Abstract Surface topography impacts on cell growth and differentiation, but it is not trivial to generate defined surface structures and to assess the relevance of specific topographic parameters. In ...this study, we have systematically compared in vitro differentiation of mesenchymal stem cells (MSCs) on a variety of groove/ridge structures. Micro- and nano-patterns were generated in polyimide using reactive ion etching or multi beam laser interference, respectively. These structures affected cell spreading and orientation of human MSCs, which was also reflected in focal adhesions morphology and size. Time-lapse demonstrated directed migration parallel to the nano-patterns. Overall, surface patterns clearly enhanced differentiation of MSCs towards specific lineages: 15 μm ridges increased adipogenic differentiation whereas 2 μm ridges enhanced osteogenic differentiation. Notably, nano-patterns with a periodicity of 650 nm increased differentiation towards both osteogenic and adipogenic lineages. However, in absence of differentiation media surface structures did neither induce differentiation, nor lineage-specific gene expression changes. Furthermore, nanostructures did not affect the YAP/TAZ complex, which is activated by substrate stiffness. Our results provide further insight into how structuring of tailored biomaterials and implant interfaces – e.g. by multi beam laser interference in sub-micrometer scale – do not induce differentiation of MSCs per se , but support their directed differentiation.
Neonatal sepsis is characterized by hyperinflammation causing enhanced morbidity and mortality compared to adults. This suggests differences in the response towards invading threats. Here we ...investigate activated cord blood macrophages (CBMΦ) in comparison to adult macrophages (PBMΦ), indicating incomplete interferon gamma (IFN-γ) and interleukin 10 (IL-10)-induced activation of CBMΦ. CBMΦ show reduced expression of phagocytosis receptors and cytokine expression in addition to altered energy metabolism. In particular, IFN-γ as well as IL-10-activated CBMΦ completely fail to increase glycolysis and furthermore show reduced activation of the mTOR pathway, which is important for survival in sepsis. MTOR inhibition by rapamycin equalizes cytokine production in CBMΦ and PBMΦ. Finally, incubation of PBMΦ with cord blood serum or S100A8/A9, which is highly expressed in neonates, suppresses mTOR activation, prevents glycolysis and the expression of an PBMΦ phenotype. Thus, a metabolic alteration is apparent in CBMΦ, which might be dependent on S100A8/A9 expression.
Apoptosis is a pivotal process in embryogenesis and postnatal cell homeostasis and involves the shedding of membranous microvesicles termed apoptotic bodies. In response to tissue damage, the CXC ...chemokine CXCL12 and its receptor CXCR4 counteract apoptosis and recruit progenitor cells. Here, we show that endothelial cell-derived apoptotic bodies are generated during atherosclerosis and convey paracrine alarm signals to recipient vascular cells that trigger the production of CXCL12. CXCL12 production was mediated by microRNA-126 (miR-126), which was enriched in apoptotic bodies and repressed the function of regulator of G protein (heterotrimeric guanosine triphosphate-binding protein) signaling 16, an inhibitor of G protein-coupled receptor (GPCR) signaling. This enabled CXCR4, a GPCR, to trigger an autoregulatory feedback loop that increased the production of CXCL12. Administration of apoptotic bodies or miR-126 limited atherosclerosis, promoted the incorporation of Sca-1+ progenitor cells, and conferred features of plaque stability on different mouse models of atherosclerosis. This study highlights functions of microRNAs in health and disease that may extend to the recruitment of progenitor cells during other forms of tissue repair or homeostasis.
Recent findings suggested a benefit of anti-EGFR therapy for basal-like muscle-invasive bladder cancer (MIBC). However, the impact on bladder cancer with substantial squamous differentiation ...(Sq-BLCA) and especially pure squamous cell carcinoma (SCC) remains unknown. Therefore, we comprehensively characterized pure and mixed Sq-BLCA (n = 125) on genetic and protein expression level, and performed functional pathway and drug-response analyses with cell line models and isolated primary SCC (p-SCC) cells of the human urinary bladder. We identified abundant EGFR expression in 95% of Sq-BLCA without evidence for activating EGFR mutations. Both SCaBER and p-SCC cells were sensitive to EGFR tyrosine kinase inhibitors (TKIs: erlotinib and gefitinib). Combined treatment with anti-EGFR TKIs and varying chemotherapeutics led to a concentration-dependent synergism in SCC cells according to the Chou-Talalay method. In addition, the siRNA knockdown of EGFR impaired SCaBER viability suggesting a putative "Achilles heel" of Sq-BLCA. The observed effects seem Sq-BLCA-specific since non-basal urothelial cancer cells were characterized by poor TKI sensitivity associated with a short-term feedback response potentially attenuating anti-tumor activity. Hence, our findings give further insights into a crucial, Sq-BLCA-specific role of the ERBB signaling pathway proposing improved effectiveness of anti-EGFR based regimens in combination with chemotherapeutics in squamous bladder cancers with wild-type EGFR-overexpression.
The differentiation process of embryonic stem cells is a comprehensive process regulated by a variety of factors in response to stimulus. Studies of this process can be focused on cell biology as ...well as on molecular biology level. In this paper we identified the co-regulation of molecular regulators and their interactions during cardiomyocyte specific differentiation of mouse embryonic stem cells based on parallel genome wide transcriptome analyses of mRNA and microRNA. Differentially expressed mRNAs and microRNAs were identified according to their expression profiles. Subsequently, a primary network was generated by using our genome wide profiling data, predicted sequence target information of transcription factors and microRNAs from various sources, validated microRNA target information, as well as tissue specific transcription factor binding information. Considering only validated microRNA target information and tissue specific transcription factor binding information secondary regulatory networks were extracted from the primary network to identify basic regulatory elements. Eight types of 3-node patterns were identified in this network. Novel regulatory modules, like Meis1 - Gata6 and miR-21/24 - Zic3, were discovered with high plausibility by this procedure without complicated and time-consuming experimental processes.
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•Genome wide profiling of mRNA and miRNA expression in the cardiomyocyte specific differentiation process of mESCs•Regulatory networks involving TFs and miRNAs were inferred based on expression profile and tissue specific target information.•Novel regulatory patterns were enclosed in the networks.
Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between ...studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns—particularly of DNAm patterns associated with tissue type and aging.
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•MSC-derived iPSCs are redifferentiated toward MSCs in a one-step protocol•Gene expression profiles of iPS-MSCs closely resemble those of primary MSCs•DNA methylation (DNAm) profiles of iPS-MSCs lag behind those of MSCs•Age-related and tissue-specific DNAm patterns remain erased in iPS-MSCs
Wagner and colleagues redifferentiated MSC-derived iPSCs toward MSCs. These iPS-MSCs reveal similar morphology, immunophenotype, and in vitro differentiation potential as primary MSCs, but they were impaired in suppressing T cell proliferation. Furthermore, there are marked differences in DNA methylation profiles that can, at least partially, be attributed to persistent reset of tissue-specific and age-related DNA methylation changes.
The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into ...specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair.
Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source.
Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome.
Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin gene. The neuropathology of HD is characterized by the ...decline of a specific neuronal population within the brain, the striatal medium spiny neurons (MSNs). The origins of this extreme vulnerability remain unknown. Human induced pluripotent stem cell (hiPS cell)-derived MSNs represent a powerful tool to study this genetic disease. However, the differentiation protocols published so far show a high heterogeneity of neuronal populations in vitro. Here, we compared two previously published protocols to obtain hiPS cell-derived striatal neurons from both healthy donors and HD patients. Patch-clamp experiments, immunostaining and RT-qPCR were performed to characterize the neurons in culture. While the neurons were mature enough to fire action potentials, a majority failed to express markers typical for MSNs. Voltage-clamp experiments on voltage-gated sodium (Nav) channels revealed a large variability between the two differentiation protocols. Action potential analysis did not reveal changes induced by the HD mutation. This study attempts to demonstrate the current challenges in reproducing data of previously published differentiation protocols and in generating hiPS cell-derived striatal MSNs to model a genetic neurodegenerative disorder in vitro.
Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), ...and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.
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