Viable Cell Source Research Articles

Comparison of immunodulatory properties of dental pulp stem cells derived from healthy and inflamed teeth

The aim of this study was to investigate the immunodulatory properties of dental pulp stem cells derived from healthy (SCD) and inflamed pulp deciduous (SCDIP) tissues. The overall hypothesis is that SCDIP possess equal immune properties with SCD and could be used as an alternative tissue source in regenerative medicine. An intra-oral examination was carried out to assess the status of the pulp tissues and group them according to healthy or inflamed. Primary cells were established from these groups, and basic mesenchymal stem cells (MSC) characterizations were conducted. The expression of human leukocyte antigen (HLA), namely HLA-G, HLA-DR, and HLA-ABC were examined in both cell lines using flow cytometry. We further compared the immunosuppressive effects of SCD and SCDIP on phytohemagglutinin-induced T cell proliferation. Supernatants were tested for cytokine profiling using multiplex array. While SCD exhibited typical MSC characteristics, SCDIP on the other hand, did not. Compared with SCDIP, SCD effectively suppresses mitogen-induced T cells proliferation in a dose-dependent manner, as well as express a higher percentage of HLA-ABC and HLA-G. In addition, levels of several cytokines, such as TNF-α, TNF-β, and IL-2, were drastically suppressed in SCD than SCDIP. Furthermore, a high level of IL-10, an important anti-inflammatory cytokine, was present in SCD compared with SCDIP. These findings suggest that SCDIP is highly dysfunctional in terms of their stemness and immunomodulatory properties. SCDIP is not a viable therapeutic cell source especially when used in graft versus host disease (GvHD) and organ rejection.

A chondromimetic microsphere for in situ spatially controlled chondrogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have been identified as a viable cell source for cartilage tissue engineering. However, to undergo chondrogenic differentiation hMSCs require growth factors, in particular members of the transforming growth factor beta (TGF-β) family. While in vitro differentiation is feasible through continuous supplementation of TGF-β3, mechanisms to control and drive hMSCs down the chondrogenic lineage in their native microenvironment remain a significant challenge. The release of TGF-β3 from an injectable microsphere composed of the cartilage-associated extracellular matrix molecule hyaluronan represents a readily translatable approach for in situ differentiation of hMSCs for cartilage repair. In this study, chondromimetic hyaluronan microspheres were used as a growth factor delivery source for hMSC chondrogenesis. Cellular compatibility of the microspheres (1.2 and 14.1μm) with hMSCs was shown and release of TGF-β3 from the most promising 14.1μm microspheres to control differentiation of hMSCs was evaluated. Enhanced accumulation of cartilage-associated glycosaminoglycans by hMSCs incubated with TGF-β3-loaded microspheres was seen and positive staining for collagen type II and proteoglycan confirmed successful in vitro chondrogenesis. Gene expression analysis showed significantly increased expression of the chondrocyte-associated genes, collagen type II and aggrecan. This delivery platform resulted in significantly less collagen type X expression, suggesting the generation of a more stable cartilage phenotype. When evaluated in an ex vivo osteoarthritic cartilage model, implanted hMSCs with TGF-β3-loaded HA microspheres were detected within cartilage fibrillations and increased proteoglycan staining was seen in the tissue. In summary, data presented here demonstrate that TGF-β3-bound hyaluronan microspheres provide a suitable delivery system for induction of hMSC chondrogenesis and their use may represent a clinically feasible tissue engineering approach for the treatment of articular cartilage defects.

Biochemical Properties of Tissue-Engineered Cartilage

Microtia is treated with rib cartilage sculpting and staged procedures; though aesthetically pleasing, these constructs lack native ear flexibility. Tissue-engineered (TE) elastic cartilage may bridge this gap; however, TE cartilage implants lead to hypertrophic changes with calcification and loss of flexibility. Retaining flexibility in TE cartilage must focus on increased elastin, maintained collagen II, decreased collagen X, with prevention of calcification. This study compares biochemical properties of human cartilage to TE cartilage from umbilical cord mesenchymal stem cells (UCMSCs). Our goal is to establish a baseline for clinically useful TE cartilage. Discarded cartilage from conchal bowl, microtic ears, preauricular tags, rib, and TE cartilage were evaluated for collagen I, II, X, calcium, glycosaminoglycans, elastin, and fibrillin I and III. Human UCMSCs were chondroinduced on 2D surfaces and 3D D,L-lactide-co-glycolic acid (PLGA) fibers. Cartilage samples demonstrated similar staining for collagens I, II, and X, elastin, and fibrillin I and III, but differed from rib. TE pellets and PLGA-supported cartilage were similar to auricular samples in elastin and fibrillin I staining. TE samples were exclusively stained for fibrillin III. Only microtic samples demonstrated calcium staining. TE cartilage expressed similar levels of elastin, fibrillin I, and collagens I and X when compared to native cartilage. Microtic cartilage demonstrated elevated calcium, suggesting this abnormal tissue may not be a viable cell source for TE cartilage. TE cartilage appears to recapitulate the embryonic development of fibrillin III, which is not expressed in adult tissue, possibly providing a strategy to control TE elastic cartilage phenotype.

Augmented Cord Blood Stem Cell Collection, Making Adult Single Cord Transplant a Reality?

For over 25 years, cord blood stem cell collection and clinical use as a viable stem cell source for hematologic reconstitution has continued to receive increased acceptance and more diverse applications. Recently, the utilization of cellular sub-types and differentiated populations from cord blood has been employed for a variety of conditions including those resulting from cellular and organ dysfunction or damage. Unfortunately, the usefulness of cord blood derived stem cells for these varieties of applications has been limited by the stem cell number constraints of a typical cord blood collection, as compared to other sources of stem cells such as bone marrow, peripheral blood and tissue-derived stem cells. Clearly, cord blood stem cells have been recognized to have unique advantageous characteristics, such as the ready availability of a discarded, almost limitless resource; being intrinsically and relatively free of pathogens and prior infectious burdens and sequelae and demonstrating significant pluripotential capability while demonstrating improved graft versus host tolerance. Attempts to improve the useful number of cord blood derived stem cells for a particular indication have included combined multiple cord transplants, a variety of methods to expand stem cell populations and techniques to increase the stem cell yield from a placenta and umbilical cord. Methodologies to increase cord blood collection volume and cell yield have included placental expression, perfusion techniques and placental and cord tissue maceration and cellular separation. Although some of these techniques have demonstrated potential limited usefulness, they have lacked reproducibility, routine procedural practicality and have intrinsic specific risks including infection potential, cellular byproducts and the use of non-approved pharmaceuticals. If possible, the collection of sufficient number of cord blood stem cells from a single cord blood collection, which would be suitable for either a pediatric or adult transplantation, would be of great value. Additionally, the value of the use of an abundant, low cost stem cell source such as cord blood for general use for either hematopoietic or mesenchymal cell application for all potential recipients in need is obvious. However, the procurement of such an invaluable resource needs to be reliable, reproducible and simplistic by nature, avoiding specialized equipment, facilities or expertise at the time of collection. This presented technology represents such a procedure which can result in the collection of a two part stem cell product which can provide for the needs of both an adult stem cell transplant requirements as well and serve as a biologic resource for other stem cell applications such as mesenchymal cell indications. This described technique allows for the standard primary cord blood collection, followed by the placental administration of physiologic vasodilators and stem cell releasing factors in a solution which is both nutritive and cell supportive in nature. The second augmented collection immediately follows which results in a combined and increased stem cell volume that is two to three times the typical mononuclear cell yield. Transportation of a two part collection to the processing facility is standard in nature and requires no subsequent modification in the mononuclear separation procedure prior to storage. The percent CD34+ cell population is significantly elevated in the second collection volume, demonstrating effective mobilization of marginating placental stem cells. Additionally, the procedure is technically reliable and reproducible by any experienced collector and requires only a minor modification in a standard cord blood collection procedure without the use of cumbersome equipment or materials. Disclosures: No relevant conflicts of interest to declare.

Quality of Air-Transported Human Islets for Single Islet Cell Preparations.

In new generation medical therapies for type 1 diabetes mellitus (DM), cell-based approaches using pancreatic islets have attracted significant attention worldwide. In particular, dispersed islet cells obtained from isolated pancreatic islets have been a valuable source in the cell biology and tissue engineering fields. Our experimental approach to the development of new islet-based DM therapies consisted of creating a monolithic islet cell sheet format using dispersed islet cells. In this experiment, we explored the potential of internationally transporting human islets from Alberta, Canada to Tokyo, Japan and obtaining viable dispersed islet cells. A total of 34 batches of isolated and purified human islets were transported using a commercial air courier service. Prior to shipping, the human islets had been in culture for 0-108 h at the University of Alberta. The transportation period from Alberta to Tokyo was 2-5 days. The transported human islet cells were enzymatically dispersed as single cells in Tokyo. The number of single islet cells decreased as the number of transportation days increased. In contrast, cell viability was maintained regardless of the number of transportation days. The preshipment culture time had no effect on the number or viability of single cells dispersed in Tokyo. When dispersed single islet cells were plated on laminin-5-coated temperature-responsive polymer-grafted culture dishes, the cells showed favorable attachment followed by extension as a monolithic format. The present study demonstrated that long-distance transported human islets are a viable cell source for experiments utilizing dispersed human islet cells.

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.

Autologous cartilage fragments in a composite scaffold for one stage osteochondral repair in a goat model

We propose a culture-free approach to osteochondral repair with minced autologous cartilage fragments loaded onto a scaffold composed of a hyaluronic acid (HA)-derived membrane, platelet-rich fibrin matrix (PRFM) and fibrin glue. The aim of the study was to demonstrate in vitro the outgrowth of chondrocytes from cartilage fragments onto this scaffold and, in vivo, the formation of functional repair tissue in goat osteochondral defects. Two sections were considered: 1) in vitro: minced articular cartilage from goat stifle joints was loaded onto scaffolds, cultured for 1 or 2 months, and then evaluated histologically and immunohistochemically; 2) in vivo: 2 unilateral critically-sized trochlear osteochondral defects were created in 15 adult goats; defects were treated with cartilage fragments embedded in the scaffold (Group 1), with the scaffold alone (Group 2), or untreated (Group 3). Repair processes were evaluated morphologically, histologically, immunohistochemically and biomechanically at 1, 3, 6 and 12 months. We found that in vitro, chondrocytes from cartilage fragments migrated to the scaffold and, at 2 months, matrix positive for collagen type II was observed in the constructs. In vivo, morphological and histological assessment demonstrated that cartilage fragment-loaded scaffolds led to the formation of functional hyaline-like repair tissue. Repair in Group 1 was superior to that of control groups, both histologically and mechanically. Autologous cartilage fragments loaded onto an HA/PRFM/fibrin glue scaffold provided a viable cell source and allowed for an improvement of the repair process of osteochondral defects in a goat model, representing an effective alternative for one-stage repair of osteochondral lesions.

Survival and Functional Restoration of Human Fetal Ventral Mesencephalon following Transplantation in a Rat Model of Parkinson's Disease

Cell replacement therapy by intracerebral transplantation of fetal dopaminergic neurons has become a promising therapeutic option for patients suffering from Parkinson's disease during the last decades. However, limited availability of human fetal tissue as well as ethical issues, lack of alternative nonfetal donor cells, and the absence of standardized transplantation protocols have prevented neurorestorative therapies from becoming a routine procedure in patients suffering from neurodegenerative diseases. Improvement of graft survival, surgery techniques, and identification of the optimal target area are imperative for further optimization of this novel treatment. In the present study, human primary fetal ventral mesencephalon-derived tissue from 7- to 9-week-old human fetuses was transplanted into 6-hydroxydopamine-lesioned adult Sprague-Dawley rats. Graft survival, fiber outgrowth, and drug-induced rotational behavior up to 14 weeks posttransplantation were compared between different intrastriatal transplantation techniques (full single cell suspension vs. partial tissue pieces suspension injected by glass capillary or metal cannula) and the intranigral glass capillary injection of a full (single cell) suspension. The results demonstrate a higher survival rate of dopamine neurons, a greater reduction in amphetamine-induced rotations (overcompensation), and more extensive fiber outgrowth for the intrastriatally transplanted partial (tissue pieces) suspension compared to all other groups. Apomorphine-induced rotational bias was significantly reduced in all groups including the intranigral group. The data confirm that human ventral mesencephalon-derived cells serve as a viable cell source, survive in a xenografting paradigm, and functionally integrate into the host tissue. In contrast to rat donor cells, keeping the original (fetal) neuronal network by preparing only a partial suspension containing tissue pieces seems to be beneficial for human cells, although a metal cannula that causes greater tissue trauma to the host is required for injection. In addition, homotopic intranigral grafts may represent a complimentary grafting approach to the "classical" ectopic intrastriatal target site in PD.

Neuroprotective Effect of Human Placenta-Derived Cell Treatment of Stroke in Rats

Human placenta-derived adherent (PDA001) cells are mesenchymal-like stem cells isolated from postpartum placenta. In this study, we tested whether intravenously infused PDA001 improves neurological functional recovery after stroke in rats. In addition, potential mechanisms underlying the PDA001-induced neuroprotective effect were investigated. Young adult male rats (2–3 months) were subjected to 2 h of middle cerebral artery occlusion (MCAo) and treated with PDA001 (4x10(6)) or vehicle controls [dextran vehicle or phosphate buffer saline (PBS)] via intravenous (IV) administration initiated at 4 h after MCAo. A battery of functional tests and measurements of lesion volume and apoptotic cells were performed. Immunostaining and ELISA assays for vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and brain derived neurotrophic factor (BDNF) were performed in the ischemic brain to test the potential mechanisms underlying the neuroprotective effects of PDA001 cell treatment of stroke. PDA001 cell treatment at 4 h post stroke significantly improved functional outcome and significantly decreased lesion volume, TUNEL, and cleaved caspase 3-positive cell number in the ischemic brain, compared to MCAo-vehicle and MCAo-PBS control. Treatment of stroke with PDA001 cells also significantly increased HGF and VEGF expression in the ischemic border zone (IBZ) compared to controls. Using ELISA assays, treatment of stroke with PDA001 cells significantly increased VEGF, HGF, and BDNF levels in the ischemic brain compared to controls. When administered intravenously at 4 h after MCAo, PDA001 cells promoted neuroprotective effects. These effects induced by PDA001 cell treatment may be related to the increase of VEGF, HGF, and BDNF expression,and a decrease of apoptosis. PDA001 cells may provide a viable cell source to treat stroke.

Erratum

Dear Editor, In regards to the 28(4) STEP-BY-STEP article “Guided Tissue Regeneration for Infrabony Pocket Treatment in Dogs” the authors would like to make a correction to the published manuscript. Page 282, paragraph five, sentence five reads “While both products act as scaffolding for migrating cells to form new bone (osteoconduction), the allograft is a source of cells (osteogenisis) and bone morphogenic proteins (osteoinduction) which may enhance bone production.” The demineralized ostoeoallograft is considered osteoconductive and osteoinductive, however it does not provide a source of viable cells and therefore should not be considered osteogenic. True osteogenisis could be achieved with the application of an autogenous bone graft that provides viable bone forming cells. Sincerely, Wade Gingerich

Dynamic Compressive Loading Enhances Cartilage Matrix Synthesis and Distribution and Suppresses Hypertrophy in hMSC-Laden Hyaluronic Acid Hydrogels

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair, and there is growing evidence that mechanical signals play a critical role in the regulation of stem cell chondrogenesis and in cartilage development. In this study we investigated the effect of dynamic compressive loading on chondrogenesis, the production and distribution of cartilage specific matrix, and the hypertrophic differentiation of human MSCs encapsulated in hyaluronic acid (HA) hydrogels during long term culture. After 70 days of culture, dynamic compressive loading increased the mechanical properties, as well as the glycosaminoglycan (GAG) and collagen contents of HA hydrogel constructs in a seeding density dependent manner. The impact of loading on HA hydrogel construct properties was delayed when applied to lower density (20 million MSCs/ml) compared to higher seeding density (60 million MSCs/ml) constructs. Furthermore, loading promoted a more uniform spatial distribution of cartilage matrix in HA hydrogels with both seeding densities, leading to significantly improved mechanical properties as compared to free swelling constructs. Using a previously developed in vitro hypertrophy model, dynamic compressive loading was also shown to significantly reduce the expression of hypertrophic markers by human MSCs and to suppress the degree of calcification in MSC-seeded HA hydrogels. Findings from this study highlight the importance of mechanical loading in stem cell based therapy for cartilage repair in improving neocartilage properties and in potentially maintaining the cartilage phenotype.

Viability of maxillary bone harvesting by using different osteotomy techniques. A pilot study.

The use of autogenous grafts is still considered in bone regeneration surgeries. However, the bone cell viability of such grafts after being harvested from donor sites remains a matter of debate. The aim of the present study is to evaluate particulated and block bone cell viability, in terms of presence or absence of apoptosis and necrosis, obtained from different maxillary intra-oral harvesting methods: bone scraper, rotary carbide burs and piezoelectric device. Five healthy patients were enrolled in the study. The patients required sinus augmentation by lateral window approach. The bone was harvested by the bone scraper, piezoelectric device and rotary surgical instrument. The samples were processed with the Annexin V/FITC (fluorescein isothiocyanate stain) kit and were analyzed by means of Fluoresence-Activated Cell Sorted (FACS) technique. Within the limitations of this pilot study, the results indicated that autogenous bone chips collected from the three harvesting methods presented a large percentage of apoptotic cells, although large scale production of necrotic cells was not detected. In summary, although rotary surgical instrument and piezoelectric devices are frequently used instruments for oral osteotomy, fresh autogenous bone chips collected from them did not present a viable bone cell source.

Nanofiber topography and sustained biochemical signaling enhance human mesenchymal stem cell neural commitment

Stem cells hold great promise in enhancing nerve regeneration. In particular, human mesenchymal stem cells (MSC) represent a clinically viable cell source due in part to their abundance and accessibility. Unfortunately, current methods to direct the fate of stem cells remains largely limited to biochemical-based approaches on two-dimensional substrates with restricted efficacies. Here we have evaluated a scaffold-based approach to directing stem cell differentiation. We demonstrate the combined effects of nanofiber topography and controlled drug release on enhancing MSC neural commitment. By encapsulating up to 0.3wt.% retinoic acid (RA) within aligned poly(ε-caprolactone) (PCL) nanofibers (average diameter ∼270nm, AF750), sustained released of RA was obtained for at least 14days (∼60% released). Compared with tissue culture polystyrene (TCPS), the nanofiber topography arising from plain PCL nanofibers significantly up-regulated the expressions of neural markers, Tuj-1, MAP2, GalC and RIP at the mRNA and protein levels. Combined with sustained drug availability, more significant changes in cell morphology and enhancement of neural marker expression were observed. In particular, scaffold-based controlled delivery of RA enhanced MAP2 and RIP expression compared with bolus delivery despite lower amounts of drug (>8 times lower). The generally higher expression of the mature neuronal marker MAP2 compared with glial markers at the mRNA and protein levels suggested an enhanced potential of MSC neuronal differentiation. In addition, positive staining for synaptophysin was detected only in cells cultured on aligned scaffolds in the presence of RA. Taken together, the results highlight the advantage of the scaffold-based approach in enhancing the potential of MSC neuronal differentiation and demonstrated the importance of the drug delivery approach in directing cell fate. Such biomimicking drug-encapsulating scaffolds may permit subsequent direct cell transplantation and provide guidance cues to control the fate of endogenously recruited stem cells.

Directed Differentiation of Skin-Derived Precursors Into Functional Vascular Smooth Muscle Cells

The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)-directed differentiation of Sox2(+) multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-β1 or -β3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle γ-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. SKPs are Sox2(+) progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization.

Enhanced MSC chondrogenesis following delivery of TGF-β3 from alginate microspheres within hyaluronic acid hydrogels in vitro and in vivo

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair and members of the transforming growth factor-beta (TGF-β) superfamily are a key mediator of MSC chondrogenesis. While TGF-β mediated MSC chondrogenesis is well established in in vitro pellet or hydrogel cultures, clinical translation will require effective delivery of TGF-βs in vivo. Here, we investigated the co-encapsulation of TGF-β3 containing alginate microspheres with human MSCs in hyaluronic acid (HA) hydrogels towards the development of implantable constructs for cartilage repair. TGF-β3 encapsulated in alginate microspheres with nanofilm coatings showed significantly reduced initial burst release compared to uncoated microspheres, with release times extending up to 6 days. HA hydrogel constructs seeded with MSCs and TGF-β3 containing microspheres developed comparable mechanical properties and cartilage matrix content compared to constructs supplemented with TGF-β3 continuously in culture media, whereas constructs with TGF-β3 directly encapsulated in the gels without microspheres had inferior properties. When implanted subcutaneously in nude mice, constructs containing TGF-β3 microspheres resulted in superior cartilage matrix formation when compared to groups without TGF-β3 or with TGF-β3 added directly to the gel. However, calcification was observed in implanted constructs after 8 weeks of subcutaneous implantation. To prevent this, the co-delivery of parathyroid hormone-related protein (PTHrP) with TGF-β3 in alginate microspheres was pursued, resulting in partially reduced calcification. This study demonstrates that the controlled local delivery of TGF-β3 is essential to neocartilage formation by MSCs and that further optimization is needed to avert the differentiation of chondrogenically induced MSCs towards a hypertrophic phenotype.

Evaluation of in vitro growth factor treatments on fibrochondrogenesis by synovial membrane cells from osteoarthritic and nonosteoarthritic joints of dogs

To determine the in vitro effects of selected growth factors on fibrochondrogenesis by synovial membrane cells from nonosteoarthritic (normal) and osteoarthritic joints of dogs. 5 dogs with secondary osteoarthritis of shoulder or stifle joints and 6 dogs with normal joints. Synovial membrane cells were harvested from normal and osteoarthritic joints and cultured in monolayer with or without (control) basic fibroblast growth factor, transforming growth factor-β1, and insulin-like growth factor-1. In the cultured cells, fibrochondrogenesis was measured by use of a real-time reverse transcriptase PCR assay to determine relative expressions of collagen I, collagen II, and aggrecan genes and of 3 genes involved in embryonic chondrogenesis: Sry-type homeobox protein-9 (SOX-9), frizzled-motif associated with bone development (Frzb), and regulator of G-protein signaling-10 (RGS-10). Tissue collagen content was measured via a hydroxyproline assay, and sulfated glycosaminoglycan content was measured via a 1,9-dimethylmethylene blue assay. Cellularity was determined via a double-stranded DNA assay. Immunohistochemical analysis for collagens I and II was also performed. In vitro collagen synthesis was enhanced by growth factor stimulation. Although osteoarthritic-joint synoviocytes could undergo a fibrocartilage-like phenotypic shift, their production of collagenous extracellular matrix was less than that of normal-joint synoviocytes. Gene expressions of SOX-9 and RGS-10 were highest in the osteoarthritic-joint cells; Frzb expression was highest in growth factor treated cells. Autogenous synovium may be a viable cell source for meniscal tissue engineering. Gene expressions of SOX-9 and RGS-10 may be potential future targets for in vitro enhancement of chondrogenesis.

Coculture of Human Mesenchymal Stem Cells and Articular Chondrocytes Reduces Hypertrophy and Enhances Functional Properties of Engineered Cartilage

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair; however, it still remains a challenge to recapitulate the functional properties of native articular cartilage using only MSCs. Additionally, MSCs may exhibit a hypertrophic phenotype under chondrogenic induction, resulting in calcification after ectopic transplantation. With this in mind, the objective of this study was to assess whether the addition of chondrocytes to MSC cultures influences the properties of tissue-engineered cartilage and MSC hypertrophy when cultured in hyaluronic acid hydrogels. Mixed cell populations (human MSCs and human chondrocytes at a ratio of 4:1) were encapsulated in the hydrogels and exhibited significantly higher Young's moduli, dynamic moduli, glycosaminoglycan levels, and collagen content than did constructs seeded with only MSCs or chondrocytes. Furthermore, the deposition of collagen X, a marker of MSC hypertrophy, was significantly lower in the coculture constructs than in the constructs seeded with MSCs alone. When MSCs and chondrocytes were cultured in distinct gels, but in the same wells, there was no improvement in biomechanical and biochemical properties of the engineered tissue, implying that a close proximity is essential. This approach can be used to improve the properties and prevent calcification of engineered cartilage formed from MSC-seeded hydrogels with the addition of lower fractions of chondrocytes, leading to improved clinical outcomes.

Abstract 3859: Evaluation of addition of the tumor-vascular disrupting agent ASA404 (vadimezan) to paclitaxel and carboplatin and the influence of subsequent ASA404 maintenance therapy using a human NSCLC xenograft model

Abstract The tumor vasculature is essential for maintaining tumor growth and regulating the tumor microenvironment through the supply of nutrients and oxygen. ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic acid) is a flavonoid, non-tubulin-binding Tumor-Vascular Disrupting Agent (Tumor-VDA) that induces breakdown of the established tumor vasculature resulting in the inhibition of tumor blood flow leading to tumor ischemia and extensive necrosis of the tumor core. Unlike anti-angiogenics, ASA404 (ASA) has limited effects on angiogenesis at the tumor periphery and as a consequence the remaining peripheral tumor rim represents a source of viable cells for regrowth of the tumor upon cessation of ASA treatment. Preclinical and clinical studies have highlighted the effectiveness of combining chemotherapies, particularly taxanes, with ASA for marked tumor inhibition. The ongoing ATTRACT-1 Phase III trial in NSCLC is evaluating the effect of adding ASA to the paclitaxel (P) and carboplatin (C) standard of care (SOC). In the present studies, the influence of adding ASA to P+C was examined in the human A549 NSCLC xenograft model. A549 NSCLC cells (5 × 107 in PBS containing 50% matrigel) were implanted sc into the right flank of female athymic nu/nu Harlan nude mice. Treatment schedules were as follows: ASA (18.6 mg/kg, iv on days 1, 5 and 9), P (15 mg/kg, iv qwk x3) and C (80 mg/kg, ip qwk x3). Tumor growth inhibition (TGI) was determined on day 21 and expressed as T/C (%). ASA alone resulted in a significant TGI of 17% T/C as compared to vehicle controls. P+C had a non-significant effect resulting in 82% T/C. The triple combination of ASA+P+C led to an improved TGI with a T/C of 12% being observed which was significant compared to vehicle controls and non-significant versus ASA alone, although by Clark's method, an additive effect was indicated. The influence of an additional maintenance cycle administration of ASA following ASA+P+C was then examined in a separate study. In addition to TGI (T/C %) determined at day 28, the median time to treatment endpoint (TTE) was measured based on the number of days taken until a preassigned tumor volume of 800 mm3 was reached. ASA+P+C resulted in a significant TGI of 22% T/C compared to vehicle controls. The TTE was extended from 24.8 days seen in controls to 63.5 days with the triple combination with 1 partial regression (PR). The influence of a subsequent cycle of ASA given alone (on days 21, 25 and 29) resulted in a further TGI with tumor regression being observed (T/T0 −13%), a TTE of 119 days and 3 PR. These studies demonstrate the value of the addition of ASA404 (vadimezan) to a current SOC composed of P+C and the potential of ASA404 maintenance therapy for added therapeutic benefit. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3859.

Abstract 4443: Molecular mechanistic study of ASA404 (vadimezan)-induced endothelial cell death

Abstract The tumor vasculature is a key component in maintaining tumor growth and regulating the tumor microenvironment through the supply of nutrients and oxygen. ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic acid) is a flavonoid, non-tubulin-binding Tumor-Vascular Disrupting Agent (Tumor-VDA) that induces breakdown of the established tumor vasculature through the induction of tumor endothelial cell apoptosis, resulting in the inhibition of tumor blood supply, leading to tumor ischemia and extensive necrosis of the tumor core. In contrast to antiangiogenics, ASA404 has limited effects on angiogenesis at the tumor periphery and so the remaining peripheral tumor rim represents a source of viable cells for regrowth of the tumor upon cessation of ASA404 treatment. Preclinical and clinical studies have highlighted the effectiveness of combining chemotherapies, especially taxanes, with ASA404 for marked tumor inhibition. Currently, the benefits of adding ASA404 to the standard of care in first- and second-line NSCLC indications are being evaluated in the ATTRACT-1 and ATTRACT-2 Phase III trials respectively. Despite the prior clinical efficacy observed with ASA404, the detailed molecular mechanism by which ASA404 selectively targets the tumor vasculature still remains to be defined. To investigate this, we initiated studies to explore the function of ASA404 both in vitro using human endothelial cells (HUVEC) and in ASA404-treated MDA-MB-231 breast cancer xenografts examined ex vivo. Consistent with previous reports, we observed inhibition of HUVEC cell proliferation along with rapid cell death in vitro as assessed using FACS analysis and newly demonstrated the rapid induction of caspase 3 assessed by immunocytochemistry. Treatment of the MDA-MB-231 breast xenografts in vivo with ASA404 resulted in significant tumor growth inhibition, tumor necrosis and elevation of the hypoxia marker carbonic anhydrase 9 (CAIX). The nature of ASA404-induced endothelial cell death is likely to be due to apoptosis since rapid caspase 3 cleavage was observed in the endothelial cells both in vitro and in vivo. In addition, the mitochondrial potential of cultured HUVEC cells in vitro was found to be seriously compromised after 1-2 hours treatment with ASA404 as monitored by Mito-tracker staining. This was accompanied by cytochrome C release into the cytosol as evidenced by immunohistochemical co-localization of cytochrome C and mitochondria markers. We also detected a moderate, but concentration-dependent increase in ceramide levels in HUVEC cells following 2 hours of ASA404 treatment, which may serve as a link to the observed mitochondrial depolarization phenotype. Lastly, a pooled-based shRNA screen was also conducted to search for genes that could modulate ASA404 activity in HUVEC cells. Some of the candidates identified may provide new insights into the pathways that are critical in the ASA404 mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4443.

Transdifferentiation potentiality of human Wharton's jelly stem cells towards vascular endothelial cells

Human Wharton's jelly stem cells (HWJSC) emerged as a potential source of viable cells for use in tissue engineering. In this work, we have analyzed the transdifferentiation capabilities of HWJSC towards transdifferentiated endothelial-like cells (Tr-ELC) in order to establish the potential usefulness of these cells in vascular tissue engineering. Our results show that Tr-ELC became more polygonal and less proliferative than HWJSC, resembling the structure and proliferation rate of the endothelial cells. In addition, the markers of mesenchymal undifferentiation CD9, E-cad, PODXL, and SSEA-4 are downregulated in Tr-ELC, suggesting that these cells can be in the process of adult differentiation. Besides, RT-PCR and microarray analyses revealed that some genes with a role in defining the endothelial phenotype and structure are upregulated (VEGF-R1, EDF1, AAMP, CD31, CD34, CDH5, and ICAM2) or downregulated (VEGF) in Tr-ELC, although a number of genes related to relevant endothelial cell functions (CD36, ECE2, VWF, THBD, PGI2, ECE1, and ACE) did not change or were only partially induced. All this implies that HWJSC are able to efficiently transdifferentiate towards Tr-ELC at the phenotypical level following a hierarchical pattern of gene activation, with an earlier induction of morphological and phenotypical genes.