Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells.

BACKGROUND: Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function. METHODS: Human PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses. RESULTS: GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation. CONCLUSIONS: We generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.

Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells-derived hepatic stem cells.

Pluripotent stem cells have been investigated as a renewable source of therapeutic hepatic cells, in order to overcome the lack of transplantable donor hepatocytes. Whereas different studies were able to correct hepatic defects in animal models, they focused on the most mature phenotype of hepatocyte-like cells (HLCs) derived from pluripotent stem cells and needed freshly prepared cells, which limits clinical applications of HLCs. Here, we report the production of hepatic stem cells (pHSCs) from human-induced pluripotent stem cells (hiPSCs) in xeno-free, feeder-free, and chemically defined conditions using as extracellular matrix a recombinant laminin instead of Matrigel, an undefined animal-derived matrix. Freshly prepared and frozen pHSCs were transplanted via splenic injection in Gunn rats, the animal model for Crigler-Najjar syndrome. Following cell transplantation and daily immunosuppression treatment, bilirubinemia was significantly decreased (around 30% decrease, P < .05) and remained stable throughout the 6-month study. The transplanted pHSCs underwent maturation in vivo to restore the deficient metabolic hepatic function (bilirubin glucuronidation by UGT1A1). In conclusion, we demonstrate for the first time the differentiation of hiPSCs into pHSCs that (a) are produced using a differentiation protocol compatible with Good Manufacturing Practices, (b) can be frozen, and (c) are sufficient to demonstrate in vivo therapeutic efficacy to significantly lower hyperbilirubinemia in a model of inherited liver disease, despite their immature phenotype. Thus, our approach provides major advances toward future clinical applications and would facilitate cell therapy manufacturing from human pluripotent stem cells.

Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system.

Despite decades of investigation on the proliferation of adult human primary hepatocytes, their expansion in vitro still remains challenging. To later be able to consider hepatocytes as a cell therapy alternative or bridge to liver transplantation, dramatically impeded by a shortage in liver donors, the first step is having an almost unlimited source of these cells. The banking of transplantable hepatocytes also implies a protocol for their expansion that can be compatible with large-scale production. We show that adult human primary hepatocytes when grown in 3D organoids are easily amplified, providing a substantial source of functional hepatocytes ready for transplantation. Following their plating, differentiated human hepatocytes are amplified during a transient and reversible step as liver progenitors, and can subsequently be converted back to mature differentiated hepatocytes. The protocol we propose is not only compatible with automated and high-throughput cell culture systems, thanks to the expansion of hepatocytes in suspension, but also guarantees the generation of a high number of functional cells from the same patient sample, with a relatively easy set up.

Generation of Immunodeficient Rats With Rag1 and Il2rg Gene Deletions and Human Tissue Grafting Models.

BACKGROUND: Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease, and these disease models would greatly benefit from immunodeficient rats to test different immunogenic treatments. METHODS: We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. RESULTS: As compared with Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient because they partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound immunossuppressed phenotype because they displayed undetectable levels of T, B, and NK cells. Similarly, all immunoglobulin isotypes in sera were decreased in Rag1- or Il2rg-deficient rats and undetectable in Rats Rag1 and Il2rg (RRG) animals. Rag1- or Il2rg-deficient rats rejected allogeneic skin transplants and human tumors, whereas animals not only accepted allogeneic rat skin but also xenogeneic human tumors, skin, and hepatocytes. Immune humanization of RRG animals was unsuccessful. CONCLUSIONS: Thus, immunodeficient RRG animals are useful recipients for long-term studies in which immune responses could be an obstacle, including tissue humanization of different tissues.

A single aspartate mutation in the conserved catalytic site of Rev3L generates a hypomorphic phenotype in vivo and in vitro.

Rev3, the catalytic subunit of yeast DNA polymerase ζ, is required for UV resistance and UV-induced mutagenesis, while its mammalian ortholog, REV3L, plays further vital roles in cell proliferation and embryonic development. To assess the contribution of REV3L catalytic activity to its in vivo function, we generated mutant mouse strains in which one or two Ala residues were substituted to the Asp of the invariant catalytic YGDTDS motif. The simultaneous mutation of both Asp (ATA) phenocopies the Rev3l knockout, which proves that the catalytic activity is mandatory for the vital functions of Rev3L, as reported recently. Surprisingly, although the mutation of the first Asp severely impairs the enzymatic activity of other B-family DNA polymerases, the corresponding mutation of Rev3 (ATD) is hypomorphic in yeast and mouse, as it does not affect viability and proliferation and moderately impacts UVC-induced cell death and mutagenesis. Interestingly, Rev3l hypomorphic mutant mice display a distinct, albeit modest, alteration of the immunoglobulin gene mutation spectrum at G-C base pairs, further documenting its role in this process.

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant.

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι(-/-) embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light.

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6-4) pyrimidine pyrimidone photoproducts ((6-4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6-4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6-4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.

Somatic hypermutation at A/T-rich oligonucleotide substrates shows different strand polarities in Ung-deficient or -proficient backgrounds.

A/T mutations at immunoglobulin loci are introduced by DNA polymerase η (Polη) during an Msh2/6-dependent repair process which results in A's being mutated 2-fold more often than T's. This patch synthesis is initiated by a DNA incision event whose origin is still obscure. We report here the analysis of A/T oligonucleotide mutation substrates inserted at the heavy chain locus, including or not including internal C's or G's. Surprisingly, the template composed of only A's and T's was highly mutated over its entire 90-bp length, with a 2-fold decrease in mutation from the 5' to the 3' end and a constant A/T ratio of 4. These results imply that Polη synthesis was initiated from a break in the 5'-flanking region of the substrate and proceeded over its entire length. The A/T bias was strikingly altered in an Ung(-/-) background, which provides the first experimental evidence supporting a concerted action of Ung and Msh2/6 pathways to generate mutations at A/T bases. New analysis of Pms2(-/-) animals provided a complementary picture, revealing an A/T mutation ratio of 4. We therefore propose that Ung and Pms2 may exert a mutual backup function for the DNA incision that promotes synthesis by Polη, each with a distinct strand bias.

Temporally distinct translesion synthesis pathways for ultraviolet light-induced photoproducts in the mammalian genome.

Replicative polymerases (Pols) arrest at damaged DNA nucleotides, which induces ubiquitination of the DNA sliding clamp PCNA (PCNA-Ub) and DNA damage signaling. PCNA-Ub is associated with the recruitment or activation of translesion synthesis (TLS) DNA polymerases of the Y family that can bypass the lesions, thereby rescuing replication and preventing replication fork collapse and consequent formation of double-strand DNA breaks. Here, we have used gene-targeted mouse embryonic fibroblasts to perform a comprehensive study of the in vivo roles of PCNA-Ub and of the Y family TLS Pols η, ι, κ, Rev1 and the B family TLS Polζ in TLS and in the suppression of DNA damage signaling and genome instability after exposure to UV light. Our data indicate that TLS Pols ι and κ and the N-terminal BRCT domain of Rev1, that previously was implicated in the regulation of TLS, play minor roles in TLS of DNA photoproducts. PCNA-Ub is critical for an early TLS pathway that replicates both strongly helix-distorting (6-4) pyrimidine-pyrimidone ((6-4)PP) and mildly distorting cyclobutane pyrimidine dimer (CPD) photoproducts. The role of Polη is mainly restricted to early TLS of CPD photoproducts, whereas Rev1 and, in particular, Polζ are essential for the bypass of (6-4)PP photoproducts, both early and late after exposure. Thus, structurally distinct photoproducts at the mammalian genome are bypassed by different TLS Pols in temporally different, PCNA-Ub-dependent and independent fashions.

Different sets of translesion synthesis DNA polymerases protect from genome instability induced by distinct food-derived genotoxins.

DNA lesions, induced by genotoxic compounds, block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA polymerases (Pols). TLS safeguards the completion of replication, albeit at the expense of nucleotide substitution mutations. We studied the in vivo role of individual TLS Pols in cellular responses to benzo[a]pyrene diolepoxide (BPDE), a polycyclic aromatic hydrocarbon, and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. To this aim, we used mouse embryonic fibroblasts with targeted disruptions in the TLS-associated Pols η, ι, κ, and Rev1 as well as in Rev3, the catalytic subunit of TLS Polζ. After exposure, cellular survival, replication fork progression, DNA damage responses (DDR), and the induction of micronuclei were investigated. The results demonstrate that Rev1, Rev3, and, to a lesser extent, Polη are involved in TLS and the prevention of DDR and of DNA breaks, in response to both agents. Conversely, Polκ and the N-terminal BRCT domain of Rev1 are specifically involved in TLS of BPDE-induced DNA damage. We furthermore describe a novel role of Polι in TLS of 4-HNE-induced DNA damage in vivo. We hypothesize that different sets of TLS polymerases act on structurally different genotoxic DNA lesions in vivo, thereby suppressing genomic instability associated with cancer. Our experimental approach may provide a significant contribution in delineating the molecular bases of the genotoxicity in vivo of different classes of DNA-damaging agents.

Multiple layers of B cell memory with different effector functions.

Memory B cells are at the center of longstanding controversies regarding the presence of antigen for their survival and their re-engagement in germinal centers after secondary challenge. Using a new mouse model of memory B cell labeling dependent on the cytidine deaminase AID, we show that after immunization with a particulate antigen, B cell memory appeared in several subsets, comprising clusters of immunoglobulin M-positive (IgM(+)) and IgG1(+) B cells in germinal center-like structures that persisted up to 8 months after immunization, as well as IgM(+) and IgG1(+) B cells with a memory phenotype outside of B cell follicles. After challenge, the IgG subset differentiated into plasmocytes, whereas the IgM subset reinitiated a germinal center reaction. This model, in which B cell memory appears in several layers with different functions, reconciles previous conflicting propositions.

A backup role of DNA polymerase kappa in Ig gene hypermutation only takes place in the complete absence of DNA polymerase eta.

Patients with the variant form of xeroderma pigmentosum (XPV) syndrome have a genetic deficiency in DNA polymerase (Pol) eta, and display accordingly an increased skin sensitivity to UV light, as well as an altered mutation pattern of their Ig V genes in memory B cells, alteration that consists in a reduced mutagenesis at A/T bases. We previously suggested that another polymerase with a different mutation signature, Pol kappa, is used as backup for Ig gene hypermutation in both humans and mice in cases of complete Pol eta deficiency, a proposition supported in this study by the analysis of Pol eta x Pol kappa double-deficient mice. We also describe a new XPV case, in which a splice site mutation of the first noncoding exon results in a decreased mRNA expression, a mRNA that otherwise encodes a normal Pol eta protein. Whereas the Pol eta mRNA level observed in patient's fibroblasts is one-twentieth the value of healthy controls, it is only reduced to one-fourth of the normal level in activated B cells. Memory B cells from this patient showed a 50% reduction in A/T mutations, with a spectrum that still displays a strict Pol eta signature. Pol eta thus appears as a dominant enzyme in hypermutation, its presence precluding the use of a substitute enzyme even in conditions of reduced availability. Such a dominant behavior may explain the lack of Pol kappa signature in Ig gene mutations of some XPV patients previously described, for whom residual Pol eta activity might exist.

Competitive repair pathways in immunoglobulin gene hypermutation.

This review focuses on the contribution of translesion DNA polymerases to immunoglobulin gene hypermutation, in particular on the roles of DNA polymerase eta (Poleta) in the generation of mutations at A/T bases from the initial cytosine-targeted activation-induced cytidine deaminase (AID)-mediated deamination event, and of Polkappa, an enzyme of the same polymerase family, used as a substitute when Poleta is absent. The proposition that the UNG uracil glycosylase and the MSH2-MSH6 mismatch recognition complex are two competitive rather than alternative pathways in the processing of uracils generated by AID is further discussed.

DNA polymerase eta is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse.

Mutations at A/T bases within immunoglobulin genes have been shown to be generated by a repair pathway involving the DNA-binding moiety of the mismatch repair complex constituted by the MSH2-MSH6 proteins, together with DNA polymerase eta (pol eta). However, residual A/T mutagenesis is still observed upon inactivation in the mouse of each of these factors, suggesting that the panel of activities involved might be more complex. We reported previously (Delbos, F., A. De Smet, A. Faili, S. Aoufouchi, J.-C. Weill, and C.-A. Reynaud. 2005. J. Exp. Med. 201:1191-1196) that residual A/T mutagenesis in pol eta-deficient mice was likely contributed by another enzyme not normally involved in hypermutation, DNA polymerase kappa, which is mobilized in the absence of the normal polymerase partner. We report the complete absence of A/T mutations in MSH2-pol eta double-deficient mice, thus indicating that the residual A/T mutagenesis in MSH2-deficient mice is contributed by pol eta, now recruited by uracil N-glycosylase, the second DNA repair pathway involved in hypermutation. We propose that this particular recruitment of pol eta corresponds to a profound modification of the function of uracil glycosylase in the absence of the mismatch repair complex, suggesting that MSH2-MSH6 actively prevent uracil glycosylase from error-free repair during hypermutation. pol eta thus appears to be the sole contributor of A/T mutations in the normal physiological context.

Contribution of DNA polymerase eta to immunoglobulin gene hypermutation in the mouse.

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase eta, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase iota gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of pol eta-deficient mice differs markedly. This suggests that, in the absence of pol eta, the MSH2-MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly pol kappa, to assume its function in hypermutation.

Normal immune system development in mice lacking the Deltex-1 RING finger domain.

The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1(Delta/Delta) mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.