Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

BACKGROUND AIMS: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. METHODS: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). RESULTS: Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). CONCLUSIONS: We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen.

Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.

Specific calcineurin isoforms are involved in Drosophila toll immune signaling.

Because excessive or inadequate responses can be detrimental, immune responses to infection require appropriate regulation. Networks of signaling pathways establish versatility of immune responses. Drosophila melanogaster is a powerful model organism for dissecting conserved innate immune responses to infection. For example, the Toll pathway, which promotes activation of NF-κB transcription factors Dorsal/Dorsal-related immune factor (Dif), was first identified in Drosophila. Together with the IMD pathway, acting upstream of NF-κB transcription factor Relish, these pathways constitute a central immune signaling network. Inputs in these pathways contribute to specific and appropriate responses to microbial insults. Relish activity during infection is modulated by Ca(2+)-dependent serine/threonine phosphatase calcineurin, an important target of immunosuppressants in transplantation biology. Only one of the three Drosophila calcineurin isoforms, calcineurin A1, acts on Relish during infection. However, it is not known whether there is a role for calcineurin in Dorsal/Dif immune signaling. In this article, we demonstrate involvement of specific calcineurin isoforms, protein phosphatase at 14D (Pp2B-14D)/calcineurin A at 14F (CanA-14F), in Toll-mediated immune signaling. These isoforms do not affect IMD signaling. In cell culture, pharmacological inhibition of calcineurin or RNA interference against homologous calcineurin isoforms Pp2B-14D/CanA-14F, but not against isoform calcineurin A1, decreased Toll-dependent Dorsal/Dif activity. A Pp2B-14D gain-of-function transgene promoted Dorsal nuclear translocation and Dorsal/Dif activity. In vivo, Pp2B-14D/CanA-14F RNA interference attenuated the Dorsal/Dif-dependent response to infection without affecting the Relish-dependent response. Altogether, these data identify a novel input, calcineurin, in Toll immune signaling and demonstrate involvement of specific calcineurin isoforms in Drosophila NF-κB signaling.

Dissection of a hypoxia-induced, nitric oxide-mediated signaling cascade.

Befitting oxygen's key role in life's processes, hypoxia engages multiple signaling systems that evoke pervasive adaptations. Using surrogate genetics in a powerful biological model, we dissect a poorly understood hypoxia-sensing and signal transduction system. Hypoxia triggers NO-dependent accumulation of cyclic GMP and translocation of cytoplasmic GFP-Relish (an NFkappaB/Rel transcription factor) to the nucleus in Drosophila S2 cells. An enzyme capable of eliminating NO interrupted signaling specifically when it was targeted to the mitochondria, arguing for a mitochondrial NO signal. Long pretreatment with an inhibitor of nitric oxide synthase (NOS), L-NAME, blocked signaling. However, addition shortly before hypoxia was without effect, suggesting that signaling is supported by the prior action of NOS and is independent of NOS action during hypoxia. We implicated the glutathione adduct, GSNO, as a signaling mediator by showing that overexpression of the cytoplasmic enzyme catalyzing its destruction, GSNOR, blocks signaling, whereas knockdown of this activity caused reporter translocation in the absence of hypoxia. In downstream steps, cGMP accumulated, and calcium-dependent signaling was subsequently activated via cGMP-dependent channels. These findings reveal the use of unconventional steps in an NO pathway involved in sensing hypoxia and initiating signaling.

Drosophila calcineurin promotes induction of innate immune responses.

The sophisticated adaptive immune system of vertebrates overlies an ancient set of innate immune-response pathways, which have been genetically dissected in Drosophila. Although conserved regulatory pathways have been defined, calcineurin, a Ca(2+)-dependent phosphatase, has not been previously implicated in Drosophila immunity. Calcineurin activates mammalian immune responses by activating the nuclear translocation of the vertebrate-specific transcription factors NFAT1-4. In Drosophila, infection with gram-negative bacteria promotes the activation of the Relish transcription factor through the Imd pathway. The activity of this pathway in the larva is modulated by nitric oxide (NO). Here, we show that the input by NO is mediated by calcineurin. Pharmacological inhibition of calcineurin suppressed the Relish-dependent gene expression that occurs in response to gram-negative bacteria or NO. One of the three calcineurin genes in Drosophila, CanA1, mediated NO-induced nuclear translocation of Relish in a cell-culture assay. A CanA1 RNA interference (RNAi) transgene suppressed immune induction in larvae upon infection or upon treatment with NO donors, whereas a gain-of-function CanA1 transgene activated immune responses in untreated larvae. Interestingly, CanA1 RNAi in hemocytes but not the fat body was sufficient to block immune induction in the fat body. Thus, CanA1 provides an additional input into Relish-promoted immune responses and functions in hemocytes to promote a tissue-to-tissue signaling cascade required for robust immune response.

IL-5-mediated eosinophil survival requires inhibition of GSK-3 and correlates with beta-catenin relocalization.

Interleukin (IL)-5 is a hematopoietic cytokine able to regulate differentiation, survival, and effector functions of eosinophils. It binds specifically to its receptor, which is composed of a cytokine-specific alpha-chain and a beta-chain shared with the receptors for IL-3 and the granulocyte macrophage-colony stimulating factor. The molecular mechanisms by which IL-5 modulates eosinophil survival remain unclear. In this study, we demonstrate that IL-5 withdrawal induces eosinophil apoptosis through a mitochondria-dependent pathway, independently of Fas receptor activation. The lipid kinase phosphatidylinositol-3 kinase plays a crucial role in the maintenance of eosinophil survival, as inhibition of its activity results in apoptosis. IL-5 induces phosphorylation and thus, inhibition of the Forkhead transcription factor FOXO3a and glycogen synthase kinase 3 (GSK-3). We analyzed expression of FOXO3a-dependent transcriptional targets: Fas ligand or Bim (a proapoptotic Bcl-2 family member), but neither was detected in apoptotic eosinophils. We further show that GSK-3 is activated after IL-5 withdrawal, and inhibition of its activity rescues eosinophils from apoptosis. beta-catenin, a direct GSK-3 substrate, is present in the nucleus of IL-5-stimulated eosinophils, but it is translocated to the plasma membrane in the absence of cytokine in a GSK-3-dependent manner. This is the first report describing a potential role for GSK-3 and beta-catenin in regulating eosinophil survival and suggests a novel mechanism by which IL-5 inhibits the constitutive apoptotic program in these cells.

IL-5-mediated eosinophil survival requires inhibition of GSK-3 and correlates with ß-catenin relocalization.

Interleukin (IL)-5 is a hematopoietic cytokine able to regulate differentiation, survival, and effector functions of eosinophils. It binds specifically to its receptor, which is composed of a cytokine-specific α-chain and a ß-chain shared with the receptors for IL-3 and the granulocyte macrophage-colony stimulating factor. The molecular mechanisms by which IL-5 modulates eosinophil survival remain unclear. In this study, we demonstrate that IL-5 withdrawal induces eosinophil apoptosis through a mitochondria-dependent pathway, independently of Fas receptor activation. The lipid kinase phosphatidylinositol-3 kinase plays a crucial role in the maintenance of eosinophil survival, as inhibition of its activity results in apoptosis. IL-5 induces phosphorylation and thus, inhibition of the Forkhead transcription factor FOXO3a and glycogen synthase kinase 3 (GSK-3). We analyzed expression of FOXO3a-dependent transcriptional targets: Fas ligand or Bim (a proapoptotic Bcl-2 family member), but neither was detected in apoptotic eosinophils. We further show that GSK-3 is activated after IL-5 withdrawal, and inhibition of its activity rescues eosinophils from apoptosis. ß-catenin, a direct GSK-3 substrate, is present in the nucleus of IL-5-stimulated eosinophils, but it is translocated to the plasma membrane in the absence of cytokine in a GSK-3-dependent manner. This is the first report describing a potential role for GSK-3 and ß-catenin in regulating eosinophil survival and suggests a novel mechanism by which IL-5 inhibits the constitutive apoptotic program in these cells.

Binding of protein kinase B to the plakin family member periplakin.

The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3'-phosphorylated phosphatidylinositol lipids (PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane, intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region, results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.

The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2.

The cytokine IL-2 plays a very important role in the proliferation and survival of activated T cells. These effects of IL-2 are dependent on signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. We and others have shown that PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). In this study we have investigated the role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival. We show that IL-2 regulates phosphorylation of FoxO3 in a PI3K-dependent fashion. Phosphorylation and inactivation of FoxO3 appears to play an important role in IL-2-mediated T cell survival, because mere activation of FoxO3 is sufficient to trigger apoptosis in T cells. Indeed, active FoxO3 can induce expression of IL-2-regulated genes, such as the cdk inhibitor p27(Kip1) and the proapoptotic Bcl-2 family member Bim. Furthermore, we show that IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, we propose that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival.

Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors.

AFX-like Forkhead transcription factors, which are controlled by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, are involved in regulating cell cycle progression and cell death. Both cell cycle arrest and induction of apoptosis are mediated in part by transcriptional regulation of p27(kip1). Here we show that the Forkheads AFX (FOXO4) and FKHR-L1 (FOXO3a) also directly control transcription of the retinoblastoma-like p130 protein and cause upregulation of p130 protein expression. Detailed analysis of p130 regulation demonstrates that following Forkhead-induced cell cycle arrest, cells enter G(0) and become quiescent. This is shown by a change in phosphorylation of p130 to G(0)-specific forms and increased p130/E2F-4 complex formation. Most importantly, long-term Forkhead activation causes a sustained but reversible inhibition of proliferation without a marked increase in apoptosis. As for the activity of the Forkheads, we also show that protein levels of p130 are controlled by endogenous PI3K/PKB signaling upon cell cycle reentry. Surprisingly, not only nontransformed cells, but also cancer cells such as human colon carcinoma cells, are forced into quiescence by Forkhead activation. We therefore propose that Forkhead inactivation by PKB signaling in quiescent cells is a crucial step in cell cycle reentry and contributes to the processes of transformation and regeneration.

FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity.

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim-/- mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling. These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB--mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.