The Decellularized Cell-Derived Extracellular Matrix Enhances the Paracrine Function of Human Mesenchymal Stromal/Stem Cells.

The mesenchymal stromal/stem cells (MSCs) are known to secrete pleiotropic paracrine factors, contributing to tissue regeneration. This unique ability makes MSCs promising therapeutic tools for many diseases, including even those that were previously untreatable. Thus, the development of preconditioning approaches aimed at enhancing the paracrine function of MSCs attracts great interest. In the present work, we studied how the extracellular matrix, the essential part of the native tissue microenvironment, affects the secretory capacity of MSCs of various origins. The MSC-derived decellularized extracellular matrix (dECM), used as the cell culture substrate, triggered strong upregulation of FGF-2, MMP-1, HGF, GRO-α, GRO-ß, CXCL-5, CXCL-6, IL-6, IL-8, G-CSF and MCP-1. Functional in vitro tests revealed that conditioned media derived from MSCs cultured on dECM significantly improved 3T3 fibroblast and HaCaT keratinocyte scratch wound healing, stimulated THP-1 monocyte migration and promoted capillary-like HUVEC-based tube formation compared to conditioned media from MSCs grown on plastic. In addition, we found that FAK inhibition promoted dECM-induced upregulation of paracrine factors, suggesting that this kinase participates in the MSCs' paracrine response to dECM. Together, these findings demonstrate that dECM provides cues that considerably enhance the secretory function of MSCs. Thus, dECM usage as a cell culture substrate alone or in combination with a FAK inhibitor may be viewed as a novel MSC preconditioning technique.

p53 Affects Zeb1 Interactome of Breast Cancer Stem Cells.

P53 is a critical tumor suppressor that protects the integrity of genome and prevents cells from malignant transformation, including metastases. One of the driving forces behind the onset of metastases is the epithelial to mesenchymal transition (EMT) program. Zeb1 is one of the key transcription factors that govern EMT (TF-EMT). Therefore, the interaction and mutual influence of p53 and Zeb1 plays a critical role in carcinogenesis. Another important feature of tumors is their heterogeneity mediated by the presence of so-called cancer stem cells (CSCs). To this end, we have developed a novel fluorescent reporter-based approach to enrich the population of CSCs in MCF7 cells with inducible expression of Zeb1. Using these engineered cell lines, we studied the effect of p53 on Zeb1 interactomes isolated from both CSCs and regular cancer cells. By employing co-immunoprecipitations followed by mass spectrometry, we found that the composition of Zeb1 interactome was affected not only by the p53 status but also by the level of Oct4/Sox2 expression, indicating that stemness likely affects the specificity of Zeb1 interactions. This study, together with other proteomic studies of TF-EMT interactomes, provides a framework for future molecular analyses of biological functions of Zeb1 at all stages of oncogenesis.

Targeting Brain Tumors with Mesenchymal Stem Cells in the Experimental Model of the Orthotopic Glioblastoma in Rats.

Despite multimodal approaches for the treatment of multiforme glioblastoma (GBM) advances in outcome have been very modest indicating the necessity of novel diagnostic and therapeutic strategies. Currently, mesenchymal stem cells (MSCs) represent a promising platform for cell-based cancer therapies because of their tumor-tropism, low immunogenicity, easy accessibility, isolation procedure, and culturing. In the present study, we assessed the tumor-tropism and biodistribution of the superparamagnetic iron oxide nanoparticle (SPION)-labeled MSCs in the orthotopic model of C6 glioblastoma in Wistar rats. As shown in in vitro studies employing confocal microscopy, high-content quantitative image cytometer, and xCelligence system MSCs exhibit a high migratory capacity towards C6 glioblastoma cells. Intravenous administration of SPION-labeled MSCs in vivo resulted in intratumoral accumulation of the tagged cells in the tumor tissues that in turn significantly enhanced the contrast of the tumor when high-field magnetic resonance imaging was performed. Subsequent biodistribution studies employing highly sensitive nonlinear magnetic response measurements (NLR-M2) supported by histological analysis confirm the retention of MSCs in the glioblastoma. In conclusion, MSCs due to their tumor-tropism could be employed as a drug-delivery platform for future theranostic approaches.

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

BACKGROUND: Cancer stem cells' (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs' subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. METHODS: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). RESULTS: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. CONCLUSION: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.

Paracrine senescence of human endometrial mesenchymal stem cells: a role for the insulin-like growth factor binding protein 3.

Stress-induced premature cell senescence is well recognized to be accompanied by emerging the senescence-associated secretory phenotype (SASP). Secreted SASP factors can promote the senescence of normal neighboring cells through autocrine/paracrine pathways and regulate the senescence response, as well. Regarding human endometrium-derived mesenchymal stem cells (MESCs), the SASP regulation mechanisms as well as paracrine activity of senescent cells have not been studied yet. Here, we examined the role of insulin-like growth factor binding protein 3 (IGFBP3) in the paracrine senescence induction in young MESCs. The H2O2-induced premature senescence of MESCs led to increased IGFBP3 in conditioned media (CM). The inhibitory analysis of both MAPK and PI3K signaling pathways showed that IGFBP3 releasing from senescent cells is mainly regulated by PI3K/Akt pathway activity. IGFBP3 appears to be an important senescence-mediating factor as its immunodepletion from the senescent CM weakened the pro-senescent effect of CM on young MESCs and promoted their growth. In contrast, young MESCs acquired the senescence phenotype in response to simultaneous addition of recombinant IGFBP3 (rIGFBP3). The mechanism of extracellular IGFBP3 internalization was also revealed. The present study is the first to demonstrate a significant role of extracellular IGFBP3 in paracrine senescence induction of young MESCs.

Primary cultures of human colon cancer as a model to study cancer stem cells.

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes.

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, ß- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.

Protecting a transgene expression from the HAC-based vector by different chromatin insulators.

Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors.

Derivation, characterization, and stable transfection of induced pluripotent stem cells from Fischer344 rats.

The rat represents an important animal model that, in many respects, is superior to the mouse for dissecting behavioral, cardiovascular and other physiological pathologies relevant to humans. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here, we report an improved lentivirus-based hit-and-run riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We demonstrate that the excision of proviruses does not affect either the karyotype or the differentiation ability of these cells. We show that the established riPS cells are readily amenable to genetic manipulations such as stable electroporation. Finally, we propose a genetic tool for an improvement of riPS cell quality in culture. These data may prompt iPS cell-based gene targeting in rat as well as the development of iPS cell-based therapies using disease models established in this species.

The transcription factor TCFAP2C/AP-2gamma cooperates with CDX2 to maintain trophectoderm formation.

In mammals, cell lineage specification is established at the blastocyst stage. At this stage, transcription factor Cdx2 represses pluripotency genes, thus promoting extraembryonic trophoblast fate. Recently, transcription factor Gata3 was shown to act in a parallel pathway in promoting trophoblast cell fate, suggesting that there are more factors working in the trophoblast lineage. Here, we report that the transcription factor Tcfap2c is expressed at a high level in the trophectoderm and is able to induce trophoblast fate in embryonic stem cells. Trophoblast fate induced by Tcfap2c does not require Cdx2 and vice versa, suggesting that the molecules act in alternative pathways. However, both Tcfap2c and Cdx2 are required for the upregulation of Elf5, a marker of trophoblast stem cell maintenance, suggesting that both factors are required for stable trophoblast induction. Tcfap2c-induced trophoblast-like cells are stable in long-term culture, indicating that they are capable of self-renewal. Tcfap2c-controlled trophoblast maintenance involves the induction of Cdx2 and the repression of the pluripotency factor Nanog. Tcfap2c-induced trophoblast-like cells differentiate to trophoblast derivatives in vitro and contribute to the trophectoderm in blastocysts in vivo. Taken together, these observations suggest that Tcfap2c and Cdx2 cooperate to override the pluripotency program and establish the extraembryonic trophoblast maintenance program in murine embryos.

PIAS proteins as repressors of Oct4 function.

The POU domain transcription factor Oct4 plays essential functions in the maintenance of pluripotent embryonic and germ cells of mammals. Molecular mechanisms of Oct4 action remain poorly understood. To isolate modulators of Oct4 activity, we performed a yeast two-hybrid screen with the Oct4 POU domain as a bait and isolated PIASy as an Oct4-interacting protein. Oct4 and PIASy interact in vivo via their POU domain and SAP-domain-containing N terminus, respectively. PIASy does not enhance Oct4 sumoylation but acts as a potent inhibitor of Oct4-mediated transcriptional activation, sequestering Oct4 protein from the vicinity of Cajal bodies and splicing speckles to the nuclear periphery. These modes of PIASy action are uncoupled from its sumoylation activity. Other PIAS family members, PIAS1 and PIAS3, can also interact with Oct4 in vivo and target Oct4 to the nuclear periphery, depending on cellular context. We propose that Oct4 inhibition, mediated by this new class of transcriptional partners, might be instrumental during mammalian development.

Lentivirus as a tool for lineage-specific gene manipulations.

The trophectoderm (TE) of blastocysts, the first epithelium established in mammalian development, (1) plays signaling, supportive, and patterning functions during preimplantation development, (2) ensures embryo implantation into the uterine wall, and (3) gives rise to extraembryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust nonpermeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics.

The caudal-related protein cdx2 promotes trophoblast differentiation of mouse embryonic stem cells.

Besides holding great promise in clinics, embryonic stem (ES) cells represent a valuable tool for studying regulation of early developmental processes, such as cell differentiation in preimplantation embryos. The caudal-related homeobox protein Cdx2 is a transcriptional regulator essential for trophoblast lineage, functioning as early as implantation. Using an inducible system, we show that gain of Cdx2 function in ES cells triggers trophoblast-like morphological differentiation, accompanied by ploidy increase, onset of expression of trophoblast-specific markers, and loss of pluripotency-associated gene expression. These data provide an insight into the genetic network that controls lineage specification and functioning in early mammalian development.

Oct4 is required for primordial germ cell survival.

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.

Engrailed cooperates with extradenticle and homothorax to repress target genes in Drosophila.

Engrailed is a key transcriptional regulator in the nervous system and in the maintenance of developmental boundaries in Drosophila, and its vertebrate homologs regulate brain and limb development. Here, we show that the functions of both of the Hox cofactors Extradenticle and Homothorax play essential roles in repression by Engrailed. Mutations that remove either of them abrogate the ability of Engrailed to repress its target genes in embryos, both cofactors interact directly with Engrailed, and both stimulate repression by Engrailed in cultured cells. We suggest a model in which Engrailed, Extradenticle and Homothorax function as a complex to repress Engrailed target genes. These studies expand the functional requirements for extradenticle and homothorax beyond the Hox proteins to a larger family of non-Hox homeodomain proteins.

Structure of nuclear-localized cox3 genes in Chlamydomonas reinhardtii and in its colorless close relative Polytomella sp.

Several chlorophyte algae do not have the cox3 gene, encoding subunit III of cytochrome c oxidase, in their mitochondrial genomes. The cox3 gene is nuclear-encoded in the photosynthetic alga Chlamydomonas reinhardtii and in the colorless alga Polytomella sp. In this work, the genomic sequences of the cox3 genes of these two closely related algae are reported. The cox3 genes of both C. reinhardtii and Polytomella sp. contain four introns in the region encoding the putative mitochondrial-targeting sequences. These four introns show low sequence identities, but their locations are conserved between these species. The cox3 gene of C. reinhardtii has five additional introns in the region encoding the mature subunit III of cytochrome c oxidase. Sequence analysis of intron 6 of the cox3 gene of C. reinhardtii revealed similarity with two sequence elements present in introns of several other nuclear genes from this green alga. In the majority of the genes, these conserved sequences are located either near the 3' end or near the 5' end of the introns. Based on these data, we propose that the colorless genus Polytomella separated from C. reinhardtii after the cox3 gene was transferred to the nucleus. The data also support the evolutionary hypothesis of a recent acquisition of introns in C. reinhardtii.