Deterministic Restriction on Pluripotent State Dissolution by Cell-Cycle Pathways.

During differentiation, human embryonic stem cells (hESCs) shut down the regulatory network conferring pluripotency in a process we designated pluripotent state dissolution (PSD). In a high-throughput RNAi screen using an inclusive set of differentiation conditions, we identify centrally important and context-dependent processes regulating PSD in hESCs, including histone acetylation, chromatin remodeling, RNA splicing, and signaling pathways. Strikingly, we detected a strong and specific enrichment of cell-cycle genes involved in DNA replication and G2 phase progression. Genetic and chemical perturbation studies demonstrate that the S and G2 phases attenuate PSD because they possess an intrinsic propensity toward the pluripotent state that is independent of G1 phase. Our data therefore functionally establish that pluripotency control is hardwired to the cell-cycle machinery, where S and G2 phase-specific pathways deterministically restrict PSD, whereas the absence of such pathways in G1 phase potentially permits the initiation of differentiation.

In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation.

Identifying pairwise RNA-RNA interactions is key to understanding how RNAs fold and interact with other RNAs inside the cell. We present a high-throughput approach, sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH), that maps pairwise RNA interactions in vivo with high sensitivity and specificity, genome-wide. Applying SPLASH to human and yeast transcriptomes revealed the diversity and dynamics of thousands of long-range intra- and intermolecular RNA-RNA interactions. Our analysis highlighted key structural features of RNA classes, including the modular organization of mRNAs, its impact on translation and decay, and the enrichment of long-range interactions in noncoding RNAs. Additionally, intermolecular mRNA interactions were organized into network clusters and were remodeled during cellular differentiation. We also identified hundreds of known and new snoRNA-rRNA binding sites, expanding our knowledge of rRNA biogenesis. These results highlight the underexplored complexity of RNA interactomes and pave the way to better understanding how RNA organization impacts biology.

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease.

BACKGROUND & AIMS: There are few in vitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. METHODS: We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. RESULTS: Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. CONCLUSIONS: We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

VHZ is a novel centrosomal phosphatase associated with cell growth and human primary cancers.

BACKGROUND: VHZ is a VH1-like (member Z) dual specific protein phosphatase encoded by DUSP23 gene. Some of the dual specific protein phosphatases (DSPs) play an important role in cell cycle control and have shown to be associated with carcinogenesis. Here, the expression of VHZ associated with cell growth and human cancers was investigated. RESULTS: We generated a mouse monoclonal antibody (mAb clone#209) and rabbit polyclonal antibodies (rAb) against VHZ. We performed cell proliferation assay to learn how VHZ is associated with cell cycle by retroviral transduction to express VHZ, VHZ(C95S), and control vector in MCF-7 cells. Overexpression of VHZ [but not VHZ(C95S)] in MCF-7 cells promoted cell proliferation compared to control cells. shRNA-mediated knockdown of VHZ in MCF-7 cells showed that reduction of VHZ resulted in increased G1 but decreased S phase cell populations. Using indirect immunofluorescence, we showed that both exogenous and endogenous VHZ protein was localized at the centrosome in addition to its cytoplasmic distribution. Furthermore, using immunohistochemistry, we revealed that VHZ protein was overexpressed either in enlarged centrosomes (VHZ-centrosomal-stain) of some invasive ductal carcinomas (IDC) Stage I (8/65 cases) or in entire cytoplasm (VHZ-cytosol-stain) of invasive epithelia of some IDC Stage II/III (11/47 cases) of breast cancers examined. More importantly, upregulation of VHZ protein is also associated with numerous types of human cancer, in particular breast cancer. VHZ mAb may be useful as a reagent in clinical diagnosis for assessing VHZ positive tumors. CONCLUSIONS: We generated a VHZ-specific mAb to reveal that VHZ has a novel subcellular localization, namely the centrosome. VHZ is able to facilitate G1/S cell cycle transition in a PTP activity-dependent manner. The upregulation of its protein levels in primary human cancers supports the clinical relevance of the protein in cancers.

A Chemically Defined Feeder-free System for the Establishment and Maintenance of the Human Naive Pluripotent State.

The distinct states of pluripotency in the pre- and post-implantation embryo can be captured in vitro as naive and primed pluripotent stem cell cultures, respectively. The study and application of the naive state remains hampered, particularly in humans, partially due to current culture protocols relying on extraneous undefined factors such as feeders. Here we performed a small-molecule screen to identify compounds that facilitate chemically defined establishment and maintenance of human feeder-independent naive embryonic (FINE) stem cells. The expression profile in genic and repetitive elements of FINE cells resembles the 8-cell-to-morula stage in vivo, and only differs from feeder-dependent naive cells in genes involved in cell-cell/cell-matrix interactions. FINE cells offer several technical advantages, such as increased amenability to transfection and a longer period of genomic stability, compared with feeder-dependent cells. Thus, FINE cells will serve as an accessible and useful system for scientific and translational applications of naïve pluripotent stem cells.

Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.

Klf2 is an essential factor that sustains ground state pluripotency.

The maintenance of mouse embryonic stem cells (mESCs) requires LIF and serum. However, a pluripotent "ground state," bearing resemblance to preimplantation mouse epiblasts, can be established through dual inhibition (2i) of both prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. While Gsk3 inhibition has been attributed to the transcriptional derepression of Esrrb, the molecular mechanism mediated by Mek inhibition remains unclear. In this study, we show that Krüppel-like factor 2 (Klf2) is phosphorylated by Erk2 and that phospho-Klf2 is proteosomally degraded. Mek inhibition hence prevents Klf2 protein phosphodegradation to sustain pluripotency. Indeed, while Klf2-null mESCs can survive under LIF/Serum, they are not viable under 2i, demonstrating that Klf2 is essential for ground state pluripotency. Importantly, we also show that ectopic Klf2 expression can replace Mek inhibition in mESCs, allowing the culture of Klf2-null mESCs under Gsk3 inhibition alone. Collectively, our study defines the Mek/Erk/Klf2 axis that cooperates with the Gsk3/Tcf3/Esrrb pathway in mediating ground state pluripotency.

Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast.

Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. Despite sharing the common property of pluripotency, hESCs are notably distinct from epiblast cells of the preimplantation blastocyst. Here we use a combination of three small-molecule inhibitors to sustain hESCs in a LIF signaling-dependent hESC state (3iL hESCs) with elevated expression of NANOG and epiblast-enriched genes such as KLF4, DPPA3, and TBX3. Genome-wide transcriptome analysis confirms that the expression signature of 3iL hESCs shares similarities with native preimplantation epiblast cells. We also show that 3iL hESCs have a distinct epigenetic landscape, characterized by derepression of preimplantation epiblast genes. Using genome-wide binding profiles of NANOG and OCT4, we identify enhancers that contribute to rewiring of the regulatory circuitry. In summary, our study identifies a distinct hESC state with defined regulatory circuitry that will facilitate future analysis of human preimplantation embryogenesis and pluripotency.

Engineering the first chimeric antibody in targeting intracellular PRL-3 oncoprotein for cancer therapy in mice.

Antibodies are considered as 'magic bullets' because of their high specificity. It is believed that antibodies are too large to routinely enter the cytosol, thus antibody therapeutic approach has been limited to extracellular or secreted proteins expressed by cancer cells. However, many oncogenic proteins are localized within the cell. To explore the possibility of antibody therapies against intracellular targets, we generated a chimeric antibody targeting the intracellular PRL-3 oncoprotein to assess its antitumor activities in mice. Remarkably, we observed that the PRL-3 chimeric antibody could efficiently and specifically reduce the formation of PRL-3 expressing metastatic tumors. We further found that natural killer (NK) cells were important in mediating the therapeutic effect, which was only observed in a nude mouse model (T-cell deficient), but not in a Severe Combined Immunodeficiency' (scid ) mouse model (B- and T-cell deficient), indicating the anticancer effect also depends on host B-cell activity. Our study involving 377 nude and scid mice suggest that antibodies targeting intracellular proteins can be developed to treat cancer.

Targeting intracellular oncoproteins with antibody therapy or vaccination.

Antibody-based therapies have better specificity and thus improved efficacy over standard chemotherapy regimens, which result in extended survival and improved quality of life for cancer patients. Because antibodies are viewed as too large to access intracellular locations, antibody therapy has traditionally targeted extracellular or secreted proteins expressed by cancer cells. However, many oncogenic proteins are found within the cell (such as intracellular phosphatases/kinases and transcription factors) and have therefore not been pursued for antibody therapies. Here, we explored the possibility of antibody therapy or vaccination against intracellular proteins. As proofs of concept, we selected three representative intracellular proteins as immunogens for tumor vaccine studies: PRL-3 (phosphatase of regenerating liver 3), a cancer-associated phosphatase; EGFP (enhanced green fluorescent protein), a general reporter; and mT (polyomavirus middle T), the polyomavirus middle T oncoprotein. A variety of tumors that expressed these intracellular proteins were clearly inhibited by their respective exogenous antibodies or by antigen-induced host antibodies (vaccination). These anticancer activities were reproducibly observed in hundreds of C57BL/6 tumor-bearing mice and MMTV-PymT transgenic breast tumor mice. Our in vivo data suggest that immunotherapies can target not only extracellular but also intracellular oncoproteins.

PRL-3, a metastasis associated tyrosine phosphatase, is involved in FLT3-ITD signaling and implicated in anti-AML therapy.

Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription) pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation.

PCBP1 suppresses the translation of metastasis-associated PRL-3 phosphatase.

Overexpression of phosphatase of regenerating liver (PRL)-3 is associated with the progression of diverse human cancers. We show that the overexpression of PRL-3 protein is not directly associated with its transcript levels, indicating the existence of an underlying posttranscriptional regulation. The 5' untranslanted region (UTR) of PRL-3 mRNA possesses triple GCCCAG motifs capable of suppressing mRNA translation through interaction with PolyC-RNA-binding protein 1 (PCBP1), which retards PRL-3 mRNA transcript incorporation into polyribosomes. Overexpression of PCBP1 inhibits PRL-3 expression and inactivates AKT, whereas knockdown of PCBP1 causes upregulation of PRL-3 protein levels, activation of AKT, and promotion of tumorigenesis. An inverse correlation between protein levels of PRL-3 and PCBP1 in human primary cancers supports the clinical relevance.

Monoclonal antibodies target intracellular PRL phosphatases to inhibit cancer metastases in mice.

PRL-1 (phosphatase of regenerating liver-1), PRL-2 and PRL-3 are protein tyrosine phosphatases with a C-terminal prenylation motif that are localized to the inner leaflet of the plasma membrane and early endosomes. A variety of metastatic PRL-overexpressing cancers have been reported. Therefore, the three PRL-phosphatases represent an intriguing group of proteins being validated as biomarkers and therapeutic targets in cancer. Targeting intracellular PRLs to prevent cancer metastasis by exogenous reagents is a challenging task. In an attempt to destroy PRL-overexpressing cancer cells with their respective PRL-antibodies, we generated an animal model that allows rapid formation of aggressive metastatic tumors caused by inoculation of PRL-1- or PRL-3-expressing cells. Surprisingly, mice treated with PRL-1 or PRL-3 mAbs show inhibition of tumor formation by approximately 90% compared to untreated mice. Here we provide the first examples that PRL-1 and PRL-3 mAbs are able to target their respective phosphatases specifically and efficiently despite their intracellular localization to block cancer metastasis in experimental animals. Furthermore, we also demonstrate that PRL-1 mAb specifically blocks the formation of metastatic tumors formed by PRL-1- (but not PRL-3-) expressing cells; while PRL-3 mAb specifically blocks tumor formation of PRL-3- (but not PRL-1-) expressing cells. More importantly, we show that metastatic tumor formation by A2780 human ovarian cancer cells that express endogenous PRL-3 is dramatically blocked by PRL-3 antibodies. In contrast, the PRL-3 antibody treatment has no effect on tumor formation of CT26 mouse colon cancer cells which do not naturally express PRL-3 protein. Our data provide hope for the treatment of PRL-expressing cancers and will prompt a reevaluation of a wide spectrum of intracellular oncoproteins as possible targets with mAbs for anticancer therapy.