Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease associated with progressive death of midbrain dopamine (DAn) neurons in the substantia nigra (SN). Since it has been proposed that patients with PD exhibit an overall proinflammatory state, and since astrocytes are key mediators of the inflammation response in the brain, here we sought to address whether astrocyte-mediated inflammatory signaling could contribute to PD neuropathology. For this purpose, we generated astrocytes from induced pluripotent stem cells (iPSCs) representing patients with PD and healthy controls. Transcriptomic analyses identified a unique inflammatory gene expression signature in PD astrocytes compared with controls. In particular, the proinflammatory cytokine IL-6 was found to be highly expressed and released by PD astrocytes and was found to induce toxicity in DAn. Mechanistically, neuronal cell death was mediated by IL-6 receptor (IL-6R) expressed in human PD neurons, leading to downstream activation of STAT3. Blockage of IL-6R by the addition of the FDA-approved anti-IL-6R antibody, Tocilizumab, prevented PD neuronal death. SN neurons overexpressing IL-6R and reactive astrocytes expressing IL-6 were detected in postmortem brain tissue of patients at early stages of PD. Our findings highlight the potential role of astrocyte-mediated inflammatory signaling in neuronal loss in PD and pave the way for the design of future therapeutics.

iPSC-Based Modeling of Variable Clinical Presentation in Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. METHODS: We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. RESULTS: Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. CONCLUSIONS: Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.

Modeling iPSC-derived human neurofibroma-like tumors in mice uncovers the heterogeneity of Schwann cells within plexiform neurofibromas.

Plexiform neurofibromas (pNFs) are developmental tumors that appear in neurofibromatosis type 1 individuals, constituting a major source of morbidity and potentially transforming into a highly metastatic sarcoma (MPNST). pNFs arise after NF1 inactivation in a cell of the neural crest (NC)-Schwann cell (SC) lineage. Here, we develop an iPSC-based NC-SC in vitro differentiation system and construct a lineage expression roadmap for the analysis of different 2D and 3D NF models. The best model consists of generating heterotypic spheroids (neurofibromaspheres) composed of iPSC-derived differentiating NF1(-/-) SCs and NF1(+/-) pNF-derived fibroblasts (Fbs). Neurofibromaspheres form by maintaining highly proliferative NF1(-/-) cells committed to the NC-SC axis due to SC-SC and SC-Fb interactions, resulting in SC linage cells at different maturation points. Upon engraftment on the mouse sciatic nerve, neurofibromaspheres consistently generate human NF-like tumors. Analysis of expression roadmap genes in human pNF single-cell RNA-seq data uncovers the presence of SC subpopulations at distinct differentiation states.

Generation of two heterozygous GATA2 CRISPR/Cas9-edited iPSC lines, R398W and R396Q, for modeling GATA2 deficiency.

Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.

Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.

Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(-/-) cells, descendants from the tumor originating cell. These NF1(-/-)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(-/-) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

Patient-Specific iPSC-Derived Astrocytes Contribute to Non-Cell-Autonomous Neurodegeneration in Parkinson's Disease.

Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells.

Patient-Specific iPSC-Derived Endothelial Cells Provide Long-Term Phenotypic Correction of Hemophilia A.

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.

iPS Cell Cultures from a Gerstmann-Sträussler-Scheinker Patient with the Y218N PRNP Mutation Recapitulate tau Pathology.

Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient.

Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks.

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.

Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease.

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type.

A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells.

The generation of patient-specific induced pluripotent stem cells (iPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCs can be obtained in 4-5 months.

Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions.

The availability of induced pluripotent stem cells (iPSCs) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno-free cell culture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin (trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.

P-glycoprotein activity in renal clear cell carcinoma.

BACKGROUND: The mechanism by which renal cancer patients show poor response to chemotherapy has not been well understood. The aim of this study was to evaluate the functional activity of P-glycoprotein (P-gp) in renal clear cell carcinoma (RCCC) and its possible role in chemotherapy resistance. METHODS: We studied 11 patients who underwent radical nephrectomy due to RCCC; from each patient we obtained a sample from the cancer tissue, and another from normal renal tissue. These biopsies were mechanically disaggregated to allow individual cells analysis. Cells were incubated with daunorubicin (a fluorescent drug extruded by P-gp) at 37 degrees C and 4 degrees C for 30 min. P-gp activity was analyzed using flow cytometry. Results were expressed as the percentage of cells with P-gp activity (i.e., low fluorescence). RESULTS: The analysis of renal cells showed that there was no significant difference in size between normal and cancer cells; however there were clusters of cells with different granularities. We divided the cells according to their granularity. The proportion of cells capable of extruding daunorubicin was significantly higher on tumor cells than in normal renal cells independently of the cell granularity. Our results are congruent with those obtained when mRNA or immunohistochemical test were used. This is the first report quantifying the P-gp activity from fresh samples obtained from kidney cancer in humans. CONCLUSIONS: Percentage of cells extruding daunorubicin in RCCC is elevated, indicating that P-gp activity may contribute to multidrug resistance in RCCC.

An unusual multiplex systemic lupus erythematosus family with high prevalence of nephropathy, late-onset disease, and one member with disease-onset post-HIV therapy.

In the present study, we report the clinical characteristics of a unique systemic lupus erythematosus (SLE) multiplex family with 6 of its members affected by the disease, 1 of them being male. Four patients showed nephropathy, 2 of them with late-onset SLE (52 and 55-year-old), one with cutaneous and articular involvement, and another one developing lupus after 5 years undergoing highly active antiretroviral therapy (HAART) due to acquired immunodeficiency syndrome. Notwithstanding the genetic load, the fact that 2 patients showed late-onset disease, and the extreme delay of the appearance of SLE after HAART in the proband suggest that not only genetic, but other--mainly environmental--factors are necessarily required for the development of SLE.

Lupus nephritis remission, albeit with positive anti-doping test.

A 39-year-old woman developed systemic lupus erythematosus with nephropathy after a holiday in Jamaica. She was prescribed with prednisone, azathioprine and aspirin. As she was obsessed with aesthetic procedures, she decided not to take the prescription. Instead, she took her bodybuilding trainer's advice of one intramuscular injection of stanozolol for 10 weeks in order to increase her gluteus area. One week after finishing the latter regimen, there was no disease activity. Whether lupus remission in this patient was spontaneous or a consequence of stanozolol administration will remain a riddle for this fortunate outcome.

Human leukocyte antigen class I, class II, and tumor necrosis factor-alpha polymorphisms in a healthy elder Mexican Mestizo population.

BACKGROUND: There is strong evidence that an individual's genetic background is an important predisposing factor to longevity. In the present study we analysed the frequency of HLA class I, class II, as well as the TNF-alpha -308 polymorphism that may be related to an increased life span in Mexican Mestizo healthy elders. RESULTS: HLA typing was performed by polymerase chain reaction sequence specific oligonucleotide (PCR SSO) reverse dot blot. The TNF-alpha -308 polymorphism was assessed by PCR restriction fragment length polymorphism. A significant increased frequency of HLA-DRB1*11 was found in elderly women whereas this allele was not present in elderly males. The TNF2 allele was also increased in the elder group when compared to young controls. The frequencies of the remaining alleles tested were not statistically different among groups. CONCLUSION: These data suggest an ethnicity independent tendency of HLA-DRB1*11 in elder females to increase life span and a possible role of the TNF2 allele with the successful remodelling of senescent immune system.

Multidrug resistance-1 (MDR-1) in autoimmune disorders IV. P-glycoprotein overfunction in lymphocytes from myasthenia gravis patients.

Multidrug resistance (MDR) mechanisms have been widely studied in cancer. Among them, P-glycoprotein (P-gp) overfunction has been associated with resistance to several antineoplastic agents. The physiological role of P-gp involves hormone and metabolite secretion, bacterial product detoxification, and transport of several drugs to the extracellular space, thus inhibiting their toxic or therapeutic effects. The study of MDR-1 in diseases of autoimmune origin has just recently emerged. Corticosteroids remain the mainstay therapy for autoimmune diseases. As prednisone (PDN) is transported by P-gp, the aim of this study was to evaluate the P-gp function in lymphocytes from myasthenia gravis (MG) patients. Thirty MG patients and 25 healthy controls were studied. Peripheral blood mononuclear cells were isolated by gradient centrifugation and incubated with daunorubicin (DNR) (a fluorescent drug extruded by P-gp). Functional activity of P-gp was analyzed by flow cytometry. Results were expressed as percentage of gated lymphocytes able to efflux DNR. Overall, MG patients showed increased numbers of lymphocytes with functional P-gp activity when compared with controls (x = 4.92 +/- 5.26% vs. x = 0.7 +/- 0.48%, respectively) (P < 0.0001). When patients were classified as responders (n = 21) or refractory (n = 9) to treatment, the latter group exhibited higher values of functional P-gp (x = 10.18 +/- 6.39%) when compared to the responder group (x = 2.66 +/- 2.45%) (P = 0.0076). These data suggest, on the one hand, that drug resistance may be induced by long-term treatment or by high PDN doses and, on the other, emphasize the need for the study of P-gp antagonists in order to improve the current therapeutical schemes for the treatment of MG.

Class I and class II MHC polymorphisms in Mexican patients with Behçet's disease.

Behçet's disease is a multi-system inflammatory disorder of unknown etiology. The disease is more prevalent in Eastern Mediterranean countries and Japan where there is a linkage to HLA-B51. Mexican Mestizos are suitable subjects for studying the role of ethnicity in the susceptibility to Behçet's disease. High-resolution HLA class I and class II typing was performed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) reverse dot blot and PCR-single-strand polymorphism in 32 patients with Behçet's disease and 99 healthy ethnically-matched controls. A significant increased frequency of HLA-B(*)44 (P = 0.02; OR = 2.78; CI 95% = 1.1-7.7), HLA-B(*)52 (P = 0.02; OR = 5.33; CI 95% = 1.07-29.1), and HLA-B(*)56 (P = 0.003; OR = 4.19; CI 95% = 3.37-5.21) as well as HLA-DRB1(*)01 and HLA-DRB1(*)13 (p = 0.007; OR = 3.36; CI 95% = 1.22-9.27) was found in Mexican patients with Behçet's disease when compared to controls. The low frequency of native markers in Mexican Mestizo patients with Behçet's disease suggests that genetic admixture between Eastern Mediterraneans and Orientals with Amerindians is a recent event that increased the risk of developing Behçet's disease in the Mexican population.