Cumulative Population Doubling Level Research Articles

Preclinical Evaluation of the Tumorigenic and Immunomodulatory Properties of Human Bone Marrow Mesenchymal Stromal Cell Populations with Clonal Trisomy 5.

Cytogenetic aberrations may emerge in human mesenchymal stromal cells (MSC) during ex vivo expansion for cell therapy. We have detected clonal trisomy 5 in two distinct autologous MSC products expanded from bone marrow which, based on the current quality control criteria, could not be released for clinical use. Although a safety concern, it is still unclear to what extent recurrent aneuploidies detected in MSC products may affect the threshold for neoplastic transformation or the medicinal properties of these cells. We have carried out an exploratory preclinical study to evaluate these MSC products with clonal trisomy 5, regarding their oncogenic and immunomodulatory potential. Cell population growth in vitro was reduced in MSC cultures with clonal trisomy 5 compared with the population growth of their euploid MSC counterparts, based on a lower cumulative population doubling level, reduced cell proliferation index, and increased senescence-associated beta-galactosidase activity. Subcutaneous injection of clinically relevant amount of MSC population, either with or without clonal trisomy 5, did not generate tumors in immunodeficient mice within a follow-up period of six months. Most importantly, MSC population with clonal trisomy 5 kept immunomodulatory properties upon interferon gamma (IFNγ) licensing, displaying overexpression of IDO, CXCL9, CXCL10, and CXCL11, in a similar fashion than that of IFNγ-licensed euploid MSC. Our findings suggest that bone marrow MSC products with clonal trisomy 5 may retain their therapeutic potential, based on poor tumor initiating capability and preserved immunomodulatory potency. This preclinical evidence may further support the definition of release criteria of autologous MSC products for cell therapy under critical clinical scenarios. This trial is registered with Clinical Study registration number: RBR-29x2pr.

Isolation and Characterization of Feline Wharton's Jelly-Derived Mesenchymal Stem Cells.

Wharton’s jelly is a well-known mesenchymal stem cell source in many species, including humans. However, there have been no reports confirming the presence of mesenchymal stem cells in Wharton’s jelly in cats. The purpose of this study was to isolate mesenchymal stem cells (MSCs) from the Wharton’s jelly of cats and to characterize stem cells. In this study, feline Wharton’s jelly-derived mesenchymal stem cells (fWJ-MSCs) were isolated and successfully cultured. fWJ-MSCs were maintained and the proliferative potential was measured by cumulative population doubling level (CPDL) test, scratch test, and colony forming unit (CFU) test. Stem cell marker, karyotyping and immunophenotyping analysis by flow cytometry showed that fWJ-MSCs possessed characteristic mesenchymal stem cell markers. To confirm the differentiation potential, we performed osteogenic, adipogenic and chondrogenic induction under each differentiation condition. fWJ-MSCs has the ability to differentiate into multiple lineages, including osteogenic, adipogenic and chondrogenic differentiation. This study shows that Wharton’s jelly of cat can be a good source of mesenchymal stem cells. In addition, fWJ-MSCs may be useful for stem cell-based therapeutic applications in feline medicine.

Differentiation potentials of CD271 positive cells from caprine skin tissue-derived mesenchymal stem cells

Background & Aim Mesenchymal stem cells (MSCs) are undifferentiated cells, found throughout the body after development, which multiply by cell division to replenish dying cells and regenerate damaged tissues. They can be found in adult animals and humans, unlike embryonic stem cells. MSCs characterized by a fibroblast-like morphology possess self-renewal and can be directed to differentiate into cells of mesodermal lineages under specific conditions. However, MSCs are a heterogeneous population of cells with varying magnitudes of differentiation potential among single clone of MSCs. It was documented that in vitro single cell cloning of human MSCs demonstrated that approximately 30% of the clonal cells were multipotent and thus true MSCs. CD271 (low-affinity nerve growth factor receptor) has been identified as a marker of the most homogeneous MSCs subset. Methods, Results & Conclusion Using immunomagnetic selection method, we separated CD271 positive (CD271-P) or negative cells (CD271-P) from caprine ear skin tissue-derived MSCs (capESK-MSCs). Two types of cells were examined for cellular morphology, proliferation properties, expression of cell surface markers and mesodermal differentiation capabilities at early passage. CD271-P and CD271-N cells from capESK-MSCs exhibited abilities to attach to culture plate and expand in vitro. CD271-P cells had spindle-shaped morphology, but CD271-N cells had spindle and round shape morphology. CD271-P and CD271-N cells exhibited not significant difference in cumulative population doubling level during 10 consecutive passages. In addition, CD271-P and CD271-N cells displayed similar immunophenotypic markers (CD34, CD45, CD73, CD90 and CD105). However, CD271-P and CD271-N cells exhibited difference in differentiation potentials. Two types of cells exhibited adipogenic, osteogenic and chondrogenic differentiation potentials. Specifically, CD271-P cells have a greater capacity for chondrogenic differentiation potential compared to CD271-N cells at passage four. CD271 has been identified as a marker of the most homogeneous MSCs. It is capable of promoting differentiation along adipogenic, osteogenic and chondrogenic lineages and producing significantly higher levels of cytokines as compared to the total population of MSCs. In this study, we observed homogenous cell population, proliferating ability and mesodermal differentiation potentials from CD271-P cells from capESK-MSCs.

The effect of prolonged rhBMP-2 treatment on telomerase activity, replicative capacity and senescence of human nucleus pulposus cells

ABSTRACT We investigated the effect of prolonged rhBMP-2 treatment on telomerase activity, replicative capacity and senescence of nucleus pulposus cells (NPCs) during long term culture. We obtained intervertebral disc (IVD) tissues with grade III degeneration from four patients. NPCs were isolated and passaged serially in three groups: control group, low-dose rhBMP-2 group and high-dose rhBMP-2 group until the cells reached the end of their replicative lifespan. Cumulative population doubling level (CPDL), telomerase activity and senescence markers, senescence-associated β-galactosidase (SA-β-gal), p53, p21, and p16, were assessed. The replicative capacity of NPCs in the high-dose rhBMP-2 group was decreased significantly compared to the control and low-dose rhBMP-2 groups. Mean telomerase activity was significantly greater in the high-dose rhBMP-2 group compared to the control and low-dose rhBMP-2 groups. The percentage of SA-β-gal-positive NPCs increased more rapidly in the high-dose rhBMP-2 group with passaging compared to the control and low-dose rhBMP-2 groups. The expression of p53, p21, and p16 in both low and high dose rhBMP-2 groups increased in all passages compared to the control group. We found that prolonged high-dose rhBMP-2 treatment increased telomerase activity of human NPCs, but decreased replicative capacity and lifespan in long term culture. We also found that excessive growth stimulation by prolonged high-dose rhBMP-2 treatment can promote NPCs senescence and result in growth arrest.

Effect of human thymus adipose tissue-derived mesenchymal stem cells on myocardial infarction in rat model

Background and objectiveStem cell (SC) therapy exhibits promising therapeutic efficiency against cardiovascular disease. The thymus adipose tissue (TAT) is familiar to cardiac surgeons with sternotomy; however, the application of TAT in SC therapy remains unknown. We assessed the effectiveness of TAT-derived mesenchymal SCs (TAT-MSCs) in the rat myocardial infarction (MI) model.MethodsThe human TATs were obtained from the patients who underwent coronary artery bypass graft surgery. In cell studies, we performed the cumulative population doubling level assessment, fluorescence-activated cell sorting analysis, and differentiation study. In animal studies, we segregated Sprague–Dawley rats (ischemia-reperfusion model) into three (sham, vehicle, and TAT-MSC) groups based on their corresponding treatment. Trans-thoracic echocardiogram (TTE) was obtained to assess the recovery of heart function in the 1st, 4th, 8th, and 12th week after surgical manipulations. After echocardiographic study, infarcted area of the heart was measured using triphenyl tetrazolium chloride (TTC) stain.ResultsThe sham group exhibited significantly better systolic and diastolic function (SDF) than the other groups did. After one week of TAT-MSC or vehicle injection, the TAT-MSC group exhibited a significant improvement in the E/E′ value (25.75 ± 1.09 vs. 24.20 ± 0.91, p < 0.001) compared to the vehicle group. Although statistically insignificant, the trend of improvement in SDF was better in the TAT-MSC group than in the vehicle group. The infarcted area measured by TTC staining was 22.81 ± 6.41% and 29.95 ± 9.09% in the TAT-MSC and vehicle groups, respectively (p = 0.04).ConclusionAlthough TTE results exhibited insignificant variations in SDF, a trend with improvement in the SDF of the heart was observed in the TAT-MSC group compared to the vehicle group. The infarcted area of heart indicated significant reduction in the TAT-MSC group compared to the vehicle group as confirmed by histopathological study.

Evaluation of bone marrow-derived mesenchymal stem cell quality from patients with congenital pseudoarthrosis of the tibia

BackgroundThe treatment of congenital pseudoarthrosis of the tibia (CPT) remains challenging in pediatric orthopedics due to the difficulties in bone union, continuous angulation, joint stiffness, and severe limb length discrepancy. Mesenchymal stem cells (MSCs) therapy offers a complementary approach to improve the conventional surgical treatments. Although the autologous MSC treatment shows a promising strategy to promote bone healing in CPT patients, the quality of MSCs from CPT patients has not been well studied. The purpose of this study is to investigate the quality of MSCs isolated from patients with CPT.MethodsThe bone marrow-derived MSCs from the fracture site and iliac crest of six CPT patients were isolated and compared. The cumulative population doubling level (cPDL), phenotype characteristics, and trilineage differentiation potency were observed to assess the quality of both MSCs.ResultsThere were no significant differences of the MSCs derived from the fracture site and the MSCs from the iliac crest of the subjects, in terms of cPDL, phenotype characteristics, and trilineage differentiation potency (all p > 0.05). However, MSCs from the fracture site had a higher senescence tendency than those from the iliac crest.ConclusionMSC quality is not the main reason for delayed bone regeneration in those with CPT. Thus, autologous MSC is a promising source for treating CPT patients

An Effect of Culture Media on Epithelial Differentiation Markers in Breast Cancer Cell Lines MCF7, MDA-MB-436 and SkBr3.

Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines (MCF7, SkBr3 and MDA-MB-436). Materials and methods: Cell lines were conditioned in three studied media (all containing 5% fetal bovine serum (FBS) + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines (MCF7, SkBr3), than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times.

Downregulation of Pin1 in human atherosclerosis and its association with vascular smooth muscle cell senescence

ObjectivePin1 is prevalently overexpressed in human cancers and implicated to regulate cell growth and apoptosis. Thus far, however, no role for Pin1 has been described in modulating vascular smooth muscle cell (VSMC) senescence. MethodsImmunohistochemistry and Western blotting were used to assess Pin1 protein level in human normal and atherosclerotic tissues. β-galactosidase staining, cumulative population doubling level, telomerase activity, and relative telomere length measurement were used to confirm VSMC senescence. The expressions of Pin1 and other genes involved in this research were analyzed by quantitative reverse-transcription polymerase chain reaction and Western blotting in VSMCs. Apolipoprotein E gene-deleted mice (ApoE−/−) fed a high-fat diet were treated with juglone or 10% ethanol, respectively, for 3 weeks. The extent of atherosclerosis was evaluated by Oil Red O, Masson trichrome staining, and immunohistology. ResultsPin1 protein level decreased in human atherosclerotic tissues and VSMCs, synchronously with increased VSMC senescence. Adenoviral-mediated Pin1 overexpression rescued cellular senescence in atherosclerotic VSMCs, with concurrent down-regulation of P53, p21, growth arrest and DNA-damage-inducible protein 45-alpha (Gadd45a), phosphorylated retinoblastoma (p-pRb), p65 and upregulation of cyclin subfamilies (cyclin B, D, and E), and cyclin-dependent kinase subfamilies (2, 4, and 6), whereas Pin1 knockdown resulted in the converse effects, indicating that VSMC senescence mediated by Pin1 is an integrated response to diverse signals. In vivo data from ApoE−/− mice showed that treatment of juglone led to accelerated atherosclerosis development. ConclusionsAltogether this work supports a role for Pin1 as a vital modulator of VSMC senescence, thereby providing a novel target for regulation and control of atherosclerosis.

Ionizing radiation response of primary normal human lens epithelial cells.

Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.

Effect of donor age on the proliferation and multipotency of canine adipose-derived mesenchymal stem cells.

Research into adipose tissue-derived mesenchymal stem cells (AD-MSCs) has demonstrated the feasibility of their use in clinical applications due to their ease of isolation and abundance in adipose tissue. We isolated AD-MSCs from young and old dogs, and the cells were subjected to sequential sub-passaging from passage 1 (P1) to P7. Canine AD-MSCs (cAD-MSCs) were examined for proliferation kinetics, expression of molecules associated with self-renewal, expression of cell surface markers, and differentiation potentials at P3. Cumulative population doubling level was significantly higher in cAD-MSCs of young donors than in those of old donors. In addition, expressions of CD73, CD80, Oct3/4, Nanog, cell survival genes and differentiation potentials were significantly higher in young donors than in old donors. The present study suggests that donor age should be considered when developing cell-based therapies for clinical application of cAD-MSCs.

Extensive characterization of feline intra-abdominal adipose-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) isolated from various tissues have been well characterized for therapeutic application to clinical diseases. However, in contrast to MSCs from other animal species, the characteristics of feline MSCs have not been fully documented. In this study, we conducted extensive characterization of feline adipose tissue-derived MSCs (fAD-MSCs). Study fAD-MSCs were individually isolated from the intra-abdominal adipose tissues of six felines. The expression levels of cell surface markers and pluripotent markers were evaluated. Next, proliferation capacity was analyzed by performing cumulative population doubling level (CPDL) and doubling time (DT) calculation assays. Differentiation potentials of fAD-MSCs into mesodermal cell lineages were analyzed by examining specific staining and molecular markers. All fAD-MSCs positively expressed cell surface markers such as CD29, CD44, CD90, CD105, CD166, and MHC-I, while CD14, CD34, CD45, and CD73 were negatively expressed. The CPDL of the fAD-MSCs was maintained until passage 5 to 6 (P5 to P6), whereas DT increased after P3 to P4. Also, stem cell-specific pluripotent markers (Oct3/4, Nanog, and SSEA-4) were detected. Importantly, all fAD-MSCs demonstrated mesodermal differentiation capacity. These results suggest that fully characterized fAD-MSCs could be beneficial when considering the use of these cells in feline disease research.

Effects of age, replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.

Dynamic culture of mesenchymal stem cells on thermo-responsive PNIPAM substrate

Event Abstract Back to Event Dynamic culture of mesenchymal stem cells on thermo-responsive PNIPAM substrate George Altankov1 and Maria Bianchi2 1 ICREA and Institute for Bioengineering of Catalonia, Molecular Dynamics at Cell-Biomaterials Interface, Spain 2 Institute for Bioengineering of Catalonia, Molecular Dynamics at Cell-Biomaterials Interface, Spain As of today, stem cell-based therapies are still hindered by the insufficient knowledge of the signals that govern stem cell behavior. Stem cell niche is a dynamic microenvironment that balances stem cells activity to maintain tissue homeostasis and repair. The development of strategies to mimic this dynamic niche environment provides insight to our understanding how to control the behavior of stem cells. In this work we used smart thermo-responsive polymer poly(N-isopropylacrylamide (PNIPAM) for culturing of adipose derived mesenchymal stem cells (ADSCs) applying periodic temperature cycles to perturb the cell-substratum interactions. We further studied the functional behavior of stem cells following their two principal properties: the capability for self-renewal (i.ee. the symmetric growth) and to differentiate (asymmetric cell division). Material and Methods: 2.0×104 cells/well ADSCs of 2nd passage were cultured on regular tissue culture polystyrene (control) or on thermo-responsive polymer (PNIPAAm) substrate (Nunc UpCell™) and further submitted to equal thermo-cycling consisting of 20 min cooling (37ºC to 24ºC) and 20 min heating (24ºC to 37ºC), twice per day using specially designed thermo-plate with circulating water jacked fitted to a standard CO2 incubator. For the assessment of symmetric cell division the cells were propagated in basal medium with passaging at each 3rd day and counted before seeding again for the next passage in the initial density. The cumulative population doubling level (CPDL) was calculated and compared for up to 13th passage monitoring also the overall cell morphology and fibronectin matrix secretion as indirect measure for ADSCs ageing. For assessing the asymmetric cell growth the ADSC were switched to osteogenic differentiation medium and again subjected to dynamic thermo-cycling condition. The kinetics of cells growth, alkaline phosphatase activity (AP) and Ca deposition (Alisarin red) were assayed at 3, 7, 14 and 21 dais. Results and Discussion: This is the first study identifying in vitro the effect of thermos-cycling (2 cycles per day) on the behaviour of ADSCs on PNIPAM substrate. The general conclusion was that periodic alterations in the adhesive phenotype of ADSCs significantly alter their differentiation potential but not the symmetric cells renewal in an undifferentiated state. This work was supported by CIBER-BBN (Spain) and the European Commission through the FP7 Industry-Academia Partnerships and Pathways (IAPP) project FIBROGELNET; The valuable support of the project MAT2012-38359-C03-03 HEALINSYNERGY, funded by Spanish Ministry of Science and Innovation is also acknowledged. Keywords: Cell Adhesion, Cell Differentiation, stem cell, Smart material Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Adhesive biomaterials Citation: Altankov G and Bianchi M (2016). Dynamic culture of mesenchymal stem cells on thermo-responsive PNIPAM substrate. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02120 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers George Altankov Maria Bianchi Google George Altankov Maria Bianchi Google Scholar George Altankov Maria Bianchi PubMed George Altankov Maria Bianchi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Lifespan Extension and Sustained Expression of Stem Cell Phenotype of Human Breast Epithelial Stem Cells in a Medium with Antioxidants.

We have previously reported the isolation and culture of a human breast epithelial cell type with stem cell characteristics (Type I HBEC) from reduction mammoplasty using the MSU-1 medium. Subsequently, we have developed several different normal human adult stem cell types from different tissues using the K-NAC medium. In this study, we determined whether this low calcium K-NAC medium with antioxidants (N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate) is a better medium to grow human breast epithelial cells. The results clearly show that the K-NAC medium is a superior medium for prolonged growth (cumulative population doubling levels ranged from 30 to 40) of normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures. Thus, this new method of growing Type I HBECs will be very useful in future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.

Relationship between Initial Telomere Length, Initial Telomerase Activity, Age, and Replicative Capacity of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs: What Is a Predictor of Replicative Potential?

There is evidence that telomere length (TL), telomerase activity (TA), and age are related to the replicative potential of human nucleus pulposus chondrocytes (NPCs). However, it has not yet been established if any of these factors can serve as predictors of the replicative potential of NPCs. To establish predictors of the replicative potential of NPCs, we evaluated potential relationships between replicative capacity of NPCs, initial TL (telomere length at the first passage), initial TA (telomerase activity at the first passage), and age. Nucleus pulposus specimens were obtained from 14 patients of various ages undergoing discectomy. NPCs were serially cultivated until the end of their replicative lifespans. Relationships among cumulative population doubling level (PDL), initial TL, initial TA, and age were analyzed. Initial TA was negatively correlated with age (r = -0.674, P = 0.008). However, no correlation between initial TL and age was observed. Cumulative PDL was also negatively correlated with age (r = -0.585, P = 0.028). Although the cumulative PDL appeared to increase with initial TL or initial TA, this trend was not statistically significant. In conclusion, age is the sole predictor of the replicative potential of human NPCs, and replicative potential decreases with age. Initial TL and initial TA are not predictors of replicative potential, and can serve only as reference values.

Comparison of processing times for isolation of feline adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an attractive source for cell therapy, as they have the potential for differentiation into multi-lineage cells. Adipose tissue is a safe source due to its easy extraction and abundant resource, with minimal risk to the organ donor. In this study, we attempted to correlate the harvest yield and resulting multipotency of feline adipose tissue-derived mesenchymal stem cells (fAD-MSCs) in accordance with processing time. fAD-MSCs were individually isolated from the abdominal adipose tissues of 6 felines. They were divided into two groups, based on their processing times - Group 1: 0~1 day after adipose tissue harvesting; Group 2: more than 3 days after adipose tissue harvesting. In both groups, the proliferation capacity was analyzed using the cumulative population doubling level (CPDL) calculation assay. The expression levels of MSC-specific markers and differentiation potentials into mesodermal cell lineages were also evaluated. We observed that fAD-MSC isolation yields and CPDL were excellent in Group 1 compared with Group 2. We also found that the differentiation potential-specific genes (ACAN and OPN) were strongly expressed in Group 1 compared with Group 2. These results suggest that for the clinical treatments of feline diseases, fAD-MSCs should be isolated within 1 day after adipose tissue harvesting.

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

IntroductionEfficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.ResultsThe findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.ConclusionThe presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15–20 CPDL is the most adequate maturation stage.

Isolation and Characterization of Antler-Derived Multipotent Stem Cells

Recent studies have reported that stem cells can be isolated from various tissues such as bone marrow, fatty tissue, umbilical cord blood, Wharton's jelly, and placenta. These types of stem cell studies have also arisen in veterinary medicine. Deer antlers show a seasonal regrowth of tissue, an unusual feature in mammals. Antler tissue therefore might offer a source of stem cells. To explore the possibility of stem cell populations within deer antlers, we isolated and successfully cultured antler-derived multipotent stem cells (MSCs). Antler MSCs were maintained in a growth medium, and the proliferation potential was measured via an assay called the cumulative population doubling level. Immunophenotyping and immunostaining revealed the intrinsic characteristic stem cell markers of antler MSCs. To confirm the ability to differentiate, we conducted osteogenic, adipogenic, and chondrogenic induction under the respective differentiation conditions. We discovered that antler MSCs have the ability to differentiate into multiple lineages. In conclusion, our results show that deer antler tissue may contain MSCs and therefore may be a potential source for veterinary regenerative therapeutics.

Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells.

Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was significantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These findings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

Sequential sub-passage decreases the differentiation potential of canine adipose-derived mesenchymal stem cells

Adipose-derived mesenchymal stem cells (AD-MSCs) are abundant in adipose tissue from animals of all ages, are easily isolated, can differentiate into multi-lineage cells, and have a clinical application. This promising potential may only be achieved if the cells are expanding in a large number while maintaining their stemness in sequential passages. In this study, canine AD-MSCs (cAD-MSCs) were individually isolated from five dogs and subjected to proliferative culture with seven sub-passages. The cells at each sub-passage were characterized for properties associated with multipotent MSCs such as proliferation kinetics, expression of MSCs-specific surface markers, expression of molecules associated with self-renewal and differentiation capabilities into mesodermal lineage cells. Proliferation of the cells plateaued at passage 5 by cumulative population doubling level, while cell doubling time gradually increased with passage. MSCs surface markers (CD44, CD90, and CD105) and molecules (Oct 3/4, Sox-2, Nanog and HMGA2) associated with self-renewal were all expressed in the cells between passages 1 to 6 by RT-PCR. In addition, the cells at passage 1, 3 or 6 underwent adipogenic and chondrogenic differentiation under specific induction conditions. However, the level of adipogenic and chondrogenic differentiation was negatively correlated with the number of sub-passage. The present study suggests that sequential sub-passages affect multipotent properties of cAD-MSCs, which should be considered in their therapeutic application in regenerative medicine.