Rainer Saffrich

Replicative Senescence of Mesenchymal Stem Cells: A Continuous and Organized Process

Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. These cells are usually culture expanded prior to their application. However, a precise molecular definition of MSC and the sequel of long-term in vitro culture are yet unknown. In this study, we have addressed the impact of replicative senescence on human MSC preparations. Within 43 to 77 days of cultivation (7 to 12 passages), MSC demonstrated morphological abnormalities, enlargement, attenuated expression of specific surface markers, and ultimately proliferation arrest. Adipogenic differentiation potential decreased whereas the propensity for osteogenic differentiation increased. mRNA expression profiling revealed a consistent pattern of alterations in the global gene expression signature of MSC at different passages. These changes are not restricted to later passages, but are continuously acquired with increasing passages. Genes involved in cell cycle, DNA replication and DNA repair are significantly down-regulated in late passages. Genes from chromosome 4q21 were over-represented among differentially regulated transcripts. Differential expression of 10 genes has been verified in independent donor samples as well as in MSC that were isolated under different culture conditions. Furthermore, miRNA expression profiling revealed an up-regulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499 and hsa-let-7f upon in vitro propagation. Our studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards. This process includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of MSC preparations.

Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27

Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27kip1 with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2–Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad–Max complexes and de‐repressed by Myc via a single highly conserved E‐box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase‐dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc‐dependent dissociation of p27 from Cdk2 and Myc‐induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2‐deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.

Differential regulation of c-Jun by ERK and JNK during PC12 cell differentiation.

The two MAP kinases JNK and ERK direct distinct cellular activities even though they share a number of common substrates, including several transcription factors. Here we have compared JNK and ERK signalling during PC12 cell differentiation and investigated how activation of c‐Jun by the MAPKs contributes to this cellular response. Exposure to nerve growth factor, or expression of constitutively active MEK1—two treatments which cause differentiation of PC12 cells into a neuronal phenotype—result in activation of ERK‐type MAP kinases and phosphorylation of c‐Jun on several sites including Ser63 and Ser73. Constitutively activated c‐Jun, which mimics the MAPK‐phosphorylated form of the protein, can induce neuronal differentiation of PC12 cells independently of upstream signals. Conversely, expression of dominant‐negative c‐JunbZIP prevents neurite outgrowth induced by activated MEK1. Activation of MEKK1, which stimulates the JNK pathway, is not sufficient for PC12 cell differentiation but can induce apoptosis. However, neurite outgrowth is triggered when c‐Jun is co‐expressed with activated MEKK1 or SEK1. Consistently, MEK‐induced ERK activation in PC12 cells induces c‐Jun expression, while JNK signalling does not. Therefore, dual input of expression and phosphorylation of c‐Jun provided by the ERK pathway is required to direct neuronal differentiation in PC12 cells.

Structure and dynamics of human interphase chromosome territories in vivo

Abstract A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5–10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture.

VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma.

Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31+ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm.

Importin β‐type transport receptors mediate the vast majority of transport pathways between cell nucleus and cytoplasm. We identify here the translation elongation factor 1A (eEF1A) as the predominant nuclear export substrate of RanBP21/exportin 5 (Exp5). This cargo–exportin interaction is rather un usual in that eEF1A binds the exportin not directly, but instead via aminoacylated tRNAs. Exp5 thus represents the second directly RNA‐binding exportin and mediates tRNA export in parallel with exportin‐t. It was suggested recently that 10–15% of the cellular translation would occur in the nucleus. Our data rule out such a scenario and instead suggest that nuclear translation is actively suppressed by the nuclear export machinery. We found that the vast majority of translation initiation factors (eIF2, eIF2B, eIF3, eIF4A1, eIF5 and eIF5B), all three elongation factors (eEF1A, eEF1B and eEF2) and the termination factor eRF1 are strictly excluded from nuclei. Besides Exp5 and importin 13, CRM1 and as yet unidentified exportins also contribute to the depletion of translation factors from nuclei.

Cdk2-dependent phosphorylation of p27 facilitates its Myc-induced release from cyclin E/cdk2 complexes

Activation of Myc triggers a rapid induction of cyclin E/cdk2 kinase activity and degradation of p27. Overt degradation of p27 is preceded by a specific dissociation of p27 from cyclin E/cdk2, but not from cyclin D/cdk4 complexes. We now show that cyclin E/cdk2 phosphorylates p27 at a carboxy-terminal threonine residue (T187) in vitro; mutation of this residue to valine stabilises cyclin E/cdk2 complexes. This reaction is not significantly inhibited by high concentrations of p27, suggesting that cdk2 bound to p27 is catalytically active. In vivo, p27 bound to cyclins E and A, but not to D-type cyclins is phosphorylated. Myc-induced release of p27 from cdk2 requires cdk2 kinase activity and is delayed in a T187V mutant of p27. After induction of Myc, p27 phosphorylated at threonine 187 transiently accumulates in a non cdk2 bound form. Our data suggest a mechanism in which p27 is released from cyclin E/cdk2 upon phosphorylation; in Myc-transformed cells, release is efficient as phosphorylated p27 is transiently bound in a non-cdk2 containing complex and subsequently degraded.

Trimodal Cancer Treatment: Beneficial Effects of Combined Antiangiogenesis, Radiation, and Chemotherapy

It has been suggested that chemotherapy and radiotherapy could favorably be combined with antiangiogenesis in dual anticancer strategy combinations. Here we investigate the effects of a trimodal strategy consisting of all three therapy approaches administered concurrently. We found that in vitro and in vivo, the antiendothelial and antitumor effects of the triple therapy combination consisting of SU11657 (a multitargeted small molecule inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases), Pemetrexed (a multitargeted folate antimetabolite), and ionizing radiation were superior to all single and dual combinations. The superior effects in human umbilical vein endothelial cells and tumor cells (A431) were evident in cell proliferation, migration, tube formation, clonogenic survival, and apoptosis assays (sub-G1 and caspase-3 assessment). Exploring potential effects on cell survival signaling, we found that radiation and chemotherapy induced endothelial cell Akt phosphorylation, but SU11657 could attenuate this process in vitro and in vivo in A431 human tumor xenografts growing s.c. on BALB/c nu/nu mice. Triple therapy further decreased tumor cell proliferation (Ki-67 index) and vessel count (CD31 staining), and induced greater tumor growth delay versus all other therapy regimens without increasing apparent toxicity. When testing different treatment schedules for the A431 tumor, we found that the regimen with radiotherapy (7.5 Gy single dose), given after the institution of SU11657 treatment, was more effective than radiotherapy preceding SU11657 treatment. Accordingly, we found that SU11657 markedly reduced intratumoral interstitial fluid pressure from 8.8 +/- 2.6 to 4.2 +/- 1.5 mm Hg after 1 day. Likewise, quantitative T2-weighed magnetic resonance imaging measurements showed that SU11657-treated mice had reduced intratumoral edema. Our data indicates that inhibition of Akt signaling by antiangiogenic treatment with SU11657 may result in: (a) normalization of tumor blood vessels that cause prerequisite physiologic conditions for subsequent radio/chemotherapy, and (b) direct resensitization of endothelial cells to radio/chemotherapy. We conclude that trimodal cancer therapy combining antiangiogenesis, chemotherapy, and radiotherapy has beneficial molecular and physiologic effects to emerge as a clinically relevant antitumor strategy.

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum‐deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E‐ and cyclin D1‐dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin‐dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum‐starved cells. Further, in isoleucine‐deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N‐succinimidyl ester (CFSE). Co‐culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34+CD38− fraction. Without co‐culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up‐regulated after some cell divisions and then diminished in fast proliferating cells. Co‐culture with MSC maintained a primitive immunophenotype (CD34+, CD133+ and CD38−) for more population doublings, whereas up‐regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen‐activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long‐term culture initiating cells. siRNA knockdown of N‐cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self‐renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.