Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) with their versatile growth and differentiation potential are ideal candidates for use in regenerative medicine and are currently making their way into clinical trials, which requires the development of xeno-free protocols for their culture. In this study, MSCs were cultured in 10% FCS or 7.5% HPL (human platelet lysate)-supplemented media. We found that both groups of MSCs showed a comparable morphology, phenotype and proliferation. The percentage of cells in the S- and G2-/M-phases, however, was slightly up-regulated (P<0.01) in HPL group. HPL contains PDGF (platelet derived growth factor)-AB and IGF (insulin-like growth factor)-1. In addition, compared with FCS group, MSCs in HPL group showed an increase in osteogenic differentiation and a decrease in adipogenic differentiation. In conclusion, MSCs in HPL-supplemented media maintained similar growing potential and phenotype, while osteogenic potential was enhanced. HPL offers a promising alternative to FCS for MSC expansion for clinical application, especially in bone injury diseases.     

Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells

Background aimsHuman platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow–derived MSCs.MethodsPlatelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated.ResultsNanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages.ConclusionsNanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.     

Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications

Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs.     

Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components

Background aimsThe clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL).MethodsPlatelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast–colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied.ResultsPL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC.ConclusionsPL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.     

Improved proliferation and differentiation capacity of human mesenchymal stromal cells cultured with basement-membrane extracellular matrix proteins

Background aimsIn vitro cultured mesenchymal stromal cells (MSC) are characterized by a short proliferative lifespan, an increasing loss of proliferation capacity and progressive reduction of differentiation potential. Laminin-1, laminin-5, collagen IV and fibronectin are important constituents of the basement membrane extracellular matrix (ECM) that are involved in a variety of cellular activities, including cell attachment and motility.Methods and resultsThe in vitro proliferation capacity of MSC was significantly improved when the cells were incubated in the presence of basement membrane ECM proteins. For example, a mixture of proteins improved proliferation capacity 250-fold in comparison with standard conditions after five passages. Furthermore, in colony-forming unit–fibroblast (CFU-F) assays colony numbers and size were significantly extended. Blocking specific integrin cell-surface receptors, positive effects on the proliferation capacity of MSC were inhibited. Additionally, when MSC were co-cultivated with ECM proteins, cells maintained their multipotential differentiation capacity throughout many culture passages in comparison with cells cultivated on plastic. However, expansion of MSC on laminin-5 suppressed any subsequent chondrogenic differentiation.ConclusionsOur results suggest that expansion of bone marrow-derived MSC in the presence of ECM proteins is a powerful approach for generating large numbers of MSC, showing a prolonged capacity to differentiate into mesodermal cell lineages, with the exception of the lack of chondrogenesis by using laminin-5 coating.     

The influence of cardiovascular risk factors on bone marrow mesenchymal stromal cell fitness

Background aimsIn the past, cell transplantation strategies for the treatment of heart failure have shown promising results in experimental and clinical studies. Bone marrow (BM)-derived stem cells represent the most frequently used cell population. Within this heterogeneous cell population, mesenchymal stromal cells (MSC) have been identified to induce therapeutic effects, mainly through paracrine mechanisms. Because of their low frequency in native tissues, in vitro cell culture expansion is mandatory prior to transplantation. We sought to identify patient-specific cardiovascular risk factors influencing the proliferative potential of MSC.MethodsBM aspirates from 51 patients undergoing elective cardiac surgery were analyzed for MSC frequency and cell culture expansion potential. Fibroblastic colony-forming units (CFU-F) were quantified for culture conditions applying autologous (AS) or fetal bovine serum (FBS) and different basic media. Univariate and multivariate analyzes were performed in order to determine the impact of patient-specific factors on CFU-F numbers.ResultsExpanded MSC showed a specific immune phenotype and displayed adipogenic, chondrogeneic and osteogeneic differentiation potential. CFU-F numbers did not differ under AS or FBS supplementation. Elevated numbers of mononuclear cells, diabetes mellitus, steroid treatment, chronic obstructive pulmonary disease, renal failure, high euroSCORE and impaired left ventricular function were significant determinants for higher CFU-F numbers.ConclusionsThe impact of specific cardiovascular risk factors on MSC fitness could be determined. These results may help to establish patient profiling in order to identify patients suitable for autologous MSC transplantation, and might lead to the identification of disease-related mechanisms of stem cell activation.     

Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.     

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions

Background aimsMesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein–free Good Manufacturing Practice–compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL).MethodsBone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed.ResultsThe MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed.ConclusionsWe demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice–compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness.     

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients

Background aimsAdvances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources.MethodsWe obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit–fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels.ResultsMSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential.ConclusionsTrabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.     

Manufacturing of human Wharton's jelly stem cells for clinical use: selection of serum is important

BackgroundHuman Wharton's jelly–derived mesenchymal stromal cells (hWJSCs) have gained considerable attention for their use in cell therapy. Many of these applications would require manufacturing of millions of hWJSCs. It is, therefore, necessary to develop a Good Manufacturing Practice (GMP)-compliant hWJSC expansion protocol, allowing the generation of a large quantity of cells to meet both clinical and regulatory requirements. Here, we compared human platelet lysate (HPL) and human serum (HS) in supporting clinical-grade hWJSC expansion.MethodshWJSCs were successfully isolated from six different umbilical cords using GMP-compliant dissociation enzymes. Freshly isolated hWJSCs were cultured in media supplemented with 10% of one of the following sera: fetal bovine serum (FBS), HPL and HS. Properties of the expanded hWJSCs were analyzed.ResultsWe showed that GMP-compliant dissociation enzymes were as efficient as research-grade dissociation enzymes in isolating hWJSCs. hWJSC fresh cell yield and cell viability using HPL and HS supplementations were at greater advantages than FBS. Moreover, hWJSCs expanded in HPL and HS supplementations not only preserved classical MSCs phenotypes and differentiation potential to adipocytes, osteocytes and chondrocytes, they also enhanced the migration of skin fibroblasts. However, HS, unlike HPL, did not alter immunogenicity properties of hWJSCs. hWJSCs expanded in HS supplementation also exerted greater immunosuppressive action in inhibiting T-cell proliferation and increased extracellular matrix (ECM) gene expression, making them useful in tissue repair clinical application.ConclusionOur findings indicate that HS can be considered as a promising and safer alternative to FBS, and should be recommended for clinical-grade expansion of hWJSCs.     

Population dynamics of mesenchymal stromal cells during culture expansion

Background aimsMesenchymal 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.MethodsWe 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.ResultsNot 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.ConclusionsThese 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.     

Labeling of mesenchymal stromal cells with iron oxide-poly(L-lactide) nanoparticles for magnetic resonance imaging: uptake, persistence, effects on cellular function and magnetic resonance imaging properties

Background aimsMesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the non-interference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI.MethodsWe studied the effects of iron oxide–poly(l-lactide) nanoparticles in MSC with flow cytometry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant^® proliferation testing, colony-forming unit–fibroblast (CFU-F) assays, flow chamber adhesion testing, immunologic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats.ResultsIt could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks.ConclusionsThese particles qualify for studying MSC homing and trafficking via MRI.     

Efficient isolation and enrichment of mesenchymal stem cells from bone marrow

Background aimsBone marrow (BM) mesenchymal stromal cells (MSC) have been identified as a source of pluripotent stem cells used in clinical practice to regenerate damaged tissues. BM MSC are commonly isolated from BM by density-gradient centrifugation. This process is an open system that increases the risk of sample contamination. It is also time consuming and requires technical expertise that may result in variability regarding cellular recovery. The BD Vacutainer^® Cell Preparation Tube? (CPT) was conceived to separate mononuclear cells from peripheral blood. The main goal of this study was to verify whether MSC could be isolated from BM using the CPT.MethodsBM was harvested, divided into two equal aliquots and processed using either CPT or a Ficoll-Paque? PREMIUM density gradient. Both methods were compared regarding cell recovery, viability, proliferation, differentiation capacities and the presence of MSC progenitors.ResultsSimilar numbers of mononuclear cells were isolated from BM when comparing the two methods under study. No differences were found in terms of phenotypic characterization, viability, kinetics and lineage differentiation potential of MSC derived by CPT or Ficoll. Surprisingly, a fibroblast–colony-forming unit (CFU-F) assay indicated that, with CPT, the number of MSC progenitors was 1.8 times higher compared with the Ficoll gradient separation.ConclusionsThe CPT method is able to isolate MSC efficiently from BM, allowing the enrichment of MSC precursors.     

Removal of minute virus of mice-mock virus particles by nanofiltration of culture growth medium supplemented with 10% human platelet lysate

Background aimsPlatelet concentrates (PCs) are pooled to prepare human platelet lysate (HPL) supplements of growth media to expand primary human cells for transplantation; this increases the risk of contamination by known, emerging, and unknown viruses. This possibility should be of concern because viral contamination of cell cultures is difficult to detect and may have detrimental consequences for recipients of cell therapies. Viral reduction treatments of chemically defined growth media have been proposed, but they are not applicable when media contain protein supplements currently needed to expand primary cell cultures. Recently, we successfully developed a Planova 35NPlanova 20N nanofiltration sequence of growth media supplemented with two types of HPL. The nanofiltered medium was found to be suitable for mesenchymal Stromal cell (MSC) expansion.MethodsHerein, we report viral clearance achieved by this nanofiltration process used for assessing a new experimental model using non-infectious minute virus of mice-mock virus particle (MVM-MVP) and its quantification by an immunoqPCR. Then, high doses of MVM-MVP (1012 MVPs/mL) were spiked to obtain a final concentration of 1010 MVPs/mL in Planova 35N-nanofiltered growth medium supplemented with both types of HPLs [serum converted platelet lysate SCPL) and intercept human platelet lysate (I-HPL)] at 10% (v/v) and then filtering through Planova 20N.ResultsNo substantial interference of growth medium matrices by the immune-qPCR assay was first verified. Log reduction values (LRVs) were ≥ 5.43 and ≥ 5.36 respectively, SCPL and I-HPL media. MVM-MVPs were also undetectable by dynamic light scattering and transmission electron microscopy.ConclusionsThe nanofiltration of growth media supplemented with 10% HPL provides robust removal of small nonenveloped viruses, and is an option to improve the safety of therapeutic cells expanded using HPL supplements.     

GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion

Background aimsBone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque? PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.MethodsBM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.ResultsNo differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45⁺ fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.ConclusionsBoth GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.     

Efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture

Background

Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC.

Methods and Findings

UCB-derived mononuclear cells (MNC) or selected CD34⁺ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34⁺ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin⁺, CD133⁺ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages.Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies.

Conclusions

This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.     

Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use

Background aimsThe manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) ‘quality’ with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates.MethodsAspirated MSC were enumerated using the CD45^?/low CD271^bright phenotype and AccuCheck counting beads and compared with a classic colony-forming unit–fibroblast (CFU-F) assay. The phenotype of CD45^?/low CD271^bright cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined.ResultsCD45^?/low CD271^bright cells had a classic MSC phenotype (CD73⁺ CD105⁺ CD90⁺ ). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34 ⁺ cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45^?/low CD271^bright cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique.ConclusionsAbsolute quantification of CD45^?/low CD271^bright cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.     

Dexamethasone has variable effects on mesenchymal stromal cells

Background aimsDexamethasone (Dex) is a potent synthetic member of the glucocorticoid class of steroid drugs. Frequently, Dex has been used to enhance osteogenic, chondrogenic and adipogenic differentiation of mesenchymal stromal cells (MSC). Recently, Dex was applied to promote MSC proliferation, because of the rare frequency of MSC in bone marrow, and could protect the cells from apoptosis. The effects of Dex on MSC cytobiology behavior needs to be investigated.MethodsMSC were obtained from human umbilical cord. The surface phenotype and functional characterization of MSC cultured with different concentrations of Dex were investigated, in comparison with a control group, including MSC proliferation, apoptosis, cytokine expression and immunosuppression.ResultsDifferent concentrations of Dex exerted diverse effects on MSC proliferation and apoptosis. Dex was also able to affect the pattern of cytokine expression of MSC. Furthermore, Dex impaired immunosuppression of MSC on peripheral blood mononuclear cells.ConclusionsA low dose of Dex favors MSC expansion in vitro, and protects against apoptosis. It is not suitable for MSC to be pre-treated with Dex when they are to be used to treat immunologic disease. However, when MSC are applied to promote angiogenesis, it is beneficial for them to be pre-treated with 10^?9 mol/L Dex.