Amel Dudakovic

High-Resolution Molecular Validation of Self-Renewal and Spontaneous Differentiation in Clinical-Grade Adipose-Tissue Derived Human Mesenchymal Stem Cells

Improving the effectiveness of adipose‐tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi‐potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non‐proliferative state. Flow cytometry combined with high‐throughput RNA sequencing (RNASeq) and RT‐qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self‐renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto‐architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle‐related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone‐related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10‐fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT‐related genes (WISP2, SFRP2, and SFRP4). Furthermore, post‐proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self‐renewing and multi‐potent AMSCs by providing high‐resolution quality control data that support their clinical versatility. J. Cell. Biochem. 115: 1816–1828, 2014.

Histone deacetylase 3 is required for maintenance of bone mass during aging

Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKO(OCN) mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKO(OCN) mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKO(OCN) mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKO(OCN) mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging.

Epigenetic control of skeletal development by the histone methyltransferase Ezh2

Background: Osteogenic differentiation is initiated by transcriptional and post-transcriptional epigenetic mechanisms. Results: Inhibition of H3K27 methyltransferase EZH2 enhances osteogenic commitment of human mesenchymal progenitors, and its depletion in mouse mesenchymal cells causes multiple skeletal abnormalities. Conclusion: EZH2 is required for skeletal patterning and bone formation. Significance: EZH2-dependent epigenetic mechanisms control osteogenesis both in vitro and in vivo. Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation

Background: Histone deacetylase (HDAC) inhibition promotes bone formation in vitro via undefined epigenetic events. Results: Microarray and ChIP-Seq analyses revealed genomic areas where H4 acetylation is altered by HDAC inhibitors and identified differentially regulated genes. Conclusion: Suberoylanilide hydroxamic acid increases H4 acetylation and suppresses phosphorylation of insulin/Akt signaling mediators in osteoblasts. Significance: Epigenetic profiling is a powerful means to gain mechanistic insights into bone anabolic processes. Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production

BackgroundClinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL).MethodsIn this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors.ResultsWe characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells.ConclusionsOur study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria.Clinical trialsUse of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007. Registered August 26, 2013.MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540. Registered April 23, 2013.Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027. Registered October 31, 2014.Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145. Registered August 17, 2006.A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283. Registered May 18, 2012.

Autologous Mesenchymal Stem Cells, Applied in a Bioabsorbable Matrix, for Treatment of Perianal Fistulas in Patients With Crohn's Disease

In patients with Crohns disease, perianal fistulas recur frequently, causing substantial morbidity. We performed a 12-patient, 6-month, phase 1 trial to determine whether autologous mesenchymal stem cells, applied in a bioabsorbable matrix, can heal the fistula. Fistula repair was not associated with any serious adverse events related to mesenchymal stem cells or plug placement. At 6 months, 10 of 12 patients (83%) had complete clinical healing and radiographic markers of response. We found placement of mesenchymal stem cell-coated matrix fistula plugs in 12 patients with chronic perianal fistulas to be safe and lead to clinical healing and radiographic response in 10 patients. ClinicalTrials.gov Identifier: NCT01915927.

Histone deacetylase inhibition destabilizes the multi-potent state of uncommitted adipose-derived mesenchymal stromal cells

Human adipose‐derived mesenchymal stromal cells (AMSCs) grown in platelet lysate are promising agents for therapeutic tissue regeneration. Here, we investigated whether manipulation of epigenetic events by the clinically relevant histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) alters differentiation of AMSCs. The multipotency of AMSCs was validated by their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. High‐throughput RNA sequencing and RT‐qPCR established that human histone deacetylases (HDAC1 to HDAC11, and SIRT1 to SIRT7) are differentially expressed in AMSCs. SAHA induces hyper‐acetylation of histone H3 and H4, stimulates protein expression of the HDAC‐responsive gene SLC9A3R1/NHERF1 and modulates the AKT/FOXO1 pathway. Biologically, SAHA interferes with osteogenic, chondrogenic and adipogenic lineage commitment of multipotent AMSCs. Mechanistically, SAHA‐induced loss of differentiation potential of uncommitted AMSCs correlates with multiple changes in the expression of principal transcription factors that control mesenchymal or pluripotent states. We propose that SAHA destabilizes the multi‐potent epigenetic state of uncommitted human AMSCs by hyper‐acetylation and perturbation of key transcription factor pathways. Furthermore, AMSCs grown in platelet lysate may provide a useful biological model for screening of new HDAC inhibitors that control the biological fate of human mesenchymal stromal cells. J. Cell. Physiol. 230: 52–62, 2015.

Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate

Bone degenerative pathologies like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Here we show that sulforaphane (SFN), a naturally occurring isothiocyanate, promotes osteoblast differentiation by epigenetic mechanisms. SFN enhances active DNA demethylation via Tet1 and Tet2 and promotes preosteoblast differentiation by enhancing extracellular matrix mineralization and the expression of osteoblastic markers (Runx2, Col1a1, Bglap2, Sp7, Atf4, and Alpl). SFN decreases the expression of the osteoclast activator receptor activator of nuclear factor-κB ligand (RANKL) in osteocytes and mouse calvarial explants and preferentially induces apoptosis in preosteoclastic cells via up-regulation of the Tet1/Fas/Caspase 8 and Caspase 3/7 pathway. These mechanistic effects correlate with higher bone volume (∼20%) in both normal and ovariectomized mice treated with SFN for 5 weeks compared with untreated mice as determined by microcomputed tomography. This effect is due to a higher trabecular number in these mice. Importantly, no shifts in mineral density distribution are observed upon SFN treatment as measured by quantitative backscattered electron imaging. Our data indicate that the food-derived compound SFN epigenetically stimulates osteoblast activity and diminishes osteoclast bone resorption, shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorption in vivo. Thus, SFN is a member of a new class of epigenetic compounds that could be considered for novel strategies to counteract osteoporosis.

Multi-disciplinary antimicrobial strategies for improving orthopaedic implants to prevent prosthetic joint infections in hip and knee

Like any foreign object, orthopaedic implants are susceptible to infection when introduced into the human body. Without additional preventative measures, the absolute number of annual prosthetic joint infections will continue to rise, and may exceed the capacity of health care systems in the near future. Bacteria are difficult to eradicate from synovial joints due to their exceptionally diverse taxonomy, complex mechanistic attachment capabilities, and tendency to evolve antibiotic resistance. When a primary orthopaedic implant fails from prosthetic joint infection, surgeons are generally challenged by limited options for intervention. In this review, we highlight the etiology and taxonomic groupings of bacteria known to cause prosthetic joint infections, and examine their key mechanisms of attachment. We propose that antimicrobial strategies should focus on the most harmful bacteria taxa within the context of occurrence, taxonomic diversity, adhesion mechanisms, and implant design. Patient‐specific identification of organisms that cause prosthetic joint infections will permit assessment of their biological vulnerabilities. The latter can be targeted using a range of antimicrobial techniques that exploit different colonization mechanisms including implant surface attachment, biofilm formation, and/or hematogenous recruitment. We anticipate that customized strategies for each patient, joint, and prosthetic component will be most effective at reducing prosthetic joint infections, including those caused by antibiotic‐resistant and polymicrobial bacteria.

Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling

Histone deacetylase 3 prevents chondrocytes from producing inflammatory mediators that prevent bone development. Growing bones need histone deacetylase Histone deacetylase (HDAC) inhibitors may be therapeutic in various diseases, but their use causes birth defects and is detrimental to growing bones or the repair of injured bones. Cartilage provides the bone-promoting matrix and bone-forming progenitor cells required for the formation of long bones. Carpio et al. found that HDAC3 promotes pre- and postnatal bone growth by restricting the secretion of inflammatory factors from cartilage cells called chondrocytes. Mice lacking chondrocyte-specific Hdac3 died in utero, and inducible transgenic mice lacking postnatal HDAC3 in chondrocytes had impaired long bone development. Chondrocytes from these mice had increased acetylation of a proinflammatory transcription factor as well as of histones in and near loci encoding secreted proinflammatory factors that promote matrix degradation and the proliferation and activity of bone-resorbing osteoclasts. The findings explain why HDAC inhibitors cause skeletal defects and are ill-advised for children and pregnant women as well as for patients with bone fractures. Histone deacetylase (HDAC) inhibitors are efficacious epigenetic-based therapies for some cancers and neurological disorders; however, each of these drugs inhibits multiple HDACs and has detrimental effects on the skeleton. To better understand how HDAC inhibitors affect endochondral bone formation, we conditionally deleted one of their targets, Hdac3, pre- and postnatally in type II collagen α1 (Col2α1)–expressing chondrocytes. Embryonic deletion was lethal, but postnatal deletion of Hdac3 delayed secondary ossification center formation, altered maturation of growth plate chondrocytes, and increased osteoclast activity in the primary spongiosa. HDAC3-deficient chondrocytes exhibited increased expression of cytokine and matrix-degrading genes (Il-6, Mmp3, Mmp13, and Saa3) and a reduced abundance of genes related to extracellular matrix production, bone development, and ossification (Acan, Col2a1, Ihh, and Col10a1). Histone acetylation increased at and near genes that had increased expression. The acetylation and activation of nuclear factor κB (NF-κB) were also increased in HDAC3-deficient chondrocytes. Increased cytokine signaling promoted autocrine activation of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and NF-κB pathways to suppress chondrocyte maturation, as well as paracrine activation of osteoclasts and bone resorption. Blockade of interleukin-6 (IL-6)–JAK–STAT signaling, NF-κB signaling, and bromodomain extraterminal proteins, which recognize acetylated lysines and promote transcriptional elongation, significantly reduced Il-6 and Mmp13 expression in HDAC3-deficient chondrocytes and secondary activation in osteoclasts. The JAK inhibitor ruxolitinib also reduced osteoclast activity in Hdac3 conditional knockout mice. Thus, HDAC3 controls the temporal and spatial expression of tissue-remodeling genes and inflammatory responses in chondrocytes to ensure proper endochondral ossification during development.