Hypoimmunogenic Human Pluripotent Stem Cells as a Powerful Tool for Liver Regenerative Medicine.

Induced pluripotent stem cells (iPSC) have huge potential as cell therapy for various diseases, given their potential for unlimited self-renewal and capability to differentiate into a wide range of cell types. Although autologous iPSCs represents the ideal source for patient-tailored regenerative medicine, the high costs of the extensive and time-consuming production process and the impracticability for treating acute conditions hinder their use for broad applications. An allogeneic iPSC-based strategy may overcome these issues, but it carries the risk of triggering an immune response. So far, several approaches based on genome-editing techniques to silence human leukocyte antigen class I (HLA-I) or II (HLA-II) expression have been explored to overcome the immune rejection of allogeneic iPSCs. In this study, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to delete the ß2-Microglobulin (B2M) and the Class II Major Histocompatibility Complex Transactivator (CIITA) genes, essential for the correct surface expression of HLA-I and HLA-II proteins. The resulting hypoimmunogenic iPSC line has a normal karyotype, expresses the pluripotency stem cell markers, and is capable of differentiating into the three embryonic germ layers. Furthermore, we showed that it specifically retains the ability to differentiate towards different liver cells, such as endothelial-like cells, hepatocyte-like cells, and hepatic stellate-like cells. Our results indicate that hypoimmunogenic iPSCs could give a new cost-effective and off-the-shelf opportunity for cell therapy in liver diseases.

Long-term adaptive response in COVID-19 vaccine recipients and the effect of a booster dose.

We examined the immune response in subjects previously infected with SARS-CoV2 and infection-naïve 9 months after primary 2-dose COVID-19 mRNA vaccination and 3 months after the booster dose in a longitudinal cohort of healthcare workers. Nine months after primary vaccination, previously infected subjects exhibited higher residual antibody levels, with significant neutralizing activity against distinct variants compared to infection-naïve subjects. The higher humoral response was associated with higher levels of receptor binding domain (RBD)-specific IgG+ and IgA+ memory B cells. The booster dose increased neither neutralizing activity, nor the B and T cell frequencies. Conversely, infection-naïve subjects needed the booster to achieve comparable levels of neutralizing antibodies as those found in previously infected subjects after primary vaccination. The neutralizing titer correlated with anti-RBD IFNγ producing T cells, in the face of sustained B cell response. Notably, pre-pandemic samples showed high Omicron cross-reactivity. These data show the importance of the booster dose in reinforcing immunological memory and increasing circulating antibodies in infection-naïve subjects.

Post-translational modifications by SIRT3 de-2-hydroxyisobutyrylase activity regulate glycolysis and enable nephrogenesis.

Abnormal kidney development leads to lower nephron number, predisposing to renal diseases in adulthood. In embryonic kidneys, nephron endowment is dictated by the availability of nephron progenitors, whose self-renewal and differentiation require a relatively repressed chromatin state. More recently, NAD+-dependent deacetylase sirtuins (SIRTs) have emerged as possible regulators that link epigenetic processes to the metabolism. Here, we discovered a novel role for the NAD+-dependent deacylase SIRT3 in kidney development. In the embryonic kidney, SIRT3 was highly expressed only as a short isoform, with nuclear and extra-nuclear localisation. The nuclear SIRT3 did not act as deacetylase but exerted de-2-hydroxyisobutyrylase activity on lysine residues of histone proteins. Extra-nuclear SIRT3 regulated lysine 2-hydroxyisobutyrylation (Khib) levels of phosphofructokinase (PFK) and Sirt3 deficiency increased PFK Khib levels, inducing a glycolysis boost. This altered Khib landscape in Sirt3-/- metanephroi was associated with decreased nephron progenitors, impaired nephrogenesis and a reduced number of nephrons. These data describe an unprecedented role of SIRT3 in controlling early renal development through the regulation of epigenetics and metabolic processes.

Unravelling the Role of PAX2 Mutation in Human Focal Segmental Glomerulosclerosis.

No effective treatments are available for familial steroid-resistant Focal Segmental Glomerulosclerosis (FSGS), characterized by proteinuria due to ultrastructural abnormalities in glomerular podocytes. Here, we studied a private PAX2 mutation identified in a patient who developed FSGS in adulthood. By generating adult podocytes using patient-specific induced pluripotent stem cells (iPSC), we developed an in vitro model to dissect the role of this mutation in the onset of FSGS. Despite the PAX2 mutation, patient iPSC properly differentiated into podocytes that exhibited a normal structure and function when compared to control podocytes. However, when exposed to an environmental trigger, patient podocytes were less viable and more susceptible to cell injury. Fixing the mutation improved their phenotype and functionality. Using a branching morphogenesis assay, we documented developmental defects in patient-derived ureteric bud-like tubules that were totally rescued by fixing the mutation. These data strongly support the hypothesis that the PAX2 mutation has a dual effect, first in renal organogenesis, which could account for a suboptimal nephron number at birth, and second in adult podocytes, which are more susceptible to cell death caused by environmental triggers. These abnormalities might translate into the development of proteinuria in vivo, with a progressive decline in renal function, leading to FSGS.

Generation of a homozygous CIITA knockout iPS cell line using the CRISPR-Cas9 system.

Human induced pluripotent stem cells (iPSCs) have great promise in regenerative medicine. However, several limitations, including immune-incompatibility, have raised concerns regarding their clinical application. Recent studies have shown that human iPSCs and their derivatives lose their immunogenicity when major histocompatibility complex (MHC) class I and II genes are inactivated and CD47 is over-expressed. In this study, we used CRISPR-Cas9 technology to generate an isogenic iPSC line with a homozygous frameshift mutation in the MHC II transactivator (CIITA) gene. The CIITA-/- iPSCs exhibit typical morphology of pluripotent cells, normal karyotype, expression of pluripotency markers and differentiation capacity in the three germ layers.

Generation of PKD1 mono-allelic and bi-allelic knockout iPS cell lines using CRISPR-Cas9 system.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, characterised by the development of multiple fluid-filled cysts in the kidneys and other organs. PKD1 and PKD2 are the two major causative genes encoding for polycystin-1 and polycystin-2, respectively. Here, we report the generation of two isogenic induced pluripotent stem cell (iPSC) lines with either heterozygous or compound heterozygous mutations in the PKD1 gene using CRISPR-Cas9 technology. The PKD1+/- and PKD1-/- iPSCs maintain stem cell-like morphology, normal karyotype, pluripotency and differentiation capacity in the three germ layers.

Generation of two isogenic knockout PKD2 iPS cell lines, IRFMNi003-A-1 and IRFMNi003-A-2, using CRISPR/Cas9 technology.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited renal disease, characterized by multiple cysts that can lead to kidney failure resulting in end-stage renal disease. ADPKD is mainly caused by mutations in either the PKD1 and PKD2 genes, encoding for polycystin-1 and polycystin-2, respectively. In order to clarify the disease mechanisms, here we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines in which the PKD2 gene was deleted using CRISPR/Cas9 technology. The PKD2-/- iPSCs expressed the main pluripotency markers, were able to differentiate into the three germ layers and had a normal karyotype.

Sirt3 Deficiency Shortens Life Span and Impairs Cardiac Mitochondrial Function Rescued by Opa1 Gene Transfer.

Aims: Sirtuins, a family of NAD+-dependent deacetylases, are recognized as nondispensable regulators of aging processes. Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase that maintains mitochondrial bioenergetics, an essential prerequisite for healthy aging. In this study, using Sirt3 knockout (Sirt3-/-) mice, we sought to establish whether Sirt3 deficiency affected life span, an endpoint that has never been tested formally in mammals, and uncover the mechanisms involved in organ damage associated with aging. Results:Sirt3-/- mice experienced a shorter life span than wild-type mice and severe cardiac damage, characterized by hypertrophy and fibrosis, as they aged. No alterations were found in organs other than the heart. Sirt3 deficiency altered cardiac mitochondrial bioenergetics and caused hyperacetylation of optic atrophy 1 (OPA1), a SIRT3 target. These changes were associated with aberrant alignment of trans-mitochondrial cristae in cardiomyocytes, and cardiac dysfunction. Gene transfer of deacetylated Opa1 restored cristae alignment in Sirt3-/- mice, ameliorated cardiac reserve capacity, and protected the heart against hypertrophy and fibrosis. The translational relevance of these findings is in the data showing that SIRT3 silencing in human-induced pluripotent stem cell-derived cardiomyocytes led to mitochondrial dysfunction and altered contractile phenotype, both rescued by Opa1 gene transfer. Innovation: Our findings indicate that future approaches to heart failure could include SIRT3 as a plausible therapeutic target. Conclusion: SIRT3 has a major role in regulating mammalian life span. Sirt3 deficiency leads to cardiac abnormalities, due to defective trans-mitochondrial cristae alignment and impaired mitochondrial bioenergetics. Correcting cardiac OPA1 hyperacetylation through gene transfer diminished heart failure in Sirt3-/- mice during aging. Antioxid. Redox Signal. 31, 1255-1271.

CRISPR-Cas9-Mediated Correction of the G189R-PAX2 Mutation in Induced Pluripotent Stem Cells from a Patient with Focal Segmental Glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is defined by focal (involving few glomeruli) and segmental sclerosis of the glomerular tuft that manifests with nephrotic syndrome. Mutations in genes involved in the maintenance of structure and function of podocytes have been found in a minority of these patients. A family with adult-onset autosomal dominant FSGS was recently found to carry a new germline missense heterozygous mutation (p.G189R) in the octapeptide domain of the transcription factor PAX2. Here, we efficiently corrected this point mutation in patient-derived induced pluripotent stem cells (iPSCs) by means of CRISPR-Cas9-based homology-directed repair. The iPSC lines were differentiated into podocytes, which were tested for their motility. Editing the PAX2 p.G189R mutation restored podocyte motility, which was altered in podocytes derived from patient iPSCs.

MicroRNA-184 is a downstream effector of albuminuria driving renal fibrosis in rats with diabetic nephropathy.

AIMS/HYPOTHESIS: Renal fibrosis is a common complication of diabetic nephropathy and is a major cause of end-stage renal disease. Despite the suggested link between renal fibrosis and microRNA (miRNA) dysregulation in diabetic nephropathy, the identification of the specific miRNAs involved is still incomplete. The aim of this study was to investigate miRNA profiles in the diabetic kidney and to identify potential downstream targets implicated in renal fibrosis. METHODS: miRNA expression profiling was investigated in the kidneys of 8-month-old Zucker diabetic fatty (ZDF) rats during overt nephropathy. Localisation of the most upregulated miRNA was established by in situ hybridisation. The candidate miRNA target was identified by in silico analysis and its expression documented in the diabetic kidney associated with fibrotic markers. Cultured tubule cells served to assess which of the profibrogenic stimuli acted as a trigger for the overexpressed miRNA, and to investigate underlying epigenetic mechanisms. RESULTS: In ZDF rats, miR-184 showed the strongest differential upregulation compared with lean rats (18-fold). Tubular localisation of miR-184 was associated with reduced expression of lipid phosphate phosphatase 3 (LPP3) and collagen accumulation. Transfection of NRK-52E cells with miR-184 mimic reduced LPP3, promoting a profibrotic phenotype. Albumin was a major trigger of miR-184 expression. Anti-miR-184 counteracted albumin-induced LPP3 downregulation and overexpression of plasminogen activator inhibitor-1. In ZDF rats, ACE-inhibitor treatment limited albuminuria and reduced miR-184, with tubular LPP3 preservation and tubulointerstitial fibrosis amelioration. Albumin-induced miR-184 expression in tubule cells was epigenetically regulated through DNA demethylation and histone lysine acetylation and was accompanied by binding of NF-κB p65 subunit to miR-184 promoter. CONCLUSIONS/INTERPRETATION: These results suggest that miR-184 may act as a downstream effector of albuminuria through LPP3 to promote tubulointerstitial fibrosis, and offer the rationale to investigate whether targeting miR-184 in association with albuminuria-lowering drugs may be a new strategy to achieve fully anti-fibrotic effects in diabetic nephropathy.

MicroRNAs as Master Regulators of Glomerular Function in Health and Disease.

MicroRNAs (miRNAs) are important regulators of gene expression, and the dysregulation of miRNAs is a common feature of several diseases. More miRNAs are identified almost daily, revealing the complexity of these transcripts in eukaryotic cellular networks. The study of renal miRNAs, using genetically modified mice or by perturbing endogenous miRNA levels, has revealed the important biologic roles miRNAs have in the major cell lineages that compose the glomerulus. Here, we provide an overview of miRNA biogenesis and function in regulating key genes and cellular pathways in glomerular cells during development and homeostasis. Moreover, we focus on the emerging mechanisms through which miRNAs contribute to different diseases affecting the glomerulus, such as FSGS, IgA nephropathy, lupus nephritis, and diabetic nephropathy. In-depth knowledge of miRNA-based gene regulation has made it possible to unravel pathomechanisms, enabling the design of new therapeutic strategies for glomerular diseases for which available therapies are not fully efficacious.

MicroRNAs in kidney physiology and disease.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression. They have important roles during kidney development, homeostasis and disease. In particular, miRNAs participate in the onset and progression of tubulointerstitial sclerosis and end-stage glomerular lesions that occur in various forms of chronic kidney disease (CKD). Therefore, miRNAs represent potential new therapeutic targets for a debilitating disease that continues to increase in prevalence worldwide and for which fully effective therapies are lacking. Several lines of research aimed at improving common CKD diagnostic tools and avoiding invasive kidney biopsies have also identified circulating miRNAs as possible diagnostic and even prognostic biomarkers of kidney disease. This Review discusses current understanding of the function of miRNAs in CKD, focusing on functions specifically involved in the transforming growth factor ß1 pathway, which is activated in CKD. miRNAs that, according to available evidence, seem to be involved in diabetic nephropathy, IgA nephropathy, lupus nephritis, polycystic kidney disease and graft rejection, are also discussed.

MicroRNA-324-3p promotes renal fibrosis and is a target of ACE inhibition.

The contribution of microRNA (miRNA) to the pathogenesis of renal fibrosis is not well understood. Here, we investigated whether miRNA modulates the fibrotic process in Munich Wistar Fromter (MWF) rats, which develop spontaneous progressive nephropathy. We analyzed the expression profile of miRNA in microdissected glomeruli and found that miR-324-3p was the most upregulated. In situ hybridization localized miR-324-3p to glomerular podocytes, parietal cells of Bowman's capsule, and most abundantly, cortical tubules. A predicted target of miR-324-3p is prolyl endopeptidase (Prep), a serine peptidase involved in the metabolism of angiotensins and the synthesis of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). In cultured tubular cells, transient transfection with a miR-324-3p mimic reduced Prep protein and activity, validating Prep as a target of this miRNA. In MWF rats, upregulation of miR-324-3p associated with markedly reduced expression of Prep in both glomeruli and tubules, low urine Ac-SDKP, and increased deposition of collagen. ACE inhibition downregulated glomerular and tubular miR-324-3p, promoted renal Prep expression, increased plasma and urine Ac-SDKP, and attenuated renal fibrosis. In summary, these results suggest that dysregulation of the miR-324-3p/Prep pathway contributes to the development of fibrosis in progressive nephropathy. The renoprotective effects of ACE inhibitors may result, in part, from modulation of this pathway, suggesting that it may hold other potential therapeutic targets.

The Toll-IL-1R member Tir8/SIGIRR negatively regulates adaptive immunity against kidney grafts.

Members of the TLR/IL-1R superfamily mediate ischemia/reperfusion injury and initiate immune response in transplanted organs. In this study, we tested the hypothesis that Toll-IL-1R8 (TIR8), a negative regulator of TLR/IL-1R highly expressed in the kidney, modulates immune cell activation underlying kidney rejection. In a mouse model of fully mismatched kidney allotransplantation in which the graft is spontaneously accepted, intragraft Tir8 expression was enhanced compared with naive kidneys. Targeted deletion of Tir8 in the graft exerted a powerful antitolerogenic action leading to acute rejection. Similarly, in a mouse model of kidney graft acceptance induced by costimulation blockade, most Tir8(-/-) grafts were acutely rejected. Despite similar levels of TLR4, IL-1R, and their ligands, the posttransplant ischemia/reperfusion-induced inflammatory response was more severe in Tir8(-/-) than in Tir8(+/+) grafts and was followed by expansion and maturation of resident dendritic cell precursors. In vitro, Tir8(-/-) dendritic cell precursors acquired higher allostimulatory activity and released more IL-6 upon stimulation with a TLR4 ligand and TNF-alpha than Tir8(+/+) cells, which may explain the increased frequency of antidonor-reactive T cells and the block of regulatory T cell formation in recipients of a Tir8(-/-) kidney. Thus, TIR8 acts locally as a key regulator of allogeneic immune response in the kidney. Tir8 expression and/or signaling in donor tissue are envisaged as a novel target for control of innate immunity and amelioration of graft survival.

Targeting of the small GTPase Rap2b, but not Rap1b, to lipid rafts is promoted by palmitoylation at Cys176 and Cys177 and is required for efficient protein activation in human platelets.

Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction. We found that Rap1b was not associated to lipid rafts in resting platelets, and did not translocate to these microdomains in stimulated cells. By contrast, about 20% of Rap2b constitutively associated to lipid rafts, and this percentage did not increase upon platelet stimulation. Rap2b interaction with lipid rafts also occurred in transfected HEK293T cell. Upon metabolic labelling with [(3)H]palmitate, incorporation of the label into Rap2b was observed. Palmitoylation of Rap2b did not occur when Cys176 or Cys177 were mutated to serine, or when the C-terminal CAAX motif was deleted. Contrary to CAAX deletion, Cys176 and Cys177 substitution did not alter the membrane localization of Rap2b, however, relocation of the mutants within lipid rafts was completely prevented. In intact platelets, disruption of Rap2b interaction with lipid rafts obtained by cholesterol depletion caused a significant inhibition of aggregation. Importantly, agonist-induced activation of Rap2b was concomitantly severely impaired. These results demonstrate that Rap2b, but not the more abundant Rap1b, is associated to lipid rafts in human platelets. This interaction is supported by palmitoylation of Rap2b, and is important for a complete agonist-induced activation of this GTPase.

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins.

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.