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In vivo bioluminescent tracking of mesenchymal stem cells within large hydrogel constructs.

ABSTRACT: The use of multicomponent scaffolds for cell implantation has necessitated sophisticated techniques for tracking of cell survival in vivo. Bioluminescent imaging (BLI) has emerged as a noninvasive tool for evaluating the therapeutic potential of cell-based tissue engineering strategies. However, the ability to use BLI measurements to longitudinally assess large 3D cellular constructs in vivo and the effects of potential confounding factors are poorly understood. In this study, luciferase-expressing human mesenchymal stem cells (hMSCs) were delivered subcutaneously within agarose and RGD-functionalized alginate hydrogel vehicles to investigate the impact of construct composition and tissue formation on BLI signal. Results showed that alginate constructs exhibited twofold greater BLI counts than agarose constructs at comparable hMSC doses. However, each hydrogel type produced a linear correlation between BLI counts and live cell number, indicating that within a given material, relative differences in cell number could be accurately assessed at early time points. The survival efficiency of delivered hMSCs was highest for the lower cell doses embedded within alginate matrix. BLI signal remained predictive of live cell number through 1 week in vivo, although the strength of correlation decreased over time. Irrespective of hydrogel type or initial hMSC seeding dose, all constructs demonstrated a degree of vascularization and development of a fibrotic capsule after 1 week. Formation of tissue within and adjacent to the constructs was accompanied by an attenuation of BLI signal during the initial period of the image acquisition time-frame. In alginate constructs only, greater vessel volume led to a delayed rise in BLI signal following luciferin delivery. This study identified vascular and fibrotic tissue ingrowth as potential confounding variables for longitudinal BLI studies. Further investigation into the complexities of noninvasive BLI data acquisition from multicomponent constructs, following implantation and subsequent tissue formation, is warranted.

SUBMITTER: Allen AB

PROVIDER: S-EPMC4186662 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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In vivo bioluminescent tracking of mesenchymal stem cells within large hydrogel constructs.

Allen Ashley B AB Gazit Zulma Z Su Susan S Stevens Hazel Y HY Guldberg Robert E RE

Tissue engineering. Part C, Methods 20140403 10

The use of multicomponent scaffolds for cell implantation has necessitated sophisticated techniques for tracking of cell survival in vivo. Bioluminescent imaging (BLI) has emerged as a noninvasive tool for evaluating the therapeutic potential of cell-based tissue engineering strategies. However, the ability to use BLI measurements to longitudinally assess large 3D cellular constructs in vivo and the effects of potential confounding factors are poorly understood. In this study, luciferase-express ...[more]

PMID: 24576050

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Project description:The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However, the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular, interferon-γ (IFN-γ)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-γ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation, the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore, the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-γ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-γ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture, whereas IDO expression by IFN-γ-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore, sustained IDO expression induced by IFN-γ-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-γ-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether, microparticle delivery of IFN-γ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237.

Project description:Three-dimensional hydrogel constructs incorporated with live stem cells that support chondrogenic differentiation and maintenance offer a promising regenerative route towards addressing the limited self-repair capabilities of articular cartilage. In particular, hydrogel scaffolds that augment chondrogenesis and recapitulate the native physical properties of cartilage, such as compressive strength, can potentially be applied in point-of-care procedures. We report here the synthesis of two new materials, [poly-l-lactic acid/polyethylene glycol/poly-l-lactic acid] (PLLA-PEG 1000) and [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid] (PDLLA-PEG 1000), that are biodegradable, biocompatible (>80% viability post fabrication), and possess high, physiologically relevant mechanical strength (∼1500 to 1800kPa). This study examined the effects of physiologically relevant cell densities (4, 8, 20, and 50×106/mL) and hydrogel stiffnesses (∼150kPa to∼1500kPa Young's moduli) on chondrogenesis of human bone marrow stem cells incorporated in hydrogel constructs fabricated with these materials and a previously characterized PDLLA-PEG 4000. Results showed that 20×106cells/mL, under a static culture condition, was the most efficient cell seeding density for extracellular matrix (ECM) production on the basis of hydroxyproline and glycosaminoglycan content. Interestingly, material stiffness did not significantly affect chondrogenesis, but rather material concentration was correlated to chondrogenesis with increasing levels at lower concentrations based on ECM production, chondrogenic gene expression, and histological analysis. These findings establish optimal cell densities for chondrogenesis within three-dimensional cell-incorporated hydrogels, inform hydrogel material development for cartilage tissue engineering, and demonstrate the efficacy and potential utility of PDLLA-PEG 1000 for point-of-care treatment of cartilage defects.Statement of significanceEngineering cartilage with physiologically relevant mechanical properties for point-of-care applications represents a major challenge in orthopedics, given the generally low mechanical strengths of traditional hydrogels used in cartilage tissue engineering. In this study, we characterized a new material that possesses high mechanical strength similar to native cartilage, and determined the optimal cell density and scaffold stiffness to achieve the most efficient chondrogenic response from seeded human bone marrow stem cells. Results show robust chondrogenesis and strongly suggest the potential of this material to be applied clinically for point-of-care repair of cartilage defects.

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In vivo bioluminescent tracking of mesenchymal stem cells within large hydrogel constructs.

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In vivo bioluminescent tracking of mesenchymal stem cells within large hydrogel constructs.

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