Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Oestrogen retains human periodontal ligament stem cells stemness in long‐term culture

Objectives

During long‐term culture, loss of stemness is observed which greatly restricts the application of human periodontal ligament stem cells (hPDLSCs) in tissue regeneration. Oestrogen (E2) was found to significantly enhance the proliferation and osteogenic differentiation capacity in mesenchymal stem cells. Therefore, in this study, we investigated effects of E2 on hPDLSCs stemness in long‐term culture.

Materials and methods

Effects of E2 on hPDLSCs stemness were systematically evaluated. To characterize underlying the mechanisms, its effects on PI3K/AKT signalling pathway were determined.

Results

Our results showed that E2 was able to enhance the proliferation, modify cell cycle, up‐regulate stemness‐related genes expression, promote osteogenic differentiation and elevate the positive rate of CD146 and STRO‐1 over 10 passages in hPDLSCs. Importantly, PI3K/AKT signing pathway might play a role in these effects.

Conclusions

These findings suggest that E2 retains hPDLSCs stemness in long‐term culture, which might enhance its application in tissue engineering.     

The role of hair follicle nestin‐expressing stem cells during whisker sensory‐nerve growth in long‐term 3D culture

We have previously reported that nestin‐expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND‐GFP mice), and were placed in 3D histoculture supported by Gelfoam®. β‐III tubulin‐positive fibers, consisting of ND‐GFP‐expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F‐actin. These findings indicate that β‐III tubulin‐positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND‐GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin‐expressing stem cells in the hair follicle is for growth of the follicle sensory nerve. J. Cell. Biochem. 114: 1674–1684, 2013. © 2013 Wiley Periodicals, Inc.     

Vitamin C alleviates the senescence of periodontal ligament stem cells through inhibition of Notch3 during long‐term culture

Periodontal ligament stem cells (PDLSCs), as potential “seed cells” for periodontal tissue repair and regeneration, require to be expanded in vitro for a large scale. Senescence of PDLSCs occurred during long‐term culture may compromise the therapeutic effects of PDLSCs. Medium supplements may be useful in antisenescence. However, the effects and mechanisms of vitamin C (Vc) treatment on PDLSCs during long‐term culture are still unclear. In this study, we identified that Vc‐treated PDLSCs cells maintained a slender morphology, higher growth rate and migration capacity, stemness, and osteogenic differentiation capability during a long‐term culture. Moreover, we also identified that Notch3 was significantly upregulated during the cell senescence, and Vc treatment alleviated the senescence of PDLSCs through inhibition of Notch3 during long‐term culture. In summary, Vc treatment suppressed PDLSCs senescence by reducing the expression of Notch3 and might be a simple and useful strategy to inhibit cellular senescence during the cell long‐term culture.     

Long‐term culture optimization of human omentum fat‐derived mesenchymal stem cells

Recent scientific explorations in search of novel sources for autologous transplantation transpired an alternative source of MSCs (mesenchymal stem cells) derived from omentum fat. The scarcity of experimental evidences probing into the biosafety concerns of omentum fat‐derived MSC under prolonged culture conditions limits its applicability as an efficient tool in regenerative medicine. This study, thus, aims to optimize human omentum fat‐derived MSC in four different media [DMEM (Dulbecco's modified Eagle's medium) LG (low glucose), DMEM KO (knock out), α‐MEM (α‐minimal essential media) and DMEM F12] in the facets of phenotypic characterization, growth kinetics, differentiation and karyotyping under prolonged culture. The cells exhibited a similarity in expression profile for the majority of markers with evidential variations in certain markers. The relevance of omentum fat‐derived MSCs became evident from its triumphant differentiation potential and karyotypic stability substantiated even at later passage. The results obtained from growth curve and PDT (population doubling time) lead to optimization of appropriate media for omentum fat‐derived stem cell research, thereby bringing omentum fat into the forefront of regenerative medicine.     

Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

α8‐Integrins are required for hippocampal long‐term potentiation but not for hippocampal‐dependent learning

Integrins are heterodimeric transmembrane cell adhesion receptors that are essential for a wide range of biological functions via cell–matrix and cell–cell interactions. Recent studies have provided evidence that some of the subunits in the integrin family are involved in synaptic and behavioral plasticity. To further understand the role of integrins in the mammalian central nervous system, we generated a postnatal forebrain and excitatory neuron‐specific knockout of α8‐integrin in the mouse. Behavioral studies showed that the mutant mice are normal in multiple hippocampal‐dependent learning tasks, including a T‐maze, non‐match‐to‐place working memory task for which other integrin subunits like α3‐ and β1‐integrin are required. In contrast, mice mutant for α8‐integrin exhibited a specific impairment of long‐term potentiation (LTP) at Schaffer collateral–CA1 synapses, whereas basal synaptic transmission, paired‐pulse facilitation and long‐term depression (LTD) remained unaffected. Because LTP is also impaired in the absence of α3‐integrin, our results indicate that multiple integrin molecules are required for the normal expression of LTP, and different integrins display distinct roles in behavioral and neurophysiological processes like synaptic plasticity.     

Mechanism of TNF‐α‐induced migration and hepatocyte growth factor production in human mesenchymal stem cells

Accumulating evidence suggests that mesenchymal stem cells (MSCs) may decrease destructive inflammation and reduce tissue loss. Tumor necrosis factor‐α (TNF‐α) plays a central role in induction of proinflammatory signaling and paradoxically activates intracellular anti‐inflammatory survival pathways. In this study, we investigated whether TNF‐α could induce a chemotactic effect on human MSCs and stimulate their production of anti‐inflammatory factors in vitro, as well as determined mechanisms that mediated this effect. Migration assays demonstrated that TNF‐α had a chemotactic effect on MSCs. TNF‐α increased both hepatocyte growth factor (HGF) mRNA expression in MSCs and HGF secretion in conditioned medium. These effects were dependent on the p38 MAPK and PI3K/Akt, but not JNK and ERK signaling pathways. Furthermore, these effects were inhibited by a specific neutralizing antibody to TNF receptor II, but not TNF receptor I. We conclude that TNF‐α can enhance human MSCs migration and stimulate their production of HGF. These effects are mediated via a specific TNF receptor and signaling pathways. J. Cell. Biochem. 111: 469–475, 2010. © 2010 Wiley‐Liss, Inc.     

Value of long‐term ecological studies

Long‐term ecological studies are critical for providing key insights in ecology, environmental change, natural resource management and biodiversity conservation. In this paper, we briefly discuss five key values of such studies. These are: (1) quantifying ecological responses to drivers of ecosystem change; (2) understanding complex ecosystem processes that occur over prolonged periods; (3) providing core ecological data that may be used to develop theoretical ecological models and to parameterize and validate simulation models; (4) acting as platforms for collaborative studies, thus promoting multidisciplinary research; and (5) providing data and understanding at scales relevant to management, and hence critically supporting evidence‐based policy, decision making and the management of ecosystems. We suggest that the ecological research community needs to put higher priority on communicating the benefits of long‐term ecological studies to resource managers, policy makers and the general public. Long‐term research will be especially important for tackling large‐scale emerging problems confronting humanity such as resource management for a rapidly increasing human population, mass species extinction, and climate change detection, mitigation and adaptation. While some ecologically relevant, long‐term data sets are now becoming more generally available, these are exceptions. This deficiency occurs because ecological studies can be difficult to maintain for long periods as they exceed the length of government administrations and funding cycles. We argue that the ecological research community will need to coordinate ongoing efforts in an open and collaborative way, to ensure that discoverable long‐term ecological studies do not become a long‐term deficiency. It is important to maintain publishing outlets for empirical field‐based ecology, while simultaneously developing new systems of recognition that reward ecologists for the use and collaborative sharing of their long‐term data sets. Funding schemes must be re‐crafted to emphasize collaborative partnerships between field‐based ecologists, theoreticians and modellers, and to provide financial support that is committed over commensurate time frames.     

Identification of human placenta‐derived mesenchymal stem cells involved in re‐endothelialization

Human placenta is an attractive source of mesenchymal stem cells (MSC) for regenerative medicine. The cell surface markers expressed on MSC have been proposed as useful tools for the isolation of MSC from other cell populations. However, the correlation between the expression of MSC markers and the ability to support tissue regeneration in vivo has not been well examined. Here, we established several MSC lines from human placenta and examined the expression of their cell surface markers and their ability to differentiate toward mesenchymal cell lineages. We found that the expression of CD349/frizzled‐9, a receptor for Wnt ligands, was positive in placenta‐derived MSC. So, we isolated CD349‐negative and ‐positive fractions from an MSC line and examined how successfully cell engraftment repaired fractured bone and recovered blood flow in ischemic regions using mouse models. CD349‐negative and ‐positive cells displayed a similar expression pattern of cell surface markers and facilitated the repair of fractured bone in transplantation experiments in mice. Interestingly, CD349‐negative, but not CD349‐positive cells, showed significant effects on recovering blood flow following vascular occlusion. We found that induction of PDGFβ and bFGF mRNAs by hypoxia was greater in CD349‐negative cells than in CD349‐positive cells while the expression of VEGF was not significantly different in CD349‐negative and CD349‐positive cells. These findings suggest the possibility that CD349 could be utilized as a specialized marker for MSC isolation for re‐endothelialization. J. Cell. Physiol. 226: 224–235, 2010. © 2010 Wiley‐Liss, Inc.     

Successful human long‐term application of in situ bone tissue engineering

Tissue Engineering (TE) and Regenerative Medicine (RM) have gained much popularity because of the tremendous prospects for the care of patients with tissue and organ defects. To overcome the common problem of donor‐site morbidity of standard autologous bone grafts, we successfully combined tissue engineering techniques for the first time with the arteriovenous loop model to generate vascularized large bone grafts. We present two cases of large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. For osseus reconstruction, arteriovenous loops were created as vascular axis, which were placed in the bony defects. In case 1, the bone generation was achieved using cancellous bone from the iliac crest and fibrin glue and in case 2 using a clinically approved β‐tricalciumphosphate/hydroxyapatite (HA), fibrin glue and directly auto‐transplanted bone marrow aspirate from the iliac crest. The following post‐operative courses were uneventful. The final examinations took place after 36 and 72 months after the initial operations. Computer tomogrphy (CT), membrane resonance imaging (MRI) and doppler ultrasound revealed patent arterio‐venous (AV) loops in the bone grafts as well as completely healed bone defects. The patients were pain‐free with normal ranges of motion. This is the first study demonstrating successfully axially vascularized in situ tissue engineered bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without creation of a significant donor‐site defect utilizing TE and RM techniques in human patients with long‐term stability.     

Identification and expansion of pancreatic stem/progenitor cells

Pancreatic islet transplantation represents an attractive approach for the treatment of diabetes. However, the limited availability of donor islets has largely hampered this approach. In this respect, the use of alternative sources of islets such as the ex vivo expansion and differentiation of functional endocrine cells for treating diabetes has become the major focus of diabetes research. Adult pancreatic stem cells /progenitor cells have yet to be recognized because limited markers exist for their identification. While the pancreas has the capacity to regenerate under certain circumstances, questions where adult pancreatic stem/progenitor cells are localized, how they are regulated, and even if the pancreas harbors a stem cell population need to be resolved. In this article, we review the recent achievements both in the identification as well as in the expansion of pancreatic stem/progenitor cells.     

Adenosine‐5′‐triphosphate suppresses proliferation and migration capacity of human endometrial stem cells

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP‐induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X₇ receptor in eMSCs. In spite of this, cell activation with specific P2X₇ receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X₇ receptor inhibitor, AZ10606120 also did not prevent ATP‐induced inhibition of cell migration, confirming that inhibition occurs without P2X₇ receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells.     

Rethinking long‐term vegetation dynamics: multiple glacial refugia and local expansion of a species complex

Evidence is accumulating that some arcto‐boreal plant taxa persisted through the last glacial maximum (LGM) in Alaska and adjacent Canada. However, the spatial patterns of glacial persistence and associated postglacial colonization remain largely unknown. In this study, we investigated the LGM refugia of an alder (Alnus) species complex (n = 3 taxa) and assess the spatiotemporal dynamics of Alnus in this vast region. Specifically, we conducted high‐throughput DNA sequencing (ddRADseq) on Alnus foliar samples collected from a dense population network to investigate patterns of genetic structure and infer the presence of glacial lineages. Species distribution modeling (SDM) was used to investigate the probability and possible locations of glacial persistence. These analyses were integrated and then compared with fossil pollen data to identify the locations of refugial populations and spatial patterns of postglacial colonization. Our genetic analyses revealed two glacial lineages with separate geographic origins for each Alnus taxon, suggesting that the genus persisted in multiple LGM refugia. Non‐overlapping hindcast distributions based on SDMs further support the presence of multiple, spatially distinct refugia. These ddRADseq and SDM results, in conjunction with reassessment of fossil pollen records, suggest that Alnus expanded from several population nuclei that existed during the LGM and coalesced during the Holocene to form its present range. These results challenge the unidirectional model for postglacial vegetation expansion, implying that climate buffering associated with landscape heterogeneity and adaptation to millennial‐scale environmental variability played important roles in driving late‐Quaternary population dynamics.