Tryptophan Hydroxylase-2-Mediated Serotonin Biosynthesis Suppresses Cell Reprogramming into Pluripotent State.

The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has important functions both in the neural system and during embryonic development in mammals. In this study, we set out to investigate whether and how endogenous serotonin affects reprogramming to pluripotency. As serotonin is synthesized from tryptophan by the rate limiting enzymes tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), we have assessed the reprogramming of TPH1- and/or TPH2-deficient mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). The reprogramming of the double mutant MEFs showed a dramatic increase in the efficiency of iPSC generation. In contrast, ectopic expression of TPH2 alone or in conjunction with TPH1 reverted the rate of reprogramming of the double mutant MEFs to the wild-type level and besides, TPH2 overexpression significantly suppressed reprogramming of wild-type MEFs. Our data thus suggest a negative role of serotonin biosynthesis in the reprogramming of somatic cells to a pluripotent state.

Metabolic control of induced pluripotency.

Pluripotent stem cells of the mammalian epiblast and their cultured counterparts-embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs)-have the capacity to differentiate in all cell types of adult organisms. An artificial process of reactivation of the pluripotency program in terminally differentiated cells was established in 2006, which allowed for the generation of induced pluripotent stem cells (iPSCs). This iPSC technology has become an invaluable tool in investigating the molecular mechanisms of human diseases and therapeutic drug development, and it also holds tremendous promise for iPSC applications in regenerative medicine. Since the process of induced reprogramming of differentiated cells to a pluripotent state was discovered, many questions about the molecular mechanisms involved in this process have been clarified. Studies conducted over the past 2 decades have established that metabolic pathways and retrograde mitochondrial signals are involved in the regulation of various aspects of stem cell biology, including differentiation, pluripotency acquisition, and maintenance. During the reprogramming process, cells undergo major transformations, progressing through three distinct stages that are regulated by different signaling pathways, transcription factor networks, and inputs from metabolic pathways. Among the main metabolic features of this process, representing a switch from the dominance of oxidative phosphorylation to aerobic glycolysis and anabolic processes, are many critical stage-specific metabolic signals that control the path of differentiated cells toward a pluripotent state. In this review, we discuss the achievements in the current understanding of the molecular mechanisms of processes controlled by metabolic pathways, and vice versa, during the reprogramming process.

Dual Mode of Mitochondrial ROS Action during Reprogramming to Pluripotency.

Essential changes in cell metabolism and redox signaling occur during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this paper, using genetic and pharmacological approaches, we have investigated the role of electron transport chain (ETC) complex-I (CI) of mitochondria in the process of cell reprogramming to pluripotency. Knockdown of NADH-ubiquinone oxidoreductase core subunits S1 (Ndufs1) or subunit B10 (Ndufb10) of the CI or inhibition of this complex with rotenone during mouse embryonic fibroblast (MEF) reprogramming resulted in a significantly decreased number of induced pluripotent stem cells (iPSCs). We have found that mitochondria and ROS levels due course of the reprogramming tightly correlate with each other, both reaching peak by day 3 and significantly declining by day 10 of the process. The transient augmentation of mitochondrial reactive oxygen species (ROS) could be attenuated by antioxidant treatment, which ameliorated overall reprogramming. However, ROS scavenging after day 3 or during the entire course of reprogramming was suppressive for iPSC formation. The ROS scavenging within the CI-deficient iPSC-precursors did not improve, but further suppressed the reprogramming. Our data therefore point to distinct modes of mitochondrial ROS action during the early versus mid and late stages of reprogramming. The data further substantiate the paradigm that balanced levels of oxidative phosphorylation have to be maintained on the route to pluripotency.

Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man.

Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide, are generated as byproducts of oxidative phosphorylation in the mitochondria or via cell signaling-induced NADPH oxidases in the cytosol. In the recent two decades, a plethora of studies established that elevated ROS levels generated by oxidative eustress are crucial physiological mediators of many cellular and developmental processes. In this review, we discuss the mechanisms of ROS generation and regulation, current understanding of ROS functions in the maintenance of adult and embryonic stem cells, as well as in the process of cell reprogramming to a pluripotent state. Recently discovered cell-non-autonomous ROS functions mediated by growth factors are crucial for controlling cell differentiation and cellular immune response in Drosophila. Importantly, many physiological functions of ROS discovered in Drosophila may allow for deciphering and understanding analogous processes in human, which could potentially lead to the development of novel therapeutic approaches in ROS-associated diseases treatment.

Pluripotent stem cell-based gene therapy approach: human de novo synthesized chromosomes.

A novel approach in gene therapy was introduced 20 years ago since artificial non-integrative chromosome-based vectors containing gene loci size inserts were engineered. To date, different human artificial chromosomes (HAC) were generated with the use of de novo construction or "top-down" engineering approaches. The HAC-based therapeutic approach includes ex vivo gene transferring and correction of pluripotent stem cells (PSCs) or highly proliferative modified stem cells. The current progress in the technology of induced PSCs, integrating with the HAC technology, resulted in a novel platform of stem cell-based tissue replacement therapy for the treatment of genetic disease. Nowadays, the sophisticated and laborious HAC technology has significantly improved and is now closer to clinical studies. In here, we reviewed the achievements in the technology of de novo synthesized HACs for a chromosome transfer for developing gene therapy tissue replacement models of monogenic human diseases.

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice.

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/- mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/- iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector-the alphoidtetO-HAC.

Human artificial chromosomes for pluripotent stem cell-based tissue replacement therapy.

The gene therapy approach aiming at curing various human diseases began to develop as a technology from early eighties of the last century. To date the delivery of therapeutic genes are mainly mediated by virus-based, predominantly, non-integrated virus vectors. These gene delivery approaches have several fundamental limitations on the way of efficient deployment in clinical gene therapy. A totally different approach was suggested about 20 years ago when episomal non-integrative artificial chromosome-based vectors featuring large size inserts (even native gene loci) advanced to the stage. Since then numerous human artificial chromosome (HAC) vectors were developed by both de novo synthesis and top-down engineering technology. This approach so far is limited to ex vivo gene transfer and correction within highly proliferative or reversibly immortalized precursor stem cells or pluripotent stem cells. Recent breakthrough in generation of induced pluripotent stem cells and embryonic stem cell manipulation give the additional pivotal stimuli to integrate it with the HAC technology and to develop thereby novel approaches to replacement therapies of human genetic diseases. The HAC technology is complex and time consuming while nowadays it has significantly advanced and become notably closer to medical applications. In this review we discuss current advancements in the HAC technology, in particular, in terms of improvement of chromosome transfer method and achievements in developing mouse-based gene therapy tissue replacement models for several monogenic human diseases. The main progress has been done in elaboration of top-down type HAC technology in modeling and preclinical studies of gene therapy treatment for Duchenne muscular dystrophy (DMD) disease.

Genetic tool for fate mapping of Oct4 (Pou5f1)-expressing cells and their progeny past the pluripotency stage.

BACKGROUND: Methods based on site-specific recombinases are widely used in studying gene activities in vivo and in vitro. In these studies, constitutively active or inducible variants of these recombinases are expressed under the control of either lineage-specific or ubiquitous promoters. However, there is a need for more advanced schemes that combine these features with possibilities to choose a time point from which lineage tracing starts in an autonomous fashion. For example, the key mammalian germline gatekeeper gene Oct4 (Pou5f1) is expressed in the peri-implantation epiblast which gives rise to all cells within embryos. Thus the above techniques are hardly applicable to Oct4 tracing past the epiblast stage, and the establishment of genetic tools addressing such a limitation is a highly relevant pursuit. METHODS: The CRISPR/Cas9 tool was used to manipulate the genome of mouse embryonic stem cells (ESCs), and various cell culture technics-to maintain and differentiate ESCs to neural cell, lentivirus-based reprogramming technique-to generate induced pluripotent stem cells (iPSCs). RESULTS: In this paper, we have developed a two-component genetic system (referred to as O4S) that allows tracing Oct4 gene activity past the epiblast stage of development. The first component represents a knock-in of an ubiquitous promoter-driven inducible Cre, serving as a stop signal for downstream tdTomato. Upon activation of Cre activity with 4-hydroxytamoxifen (4-OHT) at any given time point, the recombinase excises a stop signal and poses the second component of the system-the FlpO recombinase, knocked into 3'UTR of Oct4, to be expressed upon activation of the latter gene. Oct4-driven expression of FlpO, in turn, triggers the tdTomato expression and thus, permanently marks Oct4+ cells and their progeny. We have validated the O4S system in cultured ESCs and shown that it is capable, for example, to timely capture an activation of Oct4 gene during the reprogramming of somatic cells into iPSCs. CONCLUSIONS: The developed O4S system can be used to detect Oct4 activation event, both permanent and transient, in somatic cell types outside the germline. The approach can be equally adjusted to other genes, provided the first component of the system is placed under transcriptional control of these genes, thus, making it a valuable tool for cell fate mapping in mice.

hnRNP-K Targets Open Chromatin in Mouse Embryonic Stem Cells in Concert with Multiple Regulators.

The transcription factor Oct4 plays a key regulatory role in the induction and maintenance of cellular pluripotency. In this article, we show that ubiquitous and multifunctional poly(C) DNA/RNA-binding protein hnRNP-K occupies Oct4 (Pou5f1) enhancers in embryonic stem cells (ESCs) but is dispensable for the initiation, maintenance, and downregulation of Oct4 gene expression. Nevertheless, hnRNP-K has an essential cell-autonomous function in ESCs to maintain their proliferation and viability. To better understand mechanisms of hnRNP-K action in ESCs, we have performed ChIP-seq analysis of genome-wide binding of hnRNP-K and identified several thousands of hnRNP-K target sites that are frequently co-occupied by pluripotency-related and common factors (Oct4, TATA-box binding protein, Sox2, Nanog, Otx2, etc.), as well as active histone marks. Furthermore, hnRNP-K localizes exclusively within open chromatin, implying its role in the onset and/or maintenance of this chromatin state. Stem Cells 2019;37:1018-1029.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

Immortalized murine fibroblast cell lines are refractory to reprogramming to pluripotent state.

To date different cell types of various mammalian species have been reprogrammed to induced pluripotent stem cells (iPSCs) using Yamanaka's cocktail of transcription factors (Oct4, Klf4, Sox2, and cMyc). It has been shown that several primary human cancer cell lines could be reprogrammed to iPSCs. We sought if immortalized mouse fibroblast cell lines could also be reprogrammed to iPSCs. The approach of generating iPSCs from such cells should be valuable in different experimental settings as it allows clonally derive cell lines carrying mutations whose impact on reprogramming could be next evaluated. Therefore, we investigated reprogramming of widely used immortalized cell lines (NIH3T and STO), as well as of de novo immortalized fibroblast line (tKM) with the use of highly effective lentiviral polycistronic OKSM expression system. Our reprogramming experiments have shown that in contrast to mouse embryonic fibroblasts (MEFs), none of the immortalized cell lines can be reprogrammed to pluripotent state. Contrary to colonies derived from MEFs, those derived from the immortalized cells lines (1) developed much later, (2) contained large round cells, not typical for iPSCs, and (3) were negative for trusted markers of matured iPSCs, Nanog and SSEA1. Immortalized cell lines NIH3T and STO are known to be mostly aneuploid, whereas tKM population includes cells with normal karyotype, however, neither cell type can be reprogrammed. Thus our data argue that aneuploidy per se is not a reason for the observed refractoriness of mouse immortalized cells to reprogramming to pluripotent state.

Proapoptotic function of deubiquitinase DUSP31 in Drosophila.

Drosophila have been used to identify new components in apoptosis regulation. The Drosophila protein Dark forms an octameric apoptosome complex that induces the initiator caspase Dronc to trigger the caspase cell death pathway and, therefore, plays an important role in controlling apoptosis. Caspases and Dark are constantly expressed in cells, but their activity is blocked by DIAP1 E3 ligase-mediated ubiquitination and subsequent inactivation or proteasomal degradation. One of the regulatory mechanisms that stabilize proapoptotic factors is the removal of ubiquitin chains by deubiquitinases. In this study performed a modified genetic screen for deubiquitinases (dsRNA lines) to identify those involved in stabilizing proapoptotic components. Loss-of-function alleles of deubiquitinase DUSP31 were identified as suppressors of the Dronc overexpression phenotype. DUSP31 deficiency also suppresses apoptosis induced by the RHG protein, Grim. Genetic analysis revealed for the first time that DUSP31 deficiency sufficiently suppresses the Dark phenotype, indicating its involvement in the control of Dark/Dronc apoptosome function in invertebrate apoptosis.

Genetic dissection of leukemia-associated IDH1 and IDH2 mutants and D-2-hydroxyglutarate in Drosophila.

Gain-of-function mutations in nicotinamide adenine dinucleotide phosphate-dependent isocitrate dehydrogenase (IDH)1 and IDH2 frequently arise in human leukemias and other cancers and produce high levels of D-2-hydroxyglutarate (D-2HG). We expressed the R195H mutant of Drosophila Idh (CG7176), which is equivalent to the human cancer-associated IDH1-R132H mutant, in fly tissues using the UAS-Gal4 binary expression system. Idh-R195H caused a >25-fold elevation of D-2HG when expressed ubiquitously in flies. Expression of mutant Idh in larval blood cells (hemocytes) resulted in higher numbers of circulating blood cells. Mutant Idh expression in fly neurons resulted in neurologic and wing-expansion defects, and these phenotypes were rescued by genetic modulation of superoxide dismutase 2, p53, and apoptotic caspase cascade mediators. Idh-R163Q, which is homologous to the common leukemia-associated IDH2-R140Q mutant, resulted in moderately elevated D-2HG and milder phenotypes. We identified the fly homolog of D-2-hydroxyglutaric acid dehydrogenase (CG3835), which metabolizes D-2HG, and showed that coexpression of this enzyme with mutant Idh abolishes mutant Idh-associated phenotypes. These results provide a flexible model system to interrogate a cancer-related genetic and metabolic pathway and offer insights into the impact of IDH mutation and D-2HG on metazoan tissues.

Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila.

Oxidative stress induced by high levels of reactive oxygen species (ROS) is associated with the development of different pathological conditions, including cancers and autoimmune diseases. We analysed whether oxidatively challenged tissue can have systemic effects on the development of cellular immune responses using Drosophila as a model system. Indeed, the haematopoietic niche that normally maintains blood progenitors can sense oxidative stress and regulate the cellular immune response. Pathogen infection induces ROS in the niche cells, resulting in the secretion of an epidermal growth factor-like cytokine signal that leads to the differentiation of specialized cells involved in innate immune responses.

Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance.

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche. In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. Here, we show that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. Our findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche.

Genetic manipulation of AML1-ETO-induced expansion of hematopoietic precursors in a Drosophila model.

Among mutations in human Runx1/AML1 transcription factors, the t(8;21)(q22;q22) genomic translocation that creates an AML1-ETO fusion protein is implicated in etiology of the acute myeloid leukemia. To identify genes and components associated with this oncogene we used Drosophila as a genetic model. Expression of AML1-ETO caused an expansion of hematopoietic precursors in Drosophila, which expressed high levels of reactive oxygen species (ROS). Mutations in functional domains of the fusion protein suppress the proliferative phenotype. In a genetic screen, we found that inactivation of EcRB1 or activation of Foxo and superoxide dismutase-2 (SOD2) suppress the AML1-ETO-induced phenotype by reducing ROS expression in the precursor cells. Our studies indicate that ROS is a signaling factor promoting maintenance of normal as well as the aberrant myeloid precursors and suggests the importance of antioxidant enzymes and their regulators as targets for further study in the context of leukemia.

Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila.

In Drosophila, blood development occurs in a specialized larval hematopoietic organ, the lymph gland (LG), within which stem-like hemocyte precursors or prohemocytes differentiate to multiple blood cell types. Here we show that components of the Wingless (Wg) signaling pathway are expressed in prohemocytes. Loss- and gain-of-function analysis indicates that canonical Wg signaling is required for maintenance of prohemocytes and negatively regulates their differentiation. Wg signals locally in a short-range fashion within different compartments of the LG. In addition, Wg signaling positively regulates the proliferation and maintenance of cells that function as a hematopoietic niche in Drosophila, the posterior signaling center (PSC), and in the proliferation of crystal cells. Our studies reveal a conserved function of Wg signaling in the maintenance of stem-like blood progenitors and reveal an involvement of this pathway in the regulation of hemocyte differentiation through its action in the hematopoietic niche.

Expression pattern of Filamin-240 in Drosophila blood cells.

The expression pattern of Filamin-240 was studied in subsets of Drosophila blood cells by means of immunofluorescent staining and Western blot analysis with use of an antibody specific to a "filamin-folding domain", a consensus motif profile generated from the 20 existing filamin repeats. Expression of Filamin-240 is restricted to lamellocytes - a special blood cell type of the cellular immune response - and is involved in the regulation of lamellocyte development. In the cher1 homozygous larvae, which lack Filamin-240 protein, a vigorous lamellocyte differentiation occurs which is further enhanced upon in vivo immune challenge by a parasitic wasp, Leptopilina boulardi. By introducing a full-length transgene encoding the Drosophila Filamin-240 protein into the cher1 Filamin-deficient homozygous mutant, the mutant blood cell phenotype was rescued. These data demonstrate that the expression of Filamin-240 is strictly lamellocyte specific in Drosophila blood cells and that the protein is a suppressor of lamellocyte development.

Increased expression of Drosophila tetraspanin, Tsp68C, suppresses the abnormal proliferation of ytr-deficient and Ras/Raf-activated hemocytes.

Tetraspanins are evolutionary conserved transmembrane proteins thought to facilitate cell proliferation, movement or fusion by acting as organizers of different signaling events. Despite their prevalence and conservation, their specific role and functions remain largely elusive, as their redundancy in various organisms has hindered loss of function studies. Here, we take a gain of function approach to study Drosophila tetraspanin Tsp68C and its effect on larval hemocytes. We recently characterized a lethal mutation in ytr, a conserved gene that encodes a nuclear arginine-rich protein of unknown function, which is accompanied by abnormal differentiation and proliferation of the larval hematopoietic tissue in flies. A hemolectin (hml)-Gal4 construct carried by hml-Gal4 transgenic flies was sufficient by itself to abrogate the hematopoietic defects in ytr mutant larvae. This rescue correlated with the overexpression of tsp68C, a tetraspanin gene nested in the hml promoter. The suppression of abnormal proliferation by the hml-Gal4 construct was not restricted to ytr-deficient hemocytes, but was also observed in hemocytes expressing the oncogenic forms of Raf or Ras proteins. However, it had no effect on overproliferation mediated by a constitutively active form of Jak. New hml-Gal4 lines, in which the tsp68C gene was silenced or deleted from the promoter, no longer rescued the hematopoietic defect in ytr mutants nor suppressed the activated Raf-induced overproliferation. Therefore, change in tetraspanin Tsp68C expression has a strong suppressor effect on abnormal proliferation and differentiation of hemocytes in the context of specific lesions, such as overactivation of the Ras/Raf/MAPK pathway.

Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development.

To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.