Properties Of Embryonic Stem Cells Research Articles

ATG5 attenuates inflammatory signaling in mouse embryonic stem cells to control differentiation

Attenuated inflammatory response is a property of embryonic stem cells (ESCs). However, the underlying mechanisms are unclear. Moreover, whether the attenuated inflammatory status is involved in ESC differentiation is also unknown. Here, we found that autophagy-related protein ATG5 is essential for both attenuated inflammatory response and differentiation of mouse ESCs and that attenuation of inflammatory signaling is required for mouse ESC differentiation. Mechanistically, ATG5 recruits FBXW7 to promote ubiquitination and proteasome-mediated degradation of β-TrCP1, resulting in the inhibition of nuclear factor κB (NF-κB) signaling and inflammatory response. Moreover, differentiation defects observed in ATG5-depleted mouse ESCs are due to β-TrCP1 accumulation and hyperactivation of NF-κB signaling, as loss of β-TrCP1 and inhibition of NF-κB signaling rescued the differentiation defects. Therefore, this study reveals a previously uncharacterized mechanism maintaining the attenuated inflammatory response in mouse ESCs and further expands the understanding of the biological roles of ATG5.

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.

TWIST1 Plays Role in Expression of Stemness State Markers in ESCC

Stemness markers play critical roles in the maintenance of key properties of embryonic stem cells (ESCs), including the pluripotency, stemness state, and self-renewal capacities, as well as cell fate decision. Some of these features are present in cancer stem cells (CSCs). TWIST1, as a bHLH transcription factor oncogene, is involved in the epithelial-mesenchymal transition (EMT) process in both embryonic and cancer development. Our aim in this study was to investigate the functional correlation between TWIST1 and the involved genes in the process of CSCs self-renewal in human esophageal squamous cell carcinoma (ESCC) line KYSE-30. TWIST1 overexpression was enforced in the ESCC KYSE-30 cells using retroviral vector containing the specific pruf-IRES-GFP-hTWIST1 sequence. Following RNA extraction and cDNA synthesis, the mRNA expression profile of TWIST1 and the stem cell markers, including BMI1, CRIPTO1, DPPA2, KLF4, SOX2, NANOG, and MSI1, were assessed using relative comparative real-time PCR. Ectopic expression of TWIST1 in KYSE-30 cells resulted in an increased expression of TWIST1 compared to control GFP cells by nearly 9-fold. Transduction of TWIST1-retroviral particles caused a significant enhancement in BMI1, CRIPTO1, DPPA2, KLF4, and SOX2 mRNA expression, approximately 4.5-, 3.2-, 5.5-, 3.5-, and 3.7-folds, respectively, whereas this increased TWIST1 expression caused no change in the mRNA expression of NANOG and MSI1 genes. TWIST1 gene ectopic expression in KYSE-30 cells enhanced the level of cancer stem cell markers' mRNA expression. These results may emphasize the role of TWIST1 in the self-renewal process and may corroborate the involvement of TWIST1 in the stemness state capacity of ESCC cell line KYSE-30, as well as its potential as a therapeutic target.

The synthetic molecule stauprimide impairs cell growth and migration in triple-negative breast cancer

Stauprimide, a semi-synthetic derivative of staurosporine, is known mainly for its potent differentiation-enhancing properties in embryonic stem cells. Here, we studied the effects of stauprimide in cell growth and migration of triple-negative breast cancer cells in vitro, evaluating its potential antitumoral activity in an orthotopic mouse model of breast cancer in vivo. Our results from survival curves, EdU incorporation, cell cycle analysis and annexin-V detection in MDA-MB-231 cells indicated that stauprimide inhibited cell proliferation, arresting cell cycle in G2/M without induction of apoptosis. A decrease in the migratory capability of MDA-MB-231 was also assessed in response to stauprimide. In this work we pointed to a mechanism of action of stauprimide involving the modulation of ERK1/2, Akt and p38 MAPK signalling pathways, and the downregulation of MYC in MDA-MB-231 cells. In addition, orthotopic MDA-MB-231 xenograft and 4T1 syngeneic models suggested an effect of stauprimide in vivo, increasing the necrotic core of tumors and reducing metastasis in lung and liver of mice. Together, our results point to the promising role of stauprimide as a putative therapeutic agent in triple-negative breast cancer.

Structural studies of SALL family protein zinc finger cluster domains in complex with DNA reveal preferential binding to an AATA tetranucleotide motif

The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different dsDNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common preference for the AATA tetranucleotide shared by ZFC4 of SALL1, SALL3, and SALL4. Furthermore, our cell biology experiments demonstrate that the DNA-binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family proteins in controlling cell fate via preferential targeting to AT-rich sites within genomic DNA during cell differentiation.

Alternative In Vivo Models to Study Teratoma.

Embryonic stem cells give rise to teratomas when injected in vivo in experimental animal models. The characterization, the manipulation, and the breaking off of this specific characteristic are doubtlessly the last frontier for the applications of stem cells in translational medicine. Moreover, the urgency to adapt to new scientific demands drives the researcher to find alternative and faster models for testing the teratogenic properties of embryonic stem cells. Here, we compare the emerging model of the chick embryo chorioallantoic membrane (CAM) to the murine model, which represents the gold standard procedure for teratogenesis.

P21-Activated Kinase 4 Pak4 Maintains Embryonic Stem Cell Pluripotency via Akt Activation.

Exploiting the pluripotent properties of embryonic stem cells (ESCs) holds great promise for regenerative medicine. Nevertheless, directing ESC differentiation into specialized cell lineages requires intricate control governed by both intrinsic and extrinsic factors along with the actions of specific signaling networks. Here, we reveal the involvement of the p21-activated kinase 4 (Pak4), a serine/threonine kinase, in sustaining murine ESC (mESC) pluripotency. Pak4 is highly expressed in R1 ESC cells compared with embryonic fibroblast cells and its expression is progressively decreased during differentiation. Manipulations using knockdown and overexpression demonstrated a positive relationship between Pak4 expression and the clonogenic potential of mESCs. Moreover, ectopic Pak4 expression increases reprogramming efficiency of Oct4-Klf4-Sox2-Myc-induced pluripotent stem cells (iPSCs) whereas Pak4-knockdown iPSCs were largely incapable of generating teratomas containing mesodermal, ectodermal and endodermal tissues, indicative of a failure in differentiation. We further establish that Pak4 expression in mESCs is transcriptionally driven by the core pluripotency factor Nanog which recognizes specific binding motifs in the Pak4 proximal promoter region. In turn, the increased levels of Pak4 in mESCs fundamentally act as an upstream activator of the Akt pathway. Pak4 directly binds to and phosphorylates Akt at Ser473 with the resulting Akt activation shown to attenuate downstream GSK3β signaling. Thus, our findings indicate that the Nanog-Pak4-Akt signaling axis is essential for maintaining mESC self-renewal potential with further importance shown during somatic cell reprogramming where Pak4 appears indispensable for multi-lineage specification.

Evaluation of Cytocompatibility as Assessed by Genomic Stability of Canine induced Pluripotent Stem Cells Propagated on Carbon Nanotube Substrates

Background: Induced pluripotent stem cells (iPSC) are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed to an embryonic stem (ES) cell-like state through the forced expression of genes and factors important for maintaining the defined properties of ES cells. So far there are very few experiments that have been able to prove that nanomaterial-based scaffold can cultivate and maintain the iPSC as an alternative to feeder-free maintenance of iPSC. Methods: The present experiment has given us fundamental information on ex vivo canine iPSC behavior on -OH functionalized single and multi-walled carbon nanotube (CNT) scaffold. Here in we evaluated the cytocompatibility of iPSC cultured on MEF feeder, OH-SWCNT and OH-MWCNT. Result: We have seen very wonderful growth of ciPSC on CNTs similar to feeder. The cells were positive for alkaline phosphatase staining and expressed pluripotent markers. Cytotoxicity analysis revealed that -OH functionalized CNTs provide a milieu of low cytotoxicity. With this test we can interpret that -OH functionalized CNT can be used as xeno-free substrate to support the maintenance of iPSC in an undifferentiated state.

The MuvB complex safeguards embryonic stem cell identity through regulation of the cell cycle machinery

Increasing evidences indicate that unlimited capacity for self-renewal and pluripotency, two unique properties of embryonic stem cells (ESCs), are intrinsically linked to cell cycle control. However, the precise mechanisms coordinating cell fate decisions and cell cycle regulation remain to be fully explored. Here, using CRISPR/Cas9-mediated genome editing, we show that in ESCs, deficiency of components of the cell cycle regulatory MuvB complex Lin54 or Lin52, but not Lin9 or Lin37, triggers G2/M arrest, loss of pluripotency, and spontaneous differentiation. Further dissection of these phenotypes demonstrated that this cell cycle arrest is accompanied by the gradual activation of mesoendodermal lineage-specifying genes. Strikingly, the abnormalities observed in Lin54-null ESCs were partially but significantly rescued by ectopic coexpression of genes encoding G2/M proteins Cyclin B1 and Cdk1. Thus, our study provides new insights into the mechanisms by which the MuvB complex determines cell fate through regulation of the cell cycle machinery.

LIS1, an RNA-binding protein, regulates the physical properties of embryonic stem cells

LIS1 (Lissencephaly-1) was identified as the first gene involved in a neuronal migration disorder. The dosage of the LIS1 protein is important for proper brain development, whereas the absence of the protein results in early embryonic lethality. LIS1 interacts with the molecular motor cytoplasmic dynein, and this interaction impacts multiple activities. In this study, we set out to investigate the functions of LIS1 in embryonic stem cells. We found that LIS1 interacts with numerous RNA-binding proteins, including the Argonaute complex, and that LIS1 binds directly to RNA.

Generation and Analysis of Mosaic Spinal Cord Organoids Derived from Mouse Embryonic Stem Cells.

Three central cell populations play roles in morphogen action, the cells that produce, the cells that distribute, and the cells that respond to the morphogen. Taking advantage of the properties of embryonic stem cell to aggregate and readily differentiate into neural progenitor tissue, we describe an approach using genetically modified murine stem cell lines to individually address the contribution of these cells in the establishment and response to a morphogenetic gradient in mosaic spinal cord organoids.

Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas

Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.

Generation of Systemic Lupus Erythematosus Patient-Derived Induced Pluripotent Stem Cells from Blood.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by the production of multiple autoimmune antibodies and potentially involves any organ or tissue with a broad range of clinical manifestations. Conventional therapy still utilizes glucocorticoids and immunosuppressants. However, some patients show inadequate responses to glucocorticoids and immunosuppression, which may induce secondary immune dysfunction and severe infection as well as lead to an increased tumor risk. The lack of in vitro models has hampered progress in understanding and treating SLE. Patient-derived induced pluripotent stem cells (iPSCs) may provide a unique opportunity for modeling in vitro diseases as well as a platform for drug screening in individual patients. We isolated peripheral blood mononuclear cells from blood to explore the establishment of an in vitro model platform for SLE and directly purified CD34+ cells and seeded them for expansion. CD34+ cells were forced to express seven pluripotency factors, OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT, through transduction in lentiviral vectors. The morphological characteristics of induced pluripotent stem-like cells, such as prominent nucleoli and a high nucleus-to-cytoplasm ratio, were observed. The pluripotency of established SLE patient-derived iPSCs was confirmed by the expression of embryonic stem cell (ESC) markers and the ability of cells to differentiate into multiple cell lines. SLE patient-derived iPSCs exhibited human ESC properties, including morphology; growth characteristics; expression of pluripotency, genes, and surface markers; and teratoma formation. In conclusion, we generated SLE patient-derived iPSCs and validated their pluripotency. This study is a first but critical step that can provide a model platform for research aimed at understanding the SLE mechanism, which may lead to the discovery of new targets or compounds for the treatment of this disease.

Роль транскрипционных факторов коктейля Яманаки (OCT4, sOX2, KLF4, c-Myc) в дифференцировке соматических клеток, их злокачественной трансформации и прогрессировании опухолей

The paper reviews the role of transcription factors that are part of the Yamanaka cocktail (OCT4, SOX2, KLF4, and c-Myc) in the mechanisms of tumor stem cell (TSC) differentiation (a pool of pluripotent cells), their participation in the malignant transformation of somatic cells, and in tumor progression. Their increased proliferative activity, which determines their malignant potential, was believed to be the main feature of tumor cells. Currently, there is more evidence supporting another concept that explains the continued tumor growth, metastasis, and resistance to therapy of most malignant tumors with a small, rest-ing, and rarely dividing population of pluripotent tumor cells. Year 2006 is considered to be the beginning of a new direction in developmental biology when Japanese researchers K. Takahashi and S. Yamanaka published the results of their studies on the properties of embryonic stem cells and experiments on direct reprogramming of somatic terminally differentiated cells.The research proved that four transcription factors were sufficient to maintain the pluripotent properties of cells, namely Oct4, SOX2, KLF4, and c-Myc, which were later called the Yamanaka cocktail. Such factors as SOX2and Oct4 are at the top of the hierarchy of transcription factors that regulate the pluripotent proper-ties of cells, their differentiation, and dedifferentiation. TSCs represent, albeit a complex, but very promising target for the development of innovative products for the diagnosis of and targeted therapy for neoplasms. From this point of view, the factors of pluripotency, which are parts of the Yamanaka cocktail, could be such promising targets, whose effect will probably be able to suppress or reduce the malignant potential of the most aggressive tumors and even prevent carcinogenic transformation in cases of precancerous pathology. Keywords: Yamanaka cocktail, OCT4, SOX2, KLF4, c-Myc, malignant transformation

Oncofetal Protein SALL4 Is Highly Expressed in Myelodysplastic Syndrome Alongside with NAT10 and P53

Introduction: Myelodysplastic syndrome (MDS) is a group of heterogeneous diseases characterized by cytologic dysplasia and refractory cytopenias as a result of ineffective hematopoiesis. We have reported that oncofetal protein SALL4 can be used as a prognostic biomarker in MDS disease. SALL4 is a transcription factor that is important for development and embryonic stem cell properties. While SALL4 expression is down-regulated or absent in most adult tissues, SALL4 is re-expressed in various cancers. We have previously reported that aberrant expression of SALL4 can be detected in MDS and AML patients. Studies in murine models, as well as in human samples, have demonstrated that SALL4 is essential for leukemic cell survival and that SALL4 transgenic mice develop an MDS-like phenotype prior to transformation to AML, suggesting that SALL4 can be a driver of MDS/AML pathogenesis. However, SALL4 expression status and related pathway in bone marrow (BM) cells and relationship with somatic gene mutations in MDS patients has not been explored. In this study, we evaluated the expression of SALL4 and related factors using single-cell mass cytometry (CyTOF) and NanoString technology in MDS patients alongside with target sequence for MDS-related mutations. Materials and methods: We evaluated the expression of SALL4 using single-cell mass cytometry (CyTOF) utilizing 28 antibodies including surface lineage markers and intracellular proteins, such as p53, ki67, c-myc and pAKT to identify SALL4 expressed cells and related pathway in bone marrow (BM) for 10 MDS patients. SALL4 expression was also analyzed in formalin-fixed paraffin-embedded (FFPE) 14 patient samples utilizing two SALL4 probes designed for NanoString technology alongside with target sequence for MDS-related mutations. One probe only detects expression of SALL4A, which is full length SALL4, while the other probe can detect both SALL4A as well as its splicing variant SALL4B. We examined the expression of a panel of 17 known SALL4 downstream target genes. Results: First, we assessed an expression level of SALL4 using CyTOF in 10 MDS patients as compared with normal BM samples (N=5). We observed that Lin-CD34+CD38+ progenitor cells in 8 out of the 10 MDS patients had higher SALL4 expression levels, while the progenitor cells of normal BM cells did not express SALL4. Similar results were observed in Lin-CD34+CD38- hematopoietic stem cells, CD11b+ mature myeloid cells, CD19+ B cells, CD235+ erythroid cells. Conversely only 3 MDS patient had weak SALL4 expression in CD3+ T cells. Subsequently, we investigated the correlation with p53, ki67, c-myc and pAKT. SALL4 is only correlated with p53 (P=0.005), while others were not. Next, we also assessed an expression level of SALL4A and SALL4A&B in 14 MDS patient bone marrow (BM) samples using Nanostring nCounter. We observed that 12 out of 14 MDS patients had higher SALL4 expression levels as compared with control normal BM samples (N=3). Next, we evaluated whether MDS patients expressed SALL4A, SALL4B, or both. Of the 14 MDS patients, 8 predominantly expressed SALL4A Of these 8 MDS patients, 5 harbored RNA splicing-related mutations, such as SF3B1 (N=4), U2AF1 (N=1). Only 1 out of 7 patients who predominantly expressed SALL4B demonstrated a splicing related mutation. Subsequently, we investigated the correlation between SALL4 and its known downstream targets. An expression of SALL4A is moderately correlated with CBLb (γ=0.53), CDH1 (γ=0.42) and NAT10 (γ=0.41). The expression of SALLA&B is moderately correlated with NAT10 (γ=0.47) and RUNX1 (γ=0.46). NAT10 (N-acetyltransferase 10) has been reported to promote transcription of RNA polymerase I and is a critical regulator of p53 homeostasis. It was identified to be a potential SALL4 downstream target in our early Chip-seq studies. In the isogenic K562 cell lines with SALL4 overexpression, higher levels of NAT10 were observed, suggesting that NAT10 is one of the downstream targets of SALL4. Conclusions: Our study has demonstrated for the first time that 1) SALL4 was expressed in various MDS BM cells confirmed by CyTOF 2) there are SALL4 splicing variants in MDS patients, particularly with SF3B1 mutations 3) a novel SALL4/NAT10/p53 link has been identified in these MDS patients. Future studies on mechanism(s) and biological role(s) of SALL4 splicing variants in MDS are needed. Disclosures No relevant conflicts of interest to declare.

Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases, including Muscular Dystrophies.

Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krüppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close.

Rapid Processing and Drug Evaluation in Glioblastoma Patient-Derived Organoid Models with 4D Bioprinted Arrays

SummaryGlioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.

RNA-Seq analysis reveals pluripotency-associated genes and their interaction networks in human embryonic stem cells

Insight into the key genes of pluripotency in human and their interrelationships is necessary for understanding the underlying mechanism of pluripotency and hence their successful application in regenerative medicine. The recent advances in transcriptomics technologies have created new opportunities to decipher the genes involved in pluripotency, genetic network that governs the unique properties of embryonic stem cells and lineage differentiation mechanisms in a deeper scale. There are a large number of experimental studies on human embryonic stem cells (hESCs) being routinely conducted for unfolding the underlying biology of embryogenesis and their clinical prospects. However, the outcome of these studies often lacks consensus due to differences in samples, experimental techniques and/or analysis protocols. A universal stemness gene list is still lacking. Thus, we aim to identify the pluripotency-associated genes and their interaction network. In this quest, we compared transcriptomic profiles of pluripotent and non-pluripotent samples from diverse cell lines/types generated through RNA-sequencing (RNA-seq). We used a uniform pipeline for the analysis of raw RNA-seq data in order to reduce the amount of variation. Our analysis revealed a consensus set of 498 pluripotency-associated genes and 432 genes as potential pluripotent cell differentiation markers. Furthermore, we predicted 32 genes as “pluripotency critical genes”. These pluripotency critical genes formed a tightly bound co-expression network with small-world architecture. Gene ontology (GO) and pathway enrichment analysis, StemChecker and literature survey confirmed the involvement of the genes in the induction and maintenance of pluripotency, though more experimental studies are required for understanding their molecular mechanisms in human.

Cancer from the perspective of stem cells and misappropriated tissue regeneration mechanisms

Tumorigenesis can be considered as pathologically misappropriated tissue regeneration. In this review we will address some unresolved issues that support this concept. First, we will address the issue of the identity of cancer-initiating cells and the presence of cancer stem cells in growing tumors. We will also ask are there rare and distinct populations of cancer stem cells in established tumor cell lines, or are all of the cells cancer stem cells? Second, the most important clinical problem with cancer is its metastasis, and here a challenging question arises: by employing radio-chemotherapy for tumor treatment, do we unintentionally create a prometastatic microenvironment in collateral organs? Specifically, many factors upregulated in response to radio-chemotherapy-induced injury may attract highly migratory cancer cells that survived initial treatment. Third, what is the contribution of normal circulating stem cells to the growing malignancy? Do circulating normal stem cells recognize a tumor as a hypoxia-damaged tissue that needs vascular and stromal support and thereby contribute to tumor expansion? Fourth, is it reasonable to inhibit only one prometastatic ligand–receptor axis when cancer stem cells express several receptors for several chemotactic factors that may compensate for inhibition of the targeted receptor? Fifth, since most aggressive cancer cells mimic early-development stem cells, which properties of embryonic stem cells are retained in cancer cells? Would it be reasonable to inhibit cancer cell signaling pathways involved in the migration and proliferation of embryonic stem cells? We will also briefly address some new players in cancerogenesis, including extracellular microvesicles, bioactive phospholipids, and extracellular nucleotides.

Generation of induced pluripotent stem cells by a polycistronic lentiviral vector in feeder- and serum- free defined culture.

Induced pluripotent stem cells (iPSCs) have great potentials for regenerative medicine. However, serious concerns such as the use of the viral-mediated reprogramming strategies and exposure of iPSCs to animal products from feeder cells and serum-containing medium have restricted the application of iPSCs in the clinics. Therefore, the generation of iPSCs with minimal viral integrations and in non-animal sourced and serum-free medium is necessary. In this report, a polycistronic lentiviral vector carrying Yamanaka's factors was used to reprogram mouse fibroblasts into iPSCs in feeder- and xeno-free culture environment. The generated iPSCs exhibited morphology and self-renewal properties of embryonic stem cells (ESCs), expression of specific pluripotent markers, and potentials to differentiate into the three-major distinct specialized germ layers in vitro. The iPSCs were also shown to have the potential to differentiate into neural precursor and neurons in culture, with greater than 95% expression of nestin, Pax6 and βIII-tubulin. This body of work describes an alternative method of generating iPSCs by using polycistronic lentiviral vector that may minimize the risks associated with viral vector-mediated reprogramming and animal derived products in the culture media.