Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.     

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.     

Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells

Background

Mesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC.

Hypothesis and Objectives

We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation.

Results

1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis.

Conclusions/Significance

Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.     

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.     

Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells

Background aimsCulture medium for mesenchymal stromal cells (MSC) is frequently supplemented with fetal calf serum (FCS). FCS can induce xenogeneic immune reactions, transmit bovine pathogens and has a high lot-to-lot variability that hampers reproducibility of results. Several studies have demonstrated that pooled human platelet lysate (HPL) provides an attractive alternative for FCS. However, little is known about the variation between different platelet lysates.MethodsWe compared activities of individual HPL on initial fibroblastoid colony-forming units (CFU-F), proliferation, in vitro differentiation and long-term culture. These data were correlated with chemokine profiles of HPL.ResultsIsolation of MSC with either HPL or FCS resulted in similar CFU-F frequency, colony morphology, immunophenotype and adipogenic differentiation potential. Osteogenic differentiation was even more pronounced in HPL than FCS. There were significant differences in MSC proliferation with different HPL, but it was always higher in comparison with FCS. Cell growth correlated with the concentration of platelet-derived growth factor (PDGF) and there was a moderate association with platelet counts. All HPL facilitated expansion for more than 20 population doublings.ConclusionsTaken together, reliable long-term expansion was possible with all HPL, although there was some variation in platelet lysates of individual units. Therefore the use of donor recipient-matched or autologous HPL is feasible for therapeutic MSC products.     

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data

BackgroundMany data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone.MethodsKey parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone.ResultsDespite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings.ConclusionsClinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.     

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.     

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.     

Ex vivo expansion of human mesenchymal stem cells: A more effective cell proliferation kinetics and metabolism under hypoxia

The low bone marrow (BM) MSC titers demand a fast ex vivo expansion process to meet the clinically relevant cell dosage. Attending to the low oxygen tension of BM in vivo, we studied the influence of hypoxia on human BM MSC proliferation kinetics and metabolism. Human BM MSC cultured under 2% (hypoxia) and 20% O₂ (normoxia) were characterized in terms of proliferation, cell division kinetics and metabolic patterns. BM MSC cultures under hypoxia displayed an early start of the exponential growth phase, and cell numbers obtained at each time point throughout culture were consistently higher under low O₂, resulting in a higher fold increase after 12 days under hypoxia (40 ± 10 vs. 30 ± 6). Cell labeling with PKH26 allowed us to determine that after 2 days of culture, a significant higher cell number was already actively dividing under 2% compared to 20% O₂ and BM MSC expanded under low oxygen tension displayed consistently higher percentages of cells in the latest generations (generations 4–6) until the 5th day of culture. Cells under low O₂ presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O₂ favored CFU‐F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O₂, compared to normoxic conditions, associated to an earlier start of cellular division and supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings. J. Cell. Physiol. 223: 27–35, 2010. © 2009 Wiley‐Liss, Inc.     

The clonogenic potential of hematopoietic stem cells and mesenchymal stromal cells in various hematologic diseases: a pilot study

Background aimsMesenchymal stromal cells (MSC) are the most popular cells used in regenerative medicine and biotechnology. The clonogenic potential of these cells is defined by colony-forming unit-fibroblasts (CFU-F). It is well known that there is an interaction between hematopoietic cells and stromal cells in disease formation pathogenesis. Therefore we hypothesized that there should be a quantitative and qualitative relationship between MSC colonies (CFU-F) and hematopoietic stem cell colonies (colony-forming unit-granulocyte-macrophages; CFU-GM) among patients with and without hematologic diseases.MethodsForty-two patients were included in this study. Patients were divided into three groups: group A, patients with hematologic malignancies (n = 20); group B, patients with bone marrow (BM) failure (n = 11); group C, patients without hematologic diseases (n = 11). BM aspirates were plated in different densities for CFU-F culture. The plating density was the same for CFU-GM culture.ResultsCFU-GM colonies grew in 90% of group A cells and all of group B and C cells (P = 0.0001). CFU-F colonies became visible on the ninth day of plating in group A and on the eight day in groups B and C. There was no statistically significant difference between the groups for the duration of CFU-F colony formation (P = 0.12). There were differences in the morphology of the colonies among the groups.ConclusionsThis is the first study that has compared the clonogenic potential of stromal cells and hematopoietic stem cells in the same subjects with and without hematologic diseases. No correlation was shown between the clonogenic potential of stromal cells and hematopoietic cells.     

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3⁺/CD19⁺/CD14⁺/CD38⁺‐depleted MNC and CD133⁺‐ or LNGFR⁺‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90⁺/CD73⁺/CD105⁺ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.     

Expansion of human bone marrow-derived mesenchymal stromal cells: serum-reduced medium is better than conventional medium

Background aimsHuman mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation.MethodshMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks.Results and ConclusionsNo proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation.     

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.     

Canonical and non-canonical Wnts differentially affect the development potential of primary isolate of human bone marrow mesenchymal stem cells

This study examines the role of Wnt signaling events in regulating the differential potential of mesenchymal stem cells (MSCs) from adult bone marrow (BM). Immunohistochemical analysis of BM revealed co-localization of Wnt5a protein, a non-canonical Wnt, with CD45(+) cells and CD45(-) STRO-1(+) cells, while Wnt3a expression, a canonical Wnt, was associated with the underlying stroma matrix, suggesting that Wnts may regulate MSCs in their niche in BM. To elucidate the role of Wnts in MSC development, adult human BM-derived mononuclear cells were maintained as suspension cultures to recapitulate the marrow cellular environment, in serum-free, with the addition of Wnt3a and Wnt5a protein. Results showed that Wnt3a increased cell numbers and expanded the pool of MSCs capable of colony forming unit -- fibroblast (CFU-F) and CFU -- osteoblast (O), while Wnt5a maintained cell numbers and CFU-F and CFU-O numbers. However, when cells were cultured directly onto tissue culture plastic, Wnt5a increased the number of CFU-O relative to control conditions. These findings suggest the potential dual role of Wnt5a in the maintenance of MSCs in BM and enhancing osteogenesis ex vivo. Our work provides evidence that Wnts can function as mesenchymal regulatory factors by providing instructive cues for the recruitment, maintenance, and differentiation of MSCs.     

Rat marrow-derived mesenchymal stem cells developed in a medium supplemented with the autologous versus bovine serum

The controversial effect of autologous serum (AS) on human mesenchymal stem cells (MSC) was studied in rat MSC culture. Rat bone marrow cells were plated in a medium containing either FBS (fetal bovine serum) or AS were cultured to passage 3, during which the population doubling number (PDN) of both cultures were measured and compared statistically. The number of viable cells, the cell colonogic activity, and cell growth rate were also compared. In addition, mineralization in the osteogenic cultures from each system was measured. Our data indicated that AS enriched medium provided a microenvironment in which growth rate as well as bone differentiation of the isolated MSCs were significantly higher than in FBS enriched medium.     

Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum

Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors. However, late passage cultures in AS consistently display normal DNA copy numbers. Combined bisulfite restriction analysis and bisulfite sequencing show that although DNA methylation states are overall stable in culture, AS exhibits stronger propensity than FBS to maintain unmethylated states. Comparison of DNA methylation in BMMSCs with freshly isolated and cultured adipose stem cells (ASCs) also reveals that most genes unmethylated in both BMMSCs and ASCs in early passage are also unmethylated in uncultured ASCs. We conclude that (i) BMMSCs expanded in AS or FBS may display localized genetic alterations, (ii) AS tends to generate more consistent genomic backgrounds and DNA methylation patterns, and (iii) the unmethylated state of uncultured MSCs is more likely to be maintained in culture than the methylated state.     

肌间隙移植明胶海绵获取成体干细胞

通过移植生物材料到小鼠体内而建立一种新的干细胞获取方法.移植明胶海绵到小鼠后肢的大腿内侧肌间隙,12天后分离明胶海绵中的迁移细胞.海绵来源干细胞形态、增殖能力、多向分化能力与骨髓源干细胞相似,但海绵中成纤维细胞集落形成单位的比例(82.2±10.6/105)远远高于骨髓中的比例(43.7±7.4/106)(P＜0.05...     

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.     

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.