Publications by authors named "Piera Smeriglio"

22 Publications

  • Page 1 of 1

A dysfunctional TRPV4-GSK3β pathway prevents osteoarthritic chondrocytes from sensing changes in extracellular matrix viscoelasticity.

Nat Biomed Eng 2021 Mar 11. Epub 2021 Mar 11.

Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, CA, USA.

Changes in the composition and viscoelasticity of the extracellular matrix in load-bearing cartilage influence the proliferation and phenotypes of chondrocytes, and are associated with osteoarthritis. However, the underlying molecular mechanism is unknown. Here we show that the viscoelasticity of alginate hydrogels regulates cellular volume in healthy human chondrocytes (with faster stress relaxation allowing cell expansion and slower stress relaxation restricting it) but not in osteoarthritic chondrocytes. Cellular volume regulation in healthy chondrocytes was associated with changes in anabolic gene expression, in the secretion of multiple pro-inflammatory cytokines, and in the modulation of intracellular calcium regulated by the ion-channel protein transient receptor potential cation channel subfamily V member 4 (TRPV4), which controls the phosphorylation of glycogen synthase kinase 3β (GSK3β), an enzyme with pleiotropic effects in osteoarthritis. A dysfunctional TRPV4-GSK3β pathway in osteoarthritic chondrocytes rendered the cells unable to respond to environmental changes in viscoelasticity. Our findings suggest strategies for restoring chondrocyte homeostasis in osteoarthritis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41551-021-00691-3DOI Listing
March 2021

TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of and In Vitro.

JBMR Plus 2020 Aug 26;4(8):e10383. Epub 2020 Jun 26.

Department of Orthopaedic Surgery Stanford University School of Medicine Stanford CA USA.

Skeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the Ten-eleven-translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage-specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of via short-hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome-wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte-specific class II binding sites for SOX9, as compared with the tissue-agnostic class I sites. Specifically, we find that SOX9 is unable to bind to and after KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 embryos, loss of SOX9 target gene expression is more modest than upon KD in vitro Overall, our data suggest a role for TET1-mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbm4.10383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587462PMC
August 2020

The Identification of Novel Biomarkers Is Required to Improve Adult SMA Patient Stratification, Diagnosis and Treatment.

J Pers Med 2020 Jul 29;10(3). Epub 2020 Jul 29.

Centre of Research in Myology, Institute of Myology, Sorbonne Université, INSERM, 75013 Paris, France.

Spinal muscular atrophy (SMA) is currently classified into five different subtypes, from the most severe (type 0) to the mildest (type 4) depending on age at onset, best motor function achieved, and copy number of the gene. The two recent approved treatments for SMA patients revolutionized their life quality and perspectives. However, upon treatment with Nusinersen, the most widely administered therapy up to date, a high degree of variability in therapeutic response was observed in adult SMA patients. These data, together with the lack of natural history information and the wide spectrum of disease phenotypes, suggest that further efforts are needed to develop precision medicine approaches for all SMA patients. Here, we compile the current methods for functional evaluation of adult SMA patients treated with Nusinersen. We also present an overview of the known molecular changes underpinning disease heterogeneity. We finally highlight the need for novel techniques, i.e., -omics approaches, to capture phenotypic differences and to understand the biological signature in order to revise the disease classification and device personalized treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/jpm10030075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564782PMC
July 2020

AAV9-Mediated Expression of SMN Restricted to Neurons Does Not Rescue the Spinal Muscular Atrophy Phenotype in Mice.

Mol Ther 2020 08 15;28(8):1887-1901. Epub 2020 May 15.

Sorbonne Université, INSERM, Institute of Myology, Centre of Research in Myology, 75013 Paris, France. Electronic address:

Spinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by mutations or deletions in the survival of motor neuron 1 (SMN1) gene and characterized by the degeneration of motor neurons and progressive muscle weakness. A viable therapeutic approach for SMA patients is a gene replacement strategy that restores functional SMN expression using adeno-associated virus serotype 9 (AAV9) vectors. Currently, systemic or intra-cerebrospinal fluid (CSF) delivery of AAV9-SMN is being explored in clinical trials. In this study, we show that the postnatal delivery of an AAV9 that expresses SMN under the control of the neuron-specific promoter synapsin selectively targets neurons without inducing re-expression in the peripheral organs of SMA mice. However, this approach is less efficient in restoring the survival and neuromuscular functions of SMA mice than the systemic or intra-CSF delivery of an AAV9 in which SMN is placed under the control of a ubiquitous promoter. This study suggests that further efforts are needed to understand the extent to which SMN is required in neurons and peripheral organs for a successful therapeutic effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2020.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403319PMC
August 2020

Inhibition of TET1 prevents the development of osteoarthritis and reveals the 5hmC landscape that orchestrates pathogenesis.

Sci Transl Med 2020 04;12(539)

Department of Orthopaedic Surgery, School of Medicine, Stanford University, Stanford, CA 94305, USA.

Osteoarthritis (OA) is a degenerative disease of the joint, which results in pain, loss of mobility, and, eventually, joint replacement. Currently, no disease-modifying drugs exist, partly because of the multiple levels at which cartilage homeostasis is disrupted. Recent studies have highlighted the importance of epigenetic dysregulation in OA, sparking interest in the epigenetic modulation for this disease. In our previous work, we characterized a fivefold increase in cytosine hydroxymethylation (5hmC), an oxidized derivative of cytosine methylation (5mC) associated with gene activation, accumulating at OA-associated genes. To test the role of 5hmC in OA, here, we used a mouse model of surgically induced OA and found that OA onset was accompanied by a gain of ~40,000 differentially hydroxymethylated sites before the notable histological appearance of disease. We demonstrated that ten-eleven-translocation enzyme 1 (TET1) mediates the 5hmC deposition because 98% of sites enriched for 5hmC in OA were lost in mice. Loss of TET1-mediated 5hmC protected the mice from OA development, including degeneration of the cartilage surface and osteophyte formation, by directly preventing the activation of multiple OA pathways. Loss of in human OA chondrocytes reduced the expression of the matrix metalloproteinases and and multiple inflammatory cytokines. Intra-articular injections of a dioxygenases inhibitor, 2-hydroxyglutarate, on mice after surgical induction of OA stalled disease progression. Treatment of human OA chondrocytes with the same inhibitor also phenocopied loss. Collectively, these data demonstrate that TET1-mediated 5hmC deposition regulates multiple OA pathways and can be modulated for therapeutic intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aax2332DOI Listing
April 2020

Single-cell mass cytometry reveals cross-talk between inflammation-dampening and inflammation-amplifying cells in osteoarthritic cartilage.

Sci Adv 2020 03 13;6(11):eaay5352. Epub 2020 Mar 13.

Department of Orthopedic Surgery, School of Medicine, Stanford University, Stanford, CA 94303, USA.

Aging or injury leads to degradation of the cartilage matrix and the development of osteoarthritis (OA). Because of a paucity of single-cell studies of OA cartilage, little is known about the interpatient variability in its cellular composition and, more importantly, about the cell subpopulations that drive the disease. Here, we profiled healthy and OA cartilage samples using mass cytometry to establish a single-cell atlas, revealing distinct chondrocyte progenitor and inflammation-modulating subpopulations. These rare populations include an inflammation-amplifying (Inf-A) population, marked by interleukin-1 receptor 1 and tumor necrosis factor receptor II, whose inhibition decreased inflammation, and an inflammation-dampening (Inf-D) population, marked by CD24, which is resistant to inflammation. We devised a pharmacological strategy targeting Inf-A and Inf-D cells that significantly decreased inflammation in OA chondrocytes. Using our atlas, we stratified patients with OA in three groups that are distinguished by the relative proportions of inflammatory to regenerative cells, making it possible to devise precision therapeutic approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aay5352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069698PMC
March 2020

TSP1 and TSP2 Have Unique and Overlapping Roles in Protecting against Noise-Induced Auditory Synaptopathy.

Neuroscience 2019 06 28;408:68-80. Epub 2019 Mar 28.

Department of Otolaryngology - Head and Neck Surgery, Stanford University, Stanford, CA, USA. Electronic address:

Thrombospondins (TSPs) are cell adhesion molecules that play an important role in the maintenance of hearing and afferent synaptic connections. Based on their reported function in restoring synaptic connections after stroke, we tested a potential role for TSP1 and TSP2 genes in repairing cochlear synapses following noise injury. We observed a tonotopic gradient in the expression of TSP1 and TSP2 mRNA in control mouse cochleae and an upregulation of these genes following noise exposure. Examining the functional sequelae of these changes revealed that afferent synaptic counts and auditory brainstem responses (ABRs) in noise-exposed TSP1 and TSP2 knockout (-/-) mice exhibited a worst recovery when compared to controls. Consistent with their tonotopic expression, TSP1-/- mice showed greater susceptibility to noise-induced hearing loss (NIHL) at 8 kHz and 16 kHz frequencies, whereas NIHL in TSP2-/- mice occurred only at mid and high frequencies. Further analysis of the ABR waveforms indicated peripheral neuronal damage in TSP2-/- but not in TSP1-/- mice. Noise trauma affecting mid to high frequencies triggered severe seizures in the TSP2-/- mice. We found that decreased susceptibility to audiogenic seizures in TSP1-/- mice was correlated with increased TSP2 protein levels in their inner ears, suggesting that TSP2 might functionally compensate for the loss of TSP1 in these mice. Our data indicate that TSP1 and TSP2 are both involved in susceptibility to NIHL, with TSP2 playing a more prominent role.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuroscience.2019.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556133PMC
June 2019

Effect of trabecular metal on the elution of gentamicin from Palacos cement.

J Orthop Res 2019 05 28;37(5):1018-1024. Epub 2019 Mar 28.

Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Redwood City, California, 94063.

Periprosthetic joint infections continues to be a common complication in total joint arthroplasty, resulting in significant morbidity, mortality, and additional cost. Trabecular metal implants with an internal cemented interface may be customizable drug delivery devices with an ingrowth interface. Thirty-six acetabular implants were assembled in vitro, half with a trabecular metal shell and half without. The antibiotic loaded bone cement was prepared via three different mixing techniques and at two different mixing times. Mixing time had a significant effect on the total amount of gentamicin eluted. The long mixing protocol eluted up to 126% (p = 0.001) more gentamicin than the short mixing protocol at 4 h and 192% (p < 0.001) more at 7 days. Hand or mechanical mixing technique had no significant effect on elution at 4 h. At 7 days, the mechanical mixing system under vacuum eluted over 50% (p = 0.031) more gentamicin than without a vacuum and nearly 60% (p = 0.040) more gentamicin than hand mixing. The use of a trabecular metal shell had no significant effect on the bulk elution of gentamicin at 4 h (p > 0.05) but significantly reduced total gentamicin elution under certain mixing protocols at 7 days. A possible optimization strategy to improve elution kinetics would be to use a long mixing time with a mechanical mixing system under vacuum. The establishment of trabecular metal as an effective delivery vehicle for antibiotics makes possible an entirely new class of drug eluting device designs. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jor.24274DOI Listing
May 2019

Soluble Collagen VI treatment enhances mesenchymal stem cells expansion for engineering cartilage.

Bioeng Transl Med 2017 09 21;2(3):278-284. Epub 2017 Sep 21.

Dept. of Orthopaedic Surgery Stanford University School of Medicine Stanford CA 94305.

Bone Marrow-derived mesenchymal stem cells (BM-MSC) are an attractive source for cell-based therapies in cartilage injury owing to their efficient differentiation into chondrocytes and their immune-suppressive abilities. However, their clinical use is hampered by a scarcity of cells leading to compromised efficacy. While expansion of human MSC ex vivo can potentially overcome the scarcity of cells, current methods lead to a rapid loss of the stem cell properties. In this study, we report soluble Collagen VI (cartilage pericellular matrix component) as a potential biologic that can expand the MSC population while maintaining the stem cell phenotype as confirmed by expression of the stem cell markers CD105 and CD90. Short-term treatment with Collagen VI additionally retains the potential of MSC to differentiate into mature chondrocytes in pellet culture. Cartilage pellets generated from MSC treated with Collagen VI or control express comparable amounts of the chondrogenic markers Collagen II, Aggrecan and Sox9, and the extracellular glycosaminoglycans. Our observations confirm that the use of the endogenous and cartilage-specific factor Collagen VI is valuable for a rapid and efficient expansion of MSC for potential use in cartilage regeneration and osteoarthritis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/btm2.10078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689496PMC
September 2017

Human iPSC-derived chondrocytes mimic juvenile chondrocyte function for the dual advantage of increased proliferation and resistance to IL-1β.

Stem Cell Res Ther 2017 Nov 2;8(1):244. Epub 2017 Nov 2.

Department of Orthopaedic Surgery, Stanford University School of Medicine, 300 Pasteur Drive, Edwards Bldg., R164, Stanford, CA, 94305-5341, USA.

Background: Induced pluripotent stem cells (iPSC) provide an unlimited patient-specific cell source for regenerative medicine. Adult cells have had limited success in cartilage repair, but juvenile chondrocytes (from donors younger than 13 years of age) have been identified to generate superior cartilage. With this perspective, the aim of these studies was to compare the human iPSC-derived chondrocytes (hiChondrocytes) to adult and juvenile chondrocytes and identify common molecular factors that govern their function.

Methods: Phenotypic and functional characteristics of hiChondrocytes were compared to juvenile and adult chondrocytes. Analyses of global gene expression profiling, independent gene expression, and loss-of-function studies were utilized to test molecular factors having a regulatory effect on hiChondrocytes and juvenile chondrocyte function.

Results: Here, we report that the iPSC-derived chondrocytes mimic juvenile chondrocytes in faster cell proliferation and resistance to IL-1β compared to adult chondrocytes. Whole genome transcriptome analyses revealed unique ECM factors and immune response pathways to be enriched in both juvenile and iPSC-derived chondrocytes as compared to adult chondrocytes. Loss-of-function studies demonstrated that CD24, a cell surface receptor enriched in both juvenile chondrocytes and hiChondrocytes, is a regulatory factor in both faster proliferation and resistance to proinflammatory cues in these chondrocyte populations.

Conclusions: Our studies identify that hiChondrocytes mimic juvenile chondrocytes for the dual advantage of faster proliferation and a reduced response to the inflammatory cytokine IL-1β. While developmental immaturity of iPSC-derived cells can be a challenge for tissues like muscle and brain, our studies demonstrate that it is advantageous for a tissue like cartilage that has limited regenerative ability in adulthood.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13287-017-0696-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667438PMC
November 2017

CD24 enrichment protects while its loss increases susceptibility of juvenile chondrocytes towards inflammation.

Arthritis Res Ther 2016 12 12;18(1):292. Epub 2016 Dec 12.

Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Background: Diseases associated with human cartilage, including rheumatoid arthritis (RA) and osteoarthritis (OA) have manifested age, mechanical stresses and inflammation as the leading risk factors. Although inflammatory processes are known to be upregulated upon aging, we sought to gain a molecular understanding of how aging affects the tissue-specific response to inflammation. In this report, we explored the role of cluster of differentiation 24 (CD24) in regulating differential inflammatory responses in juvenile and adult human chondrocytes.

Methods: Differential cell-surface CD24 expression was assessed in juvenile and adult chondrocytes along with human induced pluripotent stem cell (hiPSC)-derived neonatal chondrocytes through gene expression and fluorescence-activated cell sorting (FACS) analyses. Loss of function of CD24 was achieved through silencing in chondrocytes and the effects on the response to inflammatory cues were assessed through gene expression and NFκB activity.

Results: CD24 expression in chondrocytes caused a differential response to cytokine-induced inflammation, with the CD24 juvenile chondrocytes being resistant to IL-1ß treatment as compared to CD24 adult chondrocytes. CD24 protects from inflammatory response by reducing NFκB activation, as an acute loss of CD24 via silencing led to an increase in NFκB activation. Moreover, the loss of CD24 in chondrocytes subsequently increased inflammatory and catabolic gene expression both in the absence and presence of IL-1ß.

Conclusions: We have identified CD24 as a novel regulator of inflammatory response in cartilage that is altered during development and aging and could potentially be therapeutic in RA and OA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13075-016-1183-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5153697PMC
December 2016

Identification of Human Juvenile Chondrocyte-Specific Factors that Stimulate Stem Cell Growth.

Tissue Eng Part A 2016 Apr 31;22(7-8):645-53. Epub 2016 Mar 31.

Department of Orthopedic Surgery, Stanford University School of Medicine , Stanford, California.

Although regeneration of human cartilage is inherently inefficient, age is an important risk factor for osteoarthritis. Recent reports have provided compelling evidence that juvenile chondrocytes (from donors below 13 years of age) are more efficient at generating articular cartilage as compared to adult chondrocytes. However, the molecular basis for such a superior regenerative capability is not understood. To identify the cell-intrinsic differences between juvenile and adult cartilage, we have systematically profiled global gene expression changes between a small cohort of human neonatal/juvenile and adult chondrocytes. No such study is available for human chondrocytes although young and old bovine and equine cartilage have been recently profiled. Our studies have identified and validated new factors enriched in juvenile chondrocytes as compared to adult chondrocytes including secreted extracellular matrix factors chordin-like 1 (CHRDL1) and microfibrillar-associated protein 4 (MFAP4). Network analyses identified cartilage development pathways, epithelial-mesenchymal transition, and innate immunity pathways to be overrepresented in juvenile-enriched genes. Finally, CHRDL1 was observed to aid the proliferation and survival of bone marrow-derived human mesenchymal stem cells (hMSC) while maintaining their stem cell potential. These studies, therefore, provide a mechanism for how young cartilage factors can potentially enhance stem cell function in cartilage repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2015.0366DOI Listing
April 2016

Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functions via epigenetic modulation of Sca-1 and Pw1 promoters.

FASEB J 2016 Apr 15;30(4):1404-15. Epub 2015 Dec 15.

*Stem Cells and Regenerative Medicine, Institute of Cardiometabolism and Nutrition Unité Mixte de Recherche en Santé 1166 INSERM/Sorbonne University (Pierre and Marie Curie University, Paris VI), Paris, France; Department of Anatomy, Histology, Forensic Medicine, and Orthopedics, Unit of Histology, Sapienza University of Rome, Rome, Italy; INSERM Unité 955 Institut Mondor de Recherche Biomédicale, Creteil, France; Université Paris-Est Créteil, Faculty of Medicine, Creteil, France; Sorbonne Universités, Pierre and Marie Curie University, Paris VI, INSERM Unité Mixte de Recherche en Santé 974, Centre National de la Recherche Scientifique FRE3617, Center for Research in Myology, Paris, France; Etablissement Français du Sang, Creteil, France; and Université Paris Est, Ecole Nationale Veterinaire d'Alfort, Maison Alfort, France

Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fatein vitro In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regenerationin vivofollowing transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potentialin vivoand replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, includingSca-1andPw1 Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functionsviaepigenetic modulation of Sca-1 and Pw1 promoters.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.15-275420DOI Listing
April 2016

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation.

J Vis Exp 2015 Oct 7(104). Epub 2015 Oct 7.

Orthopaedic Surgery Department, Stanford University.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/53085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692641PMC
October 2015

Stable 5-Hydroxymethylcytosine (5hmC) Acquisition Marks Gene Activation During Chondrogenic Differentiation.

J Bone Miner Res 2016 Mar 5;31(3):524-34. Epub 2015 Oct 5.

Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, CA, USA.

Regulation of gene expression changes during chondrogenic differentiation by DNA methylation and demethylation is little understood. Methylated cytosines (5mC) are oxidized by the ten-eleven-translocation (TET) proteins to 5-hydroxymethylcytosines (5hmC), 5-formylcytosines (5fC), and 5-carboxylcytosines (5caC), eventually leading to a replacement by unmethylated cytosines (C), ie, DNA demethylation. Additionally, 5hmC is stable and acts as an epigenetic mark by itself. Here, we report that global changes in 5hmC mark chondrogenic differentiation in vivo and in vitro. Tibia anlagen and growth plate analyses during limb development at mouse embryonic days E 11.5, 13.5, and 17.5 showed dynamic changes in 5hmC levels in the differentiating chondrocytes. A similar increase in 5hmC levels was observed in the ATDC5 chondroprogenitor cell line accompanied by increased expression of the TET proteins during in vitro differentiation. Loss of TET1 in ATDC5 decreased 5hmC levels and impaired differentiation, demonstrating a functional role for TET1-mediated 5hmC dynamics in chondrogenic differentiation. Global analyses of the 5hmC-enriched sequences during early and late chondrogenic differentiation identified 5hmC distribution to be enriched in the regulatory regions of genes preceding the transcription start site (TSS), as well as in the gene bodies. Stable gains in 5hmC were observed in specific subsets of genes, including genes associated with cartilage development and in chondrogenic lineage-specific genes. 5hmC gains in regulatory promoter and enhancer regions as well as in gene bodies were strongly associated with activated but not repressed genes, indicating a potential regulatory role for DNA hydroxymethylation in chondrogenic gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.2711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860191PMC
March 2016

Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells.

FASEB J 2015 Aug 24;29(8):3399-410. Epub 2015 Apr 24.

*Department of Orthopaedic Surgery, Department of Mechanical Engineering, and Department of Bioengineering, Stanford University School of Medicine, Stanford, California, USA

Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.14-269720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511207PMC
August 2015

Collagen VI enhances cartilage tissue generation by stimulating chondrocyte proliferation.

Tissue Eng Part A 2015 Feb 11;21(3-4):840-9. Epub 2014 Nov 11.

1 Department of Orthopedic Surgery, Stanford University , Stanford, California.

Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2014.0375DOI Listing
February 2015

Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels.

Tissue Eng Part A 2015 Jan 1;21(1-2):147-55. Epub 2014 Oct 1.

1 Department of Orthopedic Surgery, Stanford University , Stanford, California.

Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, and embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old, and diseased chondrocytes, here we examined cartilage formation by juvenile, adult, and OA chondrocytes in three-dimensional (3D) biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and glycosaminoglycan accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2014.0070DOI Listing
January 2015

Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ.

Pflugers Arch 2014 Dec 20;466(12):2215-28. Epub 2014 Mar 20.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, 70125, Bari, Italy.

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00424-014-1495-1DOI Listing
December 2014

A global increase in 5-hydroxymethylcytosine levels marks osteoarthritic chondrocytes.

Arthritis Rheumatol 2014 Jan;66(1):90-100

Stanford University, School of Medicine, Stanford, California.

Objective: To investigate the role of the newly discovered epigenetic mark 5-hydroxymethylcytosine (5hmC) and its regulators in altered gene expression in osteoarthritis (OA).

Methods: Cartilage was obtained from OA patients undergoing total knee arthroplasty and from control patients undergoing anterior cruciate ligament reconstruction. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene expression changes were monitored by quantitative polymerase chain reaction (PCR) analysis. Levels of locus-specific 5hmC and 5mC at CpG sites in the matrix metalloproteinase 1 (MMP-1), MMP-3, ADAMTS-5, and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT-1) promoters were quantified using a glucosylation and enzyme digestion-based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were monitored in normal chondrocytes stimulated with inflammatory cytokines to identify the effect of joint inflammation.

Results: A global 5-6-fold increase in 5hmC concomitant with a loss of TET1 was observed in human OA chondrocytes compared to normal chondrocytes. Enrichment of 5hmC was observed in promoters of enzymes critical to OA pathology, MMP-1 and MMP-3. Short-term treatment of normal chondrocytes with inflammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrichment.

Conclusion: This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accumulation. Our experiments identify 5hmC and its regulators as potential diagnostic and therapeutic targets in OA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/art.38200DOI Listing
January 2014

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations.

Proc Natl Acad Sci U S A 2011 Jul 27;108(28):11470-5. Epub 2011 Jun 27.

Myology Group (Stem Cell and Muscle Biology), Unité Mixte de Recherche-S787 Institut National de la Santé et de la Recherche Médicale, University of Pierre and Marie Curie Paris VI, Paris 75634, France.

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1103873108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136256PMC
July 2011

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase.

Mol Biol Cell 2011 Apr 23;22(8):1409-19. Epub 2011 Feb 23.

Department of Anatomy, Sapienza University of Rome, Rome, Italy.

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E10-10-0821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078083PMC
April 2011