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Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model.

ABSTRACT: It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker-tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.

SUBMITTER: Zwolanek D

PROVIDER: S-EPMC5846895 | biostudies-other | 2017 Oct

REPOSITORIES: biostudies-other

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Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model.

Zwolanek Daniela D Satué María M Proell Verena V Godoy José R JR Odörfer Kathrin I KI Flicker Magdalena M Hoffmann Sigrid C SC Rülicke Thomas T Erben Reinhold G RG

JCI insight 20171019 20

It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker-tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemica ...[more]

PMID: 29046476

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Project description:While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, most tissue engineering strategies for cartilage regeneration encapsulate MSCs as single cells, partially due to the technical challenge to homogeneously encapsulate cell pellets in three-dimensional (3D) hydrogels. It remains unclear whether encapsulating MSCs as single cell suspension or cell aggregates in 3D hydrogels would enhance MSC-based cartilage formation. In this study, we determined that the optimal size of MSC micropellets (?Pellets) that can be homogeneously encapsulated in hydrogels with high cell viability is 100 cells/pellet. Using optimized ?Pellet size, MSCs were encapsulated either as single cell suspension or ?Pellets in four soft hydrogel formulations with stiffness ranging 3-6?kPa. Regardless of hydrogel formulations, single cell encapsulation resulted in more neocartilage deposition with improved mechanical functions over ?Pellet encapsulation. For single cell encapsulation, polyethylene glycol (PEG) hydrogels containing chondroitin sulfate led to the most cartilage matrix deposition, with compressive modulus reaching 211?kPa after only 21 days, a range approaching the stiffness of native cartilage. The findings from this study offer valuable insights on guiding optimal method design for MSCs and hydrogel-based cartilage regeneration. The optimized ?Pellet encapsulation method may be broadly applicable to encapsulate other stem cell types or cancer cells as aggregates in hydrogels. Impact Statement While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, it remains unclear whether encapsulating MSCs as cell pellets in three-dimensional hydrogels would enhance MSC-based cartilage formation. In this study, we determined the optimal size of MSC micropellet (?Pellet) that can be homogeneously encapsulated in hydrogels with high cell viability. Unexpectedly, single cell encapsulation resulted in more robust new cartilage formation than ?Pellet encapsulation. Furthermore, tuning hydrogel formulation led to rapid cartilage regeneration with stiffness approaching that of native cartilage. The findings from this study would facilitate clinical translation of MSCs and hydrogel-based therapies for cartilage regeneration with optimized parameters.

Project description:BackgroundSecretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage.MethodsConditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated.ResultsWe showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production.ConclusionsThis study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

Project description:BackgroundMesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo.MethodsFlow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites.ResultsFreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs' function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O'Driscoll scores showing that more hyaline cartilage was formed on the defects.ConclusionFreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy.

Dataset Information

Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model.

Publications

Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model.

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