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Background. The development of a hybrid beta-cell replacement approach, referred to as a personalized, transplantable bioartificial pancreas (BAP), holds promise to treat type 1 diabetes (T1D). This interview study aimed to explore patients’ expectations, needs, concerns, and considerations when considering to undergo a BAP transplantation. Research Design and Methods. Semistructured interviews were conducted with 24 participants diagnosed with T1D. Data collection stopped once data saturation was reached. Audio recordings of the interviews were transcribed verbatim. The interviews were independently analyzed by 2 researchers. A qualitative content analysis using an inductive approach was used. Results. Three main themes emerged as follow: (1) hoped-for benefits, (2) concerns and decision-making considerations, and (3) procedural aspects. First, the participants expected benefits across medical, psychological, and social domains. Over these 3 domains, 9 subthemes were identified, including improved clinical outcomes, a cure for diabetes, more headspace, emotional relief, a shift in responsibility, protection of privacy, improved flexibility in daily life, less visible diseases, and improved relationships with others. Second, concerns and considerations about undergoing a BAP transplant comprised adverse events, the functionality of the BAP, the surgery procedure, the biological materials used, the transplant location, and the intrusiveness associated with follow-up care. Finally, procedural considerations included equitable access, patient prioritization, and trust and control. Conclusions. Incorporating insights from this study into the clinical development and implementation of the BAP is crucial to ensure alignment of the product and procedures with the needs and expectations of people with T1D.

Abstract Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

Not available.

Liver sinusoidal endothelial cells (LSECs) are specialized endocytic cells that clear the body from blood-borne pathogens and waste macromolecules through scavenger receptors (SRs). Among the various SRs expressed by LSECs is stabilin-2 (STAB2), a class H SR that binds to several ligands, among which endogenous coagulation products. Given the well-established tolerogenic function of LSECs, we asked whether the STAB2 promoter (STAB2p) would enable us to achieve LSEC-specific lentiviral vector (LV)-mediated transgene expression, and whether the expression of this transgene would be maintained over the long term due to tolerance induction. Here, we show that STAB2p ensures LSEC-specific green fluorescent protein (GFP) expression by LV in the absence of a specific cytotoxic CD8+ T cell immune response, even in the presence of GFP-specific CD8+ T cells, confirming the robust tolerogenic function of LSECs. Finally, we show that our delivery system can partially and permanently restore FVIII activity in a mouse model of severe hemophilia A without the formation of anti-FVIII antibodies. Overall, our findings establish the suitability of STAB2p for long-term LSEC-restricted expression of therapeutic proteins, such as FVIII, or to achieve antigen-specific immune tolerance in auto-immune diseases.

Hemostasis is a sophisticated sequence of events aimed to repair vessel injury. This process occurs in combination with angiogenesis, which leads to new blood vessel formation helping in the wound repair and facilitating tissue healing. The fine mechanisms that regulate hemostasis and angiogenesis are well described, but for long time, coagulation factors (CFs) have been considered merely players in the coagulation cascade. However, several experimental evidences highlight the crucial functions of these CFs in regulating endothelial functionality, especially in the angiogenic process. Some of these CFs (e.g. thrombin and tissue factor) have been widely investigated and have been described to trigger intracellular signaling related to endothelial cell (EC) functionality. For others (e.g. factor VIII and thrombomodulin), potential receptors and molecular mechanisms have not been fully elucidated but some data show their potential to induce EC response. This review focuses on the emerging roles of selected CFs in regulating EC functions, especially highlighting their ability to activate signaling pathways involved in the angiogenesis, migration, proliferation and endothelial barrier stability.

Abstract Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, 23Na-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.

Cell therapy for diabetes requires the combination of bioengineering and new sources of beta cells. Here, the authors report a self-assembly platform based on neonatal pig islets, human endothelial cells and native organ extracellular matrix, which improves ex vivo islet maturation and in vivo function in a mouse model of diabetes.

BackgroundChronic systemic inflammation reduces the bioavailability of circulating endothelial progenitor cells (EPCs). Indoleamine 2,3-dioxygenase 1 (IDO1), a key enzyme of immune tolerance catalyzing the initial step of tryptophan degradation along the so-called l-kynurenine (l-kyn) pathway, that is induced by inflammatory stimuli and exerts anti-inflammatory effects. A specific relationship between IDO1 activity and circulating EPC numbers has not yet been investigated.MethodsIn this study, circulating EPCs were examined in mice treated with low doses of lipopolysaccharide (LPS) to mimic low-grade inflammation. Moreover, the association between IDO1 activity and circulating EPCs was studied in a cohort of 277 patients with variable systemic low-grade inflammation.ResultsRepeated low doses of LPS caused a decrease in circulating EPCs and l-kyn supplementation, mimicking IDO1 activation, significantly increased EPC numbers under homeostatic conditions preventing EPC decline in low-grade endotoxemia. Accordingly, in patients with variable systemic low-grade inflammation, there was a significant interaction between IDO1 activity and high-sensitivity C-reactive protein (hs-CRP) in predicting circulating EPCs, with high hs-CRP associated with significantly lower EPCs at low IDO1 activity but not at high IDO1 activity.InterpretationOverall, these findings demonstrate that systemic low-grade inflammation reduces circulating EPCs. However, high IDO1 activity and l-kyn supplementation limit circulating EPC loss in low-grade inflammation.

Abstract Liver gene therapy with adeno‐associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X‐linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti‐F8 antibodies which neutralise F8 activity. Taking advantage of split‐intein‐mediated protein trans‐splicing, we divided the coding sequence of the large and highly secreted F8‐N6 variant in two separate AAV‐intein vectors whose co‐administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti‐F8 antibodies unlike animals treated with the single oversized AAV‐F8 vector under clinical development. Therefore, liver gene therapy with AAV‐F8‐N6 intein should be considered as a potential therapeutic strategy for HemA.

“Lentiviral gene therapy to the liver establishes stable long-term normal to supra-normal coagulation factor VIII activity in mouse models of hemophilia A and in non-human primates, representing a potential new treatment option for people with hemophilia A.”.

Hemophilia A (HA) cell therapy approaches in pediatric individuals require suitable factor (F)VIII-producing cells for stable engraftment. Liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells (HSC) have been demonstrated to be suitable for the treatment of adult HA mice. However, after transplantation in busulfan (BU)-conditioned newborn mice, adult LSEC/HSC cannot efficiently engraft, while murine fetal liver (FL) hemato/vascular cells from embryonic day 11-13 of gestation (E11-E13), strongly engraft the hematopoietic and endothelial compartments while also secreting FVIII. Our aim was to investigate the engraftment of FL cells in newborn HA mice to obtain a suitable “proof of concept” for the development of a new HA treatment in neonates. Hence, we transplanted FL E11 or E13 cells and adult bone marrow (BM) cells into newborn HA mice with or without BU preconditioning. Engraftment levels and FVIII activity were assessed starting from 6 weeks after transplantation. FL E11-E13+ BU transplanted newborns reached up to 95% engraftment with stable FVIII activity levels observed for 16 months. FL E13 cells showed engraftment ability even in the absence of BU preconditioning, while FL E11 cells did not. BM BU transplanted newborn HA mice showed high levels of engraftment; nevertheless, in contrast to FL cells, BM cells cannot engraft HA newborns in BU non-conditioning regimen. Finally, none of the transplanted mice developed anti-FVIII antibodies. Overall, this study sheds some light on the therapeutic potential of healthy FL cells in the cure of HA neonatal/pediatric patients.

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA.

The eggshell is a biomineral consisting of CaCO3 in the form of calcite phase and a pervading organic matrix (1–3.5 wt.%). Transforming eggshell calcite particles into calcium phosphate (apatite) micro-nanoparticles opens the door to repurposing the eggshell waste as materials with potential biomedical applications, fulfilling the principles of the circular economy. Previous methods to obtain these particles consisted mainly of two steps, the first one involving the calcination of the eggshell. In this research, direct transformation by a one-pot hydrothermal method ranging from 100–200 °C was studied, using suspensions with a stoichiometric P/CaCO3 ratio, K2HPO4 as P reagent, and eggshells particles (Ø < 50 μm) both untreated and treated with NaClO to remove surface organic matter. In the untreated group, the complete conversion was achieved at 160 °C, and most particles displayed a hexagonal plate morphology, eventually with a central hole. In the treated group, this replacement occurred at 180 °C, yielding granular (spherulitic) apatite nanoparticles. The eggshell particles and apatite micro-nanoparticles were cytocompatible when incubated with MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells and promoted the osteogenic differentiation of m17.ASC cells. The study results are useful for designing and fabricating biocompatible microstructured materials with osteoinductive properties for applications in bone tissue engineering and dentistry.

New therapeutic strategies are required in cancer therapy. Considering the prominent role of tumor-associated macrophages (TAMs) in the development and progression of cancer, the re-education of TAMs in the tumor microenvironment (TME) could represent a potential approach for cancer immunotherapy. TAMs display an irregular unfolded protein response (UPR) in their endoplasmic reticulum (ER) to endure environmental stress and ensure anti-cancer immunity. Therefore, nanotechnology could be an attractive tool to modulate the UPR in TAMs, providing an alternative strategy for TAM-targeted repolarization therapy. Herein, we developed and tested polydopamine-coupled magnetite nanoparticles (PDA-MNPs) functionalized with small interfering RNAs (siRNA) to downregulate the protein kinase R (PKR)-like ER kinase (PERK) expression in TAM-like macrophages derived from murine peritoneal exudate (PEMs). After the evaluation of the cytocompatibility, the cellular uptake, and the gene silencing efficiency of PDA-MNPs/siPERK in PEMs, we analyzed their ability to re-polarize in vitro these macrophages from M2 to the M1 inflammatory anti-tumor phenotype. Our results indicate that PDA-MNPs, with their magnetic and immunomodulator features, are cytocompatible and able to re-educate TAMs toward the M1 phenotype by PERK inhibition, a UPR effector contributing to TAM metabolic adaptation. These findings can provide a novel strategy for the development of new tumor immunotherapies in vivo.

Maria Feola et al., elucidate the compensatory role of pleckstrin-2 in ineffective erythropoiesis in β-thalassemic mice by reducing cofilin-mediated apoptosis and enhancing enucleation by activating RacGTPases. These findings could support future therapeutic interventions.

Summary: Liver sinusoidal endothelial cells (LSECs) form the predominant microvasculature in the liver where they carry out many functions including the secretion of coagulation factor VIII (FVIII). To investigate the early origins of this lineage, we develop an efficient and scalable protocol to produce human pluripotent stem cell (hPSC)-derived LSEC progenitors characterized as venous endothelial cells (VECs) from different mesoderm subpopulations. Using a sensitive and quantitative vascular competitive transplantation assay, we demonstrate that VECs generated from BMP4 and activin A-induced KDR+CD235a/b+ mesoderm are 50-fold more efficient at LSEC engraftment than venous cells from BMP4 and WNT-induced KDR+CD235a/b− mesoderm. When transplanted into immunocompromised hemophilia A mice (NSG-HA), these VECs engraft the liver, proliferate, and mature to functional LSECs that secrete bioactive FVIII capable of correcting the bleeding phenotype. Together, these findings highlight the importance of appropriate mesoderm induction for generating hPSC-derived LSECs capable of functioning in a preclinical model of hemophilia A.

The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 × 106 intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 × 103 to 1:1 × 106 human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies.

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.

Abstract Background SHOX enhancer CNVs, affecting one or more of the seven recognized evolutionary conserved non‐coding elements (CNEs) represent one of the most frequent cause of SHOX‐haploinsufficiency. During the diagnostic workflow deletions/duplications have been identified downstream SHOX not including any of the these CNEs. Methods Fine tiling aCGH and breakpoint PCR were used to characterize the critical interval and to search for novel alterations in a cohort of selected patients. Results Screening of 252 controls provided evidence that duplications in this area represent likely benign variants whereas none of the deletions were detected. These findings suggested that other alterations relevant for SHOX‐haploinsufficiency might be missed by the standard diagnostic methods. To identify such undisclosed elements, the aCGH was used to reanalyze 52 unresolved cases with clinical features strongly suggestive of SHOX‐haploinsufficiency. This analysis followed by the screening of 210 patients detected two partially overlapping small deletions of ~12 and ~8 kb in four unrelated individuals, approximately 15 kb downstream SHOX, that were absent in 720 normal stature individuals. Conclusion Our results strengthen the hypothesis that alterations of yet unidentified cis‐regulatory elements residing outside those investigated through conventional methods, might explain the phenotype in ISS/LWD patients thus enlarging the spectrum of variants contributing to SHOX‐haploinsufficiency.

Cells of the cardiovascular system are physiologically exposed to a variety of mechanical forces fundamental for both cardiac development and functions. In this context, forces generated by actomyosin networks and those transmitted through focal adhesion (FA) complexes represent the key regulators of cellular behaviors in terms of cytoskeleton dynamism, cell adhesion, migration, differentiation, and tissue organization. In this study, we investigated the involvement of FAs on cardiomyocyte differentiation. In particular, vinculin and focal adhesion kinase (FAK) family, which are known to be involved in cardiac differentiation, were studied. Results revealed that differentiation conditions induce an upregulation of both FAK-Tyr397 and vinculin, resulting also in the translocation to the cell membrane. Moreover, the role of mechanical stress in contractile phenotype expression was investigated by applying a uniaxial mechanical stretching (5% substrate deformation, 1 Hz frequency). Morphological evaluation revealed that the cell shape showed a spindle shape and reoriented following the stretching direction. Substrate deformation resulted also in modification of the length and the number of vinculin-positive FAs. We can, therefore, suggest that mechanotransductive pathways, activated through FAs, are highly involved in cardiomyocyte differentiation, thus confirming their role during cytoskeleton rearrangement and cardiac myofilament maturation.

Islet transplantation is a feasible approach to treat type I diabetes, however inflammation and poor vascularisation impair long-term engraftment. Here the authors show that incorporating human amniotic epithelial cells into islet organoids improves engraftment and function of organoids, through enhanced revascularisation.

Gene expression regulation is the result of complex interactions between transcriptional and post-transcriptional controls, resulting in cell-type-specific gene expression patterns that are determined by the developmental and differentiation stage of pathophysiological conditions. Understanding the complexity of gene expression regulatory networks is fundamental to gene therapy, an approach which has the potential to treat and cure inherited disorders by delivering the correct gene to patient specific cells or tissues by means of both viral and non-viral vectors. Besides the issues of biosafety, in recent years efforts have focused on achieving a robust and sustained transgene expression, which attains a phenotypic correction in several diseases, while avoiding transgene-related adverse effects, such as overexpression-associated cytotoxicity and/or immune responses to the transgene. In this sense, the use of cell-type-specific promoters and microRNA target sequences (miRTs) in gene transfer expression cassettes have allowed for a restricted expression after gene transfer in several studies. This review will focus on the use of transcriptional and post-transcriptional regulation to achieve a highly specific and safe transgene expression, as well as their application in ex vivo and in vivo gene therapeutic approaches.

Summary: We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an optimized protocol. FVIII-expressing ECs were intraportally transplanted in monocrotaline-conditioned non-obese diabetic (NOD) severe combined immune-deficient (scid)-IL2rγ null HA mice generating a chimeric liver with functional human ECs. Transplanted cells engrafted and proliferated in the liver along sinusoids, in the long term showed stable therapeutic FVIII activity (6%). These results demonstrate that the hemophilic phenotype can be rescued by transplantation of ECs derived from HA FVIII-corrected iPSCs, confirming the feasibility of cell-reprogramming strategy in patient-derived cells as an approach for HA gene and cell therapy. : Follenzi and colleagues show that endothelial cells can be obtained starting from blood CD34+ cells of both healthy and hemophilic donors passing through iPSC generation. During the differentiation process the cells can be genetically modified by LV to express FVIII; corrected endothelial cells secrete FVIII in vitro and ameliorate the hemophilic phenotype in a mouse model of the disease. Keywords: hemophilia A, induced pluripotent stem cells (iPSCs), endothelial cells, cell and gene therapy, FVIII, chimeric vasculature, lentiviral vectors

A major challenge in the development of a gene therapy for hemophilia A (HA) is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 alone was ineffective. Moreover, Ets-1/Ets-2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of GFP or FVIII cassettes driven by the shortened promoters led to GFP expression mainly in endothelial cells in the liver and to long-term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in HA gene therapy.

Replacement therapy with coagulation factor VIII (FVIII) represents the current clinical treatment for patients affected by hemophilia A (HA). This treatment while effective is, however, hampered by the formation of antibodies which inhibit the activity of infused FVIII in up to 30% of treated patients. Immune tolerance induction (ITI) protocols, which envisage frequent infusions of high doses of FVIII to confront this side effect, dramatically increase the already high costs associated to a patient's therapy and are not always effective in all treated patients. Therefore, there are clear unmet needs that must be addressed in order to improve the outcome of these treatments for HA patients. Taking advantage of preclinical mouse models of hemophilia, several strategies have been proposed in recent years to prevent inhibitor formation and eradicate the pre-existing immunity to FVIII inhibitor positive patients. Herein, we will review some of the most promising strategies developed to avoid and eradicate inhibitors, including the use of immunomodulatory drugs or molecules, oral or transplacental delivery as well as cell and gene therapy approaches. The goal is to improve and potentiate the current ITI protocols and eventually make them obsolete.

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3-mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3-mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P

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Liver transplantation is the most common treatment for patients suffering from liver failure that is caused by congenital diseases, infectious agents, and environmental factors. Despite a high rate of patient survival following transplantation, organ availability remains the key limiting factor. As such, research has focused on the transplantation of different cell types that are capable of repopulating and restoring liver function. The best cellular mix capable of engrafting and proliferating over the long-term, as well as the optimal immunosuppression regimens, remain to be clearly well-defined. Hence, alternative strategies in the field of regenerative medicine have been explored. Since the discovery of induced pluripotent stem cells (iPSC) that have the potential of differentiating into a broad spectrum of cell types, many studies have reported the achievement of iPSCs differentiation into liver cells, such as hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. In parallel, an increasing interest in the study of self-assemble or matrix-guided three-dimensional (3D) organoids have paved the way for functional bioartificial livers. In this review, we will focus on the recent breakthroughs in the development of iPSCs-based liver organoids and the major drawbacks and challenges that need to be overcome for the development of future applications.

Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.

Tumor-targeted drug-loaded nanocarriers represent innovative and attractive tools for cancer therapy. Several magnetic nanoparticles (MNPs) were analyzed as potential tumor-targeted drug-loaded nanocarriers after functionalization with anti-Met oncogene (anti-Met/HGFR) monoclonal antibody (mAb) and doxorubicin (DOXO). Their cytocompatibility, stability, immunocompetence (immunoprecipitation), and their interactions with cancer cells in vitro (Perl’s staining, confocal microscopy, cytotoxic assays: MTT, real time toxicity) and with tumors in vivo (Perl’s staining) were evaluated. The simplest silica- and calcium-free mAb-loaded MNPs were the most cytocompatible, the most stable, and showed the best immunocompetence and specificity. These mAb-functionalized MNPs specifically interacted with the surface of Met/HGFR-positive cells, and not with Met/HGFR-negative cells; they were not internalized, but they discharged in the targeted cells DOXO, which reached the nucleus, exerting cytotoxicity. The presence of mAbs on DOXO-MNPs significantly increased their cytotoxicity on Met/HGFR-positive cells, while no such effect was detectable on Met/HGFR-negative cells. Bare MNPs were biocompatible in vivo; mAb presence on MNPs induced a better dispersion within the tumor mass when injected in situ in Met/HGFR-positive xenotumors in NOD/SCID-γnull mice. These MNPs may represent a new and promising carrier for in vivo targeted drug delivery, in which applied gradient and alternating magnetic fields can enhance targeting and induce hyperthermia respectively.

Research on mesenchymal stem cells from adipose tissue shows promising results for cell-based therapy in cartilage lesions. In these studies, cells have been isolated, expanded, and differentiated in vitro before transplantation into the damaged cartilage or onto materials used as scaffolds to deliver cells to the impaired area. The present study employed in vitro assays to investigate the potential of intra-articular injection of microfragmented lipoaspirate as a one-step repair strategy; it aimed to determine whether adipose tissue can act as a scaffold for cells naturally present at their anatomical site. Cultured clusters of lipoaspirate showed a spontaneous outgrowth of cells with a mesenchymal phenotype and with multilineage differentiation potential. Transduction of lipoaspirate clusters by lentiviral vectors expressing GFP evidenced the propensity of the outgrown cells to repopulate fragments of damaged cartilage. On the basis of the results, which showed an induction of proliferation and ECM production of human primary chondrocytes, it was hypothesized that lipoaspirate may play a paracrine role. Moreover, the structure of a floating culture of lipoaspirate, treated for 3 weeks with chondrogenic growth factors, changed: tissue with a high fat component was replaced by a tissue with a lower fat component and connective tissue rich in GAG and in collagen type I, increasing the mechanical strength of the tissue. From these promising in vitro results, it may be speculated that an injectable autologous biologically active scaffold (lipoaspirate), employed intra-articularly, may 1) become a fibrous tissue that provides mechanical support for the load on the damaged cartilage; 2) induce host chondrocytes to proliferate and produce ECM; and 3) provide cells at the site of injury, which could regenerate or repair the damaged or missing cartilage.

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbbth1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

A large fraction of factor VIII in blood originates from liver sinusoidal endothelial cells although extrahepatic sources also contribute to plasma factor VIII levels. Identification of cell-types other than endothelial cells with the capacity to synthesize and release factor VIII will be helpful for therapeutic approaches in hemophilia A. Recent cell therapy and bone marrow transplantation studies indicated that Küpffer cells, monocytes and mesenchymal stromal cells could synthesize factor VIII in sufficient amount to ameliorate the bleeding phenotype in hemophilic mice. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We identified factor VIII in cells isolated from peripheral and cord blood, as well as bone marrow. Co-staining for cell type-specific markers verified that factor VIII was expressed in monocytes, macrophages and megakaryocytes. We additionally verified that factor VIII was expressed in liver sinusoidal endothelial cells and endothelial cells elsewhere, e.g., in the spleen, lungs and kidneys. Factor VIII was well expressed in sinusoidal endothelial cells and Küpffer cells isolated from human liver, whereas by comparison isolated human hepatocytes expressed factor VIII at very low levels. After transplantation of CD34+ human cord blood cells into NOD/SCIDγNull-hemophilia A mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in the majority of mice. In these animals, plasma factor VIII activity 12 weeks after cell transplantation was up to 5% and nine of 12 mice survived after a tail clip-assay. In conclusion, hematopoietic cells, in addition to endothelial cells, express and secrete factor VIII: this information should offer further opportunities for understanding mechanisms of factor VIII synthesis and replenishment.

A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.