Early Passage Mesenchymal Stem Cells Research Articles

(58) Repeated but Not Single Dose of Early Passage Mesenchymal Stem Cells Regenerated Aspirated Damaged Donor Lungs and Reduced Primary Graft Dysfunction

(58) Repeated but Not Single Dose of Early Passage Mesenchymal Stem Cells Regenerated Aspirated Damaged Donor Lungs and Reduced Primary Graft Dysfunction

Nicotinamide Mononucleotide Supplementation Improves Mitochondrial Dysfunction and Rescues Cellular Senescence by NAD+/Sirt3 Pathway in Mesenchymal Stem Cells.

In vitro expansion-mediated replicative senescence has severely limited the clinical applications of mesenchymal stem cells (MSCs). Accumulating studies manifested that nicotinamide adenine dinucleotide (NAD+) depletion is closely related to stem cell senescence and mitochondrial metabolism disorder. Promoting NAD+ level is considered as an effective way to delay aging. Previously, we have confirmed that nicotinamide mononucleotide (NMN), a precursor of NAD+, can alleviate NAD+ deficiency-induced MSC senescence. However, whether NMN can attenuate MSC senescence and its underlying mechanisms are still incompletely clear. The present study herein showed that late passage (LP) MSCs displayed lower NAD+ content, reduced Sirt3 expression and mitochondrial dysfunction. NMN supplementation leads to significant increase in intracellular NAD+ level, NAD+/ NADH ratio, Sirt3 expression, as well as ameliorated mitochondrial function and rescued senescent MSCs. Additionally, Sirt3 over-expression relieved mitochondrial dysfunction, and retrieved senescence-associated phenotypic features in LP MSCs. Conversely, inhibition of Sirt3 activity via a selective Sirt3 inhibitor 3-TYP in early passage (EP) MSCs resulted in aggravated cellular senescence and abnormal mitochondrial function. Furthermore, NMN administration also improves 3-TYP-induced disordered mitochondrial function and cellular senescence in EP MSCs. Collectively, NMN replenishment alleviates mitochondrial dysfunction and rescues MSC senescence through mediating NAD+/Sirt3 pathway, possibly providing a novel mechanism for MSC senescence and a promising strategy for anti-aging pharmaceuticals.

Porcine somatic cell nuclear transfer using telomerase reverse transcriptase-transfected mesenchymal stem cells reduces apoptosis induced by replicative senescence

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

Glia-Like Cells from Late-Passage Human MSCs Protect Against Ischemic Stroke Through IGFBP-4.

Stem cell therapy is considered to be a promising future treatment for intractable neurological diseases, although all the clinical trials using stem cells have not yet shown any good results. Early passage mesenchymal stem cells (MSCs) have been used in most clinical trials because of the issues on safety and efficacy. However, it is not easy to get plenty of cells enough for the treatment and it costs too much. Lots of late passage MSCs can be obtained at lower cost but their efficacy would be a big hurdle for clinical trials. If late passage MSCs with better efficacy could be used in clinical trials, it could be a new and revolutionary solution to reduce cost and enhance easier clinical trials. In the present study, it was investigated whether late passage MSCs could be induced into glia-like cells (ghMSCs); ghMSCs had better efficacy and they protected neurons and the brain from ischemia, and insulin-like growth factor binding protein-4 (IGFBP-4) played a critical role in beneficial effect of ghMSCs. ghMSCs were induced from MSCs and treated in in vitro and in vivo models of ischemia. They effectively protected neurons from ischemia and restored the brain damaged by cerebral infarction. These beneficial effects were significantly blocked by IGFBP-4 antibody. The current study demontsrated that late passage hMSCs can be efficiently induced into ghMSCs with better neuroprotective effect on ischemic stroke. Moreover, the results indicate that IGFBP-4 released from ghMSCs may serve as one of the key neuronal survival factors secreted from ghMSCs.

In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects

In vitro cultured autologous mesenchymal stem cells (MSCs) within passage 5 have been approved for clinical application in stem cell-based treatment of cartilage defects. However, their chondrogenic potential has not yet been questioned or verified. In this study, the chondrogenic potential of bone marrow MSCs at passage 3 (P3 BMSCs) was investigated both in cartilage repair and in vitro, with freshly isolated bone marrow mononuclear cells (BMMNCs) as controls. The results showed that P3 BMSCs were inferior to BMMNCs not only in their chondrogenic differentiation ability but also as candidates for long-term repair of cartilage defects. Compared with BMMNCs, P3 BMSCs presented a decay in telomerase activity and a change in chromosomal morphology with potential anomalous karyotypes, indicating senescence. In addition, interindividual variability in P3 BMSCs is much higher than in BMMNCs, demonstrating genomic instability. Interestingly, remarkable downregulation in cell cycle, DNA replication and mismatch repair (MMR) pathways as well as in multiple genes associated with telomerase activity and chromosomal stability were found in P3 BMSCs. This result indicates that telomerase and chromosome anomalies might originate from expansion, leading to impaired stemness and pluripotency of stem cells. In vitro culture and expansion are not recommended for cell-based therapy, and fresh BMMNCs are the first choice.

Early Passage Mesenchymal Stem Cells Display Decreased Radiosensitivity and Increased DNA Repair Activity.

Cell therapies using human mesenchymal stem cells (MSCs) have received much attention in the past decade. In pursuit of the therapeutic potential of MSCs, cell expansion is required to generate a great number of cells with desired phenotype and functionality. Long‐term expansion in vitro, however, can lead to altered functions. To explore the changes in DNA damage responses (DDR) in MSCs expanded, DDR pathways following irradiation were characterized in early‐ and late‐passage bone marrow MSCs. Seventy‐two hours after irradiation, the percentage of sub‐G1 cells in early‐passage MSCs did not change significantly. Reduced TUNEL staining was observed in early‐passage MSCs compared to late‐passage MSCs 4 h after irradiation. Comet assay also revealed that early‐passage MSCs were more resistant to irradiation or DNA damages induced by genotoxic agents than late‐passage MSCs. ATM phosphorylation and γ‐H2AX and phospho‐p53 increased in early‐passage MSCs while decreased in late‐passage MSCs. Through inhibition by KU55933, DDR pathway in early‐passage MSCs was shown to be ATM‐dependent. Higher levels of poly (ADP‐ribose) polymerase‐1 (PARP‐1) and PAR synthesis were observed in early‐passage MSCs than in late‐passage MSCs. Knockdown of PARP‐1 in early‐passage MSCs resulted in sensitization to irradiation‐induced apoptosis. Overexpression of PARP‐1 in late passage MSCs could render irradiation resistance. Lower activity of DDR in late‐passage MSCs was associated with rapid proteasomal degradation of PARP‐1. In conclusion, early‐passage MSCs are more irradiation‐resistant and have increased DDR activity involving PARP‐1, ATM and their downstream signals. Stem Cells Translational Medicine 2017;6:1504–1514

Therapeutic Benefit for Late, but Not Early, Passage Mesenchymal Stem Cells on Pain Behaviour in an Animal Model of Osteoarthritis.

Background Mesenchymal stem cells (MSCs) have a therapeutic potential for the treatment of osteoarthritic (OA) joint pathology and pain. The aims of this study were to determine the influence of a passage number on the effects of MSCs on pain behaviour and cartilage and bone features in a rodent model of OA. Methods Rats underwent either medial meniscal transection (MNX) or sham surgery under anaesthesia. Rats received intra-articular injection of either 1.5 × 106 late passage MSCs labelled with 10 μg/ml SiMAG, 1.5 × 106 late passage mesenchymal stem cells, the steroid Kenalog (200 μg/20 μL), 1.5 × 106 early passage MSCs, or serum-free media (SFM). Sham-operated rats received intra-articular injection of SFM. Pain behaviour was quantified until day 42 postmodel induction. Magnetic resonance imaging (MRI) was used to localise the labelled cells within the knee joint. Results Late passage MSCs and Kenalog attenuated established pain behaviour in MNX rats, but did not alter MNX-induced joint pathology at the end of the study period. Early passage MSCs exacerbated MNX-induced pain behaviour for up to one week postinjection and did not alter joint pathology. Conclusion Our data demonstrate for the first time the role of a passage number in influencing the therapeutic effects of MSCs in a model of OA pain.

Following Autologous Stem Cell Transplantation (1 Yr) Mesenchymal Stem Cells Are Functionally Impaired with Reduced Hematopoietic Support

Autologous stem cell transplantation (ASCT) is frequently applied in patients with multiple myeloma and malignant lymphoma. Although adequate steady state hematopoiesis with normal peripheral blood counts is attained after ASCT, marked cytopenias may occur in times of stress such as sepsis or re-exposure to chemotherapy. Our group has previously shown impairment of the hematopoietic stem cell (HSC) compartment 1 year post ASCT (pASCT), reflected by reduced HSC frequency and quiescence, and increased ROS production (Haematologica 2013;98:1264). Considering the essential role for mesenchymal stem cells (MSCs) in supporting hematopoiesis, we studied the MSC compartment 1 year post ASCT.Bone marrow biopsies from pASCT patients (n=17) were studied and compared to normal bone marrow from healthy donors (NBM, n=20) by performing immunohistochemistry staining of endothelial cells by CD34 (indicating microvessel density, MVD) and MSCs by nestin, CD146 (Melanoma Cell Adhesion Molecule, MCAM) and CD271 (Nerve Growth Factor Receptor, NGFR). A significant increase in CD271+ MSCs was observed in pASCT bone marrow biopsies compared to NBM (p<0.0001), while the expression of additional markers did not differ between pASCT vs. NBM. MSCs were cultured from the CD34- fraction of bone marrow mononuclear cells, obtained from pASCT patients (n=17) and MSCs derived from NBM (n=20). MSCs were selected by their plastic-adherency and replated to generate MSCs. Although pASCT MSCs and NBM MSCs had similar population doubling times (1.92±0.22 and 3.52±1.02 in passage 4 (P4), pASCT MSCs cultured in vitro demonstrated a change in morphology from the onset of P4. We also observed premature exhaustion of growth in 45% of the studied patients at P5 (vs. 18% in NBM) and increased senescence shown by B-galactosidase staining in P5/P6 (p=0.04). Differentiation assays did not show impairment in differentiation towards osteoblasts or adipocytes of pASCT MSCs. Gene expression analysis on early passage MSCs showed upregulation of pro-inflammatory and cell cycle genes, such as IL6 and p21, in pASCT MSCs compared to NBM MSCs. Co-culture studies with cord blood-derived CD34+ cells on pASCT MSCs showed a significant reduction in output in CFC assays and significant reduction in number of cobblestone-area forming cells in pASCT co-cultures versus NBM (p < 0.05).Given the higher incidence of MDS and AML after ASCT, we questioned whether the observed phenotype of pASCT MSCs resembles MSCs from patients with MDS and AML. Therefore the endothelial and mesenchymal compartments of MDS (n=20) and AML (n=23) patients were studied. An increase in MVD was detected in MDS/AML bone marrow biopsies in contrast to NBM and pASCT (p < 0.05), while the expression of CD146, CD271 and nestin in MDS/AML patients was not significantly increased. 25% of AML MSC cultures showed no growth in the first passage. When MSC growth did occur, the remaining cultures did not show a difference in population doubling time or expansion. However, a change in morphology of MDS/AML MSCs similar to pASCT MSCs was observed. Studies of early passages of MDS/AML MSCs demonstrated a significantly increased gene expression of IL-6 and p21 comparable to pASCT MSCs. In addition PITX2 and Foxc1 expression was increased but no difference was observed in pASCT vs. MDS/AML MSCs. PITX2 has been linked to increased senescence of MDS MSCs while Foxc1 is linked to adipo-osteoprogenitor cell differentiation thereby affecting the HSC compartment.Since none of the pASCT patients did develop MDS, immunohistochemical stainings were also performed on bone marrow biopsies of patients that developed therapy related (t-)MDS/AML following ASCT for lymphoma and myeloma (n=7), after a mean of 117 (MDS) and 50 months (AML). An increase in MVD was observed shortly before or during MDS/AML development, which is probably related to the emergence of malignant cells. No major changes in the phenotype of the MSC compartment were observed before or during the emergence of t-MDS/AML, indicating that t-MDS/AML is preceded by an increase in MVD without distinct changes in the MSC compartment.In summary our results demonstrate that MSCs are affected after ASCT, as shown by expression pattern and functionality. These changes result in a pro-inflammatory phenotype with premature senescence and impaired support of hematopoietic cells, which may account for the reduced bone marrow reserve observed in pASCT patients. DisclosuresNo relevant conflicts of interest to declare.

Different effects of resveratrol on early and late passage mesenchymal stem cells through β-catenin regulation

Resveratrol is a sirtuin 1 (SIRT1) activator and can function as an anti-inflammatory and antioxidant factor. In mesenchymal stem cells (MSCs), resveratrol enhances the proliferation and differentiation potential and has an anti-aging effect. However, contradictory effects of resveratrol on MSC cultures have been reported. In this study, we found that resveratrol had different effects on MSC cultures according to their cell passage and SIRT1 expression. Resveratrol enhanced the self-renewal potential and multipotency of early passage MSCs, but accelerated cellular senescence of late passage MSCs. In early passage MSCs expressing SIRT1, resveratrol decreased ERK and GSK-3β phosphorylation, suppressing β-catenin activity. In contrast, in late passage MSCs, which did not express SIRT1, resveratrol increased ERK and GSK-3β phosphorylation, activating β-catenin. We confirmed that SIRT1-deficient early passage MSCs treated with resveratrol lost their self-renewal potential and multipotency, and became senescent due to increased β-catenin activity. Sustained treatment with resveratrol at early passages maintained the self-renewal potential and multipotency of MSCs up to passage 10. Our findings suggest that resveratrol can be effectively applied to early passage MSC cultures, whereas parameters such as cell passage and SIRT1 expression must be taken into consideration before applying resveratrol to late passage MSCs.

Whole-Genome Sequencing Identifies Genetic Variances in Culture-Expanded Human Mesenchymal Stem Cells

SummaryCulture-expanded human mesenchymal stem cells (MSCs) are increasingly used in clinics, yet full characterization of the genomic compositions of these cells is lacking. We present a whole-genome investigation on the genetic dynamics of cultured MSCs under ex vivo establishment (passage 1 [p1]) and serial expansion (p8 and p13). We detected no significant changes in copy-number alterations (CNAs) and low levels of single-nucleotide changes (SNCs) until p8. Strikingly, a significant number (677) of SNCs were found in p13 MSCs. Using a sensitive Droplet Digital PCR assay, we tested the nonsynonymous SNCs detected by whole-genome sequencing and found that they were preexisting low-frequency mutations in uncultured mononuclear cells (∼0.01%) and early-passage MSCs (0.1%–1% at p1 and p8) but reached 17%–36% in p13. Our data demonstrate that human MSCs maintain a stable genomic composition in the early stages of ex vivo culture but are subject to clonal growth upon extended expansion.

Do the Fibroblasts Contained in Early Passage MSC Population Adversely Affect the Characteristics of Stem Cell Population Obtained from Human Placenta?

Mesenchymal stem cells (MSCs) have been obtained from various human tissues by harvesting plastic adherent fibroblast-like cell population. For potential use in regeneration medicine, early passage MSC population is preferred to avoid cell senescence. The early passage adherent cell population contains MSCs as well as fibroblasts, however, the significance of the contained fibroblasts has not been well investigated. Thus, we investigated the stem cell characteristics of the early passage MSC population with and without fibroblasts depletion. We obtained adherent cell populations from full term placenta at passage 2∼3 and divided them into two subpopulations: fibroblasts depleted (popFD) and non-depleted population (popFND) using magnetic cell sorting method. The two subpopulations were compared in terms of cell morphology, potential for long term culture, colony forming ability, and tri-lineage differentiation for adipogenic, chondrogenic, and osteogenic differentiation. The percentage of fibroblasts contained in the early passage MSC population was 5.3% (2.9∼8.4). Both the popFD and popFND was spindle shaped from early passages and maintained long term culture up to 20∼22 passages. CFU-F assay showed no difference between the subpopulations. Overall, tri-lineage differentiation showed a tendency of better differentiation potential of popFND than popFD. We confirmed that fibroblasts are contained in early population of placenta-derived MSCs obtained by current method. This study revealed that the contained fibroblasts in early passage MSC population do not adversely affect the properties of MSCs in terms of cell morphology, potential for long term culture, colony forming ability, and tri-lineage differentiation.

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages

Bone marrow mesenchymal stem cells support proliferation and differentiation of hematopoietic progenitor cells in vitro. Since these cells constitute a rare subset of bone marrow cells, mesenchymal stem cell preparations for clinical purposes require a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on mesenchymal stem cell supportive activity. Mesenchymal stem cells were expanded for up to ten passages. These cells and CD34+ cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed. Early passage mesenchymal stem cells supported hematopoietic progenitor cell expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage mesenchymal stem cells did not support hematopoietic progenitor cell and myeloid cell outgrowth but maintained B-cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for 1 week in contact with mesenchymal stem cells was effective until the fourth passage of the mesenchymal cells and declined thereafter. The levels of engraftment of CD34(+) cells in NOD/SCID mice was higher when these cells were co-injected with early passage mesenchymal stem cells; however mesenchymal cells expanded beyond nine passages were ineffective in promoting CD34(+) cell engraftment. Non-contact cultures indicated that mesenchymal stem cell supportive activity involved diffusible factors. Among these, interleukins 6 and 8 contributed to the supportive activity of early passage mesenchymal stem cells but not to those of late passage cells. The phenotype, as well as fat, bone and cartilage differentiation capacity, of mesenchymal stem cells did not change during their culture. Conclusions Extended culture of mesenchymal stem cells alters the ability of these cells to support hematopoietic progenitor cells without causing concomitant changes in their phenotype or differentiation capacity.