Taylor Wearda

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.

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.

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.

Identification and characterization of an injury-induced skeletal progenitor.

Significance Here, we characterize the injury-induced activation of a specific, highly purified population of multipotent skeletal progenitor cells. These activated progenitors show increased cell frequency, increased viability, and enhanced osteogenic potential. They also possess a unique transcriptional profile that distinguishes them from progenitors found in uninjured bone. We report that these features improve regenerative capacity, suggesting that activated progenitors play a principal role in bone healing. We hope that a better understanding of stem and progenitor activation will inspire novel therapies that restore impaired skeletal regeneration. 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.

Pharmacological rescue of diabetic skeletal stem cell niches

Local delivery of a missing growth factor to the skeletal stem cell niche restores bone healing in diabetic mice. Stem cells: The key to boosting bone healing in diabetes Among a myriad of difficulties, people with diabetes have problems with their bones; after a break, their bones do not heal well. Tevlin et al. use mice to investigate the cause and to devise a solution. In several models of diabetes, skeletal stem cells, which normally multiply to repair a bone injury, failed to do so. The high blood concentrations of TNFα in these diabetic mice inhibited a growth factor within the stem cell niche. The authors succeeded in reversing this deficit; delivery of the missing factor directly to the niche restored the expansion of stem cells after injury and normalized bone healing. Correction of the inhospitable niche environment for skeletal stem cells is a promising approach for this complication of diabetes and perhaps for other stem cell–based diseases. Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche–related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor–α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

Emerging drugs for the treatment of wound healing

Introduction: 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. Areas covered: 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. Expert opinion: 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.

Osteoclast derivation from mouse bone marrow.

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.

Dynamic Rheology for the Prediction of Surgical Outcomes in Autologous Fat Grafting

Background: Because of the abundance and biocompatibility of fat, lipotransfer has become an attractive method for treating soft-tissue deficits. However, it is limited by unpredictable graft survival and retention. Currently, little is known about the viscoelastic properties of fat after various injection methods. Here, the authors assess the effects of cannula diameter, length, and shape on the viscoelastic properties, structure, and retention of fat. Methods: Human lipoaspirate was harvested using suction-assisted liposuction and prepared for grafting. A syringe pump was used to inject fat at a controlled flow rate through cannulas of varying gauges, lengths, and shapes. Processed samples were tested in triplicate on an oscillatory rheometer to measure their viscoelastic properties. Fat grafts from each group were placed into the scalps of immunocompromised mice. After 8 weeks, graft retention was measured using micro–computed tomography and grafts were explanted for histologic analysis. Results: Lipoaspirate injected through narrower, longer, and bent cannulas exhibited more shear thinning with diminished quality. The storage modulus (G′) of fat processed with 18-gauge cannulas was significantly lower than when processed with 14-gauge or larger cannulas, which also corresponded with inferior in vivo histologic structure. Similarly, the longer cannula group had a significantly lower storage modulus than the shorter cannula, and was associated with decreased graft retention. Conclusions: Discrete modifications in the methods used for fat placement can have a significant impact on immediate graft integrity, and ultimately on graft survival and quality. Respecting these biomechanical influences during the placement phase of lipotransfer may allow surgeons to optimize outcomes. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Skeletal Stem Cell Niche Aberrancies Underlie Impaired Fracture Healing in a Mouse Model of Type 2 Diabetes

www.PRSJournal.com 73 Figure 1. (Left) Four-year-old female with facial infiltrating lipomatosis (FIL). (Right) Droplet digital (ddPCR) reaction showing PIK3CA mutation in muscle from patient with FIL. Left upper quadrant represents droplets with only the mutant allele. Right upper quadrant shows droplets with mutant and wild-type alleles. Left lower quadrant illustrates droplets that do not contain any alleles. Right lower quadrant has droplets with only the wild-type allele.

Isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis.

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.