Synergistic effect of umbilical cord extracellular vesicles and rhBMP-2 to enhance the regeneration of a metaphyseal femoral defect in osteoporotic rats.

BACKGROUND: The aim of this study was to evaluate potential synergistic effects of a single, local application of human umbilical cord MSC-derived sEVs in combination with a low dose of recombinant human rhBMP-2 to promote the regeneration of a metaphyseal femoral defect in an osteoporotic rat model. METHODS: 6 weeks after induction of osteoporosis by bilateral ventral ovariectomy and administration of a special diet, a total of 64 rats underwent a distal femoral metaphyseal osteotomy using a manual Gigli wire saw. Defects were stabilized with an adapted Y-shaped mini-locking plate and were subsequently treated with alginate only, or alginate loaded with hUC-MSC-sEVs (2 × 109), rhBMP-2 (1.5 µg), or a combination of sEVs and rhBMP-2 (n = 16 for each group). 6 weeks post-surgery, femora were evaluated by µCT, descriptive histology, and biomechanical testing. RESULTS: Native radiographs and µCT analysis confirmed superior bony union with callus formation after treatment with hUC-MSC-sEVs in combination with a low dose of rhBMP-2. This finding was further substantiated by histology, showing robust defect consolidation 6 weeks after treatment. Torsion testing of the explanted femora revealed increased stiffness after application of both, rhBMP-2 alone, or in combination with sEVs, whereas torque was only significantly increased after treatment with rhBMP-2 together with sEVs. CONCLUSION: The present study demonstrates that the co-application of hUC-MSC-sEVs can improve the efficacy of rhBMP-2 to promote the regeneration of osteoporotic bone defects.

Evaluation of the Potential of Umbilical Cord Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles to Improve Rotator Cuff Healing: A Pilot Ovine Study.

BACKGROUND: Despite significant advancements in surgical techniques to repair rotator cuff (RC) injuries, failure rates remain high and novel approaches to adequately overcome the natural biological limits of tendon and enthesis regeneration of the RC are required. Small extracellular vesicles (sEVs) derived from the secretome of human multipotent mesenchymal stromal cells (MSCs) have been demonstrated to modulate inflammation and reduce fibrotic adhesions, and therefore their local application could improve outcomes after RC repair. PURPOSE: In this pilot study, we evaluated the efficacy of clinical-grade human umbilical cord (hUC) MSC-derived sEVs (hUC-MSC-sEVs) loaded onto a type 1 collagen scaffold in an ovine model of acute infraspinatus tendon injury to improve RC healing. STUDY DESIGN: Controlled laboratory study. METHODS: sEVs were enriched from hUC-MSC culture media and were characterized by surface marker profiling. The immunomodulatory capacity was evaluated in vitro by T-cell proliferation assays, and particle count was determined by nanoparticle tracking analysis. Twelve skeletally mature sheep were subjected to partial infraspinatus tenotomy and enthesis debridement. The defects of 6 animals were treated with 2 × 1010 hUC-MSC-sEVs loaded onto a type 1 collagen sponge, whereas 6 animals received only a collagen sponge, serving as controls. Six weeks postoperatively, the healing of the infraspinatus tendon and the enthesis was evaluated by magnetic resonance imaging (MRI) and hard tissue histology. RESULTS: CD3/CD28-stimulated T-cell proliferation was significantly inhibited by hUC-MSC-sEVs (P = .015) that displayed the typical surface marker profile, including the presence of the MSC marker proteins CD44 and melanoma-associated chondroitin sulfate proteoglycan. The local application of hUC-MSC-sEVs did not result in any marked systemic adverse events. Histologically, significantly improved Watkins scores (P = .031) indicated improved tendon and tendon-to-bone insertion repair after sEV treatment and lower postcontrast signal of the tendon and adjacent structures on MRI suggested less residual inflammation at the defect area. Furthermore, the formation of osteophytes at the injury site was significantly attenuated (P = .037). CONCLUSION: A local, single-dose application of hUC-MSC-sEVs promoted tendon and enthesis healing in an ovine model of acute RC injury. CLINICAL RELEVANCE: Surgical repair of RC tears generally results in a clinical benefit for the patient; however, considerable rerupture rates have been reported. sEVs have potential as a cell-free biotherapeutic to improve healing outcomes after RC injury.

Calorimetry of extracellular vesicles fusion to single phospholipid membrane.

Extracellular vesicles (EVs)-mediated communication relies not only on the delivery of complex molecular cargoes as lipids, proteins, genetic material, and metabolites to their target cells but also on the modification of the cell surface local properties induced by the eventual fusion of EVs' membranes with the cells' plasma membrane. Here we applied scanning calorimetry to study the phase transition of single phospholipid (DMPC) monolamellar vesicles, investigating the thermodynamical effects caused by the fusion of doping amounts of mesenchymal stem cells-derived EVs. Specifically, we studied EVs-induced consequences on the lipids distributed in the differently curved membrane leaflets, having different density and order. The effect of EV components was found to be not homogeneous in the two leaflets, the inner (more disordered one) being mainly affected. Fusion resulted in phospholipid membrane flattening associated with lipid ordering, while the transition cooperativity, linked to membrane domains' coexistence during the transition process, was decreased. Our results open new horizons for the investigation of the peculiar effects of EVs of different origins on target cell membrane properties and functionality.

Label-free characterization of an extracellular vesicle-based therapeutic.

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers. Furthermore, analysis of QCM dissipation versus frequency allowed us to quantitatively determine the ratio of marker-specific EVs versus non-vesicular particles (NVPs) - a parameter that cannot be obtained by any other technique so far. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and NVPs in our EV preparations. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV therapeutics and opens the door to improved quality control.

First-in-human intracochlear application of human stromal cell-derived extracellular vesicles.

Extracellular vesicles (EVs) derived from the secretome of human mesenchymal stromal cells (MSC) contain numerous factors that are known to exert anti-inflammatory effects. MSC-EVs may serve as promising cell-based therapeutics for the inner ear to attenuate inflammation-based side effects from cochlear implantation which represents an unmet clinical need. In an individual treatment performed on a 'named patient basis', we intraoperatively applied allogeneic umbilical cord-derived MSC-EVs (UC-MSC-EVs) produced according to good manufacturing practice. A 55-year-old patient suffering from Menière's disease was treated with intracochlear delivery of EVs prior to the insertion of a cochlear implant. This first-in-human use of UC-MSC-EVs demonstrates the feasibility of this novel adjuvant therapeutic approach. The safety and efficacy of intracochlear EV-application to attenuate side effects of cochlea implants have to be determined in controlled clinical trials.

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

Mesenchymal Stromal Cell-Based Bone Regeneration Therapies: From Cell Transplantation and Tissue Engineering to Therapeutic Secretomes and Extracellular Vesicles.

Effective regeneration of bone defects often presents significant challenges, particularly in patients with decreased tissue regeneration capacity due to extensive trauma, disease, and/or advanced age. A number of studies have focused on enhancing bone regeneration by applying mesenchymal stromal cells (MSCs) or MSC-based bone tissue engineering strategies. However, translation of these approaches from basic research findings to clinical use has been hampered by the limited understanding of MSC therapeutic actions and complexities, as well as costs related to the manufacturing, regulatory approval, and clinical use of living cells and engineered tissues. More recently, a shift from the view of MSCs directly contributing to tissue regeneration toward appreciating MSCs as "cell factories" that secrete a variety of bioactive molecules and extracellular vesicles with trophic and immunomodulatory activities has steered research into new MSC-based, "cell-free" therapeutic modalities. The current review recapitulates recent developments, challenges, and future perspectives of these various MSC-based bone tissue engineering and regeneration strategies.

Manufacturing and characterization of extracellular vesicles from umbilical cord-derived mesenchymal stromal cells for clinical testing.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) may deliver therapeutic effects that are comparable to their parental cells. MSC-EVs are promising agents for the treatment of a variety of diseases. To reach the intermediate goal of clinically testing safety and efficacy of EVs, strategies should strive for efficient translation of current EV research. On the basis of our in vitro an in vivo findings regarding the biological actions of EVs and our experience in manufacturing biological stem cell therapeutics for routine use and clinical testing, we discuss strategies of manufacturing and quality control of umbilical cord-derived MSC-EVs. We introduce guidelines of good manufacturing practice and their practicability along the path from the laboratory to the patient. We present aspects of manufacturing and final product quality testing and highlight the principle of "The process is the product." The approach presented in this perspective article may facilitate translational research during the development of complex biological EV-based therapeutics in a very early stage of manufacturing as well as during early clinical safety and proof-of-concept testing.

An In Vitro Potency Assay for Monitoring the Immunomodulatory Potential of Stromal Cell-Derived Extracellular Vesicles.

The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs) is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs) exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.

A Good Manufacturing Practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND AIMS: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs. METHODS: Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit. RESULTS: Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV. CONCLUSIONS: The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.

Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

BACKGROUND: Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin. METHODS: We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages. RESULTS: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure. CONCLUSIONS: Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Plectin deposition at podosome rings requires myosin contractility.

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.

The Rsu-1-PINCH1-ILK complex is regulated by Ras activation in tumor cells.

The link between Ras transformation and enhanced cell migration due to altered integrin signaling is well established in tumorigenesis, however there remain gaps in our understanding of its mechanism. The Ras suppressor, Rsu-1, has recently been linked to the IPP (integrin-linked kinase {ILK}, PINCH-1/LIMS1, parvin) focal adhesion complex based on its interaction with the LIM 5 domain of PINCH1. Defining the role of the Rsu1-PINCH1-ILK-parvin complex in tumorigenesis is important because both ILK and PINCH1 are elevated in certain tumors while ectopic expression of Rsu-1 blocks tumorigenesis. Our studies previously identified an alternatively spliced isoform of Rsu-1 in high-grade gliomas. We report here the detection of a truncated (p29) Rsu-1 protein, which correlates with the presence of the alternatively spliced Rsu-1 RNA. This RNA and the respective protein were detected in human tumor cell lines that contain high levels of activated Ras, and inhibitor studies demonstrate that the Mek-ERK pathway regulates expression of this truncated Rsu-1 product. We also show that Rsu-1 co-localizes with ILK at focal contacts and co-immunoprecipitates with the ILK-PINCH1 complex in non-transformed cells, but following Ras transformation the association of Rsu-1 with the PINCH1-ILK complex is greatly reduced. Using a human breast cancer cell line, our in vitro studies demonstrate that the depletion of Rsu-1 full-length protein enhances cell migration coincident with an increase in Rac-GTP while the depletion of the p29 Rsu-1 truncated protein inhibits migration. These findings indicate that Rsu-1 may inhibit cell migration by stabilizing the IPP adhesion complex and that Ras activation perturbs this inhibitory function by modulating both Rsu-1 splicing and association of full-length Rsu-1 with IPP. Hence, our findings demonstrate that Rsu-1 links the Ras pathway with the IPP complex and the perturbations of cell attachment-dependent signaling that occur in the malignant process.

Assembly and biological role of podosomes and invadopodia.

Regulated tissue invasion via motile and lytic events is critical for physiological processes such as immune system function and inflammatory responses, wound healing, and organ development, but pathological subversion of this process drives tumour cell invasion and metastasis. Cell migration and invasion require the integration of several processes that include: first, the local modulation of cytoskeleton structure and contractile forces; second, the turnover of substrate adhesions and their associated microfilaments; and third, the generation of specialised, transient domains that mediate the protease-dependent focal degradation of the extracellular matrix. Recent work has re-discovered prominent actin-based cellular structures, termed invadopodia and podosomes, as unique structural and functional modules through which major invasive mechanisms are regulated. The stage is now set to unravel their roles in the physiology and pathology of tissue plasticity and repair.

Calponins: adaptable modular regulators of the actin cytoskeleton.

Over 20 years ago Katsuhito Takahashi isolated a heat stable, calmodulin and actin binding protein from chicken gizzard smooth muscle. Considered initially as a mainly structural component of the vertebrate smooth muscle contractile machinery, the 34-kDa calcium- and calmodulin-binding troponin T-like protein, calponin quickly appeared to also be involved in a number of regulatory and signal transduction events in the actin cytoskeleton. Calponins regulate actomyosin contraction, and reduce metastatic cell motility and tissue invasion. From these various cellular functions the biological role of calponin is now slowly emerging, namely that of an actin filament-stabilizing molecule that contributes to physiological thin filament turnover rates in different cell types.