Derrick C. Wan

Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling

Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro. In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.

Noggin Suppression Enhances in Vitro Osteogenesis and Accelerates in Vivo Bone Formation

Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.

Cranial sutures: a brief review.

Summary: Craniosynostosis, or the premature fusion of one or more cranial sutures, is a relatively common congenital defect that causes a number of morphologic and functional abnormalities. With advances in genetics and molecular biology, research of craniosynostosis has progressed from describing gross abnormalities to understanding the molecular interactions that underlie these cranial deformities. Animal models have been extremely valuable in improving our comprehension of human craniofacial morphogenesis, primarily by human genetic linkage analysis and the development of knock-out animals. This article provides a brief review of perisutural tissue interactions, embryonic origins, signaling molecules and their receptors, and transcription factors in maintaining the delicate balance between proliferation and differentiation of cells within the suture complex that determines suture fate. Finally, this article discusses the potential implications for developing novel therapies for craniosynostosis.

CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor β1 (TGF-β1) signaling.

Background: ASCs are promising for skeletal regeneration, but their heterogeneity limits their use. Results: Microfluidic analysis and FACS identified a cellular subset (CD105low) with enhanced osteogenic capacity. Conclusion: CD105 depletion was found to enhance osteogenesis through reduction of TGF-β1 signaling. Significance: We illuminate the functional relevance of hASC heterogeneity and enhance understanding of CD105 with respect to osteogenic differentiation. Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105low cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105high or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105low adipose-derived cells is likely due to identification of a subpopulation with lower TGF-β1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

In vivo directed differentiation of pluripotent stem cells for skeletal regeneration.

Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2–releasing poly-l-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

Origin Matters: Differences in Embryonic Tissue Origin and Wnt Signaling Determine the Osteogenic Potential and Healing Capacity of Frontal and Parietal Calvarial Bones

Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest–derived frontal and paraxial mesodermal–derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest–derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal–derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest–derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF‐2) treatment induces phosphorylation of GSK‐3β and increases the nuclear levels of β‐catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest–derived frontal bone.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells

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.

Cranial Suture Biology

Publisher Summary This chapter focuses on the cranial suture biology. The term “craniosynostosis” was first used in 1830 by Otto to describe the premature fusion of cranial sutures. Since this first identification of craniosynostosis as a distinct clinical entity, several theories have been proposed to explain both the pathogenesis of premature suture fusion and the resultant aberrations in calvarial growth that result in a dysmorphic skull. Recent advances in clinical genetics have resulted in the identification of genetic mutations in the major craniosynostostic syndromes. Despite these insights into the rudimentary disturbances leading to craniosynostosis, the processes by which mutations in these genes trigger premature suture fusion remain largely unknown. Rodents are proving to be extremely valuable in unraveling the cellular and molecular mechanisms of cranial suture morphogenesis and pathology. The cranial sutures include the metopic or interfrontal suture (between the frontal bones), the sagittal suture (between the parietal bones), the coronal suture (between the frontal and parietal bones), and the lambdoid sutures (between the parietal and interparietal bones). The sutures can be thought of as a complex consisting of four principal components: (1) the osteogenic fronts of the approximating bone plates; (2) the suture mesenchyme spanning the osteogenic fronts; (3) the overlying pericranium or cranial periosteum; and (4) the underlying dura mater, a tough, fibrous membrane that constitutes the outer meningeal layer that envelops the brain and forms the inner lining of cranial bones and sutures. The main objective is to obtain a thorough understanding of normal and pathological suture morphogenesis and development. Armed with this knowledge, researchers will be prepared to devise biologically based therapeutic strategies that could be used both in utero or postnatally to prevent craniosynostosis, potentially alleviating any adverse sequelae and avoiding the morbidity of current surgical approaches.

Cell-based therapies for skeletal regenerative medicine

Skeletal deficits represent a substantial biomedical burden on the US healthcare system. Current strategies for reconstructing bony defects are fraught with inadequacies. Cell-based therapies for skeletal regeneration offer a paradigm shift that may provide alternative solutions. Substantial work has identified a host of cellular sources that possess the potential for osteogenic differentiation. Significant efforts have been devoted toward characterizing the role of postnatal cellular sources that are relatively abundant and easily accessible. Among these, the potential of using adipose-derived stromal cells for skeletal regeneration has garnered much interest. Integral to these efforts directed at characterizing cellular sources are studies that seek to understand the factors that initiate and regulate osteogenic differentiation of progenitor cells. Specifically, focus has been directed on elucidating the role of bone morphogenetic protein and fibroblast growth factor signaling in regulating osteogenic differentiation of osteoprogenitor cells. Concurrent studies in the field of scaffold design have also helped to advance the potential for cell-based therapies.

Fat Grafting Versus Adipose-Derived Stem Cell Therapy: Distinguishing Indications, Techniques, and Outcomes

With adipose-derived stem cells (ASCs) at the forefront of research and potential clinical applications, it is important that clinicians be able to distinguish them from the fat grafting currently used clinically and to understand how the two approaches relate to one another. At times, there has been confusion in clinically considering the two therapies to be the same. This report is aimed at distinguishing clearly between fat grafting and ASC therapy with regard to the indications, harvesting, processing, application techniques, outcomes, and complications. Findings have shown that autologous fat transfer, a widely used procedure for soft tissue augmentation, is beneficial for reconstructive and cosmetic procedures used to treat patients with volume loss due to disease, trauma, congenital defects, or the natural process of aging. On the other hand, ASCs have been identified as an ideal source of cells for regenerative medicine, with the potential to serve as soft tissue therapy for irradiated, scarred, or chronic wounds. Recent advances in tissue engineering suggest that the supplementation of fat grafts with ASCs isolated in the stromal vascular fraction may increase the longevity and quality of the fat graft. Research suggests that ASC supplementation may be a great clinical tool in the future, but more data should be acquired before clinical applications.