Transgenic mice for in vivo epigenome editing with CRISPR-based systems.

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

Primary Cilia Exhibit Mechanosensitivity to Cyclic Tensile Strain and Lineage-Dependent Expression in Adipose-Derived Stem Cells.

Non-motile primary cilia are dynamic cellular sensory structures and are expressed in adipose-derived stem cells (ASCs). We have previously shown that primary cilia are involved in chemically-induced osteogenic differentiation of human ASC (hASCs) in vitro. Further, we have reported that 10% cyclic tensile strain (1 Hz, 4 hours/day) enhances hASC osteogenesis. We hypothesize that primary cilia respond to cyclic tensile strain in a lineage dependent manner and that their mechanosensitivity may regulate the dynamics of signaling pathways localized to the cilium. We found that hASC morphology, cilia length and cilia conformation varied in response to culture in complete growth, osteogenic differentiation, or adipogenic differentiation medium, with the longest cilia expressed in adipogenically differentiating cells. Further, we show that cyclic tensile strain both enhances osteogenic differentiation of hASCs while it suppresses adipogenic differentiation as evidenced by upregulation of RUNX2 gene expression and downregulation of PPARG and IGF-1, respectively. This study demonstrates that hASC primary cilia exhibit mechanosensitivity to cyclic tensile strain and lineage-dependent expression, which may in part regulate signaling pathways localized to the primary cilium during the differentiation process. We highlight the importance of the primary cilium structure in mechanosensing and lineage specification and surmise that this structure may be a novel target in manipulating hASC for in tissue engineering applications.

Key developments that impacted the field of mechanobiology and mechanotransduction.

Advances in mechanobiology have evolved through insights from multiple disciplines including structural engineering, biomechanics, vascular biology, and orthopaedics. In this paper, we reviewed the impact of key reports related to the study of applied loads on tissues and cells and the resulting signal transduction pathways. We addressed how technology has helped advance the burgeoning field of mechanobiology (over 33,600 publications from 1970 to 2016). We analyzed the impact of critical ideas and then determined how these concepts influenced the mechanobiology field by looking at the citation frequency of these reports as well as tracking how the overall number of citations within the field changed over time. These data allowed us to understand how a key publication, idea, or technology guided or enabled the field. Initial observations of how forces acted on bone and soft tissues stimulated the development of computational solutions defining how forces affect tissue modeling and remodeling. Enabling technologies, such as cell and tissue stretching, compression, and shear stress devices, allowed more researchers to explore how deformation and fluid flow affect cells. Observation of the cell as a tensegrity structure and advanced methods to study genetic regulation in cells further advanced knowledge of specific mechanisms of mechanotransduction. The future of the field will involve developing gene and drug therapies to simulate or augment beneficial load regimens in patients and in mechanically conditioning organs for implantation. Here, we addressed a history of the field, but we limited our discussions to advances in musculoskeletal mechanobiology, primarily in bone, tendon, and ligament tissues. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:605-619, 2018.

Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

In this study, we report for the first time that the primary cilium acts as a crucial sensor for electrical field stimulation (EFS)-enhanced osteogenic response in osteoprogenitor cells. In addition, primary cilia seem to functionally modulate effects of EFS-induced cellular calcium oscillations. Primary cilia are organelles that have recently been implicated to play a crucial sensor role for many mechanical and chemical stimuli on stem cells. Here, we investigate the role of primary cilia in EFS-enhanced osteogenic response of human adipose-derived stem cells (hASCs) by knocking down 2 primary cilia structural proteins, polycystin-1 and intraflagellar protein-88. Our results indicate that structurally integrated primary cilia are required for detection of electrical field signals in hASCs. Furthermore, by measuring changes of cytoplasmic calcium concentration in hASCs during EFS, our findings also suggest that primary cilia may potentially function as a crucial calcium-signaling nexus in hASCs during EFS.-Cai, S., Bodle, J. C., Mathieu, P. S., Amos, A., Hamouda, M., Bernacki, S., McCarty, G., Loboa, E. G. Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

Concise Review: Primary Cilia: Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies.

Directing stem cell lineage commitment prevails as the holy grail of translational stem cell research, particularly to those interested in the application of mesenchymal stem cells and adipose-derived stem cells in tissue engineering. However, elucidating the mechanisms underlying their phenotypic specification persists as an active area of research. In recent studies, the primary cilium structure has been intimately associated with defining cell phenotype, maintaining stemness, as well as functioning in a chemo, electro, and mechanosensory capacity in progenitor and committed cell types. Many hypothesize that the primary cilium may indeed be another important player in defining and controlling cell phenotype, concomitant with lineage-dictated cytoskeletal dynamics. Many of the studies on the primary cilium have emerged from disparate areas of biological research, and crosstalk amongst these areas of research is just beginning. To date, there has not been a thorough review of how primary cilia fit into the current paradigm of stem cell differentiation and this review aims to summarize the current cilia work in this context. The goal of this review is to highlight the cilium's function and integrate this knowledge into the working knowledge of stem cell biologists and tissue engineers developing regenerative medicine technologies. Stem Cells 2016;34:1445-1454.

Primary cilium mechanotransduction of tensile strain in 3D culture: Finite element analyses of strain amplification caused by tensile strain applied to a primary cilium embedded in a collagen matrix.

Human adipose-derived stem cells (hASC) exhibit multilineage differentiation potential with lineage specification that is dictated by both the chemical and mechanical stimuli to which they are exposed. We have previously shown that 10% cyclic tensile strain increases hASC osteogenesis and cell-mediated calcium accretion. We have also recently shown that primary cilia are present on hASC and that chemically-induced lineage specification of hASC concurrently results in length and conformation changes of the primary cilia. Further, we have observed cilia length changes in hASC cultured within a collagen I gel in response to 10% cyclic tensile strain. We therefore hypothesize that primary cilia may play a key mechanotransduction role for hASC exposed to tensile strain. The goal of this study was to use finite element analysis (FEA) to determine strains occurring within the ciliary membrane in response to 10% tensile strain applied parallel, or perpendicular, to cilia orientation. To elucidate the mechanical environment experienced by the cilium, several lengths were modeled and evaluated based on cilia lengths measured on hASC grown under varied culture conditions. Principal tensile strains in both hASC and ciliary membranes were calculated using FEA, and the magnitude and location of maximum principal tensile strain determined. We found that maximum principal tensile strain was concentrated at the base of the cilium. In the linear elastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane from 150% to 200%, while applying strain parallel to the cilium resulted in much higher strains, approximately 400%. In the hyperelastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane around 30%, while applying strain parallel to the cilium resulted in much higher strains ranging from 50% to 70%. Interestingly, FEA results indicated that primary cilium length was not directly related to ciliary membrane strain. Rather, it appears that cilium orientation may be more important than cilium length in determining sensitivity of hASC to tensile strain. This is the first study to model the effects of tensile strain on the primary cilium and provides newfound insight into the potential role of the primary cilium as a mechanosensor, particularly in tensile strain and potentially a multitude of other mechanical stimuli beyond fluid shear.

Age-related effects on the potency of human adipose-derived stem cells: creation and evaluation of superlots and implications for musculoskeletal tissue engineering applications.

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed "superlots," containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation.

Primary cilia: the chemical antenna regulating human adipose-derived stem cell osteogenesis.

Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.

Adipose-derived stem cells in functional bone tissue engineering: lessons from bone mechanobiology.

This review aims to highlight the current and significant work in the use of adipose-derived stem cells (ASC) in functional bone tissue engineering framed through the bone mechanobiology perspective. Over a century of work on the principles of bone mechanosensitivity is now being applied to our understanding of bone development. We are just beginning to harness that potential using stem cells in bone tissue engineering. ASC are the primary focus of this review due to their abundance and relative ease of accessibility for autologous procedures. This article outlines the current knowledge base in bone mechanobiology to investigate how the knowledge from this area has been applied to the various stem cell-based approaches to engineering bone tissue constructs. Specific emphasis is placed on the use of human ASC for this application.

Biological performance of biodegradable amino acid-based poly(ester amide)s: Endothelial cell adhesion and inflammation in vitro.

Functionalized amino-acid-based poly(ester-amide)s (PEA) are a new family of synthetic biodegradable polymers consisting of three naturally occurring building blocks (amino acids, diols, and dicarboxylic acids) that have been suggested to be promising biomaterials for therapeutic use. However, little is known about their cytotoxicity, ability to support cell growth, inflammatory properties, or mechanical properties, key aspects to most biomaterials designed for in vivo implantation and tissue engineering applications. In this study, we investigated the ability of two functionalized PEA materials (amino-functionalized and carboxylic acid functionalized) and a neutral PEA control to support endothelial cell viability, proliferation, and adhesion. Additionally, we investigated the inflammatory response elicited by these functionalized PEA materials using a macrophage cell model. Our results indicate that all forms of PEA were noncytotoxic and noninflammatory in vitro. The amino-functionalized PEA bests supports endothelial cell adhesion, growth, and monolayer formation. Mechanical testing indicates that the elastic moduli of these materials are strongly dependent on the charge formulation, but do exhibit linearly elastic behavior at small strains (<10%). Our data suggest that PEA may be a viable biomaterial for use in tissue engineering applications, particularly for use as a vascular graft.