Bone marrow- and subcutaneous adipose tissue-derived mesenchymal stem cells: Differences and similarities

Bone marrow (BM) and subcutaneous adipose tissue (Ad) are both considered being prospective sources of MSC for therapeutic applications. However, functional properties and therapeutic efficacy of MSC derived from different tissues of the same patient are still poorly investigated. In our study, BM-MSC and F-MSC cultures from 43 adult donors were evaluated in successive passages for immunophenotype, secretion of VEGF, SDF1, MCP1, IL6 and TGFβ1, frequency of colony-forming units (CFU-F), frequency of adipo- and osteo-progenitors (CFU-Ad, CFU-Ost), and for onset of in vitro replicative senescence. We have demonstrated that at early passages (P2-P4 or up to 14-15 in vitro population doublings) BM- and Ad- derived MSC cultures are comparable in such important characteristics as proliferation rate (population doubling time: 3,4±0,2% in BM-MSC, 3±0,3 % in F-MSC), clonogenity (CFU-F frequency: 32±5% in BM-MSC, 31±5% in F-MSC), differentiation potential (CFU-Ad frequency: 10,4±2% in BM-MSC, 13±3% in F-MSC; CFU-Ost frequency: 18,5±5,5% in BM-MSC, 18±5% in F-MSC), but differ significantly in abundance of CD146+ fraction within the sample (25±5% in BM-MSC, 7±3 % in F-MSC) and in a level of VEGF, SDF-1, MCP1 and TGFβ1 secretion. We have also demonstrated that BM-MSC enter senescence after P3-4 while most of F-MSC did not show senescence features up to P6-8. Together, these data demonstrate that specific properties of MSC from different sources should be always taken into account, when developing and optimizing the specific protocols for MSC expansion and evaluation for each particular clinical application.     

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow,umbilical cord,and placenta: Implication in the migration

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC‐ and P‐MSC possess immunophenotypic and functional characteristics similar to BM‐MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM‐ and P‐MSC was found 5.9‐ and 3.2‐folds higher than that of UC‐MSC, respectively. By the use of 2‐DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC‐MSC when compared with those in BM‐ and P‐MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor‐1 (PAI‐1) and manganese superoxide dismutase, higher expression was found in the UC‐MSC. We also showed that the overexpression of the PAI‐1 impaired the migration capacity of BM‐ and P‐MSC while silencing of PAI‐1 enhanced the migration capacity of UC‐MSC. Our study indicates that PAI‐1 and other migration‐related proteins are pivotal in governing the migration capacity of MSC.     

Cryopreserved mesenchymal stromal cell treatment is safe and feasible for severe dilated ischemic cardiomyopathy

Background aimsBone marrow (BM) mesenchymal stromal cells (MSC) represent a novel therapy for severe heart failure with extensive myocardial scarring, especially when performed concurrently with conventional revascularization. However, stem cells are difficult to transport in culture media without risk of contamination, infection and reduced viability. We tested the feasibility and safety of off-site MSC culture and expansion with freeze-controlled cryopreservation and subsequent rapid thawing of cells immediately prior to implantation to treat severe dilated ischemic cardiomyopathy.MethodsWe recruited three consecutive patients with end-stage ischemic heart failure with evidence of full-thickness myocardial scarring. MSC was isolated from 20 mL BM aspiration, expanded and cryopreserved using 10% dimethyl sulfoxide (DMSO). Cells were transported in a cryoshipper. Patients underwent concurrent coronary artery bypass graft (CABG) with intramyocardial MSC injection.ResultsThe cell viability after thawing exceeded 90% for all samples. The supernatant was free from bacterial and fungal growth. All patients underwent the procedure safely. There were no arrhythmias noted. There was significant improvement in cardiac function and volume, resolution of scarring and increased wall thickness for all patients on cardiac magnetic resonance imaging at 6 months compared with baseline. The magnitude of improvement was more than was expected with CABG alone. Patients remained well at 1 year.ConclusionsRate-controlled freezing with 10% DMSO is a safe, feasible and practical method of cryopreserving MSC for cell storage and transportation without risk of contamination or cell death. Direct MSC injection may be beneficial as an adjunct to cardiac revascularization.     

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.     

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 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.     

Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential

BACKGROUND: BM mesenchymal stem cells (MSC) have the capacity for renewal and the potential to differentiate into multiple tissues. In this study, we compared different enrichment methods to obtain MSC from BM. METHODS: Three different methods were compared with a view to obtaining MSC more rapidly from BM: negative selection (RosetteSep and MACS) and plastic adhesion. The three cell fractions were grown in complete alpha-minimum essential medium in order to evaluate their proliferative capacity, their phenotype during culture and their potential to differentiate into adipocytes, osteocytes and chondrocytes. Identification of MSC was performed by immunofluorescence with putative mesenchymal markers SH2 and SH3 but also with hematopoietic markers. RESULTS: After negative selection, only 1+/-0.2% and 2.9+/-0.8% of cells were recovered from BM with the RosetteSep and MACS methods, respectively. However, negative depletion permitted a homogeneous population of MSC, with more than 90% SH2+ and SH3+ cells, to be obtained rapidly and in large quantities after 10 days of culture. Similar homogeneity was observed after three passages if the plastic adhesion was used as selection method and after an average of 25-30 days of culture. Different levels of MSC maturity were also suggested by the variable level expression of Stro-1. DISCUSSION: Depleting selection by RosetteSep may represent an easy method of obtaining MSC rapidly from BM with the aim of potential therapeutic use.     

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB‐MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper‐exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB‐MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB‐MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM‐MSC) and adipose tissue (AT‐MSC) as reference controls, we observed that CB‐MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10⁹ cells required for cell therapies. CB‐MSC showed karyotype stability after prolonged expansion. Functionally, CB‐MSC could be more readily induced to differentiate into chondrocytes than could BM‐MSC and AT‐MSC. CB‐MSC showed immunosuppressive activity equal to that of BM‐MSC and AT‐MSC. Collectively, our data indicate that viable CB‐MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system. J. Cell. Biochem. 112: 1206–1218, 2011. © 2011 Wiley‐Liss, Inc.     

Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes

The post-thaw survival and fertility of ram spermatozoa frozen in pellets, 0.25- and 0.5-ml PVC straws, and 0.25-ml minitubes were examined. In 5 experiments, a freezing height of 6 cm above the level of liquid nitrogen was optimal for 0.25- and 0.5-ml straws, whereas 4 cm was best for the 0.25-ml minitubes. Post-thaw motility of spermatozoa was lower for semen frozen in straws and minitubes than in pellets (Experiment 1: 43.7 vs 53.4%, P < 0.001), but after freezing was better in 0.5-ml straws and 0.25-ml minitubes than in 0.25-ml straws (Experiment 1: 44.9 vs 41.3%, P < 0.05; Experiment 2: 49.6 vs 46.8%, P < 0.01). Sperm motility was also better for 1:8 (semen:diluent) pre-freezing dilution rate (50.5%) than for 1:4 (45.6%, P < 0.01) and 1:2 (39.8%, P < 0.001) but not the 1:16 (49.5%) dilution rate. Dry ice was a better freezing medium than liquid nitrogen vapor (49.2 vs 46.9% motile spermatozoa, P < 0.001). The post-thaw motility of spermatozoa was similar for the three freezing packages if the semen was loaded at 5 degrees C, but motility was poorer for semen loaded into 0.25-ml straws than 0.25-ml minitubes at 30 degrees C (P < 0.05). In a fertility test, pregnancy rates were influenced by rams (3 rams, P < 0.05) and freezing package (pellets vs 0.25-ml minitube vs 0.25-ml straw vs 0.5-ml straw, P < 0.05) but not freezing medium (liquid nitrogen vapor vs dry ice). More ewes were pregnant after insemination with pellet-frozen semen (106/150, 71%) than with semen frozen in 0.25-ml straws (85/150, 57%; P < 0.05) and in 0.5-ml straws (83/150, 55%; P < 0.01) but not minitubes (98/150, 65%). It was concluded that minitubes provide a useful alternative to pellets as a storage package for ram spermatozoa, allowing for individual dose identification and easier storage while maintaining a fertility rate indistinguishable from that obtained with pellet-frozen semen.     

Stability of salivary steroids: the influences of storage, food and dental care

We studied influences of dental care, food and storage on the reproducibility of salivary steroid levels. Cortisol (F), 17OH-progesterone (17OHP) and Progesterone (P) were measured using adapted commercial radioimmunoassays. Saliva samples of healthy adults (n = 15; m:8; f:7) were collected directly before and after dental care, and directly before and after breakfast with various foodstuffs. A second experiment investigated stability of steroids under different storage conditions. Four series of identical saliva portions (I: Native saliva; II: Centrifuged saliva; III: Saliva with trifluor acetate (TFA); IV: Saliva with 0.5% NaN(3)) were stored at room temperature and at 4 degrees C for up to three weeks. To demonstrate influences of repeated thawing and re-freezing of saliva on steroid values, saliva samples (n = 15) were divided into identical portions. These portions were frozen and re-thawed up to 5 times before measurement. Neither dental care nor intake of bread or milk effected the reproducibility of F, 170HP, and P. Steroid levels decreased significantly in the course of three weeks under different storage conditions (P < 0.001). This decrease was clinically relevant from the second week onward, with exception of NaN(3) treated samples. After repeated freezing and re-thawing 17OHP and P decreased slightly (about 5%). Only F decreased significantly after the third thawing (P < 0.001). The results show the usefulness of standardized handling of saliva samples for improving reproducibility and reliability of salivary steroid measurements.     

Chronological change and the potential of recovery on the photosynthetic efficiency of Pyropia yezoensis f. narawaensis (Bangiales) during the sporelings frozen storage treatment in the Japanese Nori cultivation

The chronological change of photosynthetic efficiency in a frozen storage treatment of the Japanese Nori cultivation industry was examined in the cultivated red alga, Pyropia yezoensis f. narawaensis (Saga‐#5 Strain, Bangiales) by using pulse‐amplitude fluorometry. During the desiccation process that was conducted after the nursery cultivation season in November, the maximum quantum yield (F _v/F _m) of the gametophytic sporelings growing on the Nori‐net decreased monotonically with decreasing absolute water content (AWC), and was around 0.1 at 20% AWC. During frozen storage of the Nori‐net, the F _v/F _m of the frozen gametophyte was low but stable, and ranged between 0.10 ± 0.02 SD and 0.14 ± 0.05 SD. The magnitude of F _v/F _m for the gametophyte of the freezing treatment, after 10 min and 3 h of immersion in seawater, recovered quickly. After 10 min and 3 h of immersion, these values were 0.29 ± 0.12 SD and 0.47 ± 0.05 SD during the 14 days of freezing treatment, and 0.15 ± 0.02 SD and 0.29 ± 0.04 SD after 71 days of freezing treatment, and suggest that the ability to recover gradually decreased as the storage duration increased. The response of F _v/F _m from general cultivation (i.e., directly cultivated from the nursery cultivation season) and those after 47 days of freezing were almost identical, suggesting that the current Nori ‐net frozen storage period (6 or 7 weeks) was not detrimental to the gametophyte.     

Cytokine-induced stable neuronal differentiation of human bone marrow mesenchymal stem cells in a serum/feeder cell-free condition

The characteristics and multilineage differentiation potential of bone marrow mesenchymal stem cells (BM MSC) remain controversial. This study aimed to characterize human BM MSC isolated by plastic adherent or antibody selection and their neuronal differentiation potential using growth factors or chemical inducing agents. MSC were found to express low levels of neuronal markers: neurofilament-M, beta tubulin III, and neuron specific enolase. Under a serum- and feeder cell-free condition, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor induced neuronal morphology in MSC. In addition to the above markers, these cells expressed neurotransmitters or associated proteins: gamma-aminobutyric acid, tyrosine hydroxylase and serotonin. These changes were maintained for up to 3 months in all bone marrow specimens (N = 6). In contrast, butylated hydroxyanisole and dimethylsulfoxide were unable to induce sustained neuronal differentiation. Our results show that MSC isolated by two different procedures produced identical lineage differentiation with defined growth factors in a serum- and feeder cell-free condition.     

Effect of cryopreservation on human articular chondrocyte viability, proliferation, and collagen expression

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3-4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to -80 degrees C (Protocol I). Another fraction was directly frozen to -80 degrees C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of -1 degrees C/min to a temperature of -40 degrees C, 2 degrees C/min to -60 degrees C, and 5 degrees C/min to -150 degrees C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70-75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.     

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. METHODS: As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. RESULTS: We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). DISCUSSION: Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.     

Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (P > 0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish.     

Oncostatin M Maintains the Hematopoietic Microenvironment in the Bone Marrow by Modulating Adipogenesis and Osteogenesis

The bone marrow (BM) is an essential organ for hematopoiesis in adult, in which proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) is orchestrated by various stromal cells. Alterations of BM hematopoietic environment lead to various hematopoietic disorders as exemplified by the linking of fatty marrow with increased adipogenesis to anemia or pancytopenia. Therefore, the composition of mesenchymal stromal cell (MSC)-derived cells in the BM could be crucial for proper hematopoiesis, but the mechanisms underlying the MSC differentiation for hematopoiesis remain poorly understood. In this study, we show that Oncostatin M (OSM) knock out mice exhibited pancytopenia advancing fatty marrow with age. OSM strongly inhibited adipogenesis from BM MSC in vitro, whereas it enhanced their osteogenesis but suppressed the terminal differentiation. Intriguingly, OSM allowed the MSC-derived cells to support the ex vivo expansion of HSPC effectively as feeder cells. Furthermore, the administration of OSM in lethally irradiated wild-type mice blocked fatty marrow and enhanced the recovery of HSPC number in the BM and peripheral blood cells after engraftment of HSPC. Collectively, OSM plays multiple critical roles in the maintenance and development of the hematopoietic microenvironment in the BM at a steady state as well as after injury.     

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.     

Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6

Background aimsMesenchymal stromal cells (MSC) have been shown to possess immunomodulatory functions and proposed as a tool for managing or preventing graft-versus-host disease (GvHD) as well as promoting clinical transplantation tolerance. We investigated the capacity of human bone marrow (BM) MSC to modulate the proliferation of T cells obtained from peripheral blood (PB) and umbilical cord blood (CB). We addressed the importance of the MSC:T-cell ratio, requirement for cell contact and impact of soluble factors on the MSC-mediated effects. We also analyzed whether regulatory T cells could be modulated by MSC in co-cultures.MethodsThe effect of different MSC concentrations on T-cell proliferation induced by allogeneic, mitogenic or CD3/CD28 stimulation was analyzed using bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate–succinimidyl ester (CFDA-SE) labeling. The level of regulatory T cells was assessed using quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis.ResultsMSC induced a dose- and contact-dependent inhibition of T-cell proliferation but lymphocytes from CB and PB were differentially affected. At low concentrations, MSC supported both CB and PB T-cell proliferation, rather than inhibiting their proliferation. This supportive effect was contact independent and soluble factors such interleukin-6 (IL-6) appeared to be involved. Interestingly, among the expanded T-cell population in both CB and PB, regulatory T cells were increased and were a part of the new cells promoted by MSC at low doses.ConclusionsMSC represent an attractive tool for reducing the lymphocyte response by inhibiting T-cell activation and proliferation as well as promoting tolerance by maintaining and promoting the expansion of regulatory cells. Nevertheless, the dual ability of MSC to either sustain or suppress T-cell proliferation according to conditions should be considered in the context of clinical applications.     

Dynamic expression of the Robo ligand Slit2 in bone marrow cell populations

The bone marrow (BM) niche is essential for lifelong hematopoietic stem cell (HSC) maintenance, proliferation and differentiation. Several BM cell types, including osteoblast lineage cells (OBC), mesenchymal stem cells (MSC) and endothelial cells (EC) have been implicated in supporting HSC location and function, but the relative importance of these cell types and their secreted ligands remain controversial. We recently found that the cell surface receptors Robo4 and CXCR4 cooperate to localize HSC to BM niches. We hypothesized that Slit2, a putative ligand for Robo4, cooperates with the CXCR4 ligand SDF1 to direct HSC to specific BM niche sites. Here, we have isolated OBC, MSC and EC by flow cytometry and determined their frequency within the bone marrow and the relative mRNA levels of Slit2, SDF1 and Robo4. We found that expression of Slit2 and SDF1 were dynamically regulated in MSC and OBC-like populations following radiation, while Robo4 expression was restricted to EC. Radiation also significantly affected the cellularity and frequency of both the non-adherent and adherent cells within the BM stroma. These data support a physiological role for Slit2 in regulating the dynamic function of Robo-expressing cells within BM niches at steady state and following radiation.     

Bone marrow mesenchymal stem cells for improving hematopoietic function: an in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.