances in scaffold design and development.

Scarless wound healing: chasing the holy grail.

Summary: 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.

Isolation of Human Adipose-Derived Stromal Cells Using Laser-Assisted Liposuction and Their Therapeutic Potential in Regenerative Medicine

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.

Wound healing: an update

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.

Craniosynostosis: molecular pathways and future pharmacologic therapy.

Craniosynostosis describes the premature fusion of one or more cranial sutures and can lead to dramatic manifestations in terms of appearance and functional impairment. Contemporary approaches for this condition are primarily surgical and are associated with considerable morbidity and mortality. The additional post-operative problems of suture refusion and bony relapse may also necessitate repeated surgeries with their own attendant risks. Therefore, a need exists to not only optimize current strategies but also to develop novel biological therapies which could obviate the need for surgery and potentially treat or even prevent premature suture fusion. Clinical studies of patients with syndromic craniosynostosis have provided some useful insights into the important signaling pathways and molecular events guiding suture fate. Furthermore, the highly conserved nature of craniofacial development between humans and other species have permitted more focused and step-wise elucidation of the molecular underpinnings of craniosynostosis. This review will describe the clinical manifestations of craniosynostosis, reflect on our understanding of syndromic and non-syndromic craniosynostoses and outline the different approaches that have been adopted in our laboratory and elsewhere to better understand the pathogenesis of premature suture fusion. Finally, we will assess to what extent our improved understanding of the pathogenesis of craniosynostosis, achieved through laboratory-based and clinical studies, have made the possibility of a non-surgical pharmacological approach both realistic and tangible.Craniosynostosis describes the premature fusion of one or more cranial sutures and can lead to dramatic manifestations in terms of appearance and functional impairment. Contemporary approaches for this condition are primarily surgical and are associated with considerable morbidity and mortality. The additional post-operative problems of suture refusion and bony relapse may also necessitate repeated surgeries with their own attendant risks. Therefore, a need exists to not only optimize current strategies but also to develop novel biological therapies which could obviate the need for surgery and potentially treat or even prevent premature suture fusion. Clinical studies of patients with syndromic craniosynostosis have provided some useful insights into the important signaling pathways and molecular events guiding suture fate. Furthermore, the highly conserved nature of craniofacial development between humans and other species have permitted more focused and step-wise elucidation of the molecular underpinnings of craniosynostosis. This review will describe the clinical manifestations of craniosynostosis, reflect on our understanding of syndromic and non-syndromic craniosynostoses and outline the different approaches that have been adopted in our laboratory and elsewhere to better understand the pathogenesis of premature suture fusion. Finally, we will assess to what extent our improved understanding of the pathogenesis of craniosynostosis, achieved through laboratory-based and clinical studies, have made the possibility of a non-surgical pharmacological approach both realistic and tangible.

Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation

Significance Despite the importance of Wnt signaling in bone biology, there is a knowledge gap in the identity of the cells that produce the Wnt ligands and the functions of Wnts produced by specific cell types. In our study, we comprehensively characterized the expression patterns of all 19 Wnts in the developing mouse bone by in situ hybridization, and further showed that Osterix-expressing cells can produce Wnts and respond to Wnt signaling. Additionally, we found that Wnts produced by these Osterix-expressing cells regulate their differentiation and proliferation. Through providing a better understanding of how Wnt signaling contributes to bone biology, our findings also have clinical implications for the mechanism of the osteoporotic drug that targets Sclerostin, a Wnt signaling antagonist. Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated—to our knowledge for the first time—that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.

Studies in fat grafting: Part I. Effects of injection technique on in vitro fat viability and in vivo volume retention.

Background: 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, the authors compare the biological properties of fat following injection using two methods. 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 12 weeks after 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. Results: 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 the 4-, 6-, 8-, and 12-week time points. This corresponded to significantly greater histologic scores for healthy fat and lower scores for injury following injection with the device. Conclusion: Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and the authors’ in vitro and in vivo data both support the use of the automated, low-shear devices compared with the modified Coleman technique.

TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose‐Derived Stem Cells by Triggering Activation of BMP‐ERK/FGF Signaling and TAZ Upregulation

Mesenchymal stem cells (MSCs) show promise for cellular therapy and regenerative medicine. Human adipose tissue‐derived stem cells (hASCs) represent an attractive source of seed cells in bone regeneration. How to effectively improve osteogenic differentiation of hASCs in the bone tissue engineering has become a very important question with profound translational implications. Numerous regulatory pathways dominate osteogenic differentiation of hASCs involving transcriptional factors and signaling molecules. However, how these factors combine with each other to regulate hASCs osteogenic differentiation still remains to be illustrated. The highly conserved developmental proteins TWIST play key roles for transcriptional regulation in mesenchymal cell lineages. This study investigates TWIST1 function in hASCs osteogenesis. Our results show that TWIST1 shRNA silencing increased the osteogenic potential of hASCs in vitro and their skeletal regenerative ability when applied in vivo. We demonstrate that the increased osteogenic capacity observed with TWIST1 knockdown in hASCs is mediated through endogenous activation of BMP and ERK/FGF signaling leading, in turn, to upregulation of TAZ, a transcriptional modulator of MSCs differentiation along the osteoblast lineage. Inhibition either of BMP or ERK/FGF signaling suppressed TAZ upregulation and the enhanced osteogenesis in shTWIST1 hASCs. Cosilencing of both TWIST1 and TAZ abrogated the effect elicited by TWIST1 knockdown thus, identifying TAZ as a downstream mediator through which TWIST1 knockdown enhanced osteogenic differentiation in hASCs. Our functional study contributes to a better knowledge of molecular mechanisms governing the osteogenic ability of hASCs, and highlights TWIST1 as a potential target to facilitate in vivo bone healing. Stem Cells 2015;33:833–847

The role of stem cells in aesthetic surgery: fact or fiction?

Background: 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. Methods: The authors review the potential and the drawbacks of incorporation of stem cells in cosmetic procedures. A review of U.S. Food and Drug Administration–approved indications and ongoing clinical trials with adipose stem cells is provided. Furthermore, a “snapshot” analysis of Web sites using the search terms “stem cell therapy” or “stem cell treatment” or “stem cell facelift” was performed. Results: Despite the protective net cast by regulatory agencies such as the U.S. Food and Drug Administration and professional societies such as the American Society of Plastic Surgeons, the authors are witnessing worrying advertisements for procedures such as stem cell face lifts, 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. Conclusions: 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 they provide an example of a rigorous approach to research, data collection, and advertising of stem cell therapies.