Epigenetic changes in umbilical cord mesenchymal stromal cells upon stimulation and culture expansion

Background

Mesenchymal stromal cells (MSCs) are studied for their immunotherapeutic potential. Prior to therapeutic use, MSCs are culture expanded to obtain the required cell numbers and, to improve their efficacy, MSCs may be primed in vitro. Culture expansion and priming induce phenotypical and functional changes in MSCs and thus standardisation and quality control measurements come in need. We investigated the impact of priming and culturing on MSC DNA methylation and examined the use of epigenetic profiling as a quality control tool.

Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System

BackgroundThe use of bone marrow–derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times.MethodsWe evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials.ResultsAfter only two passages, we were able to expand a mean of 6.6 × 10⁸ MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10⁸ cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active.ConclusionsThe Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.     

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier‐based system

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost‐effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier‐based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM‐MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM‐MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ～4.82 ± 1.18 × 10⁵ cell mL^?1 at day 7, representing a 3.9‐fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP‐compliant large‐scale production system for hMSCs for cellular therapy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:889–895, 2014     

Bioactive factors secreted from mesenchymal stromal cells protect the intestines from experimental colitis in a three-dimensional culture

Background aims

Although mesenchymal stromal cells (MSCs) have shown therapeutic potential in intestinal tissue repair, controversy concerning their short survival and poor biodistribution in recipient tissues still remains. Therefore, we investigated the paracrine role of MSC in three-dimensional culture of colon with experimental colitis.

Culture expansion induces non-tumorigenic aneuploidy in adipose tissue-derived mesenchymal stromal cells

Background aimsAdipose tissue-derived mesenchymal stromal cells (ASCs) are of interest as a cell therapeutic agent for immunologic and degenerative diseases. During in vitro expansion, ASCs may be at risk for genetic alterations, and genetic screening is a prerequisite. We examined the presence of aneuploidy in ASCs and its origin and development during culture and evaluated the implications of aneuploidy for therapeutic use of ASCs.MethodsAdipose tissue of healthy individuals was used for isolation and expansion of ASCs. Chromosome copy numbers were studied using fluorescence in situ hybridization analysis. Aneuploidy was studied in freshly isolated ASCs, in ASCs cultured for 0–16 passages and in senescent cultures. To evaluate the plasticity of ploidy, ASCs were cloned, and the variation of ploidy in the clones was examined. Tumorigenicity was studied by subcutaneous injection of aneuploid ASCs in immunodeficient NOD/SCID mice.ResultsNo aneuploidy was detected in freshly isolated ASCs. In low passages (passages 0–4), aneuploidy was detected in 3.4% of ASCs. Prolonged culture expansion of ASCs (passages 5–16) resulted in a significant increase of aneuploidy to 7.1%. With senescence, aneuploidy increased further to 19.8%. Aneuploidy was observed in clones of diploid ASCs, demonstrating the de novo development of aneuploidy. No transformation of ASCs was observed, and in contrast to cancer cell lines, aneuploid ASCs were incapable of tumor formation in immunodeficient mice.ConclusionsASC cultures contain a stable percentage of aneuploid cells. Aneuploidy was not a predecessor of transformation or tumor formation. This finding indicates that aneuploidy is culture-induced but unlikely to compromise clinical application of ASCs.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Isolation and ex vivo expansion of synovial mesenchymal stromal cells for cartilage repair

Background aimsHyaline articular cartilage is a highly specialized tissue that offers a low-friction and wear-resistant interface for weight-bearing surface articulation in diarthrodial joints, but it lacks vascularity. It displays an inherent inability to heal when injured in a skeletally mature individual. Joint-preserving treatment procedures such as mosaicplasty, débridement, perichondrium transplantation and autologous chondrocyte implantation have shown variable results, and the average long-term result is sub-standard. Because of these limitations of the treatment methods and lack of intrinsic repair capacity of mature cartilage tissue, an alternative treatment approach is needed, and synovial mesenchymal stromal cells (SMSCs) represent an attractive therapeutic alternative because of their ex vivo proliferation capacity, multipotency and ability to undergo chondrogenesis.MethodsSMSCs were isolated from tissues obtained by arthroscopy using two types of biopsies. Ex vivo cell expansion was accomplished under static and dynamic culture followed by characterization of cells according to the International Society for Cellular Therapy guidelines. Kinetic growth models and metabolite analysis were used for understanding the growth profile of these cells.ResultsFor the first time, SMSCs were expanded in stirred bioreactors and achieved higher cell density in a shorter period of time compared with static culture or with other mesenchymal stromal cell sources.ConclusionsIn this study we were able to achieve (8.8 ± 0.2) × 10⁵ cells within <2 weeks in dynamic culture under complete xeno-free conditions. Our results also provided evidence that after dynamic culture these cells had an up-regulation of chondrogenic genes, which can be a potential factor for articular cartilage regeneration in clinical settings.     

Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells

Background aimsMultipotent stromal cells, also called mesenchymal stromal cells (MSCs), are potentially valuable as a cellular therapy because of their differentiation and immunosuppressive properties. As the result of extensive heterogeneity of MSCs, quantitative approaches to measure differentiation capacity between donors and passages on a per-cell basis are needed.MethodsHuman bone marrow-derived MSCs were expanded to passages P3, P5 and P7 from eight different donors and were analyzed for colony-forming unit capacity (CFU), cell size, surface marker expression and forward/side-scatter analysis by flow cytometry. Adipogenic differentiation potential was quantified with the use of automated microscopy. Percentage of adipogenesis was determined by quantifying nuclei and Nile red–positive adipocytes after automated image acquisition.ResultsMSCs varied in expansion capacity and increased in average cell diameter with passage. CFU capacity decreased with passage and varied among cell lines within the same passage. The number of adipogenic precursors varied between cell lines, ranging from 0.5% to 13.6% differentiation at P3. Adipogenic capacity decreased significantly with increasing passage. MSC cell surface marker analysis revealed no changes caused by passaging or donor differences.ConclusionsWe measured adipogenic differentiation on a per-cell basis with high precision and accuracy with the use of automated fluorescence microscopy. We correlated these findings with other quantitative bioassays to better understand the role of donor variability and passaging on CFU, cell size and adipogenic differentiation capacity in vitro. These quantitative approaches provide valuable tools to measure MSC quality and measure functional biological differences between donors and cell passages that are not revealed by conventional MSC cell surface marker analysis.     

DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells

Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo . This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.     

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells

BackgroundThere is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use.MethodsAdipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls.ResultsAd-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS.ConclusionsThe defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.     

Anti-inflammatory roles of mesenchymal stromal cells during acute Streptococcus pneumoniae pulmonary infection in mice

Background

Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia.

Umbilical cord derived mesenchymal stromal cells in microcarrier based industrial scale culture sustain the immune regulatory functions

Mesenchymal stromal cells (MSCs) have been isolated from numerous sources and are potentially therapeutic against various diseases. Umbilical cord-derived MSCs (UC-MSCs) are considered superior to other tissue-derived MSCs since they have a higher proliferation rate and can be procured using less invasive surgical procedures. However, it has been recently reported that 2D culture systems, using conventional cell culture flasks, limit the mass production of MSCs for cell therapy. Therefore, the development of alternative technologies, including microcarrier-based cell culture in bioreactors, is required for the large-scale production and industrialization of MSC therapy. In this study, we aimed to optimize the culture conditions for UC-MSCs by using a good manufacturing practice (GMP)-compatible serum-free medium, developed in-house, and a small-scale (30 mL) bioreactor, which was later scaled up to 500 mL. UC-MSCs cultured in microcarrier-based bioreactors (MC-UC-MSCs) showed characteristics equivalent to those cultured statically in conventional cell culture flasks (ST-UC-MSCs), fulfilling the minimum International Society for Cellular Therapy criteria for MSCs. Additionally, we report, for the first time, the equivalent therapeutic effect of MC-UC-MSCs and ST-UC-MSCs in immunodeficient mice (graft-versus-host disease model). Lastly, we developed a semi-automated cell dispensing system, without bag-to-bag variation in the filled volume or cell concentration. In summary, our results show that the combination of our GMP-compatible serum-free and microcarrier-based culture systems is suitable for the mass production of MSCs at an industrial scale. Further improvements in this microcarrier-based cell culture system can contribute to lowering the cost of therapy and satisfying several unmet medical needs.     

Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells

The ex vivo expansion of hematopoietic progenitor cells is of great interest for a variety of clinical applications, e.g. bone marrow transplantation or gene therapy. Therefore it is of general interest to develop a culture system, able to mimic the in vivo hematopoesis, which is a prerequisite for long-term hematopoietic culture. Our approach was to modify a continuously perfused bioreactor for cultivation and expansion of human hematopoietic stem cells. Therefore we immobilized stromal cells (human primary stromal cells or the murine cell line M2-10B4) in porous glass carriers in a fixed bed reactor and cocultivated human hematopoietic progenitor cells for several weeks. After inoculation of mononuclear cells derived from umbilical cord blood or peripheral blood stem cells both adherent and non adherent cells were harvested and analyzed by flow cytometry and short-term colony assays. During cultivation there was a permanent production of progenitor cells and mature blood cells derived from the immobilized cells in the carriers. We could demonstrate the immobilization of hematopoietic progenitor cells of the myeloid system detectable in short-term colony assays. Additionally we could observe the expansion of very early progenitor cells (CFU-GEMM) up to 4.2-fold and later progenitor cells (CFU-GM and BFU-E) up to 7-fold and 1.8-fold, respectively. P.M. and B.S. contributed equal parts to this work. This revised version was published online in July 2006 with corrections to the Cover Date.     

Multiomics characterization of mesenchymal stromal cells cultured in monolayer and as aggregates

Mesenchymal stromal cells (MSCs) have failed to consistently demonstrate their therapeutic efficacy in clinical trials, due in part to variability in culture conditions used for their production. Of various culture conditions used for MSC production, aggregate culture has been shown to improve secretory capacity (a putative mechanism of action in vivo) compared with standard monolayer culture. The purpose of this study was to perform multiomics characterization of MSCs cultured in monolayer and as aggregates to identify aspects of cell physiology that differ between these culture conditions to begin to understand cellular-level changes that might be related to secretory capacity. Targeted secretome characterization was performed on multiple batches of MSC-conditioned media, while nontargeted proteome and metabolome characterization was performed and integrated to identify cellular processes differentially regulated between culture conditions. Secretome characterization revealed a reduction in MSC batch variability when cultured as aggregates. Proteome and metabolome characterization showed upregulation of multiple protein and lipid metabolic pathways, downregulation of several cytoskeletal processes, and differential regulation of extracellular matrix synthesis. Integration of proteome and metabolome characterization revealed individual lipid metabolites and vesicle-trafficking proteins as key features for discriminating between culture conditions. Overall, this study identifies several aspects of MSC physiology that are altered by aggregate culture. Further exploration of these processes and pathways is needed to determine their potential role in regulating cell secretory capacity.     

Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.