Lia S. Campos

Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion

One Two T T cells develop in the thymus, where they proceed through several developmental stages, losing alternative lineage potential as they progress. The molecular regulation of this developmental process, however, is not fully understood (see the Perspective by Di Santo). P. Li et al. (p. 85, published online 10 June), L. Li et al. (p. 89), and Ikawa et al. (p. 93) now identify expression of the zinc finger transcription factor Bcl11b as the earliest checkpoint in T cell development in mice. Genetic deletion of Bcl11b in developing T cells inhibited commitment to the T cell lineage. Under conditions that should have stimulated T lineage differentiation, Bcl11b-deficient T cell progenitors failed to up-regulate genes associated with lineage-committed T cells and maintained stem cell– and progenitor cell–associated gene expression. In both developing and committed T cells, loss of Bcl11b resulted in the generation of cells that resembled natural killer (NK) cells in both phenotype and function. These NK-like cells could be expanded easily in vitro and possessed antitumor cytotoxicity, but they did not exhibit cytotoxicity against normal cells and were not tumorigenic. Because T cells are much easier to obtain from human patients than NK cells, deletion of Bcl11b in T cells may thus provide a source of easy-to-grow NK cells for cell-based antitumor therapies. A transcription factor is essential for maintenance of T cell identity. T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction, and defense against microbes and viruses. Here, we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell–associated gene expression. These induced T-to–natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro, and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.

Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome.

PiggyBac Transposon Mutagenesis: A Tool for Cancer Gene Discovery in Mice

Piggybacking on Cancer Genes Transposons are mobile segments of DNA that can insert in or near important genes to cause mutations that disrupt gene function. Rad et al. (p. 1104, published online 14 October) adapted a mutagenic transposon called Piggybac, originally derived from a moth, into a tool for discovery of cancer-causing genes in mice. Mobilization of Piggybac in mice was associated with the development of leukemias and solid tumors. In many instances the causative mutations, which were identified by mapping the Piggybac integration sites, were within genes not previously implicated in cancer. Mutations induced by a transposable element in mice can be used to identify cancer-causing genes. Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.

Neurospheres: insights into neural stem cell biology.

Neural stem cells (NSC) are a tissue‐specific subtype of self‐renewing and multipotent cells that can give rise to all neural populations. In this review, the importance of maintaining cell–cell contacts in the study of NSC is highlighted, and data obtained from some crucial single‐cell studies is compared to results obtained from neurospheres, where aggregates of NSC are grown in suspension. In particular, results that indicate how this culture system may be well suited to analyze NSC plasticity, cell–cell, and cell–extracellular matrix (ECM) interactions are pointed out, and the hypothesis that cell–cell and cell–ECM contacts may be essential for NSC maintenance, survival, and proliferation is highlighted. Finally, it is suggested that neurospheres might play a role in the study of context‐dependent behavior of NSC in niches by providing a system where NSC can be challenged chemically or biologically and analyzed in vitro, in a time‐ and context‐dependent manner.

Notch, Epidermal Growth Factor Receptor, and β1-Integrin Pathways Are Coordinated in Neural Stem Cells

Notch1 and β1-integrins are cell surface receptors involved in the recognition of the niche that surrounds stem cells through cell-cell and cell-extracellular matrix interactions, respectively. Notch1 is also involved in the control of cell fate choices in the developing central nervous system (Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583-589). Here we report that Notch and β1-integrins are co-expressed and that these proteins cooperate with the epidermal growth factor receptor in neural progenitors. We describe data that suggests that β1-integrins may affect Notch signaling through 1) physical interaction (sequestration) of the Notch intracellular domain fragment by the cytoplasmic tail of the β1-integrin and 2) affecting trafficking of the Notch intracellular domain via caveolin-mediated mechanisms. Our findings suggest that caveolin 1-containing lipid rafts play a role in the coordination and coupling of β1-integrin, Notch1, and tyrosine kinase receptor signaling pathways. We speculate that this will require the presence of the adequate β1-activating extracellular matrix or growth factors in restricted regions of the central nervous system and namely in neurogenic niches.

Single-cell RNA-seq identifies a PD-1 hi ILC progenitor and defines its development pathway

Innate lymphoid cells (ILCs) functionally resemble T lymphocytes in cytotoxicity and cytokine production but lack antigen-specific receptors, and they are important regulators of immune responses and tissue homeostasis. ILCs are generated from common lymphoid progenitors, which are subsequently committed to innate lymphoid lineages in the α-lymphoid progenitor, early innate lymphoid progenitor, common helper innate lymphoid progenitor and innate lymphoid cell progenitor compartments. ILCs consist of conventional natural killer cells and helper-like cells (ILC1, ILC2 and ILC3). Despite recent advances, the cellular heterogeneity, developmental trajectory and signalling dependence of ILC progenitors are not fully understood. Here, using single-cell RNA-sequencing (scRNA-seq) of mouse bone marrow progenitors, we reveal ILC precursor subsets, delineate distinct ILC development stages and pathways, and report that high expression of programmed death 1 (PD-1hi) marked a committed ILC progenitor that was essentially identical to an innate lymphoid cell progenitor. Our data defined PD-1hiIL-25Rhi as an early checkpoint in ILC2 development, which was abolished by deficiency in the zinc-finger protein Bcl11b but restored by IL-25R overexpression. Similar to T lymphocytes, PD-1 was upregulated on activated ILCs. Administration of a PD-1 antibody depleted PD-1hi ILCs and reduced cytokine levels in an influenza infection model in mice, and blocked papain-induced acute lung inflammation. These results provide a perspective for exploring PD-1 and its ligand (PD-L1) in immunotherapy, and allow effective manipulation of the immune system for disease prevention and therapy.

MiR-25 regulates Wwp2 and Fbxw7 and promotes reprogramming of mouse fibroblast cells to iPSCs

Background miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs). Methods We screened 52 miRNAs cloned in a piggybac (PB) vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES) cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation. Results and conclusion We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.

mDll1 and mDll3 expression in the developing mouse brain: Role in the establishment of the early cortex

The Delta/Notch signalling system is involved in several developmental processes. During fly neurogenesis, Delta expression defines the fate of neuronal precursors and inhibits neighboring Notch‐expressing cells from acquiring a neural fate, a process known as lateral inhibition. In vertebrates, recent evidence demonstrates that Notch activation can positively determine cell fate and affect neuronal process extension. Nevertheless, Delta‐like expression patterns during brain development are relatively unknown. Using a transgenic mouse, which expresses LacZ under the mDll1 promoter, we show by immunofluorescence that in the developing telencephalon mDll1 is expressed in undifferentiated cells in close contact with radial glial cells. Based on in situ hybridization data on mDll1 and mDll3 mRNA expression and on the immunohistochemical detection of β‐galactosidase in the Dll1‐lacZ transgenic mouse, we suggest that mDll1 and mDll3 are involved in the establishment of the early cortical plate and that mDll1‐expressing cells are in close contact with radial glial cells, thereby modulating the latter population, which is known to express Notch1. Furthermore, we suggest that the decrease in mDll1 mRNA found toward the end of gestation could be related, first, to the slowing of neurogenesis and, second, to the differentiation of the radial glial cell population into astrocytes. J. Neurosci. Res. 64:590–598, 2001.

A DNA transposon-based approach to validate oncogenic mutations in the mouse

Large-scale cancer genome projects will soon be able to sequence many cancer genomes to comprehensively identify genetic changes in human cancer. Genome-wide association studies have also identified putative cancer associated loci. Functional validation of these genetic mutations in vivo is becoming a challenge. We describe here a DNA transposon-based platform that permits us to explore the oncogenic potential of genetic mutations in the mouse. Briefly, promoter-less human cancer gene cDNAs were first cloned into Sleeping Beauty (SB) transposons. DNA transposition in the mouse that carried both the transposons and the SB transposase made it possible for the cDNAs to be expressed from an appropriate endogenous genomic locus and in the relevant cell types for tumor development. Consequently, these mice developed a broad spectrum of tumors at very early postnatal stages. This technology thus complements the large-scale cancer genome projects.

Establishment of mouse expanded potential stem cells

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.