A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing.

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

Effect of heparin on the biological properties and molecular signature of human mesenchymal stem cells.

Chronic use of heparin as an anti-coagulant for the treatment of thrombosis or embolism invokes many adverse systemic events including thrombocytopenia, vascular reactions and osteoporosis. Here, we addressed whether adverse effects might also be directed to mesenchymal stem cells that reside in the bone marrow compartment. Harvested human bone marrow-derived mesenchymal stem cells (hMSCs) were exposed to varying doses of heparin and their responses profiled. At low doses (<200 ng/ml), serial passaging with heparin exerted a variable effect on hMSC proliferation and multipotentiality across multiple donors, while at higher doses (≥ 100 µg/ml), heparin supplementation inhibited cell growth and increased both senescence and cell size. Gene expression profiling using cDNA arrays and RNA-seq analysis revealed pleiotropic effects of low-dose heparin on signaling pathways essential to hMSC growth and differentiation (including the TGFß/BMP superfamily, FGFs, and Wnts). Cells serially passaged in low-dose heparin possess a donor-dependent gene signature that reflects their altered phenotype. Our data indicate that heparin supplementation during the culturing of hMSCs can alter their biological properties, even at low doses. This warrants caution in the application of heparin as a culture supplement for the ex vivo expansion of hMSCs. It also highlights the need for careful evaluation of the bone marrow compartment in patients receiving chronic heparin treatment.

Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.

The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.

FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21(Waf1) and p27(Kip1).

Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential.

Heparan sulfate enhances the self-renewal and therapeutic potential of mesenchymal stem cells from human adult bone marrow.

Insufficient cell number hampers therapies utilizing adult human mesenchymal stem cells (hMSCs) and current ex vivo expansion strategies lead to a loss of multipotentiality. Here we show that supplementation with an embryonic form of heparan sulfate (HS-2) can both increase the initial recovery of hMSCs from bone marrow aspirates and increase their ex vivo expansion by up to 13-fold. HS-2 acts to amplify a subpopulation of hMSCs harboring longer telomeres and increased expression of the MSC surface marker stromal precursor antigen-1. Gene expression profiling revealed that hMSCs cultured in HS-2 possess a distinct signature that reflects their enhanced multipotentiality and improved bone-forming ability when transplanted into critical-sized bone defects. Thus, HS-2 offers a novel means for decreasing the expansion time necessary for obtaining therapeutic numbers of multipotent hMSCs without the addition of exogenous growth factors that compromise stem cell fate.

Synergism between Wnt3a and heparin enhances osteogenesis via a phosphoinositide 3-kinase/Akt/RUNX2 pathway.

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

The osteogenic transcription factor Runx2 regulates components of the fibroblast growth factor/proteoglycan signaling axis in osteoblasts.

Heparan sulfate proteoglycans cooperate with basic fibroblast growth factor (bFGF/FGF2) signaling to control osteoblast growth and differentiation, as well as metabolic functions of osteoblasts. FGF2 signaling modulates the expression and activity of Runt-related transcription factor 2 (Runx2/Cbfa1), a key regulator of osteoblast proliferation and maturation. Here, we have characterized novel Runx2 target genes in osteoprogenitors under conditions that promote growth arrest while not yet permitting sustained phenotypic maturation. Runx2 enhances expression of genes related to proteoglycan-mediated signaling, including FGF receptors (e.g., FGFR2 and FGFR3) and proteoglycans (e.g., syndecans [Sdc1, Sdc2, Sdc3], glypicans [Gpc1], versican [Vcan]). Runx2 increases expression of the glycosyltransferase Exostosin-1 (Ext1) and heparanase, as well as alters the relative expression of N-linked sulfotransferases (Ndst1 = Ndst2 > Ndst3) and enzymes mediating O-linked sulfation of heparan sulfate (Hs2st > Hs6st) or chondroitin sulfate (Cs4st > Cs6st). Runx2 cooperates with FGF2 to induce expression of Sdc4 and the sulfatase Galns, but Runx2 and FGF2 suppress Gpc6, thus suggesting intricate Runx2 and FGF2 dependent changes in proteoglycan utilization. One functional consequence of Runx2 mediated modulations in proteoglycan-related gene expression is a change in the responsiveness of bone markers to FGF2 stimulation. Runx2 and FGF2 synergistically enhance osteopontin expression (>100 fold), while FGF2 blocks Runx2 induction of alkaline phosphatase. Our data suggest that Runx2 and the FGF/proteoglycan axis may form an extracellular matrix (ECM)-related regulatory feed-back loop that controls osteoblast proliferation and execution of the osteogenic program.

Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma.

To understand the molecular etiology of osteosarcoma, we isolated and characterized a human osteosarcoma cell line (OS1). OS1 cells have high osteogenic potential in differentiation induction media. Molecular analysis reveals OS1 cells express the pocket protein pRB and the runt-related transcription factor Runx2. Strikingly, Runx2 is expressed at higher levels in OS1 cells than in human fetal osteoblasts. Both pRB and Runx2 have growth suppressive potential in osteoblasts and are key factors controlling competency for osteoblast differentiation. The high levels of Runx2 clearly suggest osteosarcomas may form from committed osteoblasts that have bypassed growth restrictions normally imposed by Runx2. Interestingly, OS1 cells do not exhibit p53 expression and thus lack a functional p53/p21 DNA damage response pathway as has been observed for other osteosarcoma cell types. Absence of this pathway predicts genomic instability and/or vulnerability to secondary mutations that may counteract the anti-proliferative activity of Runx2 that is normally observed in osteoblasts. We conclude OS1 cells provide a valuable cell culture model to examine molecular events that are responsible for the pathologic conversion of phenotypically normal osteoblast precursors into osteosarcoma cells.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells.

The growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated glycosaminoglycans for the fibroblast growth factor type 2 (FGF-2)-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of HS to cultures of primary rat MSCs stimulates their proliferation, leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation, and osteogenic differentiation of rat bone marrow stem cells (rMSCs) when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. We show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth, resulting in the cells being unable to reach the critical density necessary to induce differentiation. Blocking FGFR1 signaling in postconfluent osteogenic cultures significantly increased calcium deposition. Taken together our data suggest that FGFR1 signaling plays an important role during osteogenic differentiation, first by stimulating cell growth that is closely followed by an inhibitory effect once the cells have reached confluence. It also confirms the importance of HS as a coreceptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells.

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression.

Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.

Isolation of a native osteoblast matrix with a specific affinity for BMP2.

During their commitment and differentiation toward the osteoblast lineage, mesenchymal stem cells secrete a unique extracellular matrix (ECM) that contains large quantities of glycosaminoglycans (GAGs). Proteoglycans (PGs) are major structural and functional components of the ECM and are composed of a core protein to which one or more glycosaminoglycan sugar chains (GAGs) attach. The association of BMP2, a member of the TGF-beta super-family of growth factors, and a known heparin-binding protein, with GAGs has been implicated as playing a significant role in modulating the growth factor's in vitro bioactivity. Here we have characterised an osteoblast-derived matrix (MX) obtained from decellularised MC3T3-E1 cell monolayers for its structural attributes, using SEM and histology, and for its functional ability to maintain cell growth and viability. Using a combination of histology and anion exchange chromatography, we first confirmed the retention of GAGs within MX following the decellularisation process. Then the binding specificity of the retained GAG species within the MX for BMP2 was examined using a BMP2-HBP/EGFP (BMP2 Heparin-Binding Peptide/Enhanced Green Fluorescent Protein) fusion protein. The results of this study provide further evidence for a central role of the ECM in the regulation of BMP2 bioactivity, hence on mesenchymal stem cell commitment to the osteoblast lineage.

Osteogenic differentiation of murine embryonic stem cells is mediated by fibroblast growth factor receptors.

The mechanisms involved in the control of embryonic stem (ES) cell differentiation are yet to be fully elucidated. However, it has become clear that the family of fibroblast growth factors (FGFs) are centrally involved. In this study we examined the role of the FGF receptors (FGFRs 1-4) during osteogenesis in murine ES cells. Single cells were obtained after the formation of embryoid bodies, cultured on gelatin-coated plates, and coaxed to differentiate along the osteogenic lineage. Upregulation of genes was analyzed at both the transcript and protein levels using gene array, relative-quantitative PCR (RQ-PCR), and Western blotting. Deposition of a mineralized matrix was evaluated with Alizarin Red staining. An FGFR1-specific antibody was generated and used to block FGFR1 activity in mES cells during osteogenic differentiation. Upon induction of osteogenic differentiation in mES cells, all four FGFRs were clearly upregulated at both the transcript and protein levels with a number of genes known to be involved in osteogenic differentiation including bone morphogenetic proteins (BMPs), collagen I, and Runx2. Cells were also capable of depositing a mineralized matrix, confirming the commitment of these cells to the osteogenic lineage. When FGFR1 activity was blocked, a reduction in cell proliferation and a coincident upregulation of Runx2 with enhanced mineralization of cultures was observed. These results indicate that FGFRs play critical roles in cell recruitment and differentiation during the process of osteogenesis in mES cells. In particular, the data indicate that FGFR1 plays a pivotal role in osteoblast lineage determination.

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells.

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.

Reduced seminal parameters associated with environmental DDT exposure and p,p'-DDE concentrations in men in Chiapas, Mexico: a cross-sectional study.

In response to mounting concerns about the endocrine-disrupting influence of environmental chemicals on human health, this epidemiological study was initiated to test the hypothesis that nonoccupational exposure to the estrogenic pesticide 1,1,1-trichloro-2,2-bis(chlorodiphenyl)ethane (DDT) affects male reproductive parameters. One hundred and sixteen men aged 27 years (SD = 8.2) living in malaria endemic-areas in Chiapas (Mexico), where DDT was sprayed until 2000, participated in a cross-sectional study. Semen analyses were conducted according to World Health Organization methods and a quality control program was followed. DDT exposure was defined as the level of blood plasma p,p'-dichlorodiphenyl dichloroethylene (DDE), the major metabolite of DDT. The p,p'-DDE concentration adjusted for total lipids was 100 times higher than that reported for nonexposed populations at 45 plus or minus 32 mug/g (mean +/- SD). Crude regression analysis showed that several sperm motion parameters, including the percentage of motile sperm, decreased with higher p,p'-DDE concentrations (beta = -8.38; P = .05 for squared motility), and the percentage of sperm with morphological tail defects increased with higher plasma p,p'-DDE concentration (beta = 0.003; P = .017). Insufficient sperm chromatin condensation was observed in 46.6% of participants, and the most severe category of incomplete DNA condensation was also positively correlated with p,p'-DDE concentration (r = .223; P = .044). Therefore, nonoccupational exposure to DDT, as assessed by plasma p,p'-DDE concentrations, is associated with poorer semen parameters in men, indicating adverse effects on testicular function and/or the regulation of reproductive hormones. Previously, a causal role of environmental toxicants in human male infertility has been lacking because observed effects have been the result of unusually high exposures, either occupationally or as a result of industrial accidents, resulting in unprecedented controversy (reviewed by Cheek & McLachlan, Environmental hormones and the male reproductive system. J Androl. 1998;19:5). This is the first epidemiological study demonstrating effects after nonoccupational exposures to DDT. Based on these findings, the effect of DDT on male reproductive health should not be ignored.