Article

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells

Authors:
  • Medizinische Fakultät Mannheim der Universität Heidelberg
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Abstract

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as intrinsic heterogeneity of MSC populations has to be taken into consideration. The composition of the culture medium is a key factor for successful MSC-expansion. Aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media: StemPro(®)MSC SFM CTS (for human ex-vivo tissue and cell culture processing applications) and MSCGM(TM) (non GMP-compliant, for research only) in an academic setting as first optimization step towards GMP-compliant manufacturing. We demonstrated the feasibility of MSC expansion up to the yielded cell number with all three media. MSC showed the typical fibroblastoid morphology with distinct differences regarding cell size depending on the medium. The differentiation capacity and characteristic immunophenotype was confirmed for all MSC populations. The highest proliferation was demonstrated using StemPro(®) MSC SFM CTS, whereas HPL-medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro(®) and HPL-medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were demonstrated and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion.

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... In order to address these challenges by "in-house" serum-free media of known consistencies, newly developed proprietary serum/xeno-free, cGMP media have been developed as promising alternatives for the manufacturing of clinical-grade MSCs. Until now, there are sparse comparative studies evaluating these proprietary cGMP media and their impact on "stemness" properties of various types of MSCs [46][47][48][49]. Nevertheless, in most of these studies, StemPro (Life Technologies) is evaluated and proposed as a promising alternative to classic serum-containing media. ...
... Several commercial serum/xeno-free media have recently emerged in the market as promising tools for establishing clinical-grade MSCs with enhanced "stemness" characteristics. A few studies have investigated various serum-free proprietary media as promising alternatives to in-house-developed serum-free media [46][47][48][49] and have shown that these products are able to isolate and expand various MSC populations, adhering to the minimal criteria set by the International Society of Cellular Therapy (ISCT) and often providing an enhanced performance compared with the conventional media [52]. However, some of these studies are not standardized for several parameters, such as proliferation rates or variability in the differentiation potential of the cells among media. ...
... In contrast, aBMMSCs contained significantly more SA-β-gal-positive cells compared to DPSC cultures, which was more pronounced in the StemMacs-expanded cells, in accordance with the growth and morphological data (Figs. 1, 2 and 4). The limitation of cell division in cultures is a known phenomenon for all somatic cells unless the cells are immortalized [53]; after approximately 40-100 divisions, depending on cell type and culture conditions, the proliferation rates decay, with eventual domination by the senescent state [46]. Nevertheless, it should be noted that, despite increased numbers of SA-β-gal-positive cells, the reduction in the mean telomere length with increasing passage number, although evident, remained minor, failing to reach statistical significance for all of the cells/media studied (Fig. 5). ...
Article
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Background Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. Methods DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. Results Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions. Conclusions These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0705-0) contains supplementary material, which is available to authorized users.
... Furthermore, the increasing age of the donor is reported to be linked with a reduced expansion and multipotency (46). Details on standardization of the production of clinically applied products and further requirements have been summarized in several reviews (47,48). ...
... gov; as for 10/2016). Meanwhile, GMP-grade complete media specially developed for MSC expansion are commercially available, which also achieve higher expansion rate and thereby shorten the production time and the associated risk of product contamination (48). handling steps for sequential cell passaging are labor intensive and time consuming, as well as bearing the risk for contamination. ...
Article
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Allogeneic hematopoietic stem cell transplantation is associated with serious complications, and improvement of the overall clinical outcome of patients with hematological malignancies is necessary. During the last decades, posttransplant donor-derived adoptive cellular immunotherapeutic strategies have been progressively developed for the treatment of graft-versus-host disease (GvHD), infectious complications, and tumor relapses. To date, the common challenge of all these cell-based approaches is their implementation for clinical application. Establishing an appropriate manufacturing process, to guarantee safe and effective therapeutics with simultaneous consideration of economic requirements is one of the most critical hurdles. In this review, we will discuss the recent scientific findings, clinical experiences, and technological advances for cell processing toward the application of mesenchymal stromal cells as a therapy for treatment of severe GvHD, virus-specific T cells for targeting life-threating infections, and of chimeric antigen receptors-engineered T cells to treat relapsed leukemia.
... 20 In one study, 59 defined combinations of several recombinant GFs (TGF-b1, activin-A, bFGF, EGF, PDGF-BB, insulin-like growth factor [IGF]-1, and vascular endothelial growth factor [VEGF]) and chemokines (CCL21, CCL25, CXCL12, and RANTES) in a serum-free medium failed to achieve hMSC proliferation compared with 5% or 10% PL. Conversely, a recent study 60 reported significantly shorter expansion times to achieve clinically relevant number of hMSCs, when using a commercial CDM versus 10% PL. However, the PL-based system was found to be significantly more cost-effective than CDM (total cost of CDM was 200% that of PL) for large-scale MSC expansion. ...
... However, the PL-based system was found to be significantly more cost-effective than CDM (total cost of CDM was 200% that of PL) for large-scale MSC expansion. 60 Platelet derivatives Platelet concentrates. Platelet derivatives are attractive supplements for cell culture, because platelets contain high concentrations of physiological GFs. ...
Article
Management of degenerative spine pathologies frequently leads to the need for spinal fusion, where bone growth is induced towards stabilization of the interventioned spine. Autologous bone graft (ABG) remains the gold standard inducer, while new bone graft substitutes attempt to achieve effective de novo bone formation and solid fusion. Limited fusion outcomes have driven motivation for more sophisticated and multidisciplinary solutions, involving new biomaterials and/or biologics, through innovative delivery platforms. The present review will analyze the most recent body of literature focused on new approaches for consistent bone fusion of spinal vertebrae, including the development of new biomaterials that pursue physical and chemical aptitudes; the delivery of growth factors (GF) to accelerate new bone formation; and the use of cells to improve functional bone development. Bone graft substitutes currently in clinical practice, such as demineralized bone matrix and ceramics are still used as starting point for study of new bioactive agents. Polyesters such as polycaprolactone and polylactic acid arise as platforms for development of composites, where a mineral element and cell/growth factors constitute the delivery system. Exciting fusion outcomes were obtained in several small and large animal models with these. On what regards bioactive agents, mesenchymal stem cells, preferentially derived from the bone marrow or adipose tissue, were studied in this context. Autologous and allogeneic approaches, as well as osteogenically differentiated cells, have been tested. These cell sources have further been genetically engineered for specific growth factor expression. Nevertheless, results on fusion efficacy with cells have been inconsistent. On the other hand, delivery of growth factors (most commonly BMP-2), have provided favorable outcomes. Complications related to burst release and dosing are still target of research through development of controlled release systems or alternative GF such as NELL-1, Oxysterols or COMP-Ang1. Promising solutions with new biomaterial and GF compositions are becoming closer to the human patient, as these evidence high fusion performance, while offering cost and safety advantages. The use of cells has not yet proven solid benefits, whereas further understanding of cell behavior remains a challenge.
... Regarding GMP products, some are not xeno-free as it is the case for TheraPEAK TM MSCGM TM (Lonza) or UltraCulture (Lonza) that contains bovine proteins including transferrin and albumin. They also generally require pre-coating such as the PRIME-XV TM MSC Expansion SFM or XSFM (Fujifilm) or StemPro_MSC (Life Technologies) in which cells show low adhesion and need the presence of fibronectin (Patrikoski et al., 2013;Heathman et al., 2015;Cimino et al., 2017) and that do not allow the isolation step of MSC without the addition of human serum AB or FBS in the first passage (Hartmann et al., 2010;Chase et al., 2012;Wuchter et al., 2016). ...
Article
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Background Cell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium. Methods In this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEM α supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step. Results We showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90 ⁺ , CD73 ⁺ , CD105 ⁺ , HLADR ⁻ , CD34 ⁻ , CD45 ⁻ phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies. Conclusions The use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media.
... Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice (GMP). For GMPcompliant MSC expansion for clinical utilization, the use of xeno-free culture medium is warranted [27]. The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. ...
Article
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b> Background: The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. Summary: This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. Conclusion: Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.
... PL thus far can be considered the most used supplement for GMP-compliant MSC expansion instead of FBS. Several studies have demonstrated the efficacy of PL in supporting MSC cell growth [21,[30][31][32][33][34]. Heathman and colleagues [34] have shown that PL can be even better than FBS, delivering an increased cell growth in monolayer and also in microcarrier culture. ...
Article
Background: So far, using human blood-derived components appears to be the most efficient and safest approach available for mesenchymal stromal cell (MSC) expansion. In this paper, we report on the characterization of human AB serum (AB HS) produced by using different plasma sources, and its use as an alternative supplement to MSC expansion. Methods: Two plasma sources were used for AB HS production: plasma removed from whole blood after 24 h of collection (PC > 24 h) and plasma, cryoprecipitate reduced (PCryoR). The biochemical profile and quality of the produced AB HS batches were analyzed and their ability to support MSC cell growth after different storage times (0, 3, 6, 9 and 12 months) was evaluated. Results: The two plasma sources used showed similar characteristics regarding biochemical constituents and quality parameters and were effective in promoting MSC growth. MSCs cultured in medium supplemented with 10% AB HS presented similar doubling times and cumulative population doublings when compared to the 10% fetal bovine serum(FBS)-supplemented culture while maintaining immunophenotype, functional features, and cytogenetic profile. Conclusion: Overall, the results indicate that AB HS is an efficient FBS substitute and can be used for at least 12 months after production without impairing cell proliferation and quality.
... A promising chemically defined medium was approved by FDA (StemPro MSC SFM, Invitrogen) to isolate and expand hMSC. Different studies were done to show its suitability to culture hMSC [36,44], but due to the lack of standardization of the protocols, the results obtained are difficult to compare. Some studies reported a reduction on the potential for hMSC differentiation and on expression of some proteins, when compared with FBS-supplemented medium [36]. ...
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Due to their immunomodulatory and chemotactic properties, hMSC are being explored to treat immune-related diseases. For their use in human therapies, it is necessary to culture hMSC in xeno-free conditions. In this study, the impact that a xeno-free medium based on a human plasma derivate has on these properties was analysed. Bone marrow-derived hMSC preserved their immunosuppressive and immunostimulatory properties, as observed with in vitro assays with hMSC cocultured with mixed leukocyte reactions or with mitogen-stimulated leukocytes. Moreover, hMSC expanded in xeno-free medium were recruited by macrophages in both migration and invasion assays, which indicates that the cells maintained their chemotactic properties. These data suggest that xeno-free expanded hMSC preserved their immunomodulatory and chemotactic properties, indicating that the described xeno-free medium composition is a potential candidate to culture and expand hMSC for human cell therapies.
... Methods of scaling up stem cell manufacturing to improve availability of stem cell treatments requires the development of xeno-free approaches to ensure that the quality of the cells implanted is reproducible and to minimize the potential immunogenic responses. BM-MSC [65], ASC [66,67], UM-MSC [68], and iPSC [69] have all been cultured in xeno-free media with comparable or improved differentiation potentials and proliferation compared to xenogenic media conditions. Researchers have focused on the development of testing metrics-toxicity, pharmacodynamics, pharmacokinetics, and quality control (potency, identity, and impurities)-based on publications from regulatory agencies and the International Society for Cell Therapy and have defined key criteria for the therapeutic use of allogenic and autologous cells [70,71]. ...
Article
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Purpose of review: While the clinical potential of tissue engineering for treating joint damage has yet to be realized, research and commercialization efforts in the field are geared towards overcoming major obstacles to clinical translation, as well as towards achieving engineered grafts that recapitulate the unique structures, function, and physiology of the joint. In this review, we describe recent advances in technologies aimed at obtaining biomaterials, stem cells, and bioreactors that will enable the development of effective tissue-engineered treatments for repairing joint damage. Recent findings: 3D printing of scaffolds is aimed at improving the mechanical structure and microenvironment necessary for bone regeneration within a damaged joint. Advances in our understanding of stem cell biology and cell manufacturing processes are informing translational strategies for the therapeutic use of allogeneic and autologous cells. Finally, bioreactors used in combination with cells and biomaterials are promising strategies for generating large tissue grafts for repairing damaged tissues in pre-clinical models. Together, these advances along with ongoing research directions are making tissue engineering increasingly viable for the treatment of joint damage.
... To eliminate potential risks associated with the use of FCS, intensive research has been carried out to identify a suitable replacement of human origin. Previous findings have demonstrated the superiority of human platelet lysate (PLT) over human AB serum 8 , human plasma 9 and human autologous serum 10 for ex vivo expansion of MSCs, owing to its high content of growth factors, low cost and ease in large-scale production [11][12][13][14] . This present study focuses on validating the concept and potential benefit of replacing FCS with PLT in MSC production for clinical use rather than comparing PLT with other human blood products. ...
Article
Full-text available
Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.
... A recent meta-analysis, where most of the patients derived from the study above, showed that the therapeutic success is greater in new fistulas than in chronic fistulas, and the usage of adipose-derived MSC seems to be superior to those derived from the bone marrow [84]. In summary, the local application of MSC seems to be an attractive therapy of fistulae; however, further work has to address the standardization of these cells [85], and potential risks like carcinogenesis cannot be excluded at this point of time [82]. ...
Article
Background: Crohn's disease (CD) is a chronic immune-mediated disorder of the gastrointestinal tract. The pathophysiological understanding of this disease is limited and no curative therapy is available so far. Therefore, most patients require long-lasting or even life-long immunosuppressive therapies for the suppression of symptoms to improve quality of life and reduction of long-term risks. However, in a relevant subgroup of patients, these therapeutic goals cannot be sufficiently attained. Summary: Clinically established therapies in active CD comprise corticosteroids and immunosuppressants such as azathioprine. After the introduction of anti-TNFα (Tumor necrosis factor alpha) antibodies, other biologicals (e.g., vedolizumab and ustekinumab) have also been approved. New drugs in the pipeline like filgotinib, upadacitinib, risankizumab or rifaximin could improve the therapy of CD in the near future. Thus, an individualized therapy management, based on optimal selection of therapeutic agents will become more important. Additionally, the local application of mesenchymal stem cells might be helpful in the management of fistulas. Key Messages: The targeted biological therapeutic agents (anti-TNFα antibodies, vedolizumab, ustekinumab) are well established for therapy in CD. There are several new substances in the pipeline with promising results in phase II trials (filgotinib, rifaximin, risankizumab, upadacitinib). The upcoming extension of the therapeutic arsenal will require methods for an optimized selection of substances, thus enabling a more individualized therapy.
... The identification of formulations to replace serum in cell culture media [4][5][6] presents a complex and difficult optimization problem as the replacement culture would require a large number of factors (cell culture supplements) in complex dose combinations. Optimizing such a large problem by conventional means such as statistical design of experiments 7 and screening 8,9 would be deemed infeasible due to the large number of experiments required. ...
Article
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Substitution of serum and other clinically incompatible reagents is requisite for controlling product quality in a therapeutic cell manufacturing process. However, substitution with chemically defined compounds creates a complex, large-scale optimization problem due to the large number of possible factors and dose levels, making conventional process optimization methods ineffective. We present a framework for high-dimensional optimization of serum-free formulations for the expansion of human hematopoietic cells. Our model-free approach utilizes evolutionary computing principles to drive an experiment-based feedback control platform. We validate this method by optimizing serum-free formulations for first, TF-1 cells and second, primary T-cells. For each cell type, we successfully identify a set of serum-free formulations that support cell expansions similar to the serum-containing conditions commonly used to culture these cells, by experimentally testing less than 1 × 10⁻⁵ % of the total search space. We also demonstrate how this iterative search process can provide insights into factor interactions that contribute to supporting cell expansion.
... Very few studies have evaluated the effects of this combination on MSC viability, proliferation and function and to further complicate matters the literature reports a wide range of PRP preparation techniques, final composition, mode of activation, and volume of supplementation making it difficult to draw definitive conclusions on the biological interactions between MSCs and PRP [28,[38][39][40][41]. As an example, low volumes of homologous PRP have been used as MSC culture media substitutes to minimize the introduction of xenogenic elements prior to the clinical use of stem cell preparations [42]. Conversely, studies have shown a dose dependent effect of high volumes of PRP on cellular proliferation in MSC cultures [43]. ...
... In terms of clinical transplantation, it is important to culture MSCs in media depriving of animal components, i.e., bovine products that have a potential risk of viral transmission or prion diseases as well as immune system activation of graft recipients. Autologous or allogeneic-derived plasma or platelet lysates represent more safe substitutes than FBS and allow efficient proliferation of MSCs [43,44]. Nevertheless, some of the reports indicate the possibility of using commercially available xeno-free media for MSC culture [45,46]. ...
Article
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Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo . The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.
... MSC isolation was performed as previously described [114,115]. In brief, mononuclear cells were purified from healthy donor-derived bone marrow aspirates by density gradient centrifugation and seeded in a specific growth medium (MSCGM Bulletkit, Lonza, Walkersville, MD, USA) at 10 5 cells/cm 2 . ...
Article
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Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn−/− mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn−/− chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.
... Moreover, the cells cultured in media without FGF2 supplements were negative for mesenchymal stem cell markers and positive for non-MSC markers ( Figure 5C). Despite the merits of stem cell in cell therapy, the applications of stem cell have so far been limited to the high production cost that requires expensive commercial growth media, such as XF Medium, Essential medium etc [3,4,8,10,23,24]. Although the use of serum could reduce the cost, the unknown variables, such as existence of virus, allergens, could be problematic when entering into clinical trials [11,12]. ...
... Its ability to inhibit local joint inflammation comes from expression of IL-1 Ra and reduces pro-inflammatory cytokines by muting resident macrophages in the joint [38,39]. Meanwhile, culture and proliferation of MSC can be well-manipulated in vitro [40]. These features make the MSC therapy a possible way to be applied in clinics. ...
Article
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Purpose of Review This review aims at outlining alpha-2-macroglobulin (A2M) injection, a novel non-operation strategy which could delay the process of osteoarthritis (OA). Meanwhile, some burning issues concerning “experimental” and “applied” are also indicated in this review. Recent Findings Many researchers have found that the alpha-2-macroglobulin, a sort of broad-spectrum proteinase inhibitor, presents remarkable inhibitive effect on intra-articular inflammation. Additionally, results of animal experiments prove that the A2M can postpone cartilage degeneration. Some treatments, such as hyaluronic acid (HA), which have been applied clinically for many years proved not to be as effective; thus, the advantage of A2M is presented. Summary A2M promises to be a new strategy of non-operative treatment of OA for its excellent anti-inflammation effect and biosafety. Better improved pharmaceutical preparations and treatment strategies shall be developed with the in-depth research.
... The MSC culture media should be free of bovine components, as they transmit prion diseases and activate the immune system of organ recipients. Instead, autologous plasma or allogenic platelet lysate enhances the MSC growth and proliferation in the culture [50,51]. ...
Article
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Study Design: Meta-analysis. Objectives: We aimed to analyze the impact of cultured expansion of autologous mesenchymal stromal cells (MSCs) in the management of osteoarthritis of the knee from randomized controlled trials (RCTs) available in the literature. Materials and Methods: We conducted independent and duplicate electronic database searches including PubMed, Em-base, Web of Science, and Cochrane Library until August 2021 for RCTs analyzing the efficacy and safety of culture-expanded compared to non-cultured autologous MSCs in the management of knee osteoarthritis. The Visual Analog Score (VAS) for pain, Western Ontario McMaster University's Os-teoarthritis Index (WOMAC), Lysholm score, Knee Osteoarthritis Outcome Score (KOOS), and adverse events were the analyzed outcomes. Analysis was performed in R-platform using OpenMeta [Analyst] software. Results: Overall, 17 studies involving 767 patients were included for analysis. None of the studies made a direct comparison of the culture expanded and non-cultured MSCs, hence we pooled the results of all the included studies of non-cultured and cultured types of MSC sources and made a comparative analysis of the outcomes. At six months, culture expanded MSCs showed significantly better improvement (p < 0.001) in VAS outcome. Uncultured MSCs, on the other hand, demonstrated significant VAS improvement in the long term (12 months) in VAS (p < 0.001), WOMAC (p = 0.025), KOOS score (p = 0.016) where cultured-expanded MSCs failed to demonstrate a significant change. Culturing of MSCs did not significantly increase the complications noted (p = 0.485). On subgroup analysis, adipose-derived uncultured MSCs outperformed culture-expanded MSCs at both short term (six months) and long term (12 months) in functional Citation: Muthu, S.; Kartheek, R.R.; Jeyaraman, N.; Rajendran, R.L.; Khanna, M.; Jeyaraman, M.; Packkyarathinam, R.P.; Gangadaran, P.; Ahn, B.-C. Is Culture Expansion Necessary in Autologous Mesenchymal Stromal Cell Therapy to Obtain Superior Results in the Management of Knee Osteoarthritis?-Meta-Analysis of Randomized Controlled Trials. Bioengineering 2021, 8, 220. Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons At-tribution (CC BY) license (http://crea-tivecommons.org/licenses/by/4.0/). Bioengineering 2021, 8, 220 2 of 21 outcome parameters such as WOMAC (p < 0.001, p = 0.025), Lysholm (p < 0.006), and KOOS (p < 0.003) scores, respectively, compared to their controls. Conclusions: We identified a void in literature evaluating the impact of culture expansion of MSCs for use in knee osteoarthritis. Our indirect analysis of literature showed that culture expansion of autologous MSCs is not a necessary factor to obtain superior results in the management of knee osteoarthritis. Moreover, while using uncultured autologous MSCs, we recommend MSCs of adipose origin to obtain superior functional outcomes. However, we urge future trials of sufficient quality to validate our findings to arrive at a consensus on the need for culture expansion of MSCs for use in cellular therapy of knee osteoarthritis.
... Third, like other products of regenerative medicine, it has been well documented that mesenchymal stem cells possess robust antiinflammatory properties in that they express IL-1 Ra and can directly antagonize resident macrophages of the joint space from secreting pro-inflammatory cytokines [30,32]. Fourth, human mesenchymal stem cells can be cultured and expanded ex vivo using Good Manufacturing Practices (GMP), offering the opportunity for additional pharmacological treatment to enhance cell proliferation/maturation, as well as their repair potential [33]. Lastly, mesenchymal stem cells have been utilized in human clinical trials over the past ten years for widespread application, which demonstrates they are a safe and effective method for translational therapies [34]. ...
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Objectives: Knee osteoarthritis (OA)(1) is a debilitating condition that may ultimately require total knee arthroplasty (TKA).(2) Non-operative treatments are bracing, oral analgesics, physical therapy, and intra-articular knee injection (IAKI).(3) The objective of this paper is to provide a systematic literature review regarding intra-articular treatment of knee OA and insight into promising new products of regenerative medicine that may eventually have a substantial effect on treatment. Methods: A literature search was executed using Medline, Cochrane, and Embase with keywords "knee osteoarthritis" and "injection." Specifically, 45 articles that discussed intra-articular knee injection using corticosteroids, hyaluronic acid, analgesics, local anesthetics, and newer products of regenerative medicine, such as platelet-rich plasma (PRP)(4) and mesenchymal stem cells (MSC),(5) were analyzed. Of these, eleven were level 1, three were level 2, twelve were level 3, two were level 4, and seventeen were level 5 evidence. Papers included animal models. Results: Local anesthetics have potential side effects and may only be effective for a few hours. Morphine and ketorolac may provide significant pain relief for 24 hours. Corticosteroids may give patients weeks to months of effective analgesia, but complications may occur, such as systemic hyperglycemia, septic arthritis, and joint degradation . Hyaluronic acid is a natural component of synovial fluid, but efficacy with respect to analgesia is controversial. Platelet-rich plasma formulations, autologous conditioned serum, autologous protein solution, and mesenchymal stem cell injections contain anti-inflammatory molecules and have been proposed to attenuate joint destruction or potentially remodel the joint. Conclusions: Currently, knee OA treatment does not address the progressively inflammatory environment of the joint. More investigation is needed regarding products of regenerative medicine, but they may ultimately have profound implications in the way knee OA is managed.
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Despite huge advances in recent years, the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niches in the bone marrow is still far from being fully understood. One reason is that hematopoiesis is a multi-step maturation process leading to HSPC heterogeneity. Subpopulations of HSPCs can be identified by clonogenic assays or in serial transplantation experiments in mice following sublethal irradiation, but it is very complex to reproduce or even maintain stem cell plasticity in vitro. Advanced model systems have been developed that allow to precisely control and analyze key components of the physiologic microenvironment for not only fundamental research purposes but, as a long-term goal, also for clinical applications. In this chapter, we describe our approach of building an artificial hematopoietic stem cell niche in the form of polymer film-based microcavities with a diameter of 300 μm and a depth of up to 300 μm and arranged in a 634-cavity array. The polymer films are provided with 3 μm pores and thus allow perfusion of the culture medium. The microcavity arrays can be inserted into a microbioreactor where a closed circulation loop can be tightly controlled with regard to medium flow and gas supply. The microcavity arrays were used for a three-dimensional (3D) co-culture of MSCs and HSPCs in a defined ratio over a time period of up to 21 days. With this setup, it could be demonstrated that the HSPCs maintained their stem cell characteristics more efficiently as compared to conventional monolayer co-culture controls.
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Bone marrow provides a rich source of mesenchymal stromal cells (MSCs), which have the remarkable capacity for cell and tissue regeneration. Since their initial discovery in the guinea pig almost 50 years ago, bone-marrow-derived MSCs have been extensively studied in animals and humans. Several subpopulations have been characterized with the aim to isolate, enrich, and identify the cells with stem-cell properties and immunomodulatory actions, which are important for regenerative medicine. In this chapter, we review the properties of bone-marrow-derived MSCs with a focus on the preclinical setting and discuss their applications for bone tissue engineering and bone regeneration.
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Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem/progenitor cells that can be isolated from various tissues and have attracted increasing attention from the scientific community. This is due to MSCs showing great potential for incurable disease treatment, and most applications of MSCs involve tissue degeneration and treatment of immune- and inflammation-mediated diseases. Conventional MSC cultures contain fetal bovine serum (FBS), which is a common supplement for cell development but is also a risk factor for exposure to animal-derived pathogens. To avoid the risks resulting from the xenogeneic origin and animal-derived pathogens of FBS, xeno-free media have been developed and commercialized to satisfy MSC expansion demands for human clinical applications. This review summarized and provided an overview of xeno-free media that are currently used for MSC expansion. Additionally, we discussed the influences of different xeno-free media on MSC biology with particular regard to cell morphology, surface marker expression, proliferation, differentiation and immunomodulation. The xeno-free media can be serum-free and xeno-free media or media supplemented with some human-originating substances, such as human serum, human platelet lysates, human umbilical cord serum/plasma, or human plasma-derived supplements for cell culture medium. These media have capacity to maintain a spindle-shaped morphology, the expression of typical surface markers, and the capacity of multipotent differentiation and immunomodulation of MSCs. Xeno-free media showed potential for safe use for human clinical treatment. However, the influences of these xeno-free media on MSCs are various and any xeno-free medium should be examined prior to being used for MSC cultures. © 2020, The Korean Tissue Engineering and Regenerative Medicine Society.
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Mesenchymal stromal cells (MSCs) are multi-potent stromal-derived cells capable of self-renewal that possess several advantageous properties for wound healing, making them of interest to the field of dermatology. Research has focused on characterizing the unique properties of MSCs, which broadly revolve around their regenerative and more recently discovered immunomodulatory capacities. Because of ease of harvesting and expansion, differentiation potential and low immunogenicity, MSCs have been leading candidates for tissue engineering and regenerative medicine applications for wound healing, yet results from clinical studies have been variable, and promising pre-clinical work has been difficult to reproduce. Therefore, the specific mechanisms of how MSCs influence the local microenvironment in distinct wound etiologies warrant further research. Of specific interest in MSC-mediated healing is harnessing the secretome, which is composed of components known to positively influence wound healing. Molecules released by the MSC secretome can promote re-epithelialization and angiogenesis while inhibiting fibrosis and microbial invasion. This review focuses on the therapeutic interest in MSCs with regard to wound healing applications, including burns and diabetic ulcers, with specific attention to the genetic skin disease recessive dystrophic epidermolysis bullosa. This review also compares various delivery methods to support skin regeneration in the hopes of combating the poor engraftment of MSCs after delivery, which is one of the major pitfalls in clinical studies utilizing MSCs.
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In the present study, a protocol was developed for processing of human adipose derived mesenchymal stem cell secretome formulation of varying concentration. Its molecular composition was evaluated, and its effectiveness in vitro using breast cancer cell lines, and in vivo in a nude mice breast cancer model was studied to determine its role in suppressing triple negative breast cancer in a dose dependent manner. Because the secretome could have value as an add-on therapy along with a current drug, the effectiveness of the secretome both in monotherapy and in combination therapy along with paclitaxel was evaluated. The results showed significant cell kill when exposed to the secretome above 20 mg/ml at which concentration there was no toxicity to normal cells. 70 mg/ml of SF showed 90 ± 10% apoptosis and significant decrease in CD44+/CD24−, MDR1+ and PDL-1+ cancer cells. In vivo, the tumor showed no growth after daily intra tumor injections at 50 mg/ml and 100 mg/ml doses whereas substantial tumor growth occurred after saline intra tumor injection. The study concludes that SF is a potential biotherapeutic for breast cancer and could be used initially as an add-on therapy to other standard of care to provide improved efficacy without other adverse effects.
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Mesenchymal stem cells (hMSCs) are multipotent stem cells that have the capacity to differentiate into various lineages. These cells provide stromal support and can be utilized as a feeder layer for expansion of hematopoitic stem cells and embryonic stem cells. Furthermore, allo-transplanted MSCs are not rejected and have been shown to mediate immuno-modulatory functions in vitro. Also, MSCs have been found at the wound site at extended times. The mechanisms underlying MSC migration and immuno-modulation are still under investigation. Aim: To understand the factors involved in human MSC (hMSC) migration and their interaction with various immune cell types. Methods: Human MSCs were examined for the presence of cell surface receptors that may play a role in migration using quantitative RT-PCR. Next, hMSCs were co-cultured with purified immune cell types including dendritic cells (DCs), naïve T cells and NK cells. Following the co-culture, changes in the phenotype of the immune cells under activating conditions were analyzed using ELISA and functional assays. Results: Human MSCs express Toll receptors, especially TLR4, on their cell surface. The TLR4 on hMSCs is functional as seen by a several-fold increase in IL-6 and chemokine IL-8 upon incubation with TLR4 exogenous ligand lipopolysaccharide (LPS) and the endogenous ligand, soluble hyaluronic acid (sHA). When hMSCs were incubated with activated dendritic cells, there was a >50% decrease in TNF-α secretion and a >50% increase in IL-10 secretion. When hMSCs were incubated with naïve T cells, hMSCs decreased IFN-γ secretion and increased IL-4 secretion. Decreased IFN-γ was also seen when MSCs were incubated with NK cells. Conclusion: These results suggest that (i) hMSCs may respond to the signals generated by breakdown products of extracellular matrix (e.g. sHA) via TLR4 and assist in wound healing (ii) hMSCs immuno-modulatory effects are mediated by interacting with various immune cell types and altering their phenotypic response to a more tolerant and anti-inflammatory response.
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Compromised bone-regenerating capability following a long bone fracture is often the result of reduced host bone marrow (BM) progenitor cell numbers and efficacy. Without surgical intervention, these malunions result in mobility restrictions, deformities, and disability. The clinical application of BM-derived mesenchymal stem cells (MSCs) is a feasible, minimally invasive therapeutic option to treat non-union fractures. This review focuses on novel, newly identified cell surface markers in both the mouse and human enabling the isolation and purification of osteogenic progenitor cells as well as their direct and indirect contributions to fracture repair upon administration. Furthermore, clinical success to date is summarized with commentary on autologous versus allogeneic cell sources and the methodology of cell administration. Given our clinical success to date in combination with recent advances in the identification, isolation, and mechanism of action of MSCs, there is a significant opportunity to develop improved technologies for defining therapeutic MSCs and potential to critically inform future clinical strategies for MSC-based bone regeneration.
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Administration of bone marrow-derived mesenchymal stem cells (MSCs) is an innovative approach for the treatment of a range of diseases that are not curable by current therapies including heart failure. A number of clinical trials have been completed and many others are ongoing; more than 2,000 patients worldwide have been administered with culture-expanded allogeneic or autologous MSCs for the treatment of various diseases, showing feasibility and safety (and some efficacy) of this approach. However, protocols for isolation and expansion of donor MSCs vary widely between these trials, which could affect the efficacy of the therapy. It is therefore important to develop international standards of MSC production, which should be evidence-based, regulatory authority-compliant, of good medical practice grade, cost-effective, and clinically practical, so that this innovative approach becomes an established widely adopted treatment. This review article summarizes protocols to isolate and expand bone marrow-derived MSCs in 47 recent clinical trials of MSC-based therapy, which were published after 2007 onwards and provided sufficient methodological information. Identified issues and possible solutions associated with the MSC production methods, including materials and protocols for isolation and expansion, are discussed with reference to relevant experimental evidence with aim of future clinical success of MSC-based therapy.
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Human Mesenchymal Stem Cells (hMSCs) present a promising tool for regenerative medicine. However, ex vivo expansion is necessary to obtain sufficient cells for clinical therapy. Conventional growth media usually contain the critical component fetal bovine serum. For clinical use, chemically defined media will be required. In this study, the capability of two commercial, chemically defined, serum-free hMSC growth media (MSCGM-CD and PowerStem) for hMSC proliferation was examined and compared to serum-containing medium (MSCGM). Immunophenotyping of hMSCs was performed using flow cytometry, and they were tested for their ability to differentiate into a variety of cell types. Although the morphology of hMSCs cultured in the different media differed, immunophenotyping displayed similar marker patterns (high expression of CD29, CD44, CD73, and CD90 cell surface markers and absence of CD45). Interestingly, the expression of CD105 was significantly lower for hMSCs cultured in MSCGM-CD compared to MSCGM. Both groups maintained mesenchymal multilineage differentiation potential. In conclusion, the serum-free growth medium is suitable for hMSC culture and comparable to its serum-containing counterpart. As the expression of CD105 has been shown to positively influence hMSC cardiac regenerative potential, the impact of CD105 expression onto clinical use after expansion in MSCGM-CD will have to be tested.
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Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation.In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.
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Bone homeostasis is maintained by the balance between bone-forming osteoblasts and bone-degrading osteoclasts. Osteoblasts have a mesenchymal origin whereas osteoclasts belong to the myeloid lineage. Osteoclast and osteoblast communication occurs through soluble factors secretion, cell-bone interaction and cell-cell contact, which modulate their activities. CD200 is an immunoglobulin superfamilly member expressed on various types of cells including mesenchymal stem cells (MSCs). CD200 receptor (CD200R) is expressed on myeloid cells such as monocytes/macrophages. We assume that CD200 could be a new molecule involved in the control of osteoclastogenesis and could play a role in MSC-osteoclast communication in humans. In this study, we demonstrated that soluble CD200 inhibited the differentiation of osteoclast precursors as well as their maturation in bone-resorbing cells in vitro. Soluble CD200 did not modify the monocyte phenotype but inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL) signaling pathway as well as the gene expression of osteoclast markers such as osteoclast-associated receptor (OSCAR) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, MSCs inhibited osteoclast formation, which depended on cell-cell contact and was associated with CD200 expression on the MSC surface. Our results clearly demonstrate that MSCs, through the expression of CD200, play a major role in the regulation of bone resorption and bone physiology and that the CD200-CD200R couple could be a new target to control bone diseases.
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Mesenchymal stromal cell (MSC)-based therapy holds great promise for treating immune disorders and for regenerative medicine in agreement with their paracrine trophic and immunosuppressive activities. Various processes have been developed worldwide to produce clinical grade MSCs but, so far, it is not known if one given MSC is more efficient than another. In addition, while their broad activity on innate and adaptative immune cell subsets is now widely admitted, the precise mechanisms supporting their immunoregulatory capacities are still a matter of debate. Finally, quantitative immunological potency assays correlated to clinical efficacy and clinically relevant immunomonitoring approaches for MSC-treated patients are sorely needed. Multiple parameters could influence the immunomodulatory potential of therapeutic MSCs. The most important challenge is now to differentiate, within a high number of poorly comparable and even contradictory pre-clinical studies, the parameters that could have some clinical impact from those that are only due to uncontrolled experimental variability. Importantly, besides MSC-related differences, primarily linked to production processes, several important variables associated with immune assays themselves, including selection of effector immune cells, activation signals, and read-out techniques, should be carefully considered to obtain solid results with potential therapeutic application. In this review, we establish a core of common and reproducible immunological properties of MSCs, shed light on technical issues concerning immunomodulatory potential assessment, and put them into perspective when considering clinical application.
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Background aims: A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Methods: Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. Results: After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. Conclusions: The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.
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In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.
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Human umbilical cord stroma-derived mesenchymal stem cells (hUCS-MSCs) are considered as a remarkable and promising stem cell source to be potentially used in cellular therapies. While no graft rejection has been reported in the recipient organism even in xenotransplantation studies, they were also shown experimentally to attenuate tumor cell growth and gene transfers. In present study, we demonstrate a reliable, reproducible and efficient cryopreservation method of hUCS-MSCs resulting one of the highest cell survival rates reported so far. Conventional, computer-controlled multi-step slow freezing (MSSF), and vitrification methods were comparatively tested using cell permeable [dimethylsulfoxide (DMSO), ethylene glycol] and impermeable [trehalose, sucrose, hydroxyethyl starch (HES), human serum albumin] cryoprotectant agents (CPAs). After determining the ice nucleation point for each solution, latent heat evolution was suppressed during freezing, followed by a cooling process to -40°C at 1°C/min or 0.3°C/min. The efficiency of the cryopreservation techniques used was determined by cell viability and proliferation assays, the expression of cell surface markers, cytoskeletal proteins and chromosome alignments. The cell survival rate was found highest (87±5%) by MSSF with sucrose (0.1 M) +DMSO (10%) at 1°C/min freezing rate. In this group, no significant difference was noted before and after the cryopreservation in cell morphology, cytokeratin, vimentin, and α-smooth muscle actin profiles and the expressions of CD105, CD90, CD73, CD29 and HLA-DR. Second highest cell survival ratio (85±6%) was obtained in DMSO (10%) alone at 1°C/min freezing rate. Interestingly, poor (18±15%) cell survival rates were obtained after vitrification. Cumulatively, results indicated that MSSF favors to the other freezing protocols with an addition of sucrose or DMSO alone depending on the freezing rate used.
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Introduction Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles. Methods In this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors. Results Our data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages. Conclusions Because we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on hBM-MSC malignant transformation in the literature.
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The estimated frequency of MSCs in BM is about 0.001-0.01% of total nucleated cells. Most commonly, one applied therapeutic cell dose is about 1 -5 million MSCs/kg body weight, necessitating a reliable, fast and safe expansion system. The limited availability of MSCs demands for an extensive ex vivo amplification step to accumulate sufficient cell numbers. Human platelet lysate (PL) has proven to be a safe and feasible alternative to animal-derived serum as supplement for MSC cultivation. We have investigated the functionally closed automated cell culture hollow fiber bioreactor Quantum® cell expansion system as an alternative novel tool to conventional tissue flasks for efficient clinical-scale MSC isolation and expansion from bone marrow using PL. Cells expanded in the Quantum system fulfilled MSC criteria as shown by flow cytometry and adipogenic, chondrogenic and osteogenic differentiation capacity.Cell surface expression of a variety of chemokine receptors, adhesion molecules, and additional MSC markers was monitored for several passages by flow-cytometry. The levels of critical media components like glucose and lactate were analysed. PDGF-AA, PDGF-AB/BB, bFGF, TGF-β1, sICAM-1, sVCAM-1, RANTES, GRO, VEGF, sCD40L, IL-6 were assessed using a LUMINEX platform. Originally optimized for the use of fetal calf serum (FCS) as supplement and fibronectin as coating reagent, we succeeded to obtain an average of more than 100x10⁶ of MSCs from as little as 18.8 to 28.6 mL of BM aspirate using PL. We obtained similar yields of MSCs/μL BM in the FCS-containing and the xenogen-free expansion system. The Quantum system reliably produces a cellular therapeutic dose in a functionally closed system that requires minimal manipulation. Both isolation and expansion is possible using FCS or PL as supplement. Coating of the hollow fibres of the bioreactor is mandatory when loading MSCs. Fibronectin, PL and human plasma may serve as coating reagents.
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Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions - and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization and reprogramming into induced pluripotent stem cells (iPSC) using high density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and non-promoter regions of developmental genes. Furthermore, particularly SA-hypomethylation appears to be associated with H3K9me3, H3K27me3 and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40 TAg) result in telomere extension but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process which stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence.
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The increasing use of mesenchymal stromal cells (MSC) in clinical cellular therapy requires a safe and controlled production process compliant with Good Manufacturing Practice (GMP). Pooled blood group AB human serum (HS) has been used to replace fetal bovine serum (FBS), critically rated by the regulatory agencies, since it can support the expansion of adipose tissue-derived mesenchymal stromal cells (ASC). However, it remains unknown whether the choice of serum affects application-relevant characteristics of ASC. A microarray-based screen has revealed differentially expressed adhesion and extracellular matrix- associated molecules in HS- and FBS cultivated ASC. Since cell therapy relies on the cells´ efficacy to home and engraft, HS- and FBS-ASC were compared by analyzing adhesion, migration and transmigration as well as short-term homing in vivo. HS-cultivated ASC demonstrated a higher adhesion to plastic and extracellular matrix but reduced adhesion to endothelial cells both under static and flow conditions. Migration and transmigration assays confirmed the attraction of ASC by tumor conditioned medium irrespective of the supplement added. Co-injecting differently labeled HS- and FBS-ASC into NOD/SCID mice revealed reduced numbers of HS-ASC in lungs and liver. This has been interpreted as reduced capillary entrapment. Our data indicate that varying the serum supplement may alter application relevant characteristics of ASC, such as adhesion, as well as lung entrapment after infusion. Appropriate injury models and further molecular analyses are required to provide mechanistic insight into the differential effects of HS versus FBS on ASC cultures.
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Sjögren syndrome (SS) is a systemic autoimmune disease characterized by dry mouth and eyes, and the cellular and molecular mechanisms for its pathogenesis are complex. Here we reveal, for the first time, that bone marrow mesenchymal stem cells in SS-like NOD/Ltj mice and human patients were defective in immunoregulatory functions. Importantly, treatment with mesenchymal stem cells (MSCs) suppressed autoimmunity and restored salivary gland secretory function in both mouse models and SS patients. MSC treatment directed T cells toward Treg and Th2, while suppressing Th17 and Tfh responses, and alleviated disease symptoms. Infused MSCs migrated toward the inflammatory regions in a stromal cell-derived factor-1-dependent manner, as neutralization of stromal cell-derived factor-1 ligand CXCR4 abolished the effectiveness of bone marrow mesenchymal stem cell treatment. Collectively, our study suggests that immunologic regulatory functions of MSCs play an important role in SS pathogenesis, and allogeneic MSC treatment may provide a novel, effective, and safe therapy for patients with SS. This study was registered at www.clinicaltrials.gov as NCT00953485.
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Background: Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. Methods and findings: We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. Conclusions: The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.
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The concept that mesenchymal stromal cells (MSCs), a component of the hematopoietic microenvironment, can be a target for alloreactive effector cells in the context of graft-vs-host disease has not been investigated in detail. Mixed lymphocyte reaction (MLR) supernatant was used to mimic the inflammatory milieu induced by an allogeneic immune response in vitro. In addition to phenotype and proliferation, we monitored MSC differentiation, gene expression, and support of CD34(+) hematopoietic stem and progenitor cells after priming with MLR supernatant. Priming of MSCs with MLR supernatant led to an 11-fold decrease in cobblestone area-forming cells in the 4-week coculture (p < 0.05) and a threefold decrease of colony-forming unit macrophage in the colony-forming cell assay (p < 0.05). MSC proliferation over 8 days was increased 2.5-fold (p < 0.05). Osteogenic differentiation was enhanced, while adipogenesis was concurrently suppressed. In addition, the surface expression of HLA-DR and intercellular adhesion molecule-1 was increased 20-fold (p = 0.06) and 45-fold (p < 0.05), respectively. This was associated with increased adhesion of hematopoietic stem and progenitor cells to MLR-treated MSCs. In summary, our data shed light on the dysfunction of the stromal environment during graft-vs-host disease, possibly aggravating cytopenia and leading to an enhanced immunogenicity of MSCs.
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Due to their multi-lineage differentiation capacity, support of haematopoiesis, immunomodulation and secretion of proregenerative factors, mesenchymal stem/stromal cells (MSCs) are in the focus of intense research since decades. The literature is replete with reports on their potential in preclinical model systems. However, the heterogeneity of the primary cell population as starting material and the diverse protocols for isolation and cultivation are hampering progress in their clinical application. Consensus on common standards and harmonised isolation and characterisation protocols are important to ensure safety and efficacy. This review focuses on the recent scientific evidence of clinically relevant properties and on the speculative cardiomyogenic and hepatic differentiation potential of MSCs. Special emphasis is put on the importance of standardisation and harmonisation in clinical-scale manufacturing.
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Systemic infusion of bone marrow mesenchymal stem cells (BMMSCs) yields therapeutic benefit for a variety of autoimmune diseases, but the underlying mechanisms are poorly understood. Here we show that in mice systemic infusion of BMMSCs induced transient T cell apoptosis via the FAS ligand (FASL)-dependent FAS pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SS) and dextran-sulfate-sodium-induced experimental colitis. FASL(-/-) BMMSCs did not induce T cell apoptosis in recipients, and could not ameliorate SS and colitis. Mechanistic analysis revealed that FAS-regulated monocyte chemotactic protein 1 (MCP-1) secretion by BMMSCs recruited T cells for FASL-mediated apoptosis. The apoptotic T cells subsequently triggered macrophages to produce high levels of TGFβ, which in turn led to the upregulation of CD4(+)CD25(+)Foxp3(+) regulatory T cells and, ultimately, immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via FAS/FASL to induce T cell apoptosis.
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Mesenchymal stromal cells (MSCs) represent a heterogeneous subset of multipotent cells that can be isolated from several tissues including bone marrow and fat. MSCs exhibit immunomodulatory and anti-inflammatory properties that prompted their clinical use as prevention and/or treatment for severe graft-versus-host disease (GVHD). Although a number of phase I-II studies have suggested that MSC infusion was safe and might be effective for preventing or treating acute GVHD, definitive proof of their efficacy remains lacking thus far. Multicenter randomized studies are ongoing to more precisely assess the impact of MSC infusion on GVHD prevention/treatment, whereas further research is performed in vitro and in animal models with the aims of determining the best way to expand MSCs ex vivo as well as the most efficient dose and schedule of MSCs administration. After introducing GVHD, MSC biology, and results of MSC infusion in animal models of allogeneic hematopoietic cell transplantation, this article reviews the results of the first clinical trials investigating the use of MSC infusion as prevention or treatment of GVHD.
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Background aims: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. Methods: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. Results: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. Conclusions: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.
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Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.
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Mesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro. We monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor. Co-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34(+) cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations-and conversely their hematopoiesis supportive functions-may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor. These results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.
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Somatic cells change continuously during culture expansion-long-term culture evokes increasing cell size, declining differentiation potential, and ultimate cell cycle arrest upon senescence. These changes are of particular relevance for cellular therapy which necessitates standardized products and reliable quality control. Recently, replicative senescence has been shown to be associated with highly reproducible epigenetic modifications. Here, we describe a simple method to track the state of senescence in mesenchymal stromal cells (MSCs) or fibroblasts by monitoring continuous DNA methylation (DNAm) changes at specific sites in the genome. Six CpG sites have been identified which reveal either linear hypermethylation or hypomethylation with respect to the number of cumulative population doublings (cPDs). Conversely, the DNAm level at these CpG sites can be analyzed-for example, by pyrosequencing of bisulfite-converted DNA-and then used for linear regression models to predict cPDs. Our method provides an epigenetic biomarker to determine the state of senescence in cell preparations.
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Mesenchymal stromal cells are highly interesting candidates for clinical applications in regenerative medicine. Due to their low occurrence in human tissues, extensive in vitro expansion is necessary to obtain sufficient cell numbers applicable as a clinical dose in the context of cellular therapy. Current cell culture media formulations for the isolation and expansion of MSCs include fetal calf serum (FCS), human AB serum (ABS) or human platelet lysate (PL) as a supplement. However, these established supplements are inherently ill-defined formulations that contain a variety of bioactive molecules in varying batch-to-batch compositions and the risk of transmitting pathogens that escape routine screening procedures. In this study, we have comparatively characterized the capacity of commonly used basal media, such as αMEM, DMEM, IMDM and RPMI 1640 as well as human and animal-derived supplements, i.e. PL, ABS and FCS to stimulate cell proliferation. MSC proliferation was observed to be optimal in PL-supplemented αMEM. Using a combinatorial approach we then assessed a library of soluble factors including mitogens (TGF-β1, Activin A, bFGF, EGF, IGF-I, PDGF-BB and VEGF), chemokines (CCL21, CCL25, CXCL12, RANTES), proteins (human serum albumin), lipids (e.g. oleic acid, linoleic acid, arachidonic acid), hormones (dexamethasone, insulin, TSH), to create a defined medium as well as coating of cell culture surfaces to promote robust MSC proliferation in vitro. A combination of recombinant human factors partially met the nutritional requirements of bone marrow-derived MSCs, and was able to promote cell proliferation comparable to about 5% PL if supplemented with auxiliary 0.6 - 1.2% PL. Maximal MSC proliferation was achieved by combining 5% PL with a cocktail of recombinant factors and did not depend on coating of cell culture surfaces.
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At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of 'translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.
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Background Multipotent stromal cells exhibit immunomodulatory capacities and have been applied in transplantation and autoimmune diseases. One of the effects of MSC involves the inhibition of dendritic cell differentiation. Since IL-6 and IL-10 are known to play a role in inhibiting immature dendritic cell differentiation, we hypothesized that these cytokines also may mediate the inhibitory effect of human MSC in immature dendritic cell differentiation. Design and Methods Monocytes were cultured with IL-4 and GM-CSF in the presence or absence of culture-expanded bone marrow-derived multipotent stromal cells. Neutralization and cytokine-depletion strategies were applied to reveal the cellular source and effect of IL-6 and IL-10. Results Addition of multipotent stromal cells to monocyte cultures significantly reduced the generation of immature dendritic cells (CD14(-)CD1a(+)) and resulted in the generation of CD14(+)CD1a(-) cells that displayed a significantly reduced immunostimulatory effect. We found that culture supernatants of co-cultures of multipotent stromal cells and monocytes contained higher concentrations of IL-6 and IL-10. multipotent stromal cells produce IL-6 and neutralizing this IL-6 reversed the inhibitory effect of multipotent stromal cells. IL-10 was not produced by multipotent stromal cells, but exclusively by monocytes after exposure to multipotent stromal cells-produced IL-6. Conclusions By the constitutive production of IL-6, multipotent stromal cells prevent the differentiation of monocytes towards antigen-presenting immunogenic cells and skew differentiation towards an anti-inflammatory IL-10 producing cell type.
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Clinical-grade mesenchymal stromal cells (MSC) are usually expanded from bone marrow (BMMSC) or adipose tissue (ADSC) using processes mainly differing in the use of fetal calf serum (FCS) or human platelet lysate (PL). We aimed to compare immune modulatory properties of clinical-grade MSC using a combination of fully standardized in vitro assays. BMMSC expanded with FCS (BMMSC-FCS) or PL (BMMSC-PL), and ADSC-PL were analyzed in quantitative phenotypic and functional experiments including their capacity to inhibit the proliferation of T, B, and NK cells. The molecular mechanisms supporting T-cell inhibition were investigated. These parameters were also evaluated after pre-stimulation of MSC with inflammatory cytokines. BMMSC-FCS, BMMSC-PL, and ADSC-PL displayed significant differences in expression of immunosuppressive and adhesion molecules. Standardized functional assays revealed that resting MSC inhibited proliferation of T and NK cells, but not B cells. ADSC-PL were the most potent in inhibiting T-cell growth, a property ascribed to IFN-γ-dependent indoleamine 2,3-dioxygenase activity. MSC did not stimulate allogeneic T cell proliferation but were efficiently lysed by activated NK cells. The systematic use of quantitative and reproducible validation techniques highlights differences in immunological properties of MSC produced using various clinical-grade processes. ADSC-PL emerge as a promising candidate for future clinical trials.
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Human platelets represent a promising source of bioactive substances as growth factors not just for in vivo wound healing and tissue repair, but also for the expansion of human stem and progenitor cells in vitro. The replacement of fetal bovine serum (FBS) as a standard culture supplement by human platelet-derived growth factors now allows for the GMP-compliant implementation of various cell therapeutics in the growing field of regenerative medicine.For this purpose a protocol for the preparation of human platelet lysate (HPL) by several freeze-thaw cycles has been developed, resulting in platelet fragmentation and the release of stored growth factors. By pooling up to 15 U of HPL derived from individual blood donors, a virtually standardized product is achieved. The depletion of platelet particles and fragments in a final centrifugation step reduces the risk of alloimmunization against platelet antigens and the formation of aggregates in cell culture.The successful application of pooled human platelet lysate (pHPL) as a culture medium supplement for the ex vivo propagation of human mesenchymal stem/progenitor cells (MSPCs) and endothelial colony forming progenitor cells (ECFCs) indicates the feasibility of this animal serum-free source of growth factors. Further studies will evaluate efficacy and safety of pHPL.
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The transplantation of human stem cells seeded on biomaterials holds promise for many clinical applications in cranio-maxillo-facial tissue engineering and regenerative medicine. However, stem cell propagation necessary to produce sufficient cell numbers currently utilizes fetal calf serum (FCS) as a growth supplement which may subsequently transmit animal pathogens. Human platelet lysate (HPL) could potentially be utilized to produce clinical-grade stem cell-loaded biomaterials as an appropriate FCS substitute that is in line with clinically-applicable practice. The goal of this study was to investigate whether HPL can be successfully used to propagate human mesenchymal stem cells (HMSCs) seeded on clinically-approved collagen materials under clinically-applicable conditions using FCS as a control. HMSCs were isolated from bone marrow and cultured in the presence of 10% FCS or 10% HPL. Characterization of HMSCs was performed by flow cytometry and through osteogenic and adipogenic differentiation assays. Proliferative capacity of HMSCs on both matrices was investigated by mitochondrial dehydrogenase assays (WST) and tissue coverage scanning electron microscopy (SEM). The isolated HMSC differentiated into osteogenic and adipogenic cells authenticating the multipotentiality of the HMSCs. WST tests and the SEM images demonstrated that HPL was generally superior to FCS in promoting growth of seeded HMSCs. For all other tests HPL supported HMSCs at least equal to FCS. In conclusion, HPL is an effective growth factor to allow expansion of clinical-grade HMSCs on clinically-approved biomaterials for maxillofacial and oral implantology applications.
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Mesenchymal Stem/Stromal cells (MSCs) are increasingly applied in cell-based regenerative medicine. To yield clinically relevant cell doses, ex vivo expansion of MSCs is required to be compliant with good manufacturing practice (GMP) guidelines. A lack of standardization and harmonization seems to hamper rapid progress in the translational phase. Most protocols still use fetal bovine serum (FBS) to expand MSCs. However, the high lot-to-lot variability, risk of contamination and immunization call for xenogenic-free culture conditions. Chemically defined media are the ultimate achievement in terms of standardization. These media, however, need to maintain all key cellular and therapy-relevant features of MSCs. Because of the numerous constituents of FBS, the development of such chemically defined media with an optimal composition of the few essential factors is only beginning. Meanwhile, various human blood-derived components are under investigation, including human plasma, human serum, human umbilical cord blood serum and human platelet derivatives such as platelet lysate.
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The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo-, chondro-, and adipo-lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy.
Article
Replicative senescence has fundamental implications on cell morphology, proliferation, and differentiation potential. Here, we describe a simple method to track long-term culture based on continuous DNA-methylation changes at six specific CpG sites. This epigenetic senescence signature can be used as biomarker for various cell types to predict the state of cellular senescence with regard to the number of passages, population doublings, or days of in vitro culture. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
Article
Mesenchymal stromal cells (MSC) are promising candidates for innovative cell therapeutic applications. For clinical-scale manufacturing, different supplements have been evaluated as alternatives for the commonly used fetal bovine serum (FBS). We have reported previously that pooled human AB serum (HS) accelerates the proliferation of adipose tissue-derived MSC (ASC) while maintaining key functions of MSC biology such as differentiation, immune suppression and growth factor secretion. ASC expanded in FBS-supplemented culture media undergo replicative aging that is associated with a progressive loss of differentiation capacity but without indications of cellular transformation. The effects of HS media on ASC long-term culture, however, remain poorly characterized. Long-term cultures of ASC in FBS and HS media were analyzed with respect to proliferation, marker expression, differentiation and immune suppression. Despite signs of an accelerated proliferation, extended life span and clonogenic capacity of ASC cultivated in HS-supplemented media, HS and FBS cultures revealed no significant differences with respect to differentiation potential and expression of senescence markers. Anchorage-independent growth, which is indicative of tumorigenic properties, was not observed in either culture conditions. Similarly, immune suppressive activities were maintained. Donor variation regarding differentiation potential and marker expression became apparent in this study independent of the culture supplement or culture duration. We have demonstrated that the use of pooled allogeneic HS maintains the characteristics of ASC even after long-term expansion, further demonstrating that the use of HS is an alternative to FBS.
Article
Mesenchymal stromal cells (MSC) are heterogeneous and only a subset possesses multipotent differentiation potential. It has been proven that long-term culture has functional implications for MSC. However, little is known how the composition of subpopulation changes during culture expansion. We addressed the heterogeneity of MSC using limiting-dilution assays at subsequent passages. In addition, we used a cellular automaton model to simulate population dynamics under the assumption of mixed numbers of remaining cell divisions until replicative senescence. The composition of cells with adipogenic or osteogenic differentiation potential during expansion was also determined at subsequent passages. Not every cell was capable of colony formation upon passaging. Notably, the number of fibroblastoid colony-forming units (CFU-f) decreased continuously, with a rapid decay within early passages. Therefore the CFU-f frequency might be used as an indicator of the population doublings remaining before entering the senescent state. Predictions of the cellular automaton model suited the experimental data best if most cells were already close to their replicative limit by the time of culture initiation. Analysis of differentiated clones revealed that subsets with very high levels of adipogenic or osteogenic differentiation capacity were only observed at early passages. These data support the notion of heterogeneity in MSC, and also with regard to replicative senescence. The composition of subpopulations changes during culture expansion and clonogenic subsets, especially those with the highest differentiation capacity, decrease already at early passages.