Mouse Somatic Cell Research Articles

Robust and Reproducible Generation of Induced Neural Stem Cells from Human Somatic Cells by Defined Factors.

Background and ObjectivesRecent studies have described direct reprogramming of mouse and human somatic cells into induced neural stem cells (iNSCs) using various combinations of transcription factors. Although iNSC technology holds a great potential for clinical applications, the low conversion efficiency and limited reproducibility of iNSC generation hinder its further translation into the clinic, strongly suggesting the necessity of highly reproducible method for human iNSCs (hiNSCs). Thus, in orderto develop a highly efficient and reproducible protocol for hiNSC generation, we revisited the reprogramming potentials of previously reported hiNSC reprogramming cocktails by comparing the reprogramming efficiency of distinct factor combinations including ours.MethodsWe introduced distinct factor combinations, OSKM (OCT4+SOX2+KLF4+C-MYC), OCT4 alone, SOX2 alone, SOX2+HMGA2, BRN4+SKM+SV40LT (BSKMLT), SKLT, SMLT, and SKMLT and performed comparative analysis of reprogramming potentials of distinct factor combinations in hiNSC generation.ResultsHere we show that ectopic expression of five reprogramming factors, BSKMLT leads the robust hiNSC generation (>80 folds enhanced efficiency) from human somatic cells compared with previously described factor combinations. With our combination, we were able to observe hiNSC conversion within 7 days of transduction. Throughout further optimization steps, we found that both BRN4 and KLF4 are not essential for hiNSC conversion.ConclusionsOur factor combination could robustly and reproducibly generate hiNSCs from human somatic cells with distinct origins. Therefore, our novel reprogramming strategy might serve as a useful tool for hiNSC-based clinical application.

Composition and Genoprotective Effect of the Flavonoidal Content of Lepidium sativum L. Methanolic Seed Extract against Cyclophosphamide- Induced DNA Damage in Mice

Background: Lepidium sativum L. (Family Brassicaceae) is known to possess different pharmacological properties. Objective: The genoprotective role of flavonoids of L. sativum methanolic seed extract (LSF) against cyclophosphamide (CP)-induced DNA damage, in somatic and germ cells of mice, as well as characterization of the flavonoidal content were carried out in this study. Chromosomal aberration analysis in somatic and germ cells were also included. Materials and Methods: Six mice groups were used for this study. Group 1 served as a negative control. Group 2 received oral LSF (100 mg/kg b.wt.) for 5 consecutive days. Group 3 served as a positive control by receiving a single intraperitoneal (i.p.) CP dose (20 mg/kg b.wt.). The three other groups were orally administered 25, 50 and 100 mg/kg b.wt. LSF, respectively, for 5 consecutive days. On the last day of treatment, the three groups received i.p. injection of CP (20 mg/kg b.wt.). Flavonoids were identified using spectral analysis. Results: LSF inhibited DNA aberrations in mice caused by cyclophosphamide dose dependently in the three groups with significant difference in the two groups that received doses of 50 and 100 mg/kg b.wt. The chromosomal aberrations inhibitory indices were calculated as 18 and 31 in mice somatic cells and 27 and 48 in germ cells, respectively. LSF was found to contain the flavonoids kaempferol, quercetin, kaempferol-3-O-α-L-rhamnopyranoside, kaempferol-3-O-β-D-glucopyranoside, and quercetin-3-O-β-D-galactopyranoside. Conclusion: LSF inhibited the DNA damage induced by CP in somatic and germ cells of mice dose-dependently. The antioxidant properties associated with flavonoids might account for the genoprotective activity.

Structurally-Discovered KLF4 Variants Accelerate and Stabilize Reprogramming to Pluripotency

Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domain enable next-generation reprogramming technology with engineered reprogramming factors.

Aberrant activation of cyclin A-CDK induces G2/M-phase checkpoint in human cells

ABSTRACT Cyclin A-cyclin dependent kinase (CDK) activity is regulated by cyclin A proteolysis and CDK inhibitors (CKIs) during M and G1 phases. Our previous work has shown that constitutive activation of cyclin A-CDK in mouse somatic cells, by ectopic expression of stabilized human cyclin A2 (lacking the destruction box: CycAΔ80) in triple CKI (p21, p27, and p107)-knocked-out mouse embryonic fibroblasts, induces rapid tetraploidization. However, effects of such cyclin A-CDK hyperactivation in human cells have been unknown. Here, we show hyperactivity of cyclin A-CDK induces G2/M-phase arrest in human cell lines with relatively low expression of p21 and p27. Moreover, adenovirus E1A protein promoted CycAΔ80-derived G2/M-phase arrest by increasing the amount of cyclin A and cyclin A-CDK2 complex. This response was suppressed by an addition of ATR or Chk1 inhibitor. The amount of repressive phosphorylation of CDK1 at tyrosine 15 (Y15) was decreased by Chk1 inhibitor treatment. Moreover, we observed that co-expressing CDK1AF mutant, which is resistant to the repressive phosphorylation at threonine 14 and Y15, or cdc25A, which dephosphorylates CDK1 at Y15, suppressed the G2/M-phase arrest by CycAΔ80 with E1A. These results suggest that G2/M-phase arrest in human cells by hyperactivity of cyclin A-CDK2 is caused by repression of CDK1 via the cell cycle checkpoint ATR-Chk1 pathway.

TMOD-09. MODELING TERT PROMOTER MUTATION IN ISOGENIC NEXT GENERATION GBM MODELS

Abstract Telomere reverse transcriptase (TERT) promotor mutations increase TERT expression and are known to be the most common single genetic abnormality in Glioblastoma (GBM). Recent studies have revealed this mutation to be an early event in gliomagenesis but it is detected subclonally in a considerable number of cases. Due to the long telomere length and TERT expression in somatic cells of mice, genetically engineered mouse models of GBM do not address the function of this mutation and thus an ideal heterozygous TERT promotor mutation model is yet to be established. Recently, our lab established strategies for generating GBM models using neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) that have been CRISPR/Cas9 engineered with different combinations of authentic GBM-related genetic drivers which can be used for introduction of mutant TERT promotor (MTP) sequence and examination of its function. We obtained multiple heterozygous MTP hiPS clones in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, using a two-step targeting approach. Two single-strand guide RNAs (sgRNAs) were used to delete the TERT promotor and a portion of exon 1 followed by repair of the locus with wild type TERT promoter (WTP) and MTP sequence. Following promoter reconstitution, TERT promotor function was tested in iPSCs, NPCs, and astrocytes. This analysis revealed successful TERT expression silencing in WTP astrocytes. Furthermore, orthotopic injection of WTP and MTP NPCs into immunodeficient mice formed pathologically similar GBM-like tumors in same period of time. TERT expression was only detected in cell lines derived from MTP tumors, indicating TERT promotor mutation has little impact in initiation of tumor formation from NPCs, explaining its subclonality in patient tumors. Our model enables further research on MTP function and dependency during gliomagenesis.

Excluding Oct4 from Yamanaka Cocktail Unleashes the Developmental Potential of iPSCs.

SummaryOct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here, we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network, even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs and thereby bear major implications for further development and application of iPSC technology.

Phenolic Compounds of Codiaeum variegatum Spirale Lessened Cytotoxic and Genotoxic Effects of Mitomycin C in Mice Somatic and Germ Cells

This study evaluates the chemopreventive properties of phenolic compounds extracted from Codiaeum variegatum (PCCV) against cytotoxic and genotoxic effects of mitomycin C (MMC). Male Swiss albino mice were treated with PCCV and/or MMC, and samples were collected 24 h after the last treatments. We recorded chromosome aberrations in bone-marrow cells and spermatocytes, cell viability and DNA damage (using comet assay) in bone-marrow cells. We observed that the highest concentration of PCCV did not induce cytotoxic or genotoxic effects on mice somatic and germ cells. However, MMC reduced cell proliferation remarkably (p < 0.05), and increased chromosome aberrations and DNA damage significantly. Additionally, PCCV prohibited cytotoxicity and genotoxicity of MMC when administered to animals 2h prior to MMC. Evidently, PCCV induced a significant inhibition (p < 0.05) in the percentages of chromosome aberrations in bone-marrow cells excluding gaps (19.82, 25.23, and 42.34%) and spermatocytes (22.73, 31.82, and 48.48%) at concentrations 125, 250 and 500 mg/kg b.wt respectively. Likewise, PCCV declined tail DNA (%), tail length (μm) and tail moment, and the inhibitory index of tail DNA (%) reached to 89.44% at 500 mg/kg b.wt. In conclusion, phenolic compounds of Codiaeum variegatum reduced the cytotoxic and genotoxic effects that were induced by the mutagenic agent MMC.

Partially Reprogrammed Induced Pluripotent Stem Cells Using MicroRNA Cluster miR-302s in Guangxi Bama Minipig Fibroblasts.

Pig-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. The miR-302s cluster alone has been shown to reprogram mouse and human somatic cells into induced pluripotent stem cells (iPSCs) without exogenous transcription factors. However, miR-302s alone have not been reported to reprogram cells in large livestock. In this study, we induced pig somatic cells into partially reprogrammed piPSCs using overexpression of the miR-302s cluster (miR-302s-piPSC) and investigated the early reprogramming events during the miRNA induction process. The results showed that miR-302s-piPSCs exhibited some characteristics of pluripotent stem cells including expression of pluripotency markers-particularly, efficient activation of endogenous OCT4-and differentiation to the three germ layers in vitro. During the early reprogramming process, somatic cells first underwent epithelial-mesenchymal transition and then mesenchymal-epithelial transition to eventually form miR-302s-piPSCs. These data show, for the first time, that single factor miR-302s successfully induced pig somatic cells into miR-302s-piPSCs. This study provides a new tool and research direction for the induction of pluripotent stem cells in a large livestock.

Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance

Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC-like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram bothmouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additionalstudies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC-driven bovine technologies can be realized.

Autophagy and mTOR pathways in mouse embryonic stem cell, lung cancer and somatic fibroblast cell lines.

Embryonic developmental stages and regulations have always been one of the most intriguing aspects of science. Since the cancer stem cell discovery, striking for cancer development and recurrence, embryonic stem cells and control mechanisms, as well as cancer cells and cancer stem cell control mechanisms become important research materials. It is necessary to reveal the similarities and differences between somatic and cancer cells which are formed of embryonic stem cells divisions and determinations. For this purpose, mouse embryonic stem cells (mESCs), mouse skin fibroblast cells (MSFs) and mouse lung squamous cancer cells (SqLCCs) were grown in vitro and the differences between these three cell lines signalling regulations of mechanistic target of rapamycin (mTOR) and autophagic pathways were demonstrated by immunofluorescence and real-time polymerase chain reaction. Expressional differences were clearly shown between embryonic, cancer and somatic cells that mESCs displayed higher expressional level of Atg10, Hdac1 and Cln3 which are related with autophagic regulation and Hsp4, Prkca, Rhoa and ribosomal S6 genes related with mTOR activity. LC3 and mTOR protein levels were lower in mESCs than MSFs. Thus, the mechanisms of embryonic stem cell regulation results in the formation of somatic tissues whereas that these cells may be the causative agents of cancer in any deterioration.

Meta-analysis of trichostatin A treatment effects on mouse somatic cell nuclear transfer

Improving somatic cell nuclear transfer (SCNT) efficiency is challenging, and trichostatin A (TSA) has been implemented to improve this technique, but it does not work for porcine and monkey SCNT. Thus, a meta-analysis was done to understand the relationship between TSA and mouse SCNT. Published articles were collected using PubMed and ScienceDirect from 2000 to 2018. Total 15 studies were included that suggest TSA can improve SCNT mouse blastocyst formation and live birth. Most TSA effects studied were on histone deacetylase (HDACs), hence the impacts of TSA on the cytoplasm, specifically cancer signaling pathways, endoplasmic reticulum, and HDACs localization were investigated. It is likely that TSA benefits mouse SCNT because the nucleus is easy to remove. Using fluorescent labeling to remove nuclei and TSA incorporation, SNCT may be improved for pig and monkey studies.

Partial reprogramming as a therapeutic approach for heart disease: A state-of-the-art review.

Heart disease such as myocardial infarction is the first cause of mortality in all countries. Today, cardiac cell-based therapy using de novo produced cardiac cells is considered as a novel approach for cardiac regenerative medicine. Recently, an alchemy-like approach, known as direct reprogramming or direct conversion, has been developed to directly convert somatic cells to cardiac cells in vitro and in vivo. This cellular alchemy is a short-cut and safe strategy for generating autologous cardiac cells, and it can be accomplished through activating cardiogenesis- or pluripotency-related factors in noncardiac cells. Importantly, pluripotency factors-based direct cardiac conversion, known as partial reprogramming, is shorter and more efficient for cardiomyocyte generation in vitro. Today, this strategy is achievable for direct conversion of mouse and human somatic cells to cardiac lineage cells (cardiomyocytes and cardiac progenitor cells), using transgene free, chemical-based approaches. Although, heart-specific partial reprogramming seems to be challenging for in vivo conversion of cardiac fibroblasts to cardiac cells, but whole organism-based in vivo partial reprogramming ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in mouse. Notably, cardiac cells produced using partial reprogramming strategy can be a useful platform for disease modeling, drug screening and cardiac cell-based therapy, once the safety issues are overcome. Herein, we discuss about all progresses in de novo production of cardiac cells using partial reprogramming-based direct conversion, as well as give an overview about the potential applications of this strategy in vivo and in vitro.

Long-range interactions between topologically associating domains shape the four-dimensional genome during differentiation.

Genomic information is selectively used to direct spatial and temporal gene expression during differentiation. Interactions between topologically associating domains (TADs) and between chromatin and the nuclear lamina organize and position chromosomes in the nucleus. However, how these genomic organizers together shape genome architecture is unclear. Here, using a dual-lineage differentiation system, we report long-range TAD-TAD interactions that form constitutive and variable TAD cliques. A differentiation-coupled relationship between TAD cliques and lamina-associated domains suggests that TAD cliques stabilize heterochromatin at the nuclear periphery. We also provide evidence of dynamic TAD cliques during mouse embryonic stem-cell differentiation and somatic cell reprogramming and of inter-TAD associations in single-cell high-resolution chromosome conformation capture (Hi-C) data. TAD cliques represent a level of four-dimensional genome conformation that reinforces the silencing of repressed developmental genes.

Nuclear-cytoplasmic shuttling of class IIa histone deacetylases regulates somatic cell reprogramming.

Class IIa histone deacetylases (HDACs) are a subfamily of HDACs with important functions in development and adult tissue homeostasis. As opposed to other HDACs, they lack catalytic function and bind transcription factors to recruit transcriptional co-regulators, mostly co-repressors such as nuclear receptor co-repressor (NCoR)/silencing mediator of retinoid and thyroid hormone receptor (SMRT). Class IIa HDACs enhance mouse somatic cell reprogramming to induced pluripotent stem cells (iPSCs) by repressing the function of the pro-mesenchymal transcription factor myocyte enhancer factor 2 (MEF2), which is upregulated during this process. Here, we describe, using HDAC4 and 7 as examples, that class IIa HDACs exhibit nuclear-cytoplasmic trafficking in reprogramming, being mostly cytoplasmic in donor fibroblasts and intermediate cells but translocating to the nucleus in iPSCs. Importantly, over-expressing a mutant form of HDAC4 or 7 that becomes trapped in the nucleus enhances the early phase of reprogramming but is deleterious afterwards. The latter effect is mediated through binding to the exogenous reprogramming factors at pluripotency loci, and the subsequent recruitment of NCoR/SMRT co-repressors. Thus, our findings uncover a context-dependent function of class IIa HDACs in reprogramming and further reinforce the idea that recruitment of co-repressors by the exogenous factors is a major obstacle for reactivating the pluripotency network in this process.

X-Chromosome Dosage Modulates Multiple Molecular and Cellular Properties of Mouse Pluripotent Stem Cells Independently of Global DNA Methylation Levels.

SummaryReprogramming female mouse somatic cells into induced pluripotent stem cells (iPSCs) leads to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs compared with male iPSCs leads to differences in the properties of iPSCs is still unclear. We show that chromatin accessibility in mouse iPSCs is modulated by X-dosage. Specific sets of transcriptional regulator motifs are enriched in chromatin with increased accessibility in XX or XY iPSCs. The transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand how increased X-dosage modulates the properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene Dusp9. We show that X-dosage regulates the transcriptome, open chromatin landscape, growth, and pluripotency exit largely independently of global DNA methylation. Our results provide insights into how gene dosage modulates the epigenetic and genetic mechanisms that regulate cell identity.

Direct conversion of pig fibroblasts to chondrocyte-like cells by c-Myc

Unexpectedly, we found that c-Myc-expressing porcine embryonic fibroblasts (PEFs) subcutaneously implanted into nude mice formed cartilage-like tissues in vivo, while previous studies revealed the direct conversion of mouse and human somatic cells into chondrocytes by the combined use of several defined factors, including c-Myc, which prompted us to explore whether PEFs can be reprogrammed to become pig induced chondrocyte-like cells (piCLCs) via ectopic expression of c-Myc alone. In this study, c-Myc-expressing PEFs, designated piCLCs, which exhibited a significantly enhanced proliferation ability in vitro, displayed a chondrogenic phenotypes in vitro, as shown by the cell morphology, toluidine blue staining, alcian blue staining and chondrocyte marker gene expression. Additionally, piCLCs with a polygonal chondrocyte-like morphology were readily and efficiently converted from PEFs by enforced c-Myc expression within 10 days, while piCLCs maintained the chondrocytic phenotype and normal karyotype during long-term subculture. piCLC-derived single clones with a chondrogenic phenotype in vitro exhibited homogeneity in cell morphology and staining intensity compared with mixed piCLCs. Although the mixtures of cartilaginous tissues and tumorous tissues accounted for ~12% (6/51) of all xenografts (51), piCLCs generated stable, homogenous, hyaline cartilage-like tissues without tumour formation at 45 out of the 51 injected sites when subcutaneously injected into nude mice. The hyaline cartilage-like tissues remained for at least 16 weeks. Taken together, these findings demonstrate for the first time the direct induction of chondrocyte-like cells from PEFs with only c-Myc.

Efficient in vivo direct conversion of fibroblasts into cardiomyocytes using a nanoparticle-based gene carrier

The reprogramming of induced cardiomyocytes (iCMs) has shown potential in regenerative medicine. However, in vivo reprogramming of iCMs is significantly inefficient, and novel gene delivery systems are required to more efficiently and safely induce in vivo reprogramming of iCMs for therapeutic applications in heart injury. In this study, we show that cationic gold nanoparticles (AuNPs) loaded with Gata4, Mef2c, and Tbx5 function as nanocarriers for cardiac reprogramming. The AuNP/GMT/PEI nanocomplexes show high reprogramming efficiency in human and mouse somatic cells with low cytotoxicity and direct conversion into iCMs without integrating factors into the genome. Importantly, AuNP/GMT/PEI nanocomplexes led to efficient in vivo conversion into cardiomyocytes after myocardial infarction (MI), resulting in the effective recovery of cardiac function and scar area. Taken together, these results show that the AuNP/GMT/PEI nanocarrier can be used to develop effective therapeutics for heart regeneration in cardiac disease patients.

Human Pluripotent Stem Cell-Derived Blood Cells for Therapies

It has now been twenty years since human embryonic stem cells (hESCs) were first isolated and described in 1998. In the next decade, induced pluripotent stem cells (iPSCs) were produced first from mouse somatic cells and then from human cells. Since these landmark advances, hESCs and iPSCs have been utilized to advance our understanding of basic human developmental biology and cellular plasticity. These lessons are crucial to fulfill the goal to use human pluripotent stem cells to derive new cellular therapies to better treat and repair organs and tissues damaged by disease, trauma or aging. Clinical trials are underway to utilize differentiated cells derived from hESCs or iPSCs for treatment of retinal disease, spinal cord injury, diabetes, cardiac failure, and other disorders. Production of therapeutic blood cells such as transplantable hematopoietic stem cells (HSCs) from hESCs and iPSCs remains a key goal. However, despite intensive research efforts by our group and many others, there remain challenging to achieve long-term multi-lineage engraftment in vivo with HSCs derived from unmodified hESCs/iPSCs. More successful approaches have used genetic modification or teratoma formation, though these strategies cannot be directly translated to clinical cell products. Reasons for this continued challenge and novel solutions such as use of a Runx1 genetic reporter system will be discussed. In contrast to production of transplantable HSCs, the ability use hESCs/iPSCs to produce functional lymphocytes with anti-tumor and anti-viral activity has been quite successful. Our group has defined methods to efficiently differentiate and expand clinical-scale quantities of natural killer (NK) cells. These hESC/iPSC-derived NK cells have phenotypic and genetic profiles similar to NK cells isolated from peripheral blood. Additionally, hESC/iPSC-derived NK cells are able to kill diverse tumor cells in vitro and in vivo. The hESCs/iPSCs also serve as a versatile platform to engineer genetic enhancements to produce NK cells with improved anti-tumor activity. For example, we have produced hESC/iPSC-derived NK cells that express novel chimeric antigen receptors (CARs) that are able to better target tumors that are more refractory to NK cell-mediated killing. This optimized NK-CAR construct utilizes the NKG2D transmembrane domain, 2B4 co-stimulatory domain, and the CD3ζ signaling domain to activate key NK cell-specific intracellular signaling pathways and increase NK cell survival and expansion in vivo. In one direct comparison between CAR-expressing-iPSC-derived NK cells and “conventional” CAR-expressing T cells, demonstrates the CAR-NK cells have similar ability to kill ovarian tumors in vivo, but with less toxicity, suggesting a safer approach. We have engineered other modifications into iPSC-NK cells to enhance NK cell targeting, proliferation, expansion and survival – all key qualities to improve in vivo anti-tumor activity. These studies demonstrate that hESC/iPSC-provide an ideal platform to produce standardized, targeted, “off-the-shelf” cellular immunotherapies to treat refractory hematological malignancies and solid tumors. Finally, iPSC-derived NK cells are now being produced at clinical scale under current good manufacturing practices (cGMP) conditions with clinical trials scheduled to start by the end of 2018. DisclosuresKaufman:Fate Therapeutics: Consultancy, Research Funding.

Induced Pluripotent Stem Cells

The isolation of human embryonic stem cells in 1998 has since fueled the ideology that stem cells may eventually be used for human disease therapies as well as the regeneration of tissues and organs. The transformation of somatic cells to a pluripotent state via somatic nuclear transfer and embryonic stem cell fusion brought the scientific community nearer to understanding the molecular mechanisms that govern cellular pluripotency. In 2006, the first induced pluripotent stem (iPS) cell was reported, where a mouse somatic cell was successfully converted to a pluripotent state via transduction of four essential factors. This cellular breakthrough has allowed for robust scientific investigations of human diseases that were once extremely difficult to study. Scientists and pharmaceuticals now use iPS cells as means for disease investigations, drug development and cell or tissue transplantation. There is little doubt that scientific progress on iPS cells will change many aspects of medicine in the next couple of decades.

Exploring Confluence-Related Signalling to Modulate the Expression of Oct4 - A Role in Facilitating Mouse Somatic Cell Reprogramming?

Oct4 is uniformly expressed by all types of pluripotent cells and is essential for pluripotency. Oct4 is also the central reprogramming factor that is constant in most transcription factor cocktails used to generate iPS cells. This article discusses the mechanisms of regulating Oct4 expression by confluence-based Hgf and hypoxia signalling in mouse somatic cells. Stat3 is activated ligandindependently in confluent cells and triggers the formation of cell aggregates. Hgf signalling is preserved after confluence, stimulating β-catenin and Stat3 activity, which are both crucial for Oct4 transcription. Stat3 and β-catenin activities also help sustain cell survival and proliferation, resulting in the formation of cell spheres. Hypoxic conditions in spheres activate regulators of Oct4 and further induce Oct4 expression. Activation of the Oct4 gene depends on nuclear receptors Lrh1/SF1, Esrrb, and Rars. These interact synergistically to initiate Oct4 transcription through organizing a transcriptional initiation complex. cAMP signalling stimulates the production of the SF1/Lrh1 ligand. Esrrb agonist or estrogen might function in activating Esrrb, while Rars might be induced by hypoxia. Taken together, Oct4 expression is probably induced in mouse somatic cells by culturing post-confluence cells with media containing Hgf, Esrrb agonist, and cAMP agonist. Exploring confluence-related signalling to modulate the expression of Oct4 might be helpful in finding novel strategies for reprogramming somatic cells.