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1.  RNA-seq Analysis of Clinical-Grade Osteochondral Allografts Reveals Activation of Early Response Genes 
Preservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA-seq) and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to assess genome-wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of “early response genes” that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of “early response genes” and reduced levels of ECM-related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation.
PMCID: PMC4993686  PMID: 26909883
cartilage; allograft; osteochondral; transplant; RNA sequencing
2.  Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate 
Tissue Engineering. Part A  2015;21(21-22):2703-2713.
Purpose: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer “neoligament” seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue.
Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell–cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C).
Results: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts.
Summary: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration.
PMCID: PMC4652223  PMID: 26413793
3.  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 
Clinical 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).
In 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.
We 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.
Our 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 trials
Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): NCT01931007. Registered August 26, 2013.
MSC for Occlusive Disease of the Kidney: NCT01840540. Registered April 23, 2013.
Mesenchymal Stem Cell Therapy in Multiple System Atrophy: NCT02315027. Registered October 31, 2014.
Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. NCT00366145. Registered August 17, 2006.
A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. NCT01609283. Registered May 18, 2012.
Electronic supplementary material
The online version of this article (doi:10.1186/s13287-016-0370-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4982273  PMID: 27515308
Adipose-derived mesenchymal stromal cells; RNA-sequencing; Flow cytometry; Release criteria; CD markers; Human platelet lysate; Manufacturing
5.  Biological Strategies for Improved Osseointegration and Osteoinduction of Porous Metal Orthopedic Implants 
The biological interface between an orthopedic implant and the surrounding host tissue may have a dramatic effect upon clinical outcome. Desired effects include bony ingrowth (osseointegration), stimulation of osteogenesis (osteoinduction), increased vascularization, and improved mechanical stability. Implant loosening, fibrous encapsulation, corrosion, infection, and inflammation, as well as physical mismatch may have deleterious clinical effects. This is particularly true of implants used in the reconstruction of load-bearing synovial joints such as the knee, hip, and the shoulder. The surfaces of orthopedic implants have evolved from solid-smooth to roughened-coarse and most recently, to porous in an effort to create a three-dimensional architecture for bone apposition and osseointegration. Total joint surgeries are increasingly performed in younger individuals with a longer life expectancy, and therefore, the postimplantation lifespan of devices must increase commensurately. This review discusses advancements in biomaterials science and cell-based therapies that may further improve orthopedic success rates. We focus on material and biological properties of orthopedic implants fabricated from porous metal and highlight some relevant developments in stem-cell research. We posit that the ideal primary and revision orthopedic load-bearing metal implants are highly porous and may be chemically modified to induce stem cell growth and osteogenic differentiation, while minimizing inflammation and infection. We conclude that integration of new biological, chemical, and mechanical methods is likely to yield more effective strategies to control and modify the implant–bone interface and thereby improve long-term clinical outcomes.
PMCID: PMC4390115  PMID: 25348836
6.  Histone deacetylase inhibition destabilizes the multi-potent state of uncommitted adipose-derived mesenchymal stromal cells 
Journal of cellular physiology  2015;230(1):52-62.
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.
PMCID: PMC4225068  PMID: 24912092
osteoblast; bone; mesenchymal stem cell; histone deacetylase; osteogenesis; vorinostat; chondrocyte; adipocyte
7.  MicroRNA and mRNA Cargo of Extracellular Vesicles from Porcine Adipose Tissue-Derived Mesenchymal Stem Cells 
Gene  2014;551(1):55-64.
Mesenchymal stromal/stem cells (MSCs) are clinically useful for cell-based therapy, but concerns regarding their ability to replicate limit their human application. MSCs release extracellular vesicles (EVs) that mediate at least in part the paracrine effects of the parental cells. To understand the molecular basis of their biological properties, we characterized the RNA cargo of EVs from porcine adipose-tissue derived MSCs. Comprehensive characterization of mRNA and miRNA gene expression using high-throughput RNA sequencing (RNA-seq) revealed that EVs are selectively enriched for distinct classes of RNAs. For example, EVs preferentially express mRNA for transcription factors (e.g. MDFIC, POU3F1, NRIP1) and genes involved in angiogenesis (e.g. HGF, HES1, TCF4) and adipogenesis (e.g. CEBPA, KLF7). EVs also express Golgi apparatus genes (ARRB1, GOLGA4) and genes involved in TGF-β signaling. In contrast, mitochondrial, calcium signaling, and cytoskeleton genes are selectively excluded from EVs, possibly because these genes remain sequestered in organelles or intracellular compartments. RNA-seq generated reads for at least 386 annotated miRNAs, but only miR148a, miR532-5p, miR378, and let-7f were enriched in EVs compared to MSCs. Gene ontology analysis indicates that these miRNA target transcription factors and genes that participate in several cellular pathways, including angiogenesis, cellular transport, apoptosis, and proteolysis. Our data suggest that EVs transport gene regulatory information to modulate angiogenesis, adipogenesis, and other cell pathways in recipient cells. These observations may contribute to development of regenerative strategies using EVs to overcome potential complications of cell-based therapy.
PMCID: PMC4174680  PMID: 25158130
Mesenchymal stem cells; extracellular vesicles; microvesicles; exosomes; next generation sequencing (NGS); RNASeq; gene expression; miRNA
8.  RNA sequencing reveals a depletion of collagen targeting microRNAs in Dupuytren’s disease 
BMC Medical Genomics  2015;8:59.
Dupuytren’s disease is an inherited disorder in which patients develop fibrotic contractures of the hand. Current treatment strategies include surgical excision or enzymatic digestion of fibrotic tissue. MicroRNAs, which are key posttranscriptional regulators of genes expression, have been shown to play an important regulatory role in disorders of fibrosis. Therefore in this investigation, we apply high throughput next generation RNA sequencing strategies to characterize microRNA expression in diseased and healthy palmar fascia to elucidate molecular mechanisms responsible for pathogenic fibrosis.
We applied high throughput RNA sequencing techniques to quantify the expression of all known human microRNAs in Dupuytren’s and control palmar fascia. MicroRNAs that were differentially expressed between diseased and healthy tissue samples were used for computational target prediction using the bioinformatics tool ComiR. Molecular pathways that were predicted to be differentially expressed based on computational analysis were validated by performing RT-qPCR on RNA extracted from diseased and non-diseased palmar fascia biopsies.
A comparison of microRNAs expressed in Dupuytren’s fascia and control fascia identified 74 microRNAs with a 2-fold enrichment in Dupuytren’s tissue, and 32 microRNAs with enrichment in control fascia. Computational target prediction for differentially expressed microRNAs indicated preferential targeting of collagens and extracellular matrix related proteins in control palmar fascia. RT-qPCR confirmed the decreased expression of microRNA targeted collagens in control palmar fascia tissues.
Control palmar fascia show decreased expression of mRNAs encoding collagens that are preferentially targeted by microRNAs enriched in non-diseased fascia. Thus alterations in microRNA regulatory networks may play an important role in driving the pathogenic fibrosis seen in Dupuytren’s disease via direct regulatory effects on extracellular matrix protein synthesis.
Dupuytren’s fascia and healthy palmar fascia can be distinguished by unique microRNA profiles, which are predicted to preferentially target collagens and other extracellular matrix proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s12920-015-0135-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4597401  PMID: 26446724
Dupuytren’s disease; microRNA; fibrosis; RNA sequencing; hand
9.  High-resolution molecular validation of self-renewal and spontaneous differentiation in adipose-tissue derived human mesenchymal stem cells cultured in human platelet lysate 
Journal of cellular biochemistry  2014;115(10):1816-1828.
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 up-regulating 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.
PMCID: PMC4225070  PMID: 24905804
Mesenchymal stem cell; adipose-tissue derived stromal cells; pluripotent; multipotent; cell cycle; lineage-commitment; fibroblast; osteogenesis; chondrogenesis; adipogenesis; histone; cyclin; extracellular matrix; CD44; CD73; NT5E; CD90; THY1; CD105; ENG; NES; ACTA2; OCT4; POU5F1; NANOG; KLF4; CCND1; CCNB2; HIST1H3H; HIST1H4A; HIST2H4A; HIST2H4B; E2F1; E2F7; E2F8; HINFP; NPAT; ASPN; ECM2; FMOD; OGN; PODN; WISP2; SFRP2; SFRP4; WNT2; WNT2A; WNT5A; WNT5B; WNT7B; RARRES2; TNNT3; ADH1B; H19; CHI3L1
10.  Intra-renal delivery of mesenchymal stem cells and endothelial progenitor cells attenuates hypertensive cardiomyopathy in experimental renovascular hypertension 
Cell transplantation  2014;24(10):2041-2053.
Renovascular hypertension (RVH) leads to left ventricular (LV) hypertrophy and diastolic dysfunction, associated with increased cardiovascular mortality. Intra-renal delivery of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) improves kidney function in porcine RVH, and the potent anti-inflammatory properties of MSCs may serve to blunt inflammatory mediators in the cardio-renal axis. However, their relative efficacy in attenuating cardiac injury and dysfunction remains unknown. This study tested the hypothesis that the cardio-protective effect of EPCs and MSCs delivered into the stenotic-kidney in experimental RVH are comparable.
Pigs (n=7 per group) were studied after 10 weeks of RVH or control untreated or treated with a single intra-renal infusion of autologous EPCs or MSCs 4 weeks earlier. Cardiac and renal function (fast-CT) and stenotic-kidney release of inflammatory mediators (ELISA) were assessed in-vivo, and myocardial inflammation, remodeling, and fibrosis ex-vivo.
After 10 weeks of RVH, blood pressure was not altered in cell-treated groups, yet stenotic-kidney glomerular filtration rate (GFR), blunted in RVH, improved in RVH+EPC and normalized in RVH+MSC. Stenotic-kidney release of monocyte chemoattractant protein (MCP)-1 and its myocardial expression were elevated in RVH+EPC, but normalized only in RVH+MSC pigs. RVH-induced LV hypertrophy was normalized in both EPC and MSC-treated pigs, while diastolic function (E/A ratio) was restored to normal levels exclusively in RVH+MSC. RVH-induced myocardial fibrosis and collagen deposition decreased in RVH+EPC, but further decreased in RVH+MSC-treated pigs.
Intra-renal delivery of EPCs or MSCs attenuates RVH-induced myocardial injury, yet MSCs restore diastolic function more effectively than EPCs, possibly by greater improvement in renal function or reduction of MCP-1 release from the stenotic-kidney. These observations suggest a therapeutic potential for EPCs and MSCs in preserving the myocardium in chronic experimental RVH.
PMCID: PMC4440858  PMID: 25420012
renal hypertension; myocardium; stem cells; progenitor cells
11.  Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines 
Oncotarget  2015;6(14):12505-12519.
Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability, while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.
PMCID: PMC4494954  PMID: 25895133
isocitrate dehydrogenase; d-2-hydroxyglutarate; chondrosarcoma; sarcoma; AGI-5198
12.  Intra-renal delivery of mesenchymal stem cells attenuates myocardial injury after reversal of hypertension in porcine renovascular disease 
Percutaneous transluminal renal angioplasty (PTRA) fails to fully improve cardiac injury and dysfunction in patients with renovascular hypertension (RVH). Mesenchymal stem cells (MSCs) restore renal function, but their potential for attenuating cardiac injury after reversal of RVH has not been explored. We hypothesized that replenishment of MSCs during PTRA would improve cardiac function and oxygenation, and decrease myocardial injury in porcine RVH.
Pigs were studied after 16 weeks of RVH, RVH treated 4 weeks earlier with PTRA with or without adjunct intra-renal delivery of MSC (10^6 cells), and controls. Cardiac structure, function (fast-computed tomography (CT)), and myocardial oxygenation (Blood-Oxygen-Level-Dependent- magnetic resonance imaging) were assessed in-vivo. Myocardial microvascular density (micro-CT) and myocardial injury were evaluated ex-vivo. Kidney venous and systemic blood levels of inflammatory markers were measured and their renal release calculated.
PTRA normalized blood pressure, yet stenotic-kidney glomerular filtration rate, similarly blunted in RVH and RVH + PTRA, normalized only in PTRA + MSC-treated pigs. PTRA attenuated left ventricular remodeling, whereas myocardial oxygenation, subendocardial microvascular density, and diastolic function remained decreased in RVH + PTRA, but normalized in RVH + PTRA-MSC. Circulating isoprostane levels and renal release of inflammatory cytokines increased in RVH and RVH + PTRA, but normalized in RVH + PTRA-MSC, as did myocardial oxidative stress, inflammation, collagen deposition, and fibrosis.
Intra-renal MSC delivery during PTRA preserved stenotic-kidney function, reduced systemic oxidative stress and inflammation, and thereby improved cardiac function, oxygenation, and myocardial injury four weeks after revascularization, suggesting a therapeutic potential for adjunctive MSC delivery to preserve cardiac function and structure after reversal of experimental RVH.
PMCID: PMC4417319  PMID: 25599803
13.  Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells 
BMC Cancer  2012;12:93.
Osteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells.
2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α).
2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls.
2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment strategies in the control of osteosarcoma.
PMCID: PMC3414746  PMID: 22429849
2-Methoxyestradiol; osteosarcoma; Interferon; ISRE; GAS

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