İnfantil Osteopetrozisli Hasta Uyarılmış Pluripotent Kök Hücreleri ile Osteoklast Defektinin Modellenmesi,Osteoklast ve Hematopoietik Niş İlişkisi

117S145Malignant infantile osteopetrosis is an autosomal recessive disorder. The only available treatment is allogeneic hematopoietic stem cell transplantation. The objective of this study was to model hematopoietic niche in ostepetrosis. hiPSC lines were generated from peripheral blood mononuclear cells of three patients carrying TCIRG1. IPSC lines were differentiated first into HSCs, then into myeloid progenitors and osteoclasts using a step-wise protocol. iHSCs-derived osteoclasts were characterized by scanning-electron-microscope(SEM), flow-cytometry, IF-staining and expression of osteoclast-specific molecular markers. Different coculture conditions with bone marrow-derived-hMSCs and iHSCs were set up to study the interaction between osteopetrotic-HSCs and healthy- and/or osteopetrotics-MSCs as an in vitro hematopoietic niche model. After coculture, expression of the genes in MSCs related to HSC kinetics were analyzed and migration assays were done. All iPSC lines were showed typical ESC features with regards to morphology, pluripotency surface markers and pluripotency-associated gene expression profile. All lines were differentiated succesfully into HSCs, myeloid progenitors and osteoclasts. Osteopetrotic-iPSCs-derived osteoclasts were weak positive for Cathepsin K, and TRAP, were showed weak positive actin ring and short podosome formation, and exhibited osteoclast-spesific surface markers, but showed significantly reduced expression of Cathepsin K, Calcitonin-R, and NFATC1compared to controls. Following coculture with healthy-control iHSCs, the expression of Jagged-1,Ang-1,Kit-L,Sdf-1, and Opn increased while overexpression of N-cadherin decreased after coculture in osteopetrotic MSCs. The migration potential of osteopetrotic iHCSs were found impaired compared to control-iHSCs. In conclusion, our results indicate that dysfunctional osteoclasts in osteopetrosis lead to defective HSC niche formation resulting from altered MSC compartment, and abnormal HSC homing besides insufficient marrow space resulting from defective bone resorbtion.Malign infantil osteopetrozis otozomal resesif bir hastalıktır ve tek tedavi yöntemi allojenik hematopoetik kök hücre tedavisidir. Tez çalışmasının ana hedefi, osteopetrozis hastalığında hematopoetik nişin modellenmesidir. Bu kapsamda TCIRG1 mutasyonu taşıyan üç osteopetrozis hastasının periferik kan mononükleer hücrelerinden Uyarılmış Pluripotent Kök Hücre (uPKH) hatları elde edilmiştir. uPKH hatları ilk olarak Hematopoetik Kök Hücre’lere (HKH) daha sonra myeloid progenitor ve osteoklast hücrelerine farklılaştırılmıştır. Uyarılmış Pluripotent Kök Hücre’lerden elde edilen Hematopoetik Kök Hücre (uHKH) kaynaklı osteoklast hücreleri taramalı elektron mikroskop (SEM), akım sitometri, immünfloresan boyama ve osteoklast spesifik moleküler belirteç ifadesi değerlendirilerek karakterize edilmiştir. In vitro hematopoetik niş modelleme için, osteopetrotik–HKH ve sağlıklı-ve/veya osteopetrotik MKH’leri arasındaki etkileşimi değerlendirmek amacıyla kemik iliği MKH ve uHKH’lerin farklı kokültür kombinasyonları yapılmıştır. Kokültür deneylerinden sonra, MKH’lerde HKH’lerin kinetiği ile ilişkili genlerin ifadesi analizi edilmiş ve migrasyon deneyleri yapılmıştır. Bütün uPKH hatları morfoloji, pluripotensi yüzey belirteçleri, pluripotensi ile ilişkili gen ifade profili açısından EKH’lere benzer özellikler göstermiştir. Bütün uPKH hatları başarılı bir şekilde HKH’lere, myeloid progenitörlere ve osteoklastlara farklılaştırılmıştır. Osteopetrotik uPKH kaynaklı osteoklast hücreleri Kathepsin K ve TRAP ile zayıf boyanmış, osteoklast spesifik yüzey belirteç ifadesi gözlemlenmiş, ancak zayıf aktin halka ve kısa podozom oluşumu, Kathepsin K, Kalsitonin-R ve NFATC1 ifadesinin ise önemli ölçüde azaldığı tespit edilmiştir. Sağlıklı-kontrol uHKH’leri ile kokültür sonrası, Jagged-1, Ang-1, Kit-L, Sdf-1 ve Opn ifadesi artmıştır. Osteopetrotik MKH’lerde artmış N-kadherin ifadesi gözlemlenmiş, ancak sağlıklı uHKH’ler ile kokültür sonrası azalmıştır. Kontrol uHKH’ler ile karşılaştırıldığında osteopetrotik uHKH’lerin migrasyon potansiyelinin bozulduğu gözlemlenmiştir. Sonuç olarak, osteopetrozisde disfonksiyonel osteoklast hücreleri defektif HKH niş oluşumuna neden olmaktadır. Bunun da temel nedenleri MKH kompartmanında görülen değişim ve HKH’lerin nişteki yerleşiminin bozulmasıdır

Osteopetrotic Induced Pluripotent Stem Cells Derived From Patients With Different Disease-Associated Mutations By Non-Integrating Reprogramming Methods

Background Autosomal recessive osteopetrosis is a genetically and phenotypically heterogeneous disease, caused by defects in osteoclast formation and function. The only available treatment is allogeneic stem cell transplantation that has still high morbidity and mortality. The goal of the present study was to generate iPSCs from bone marrow-derived MSCs of osteopetrosis patients with three most common mutations by using two different integration-free gene transfer methods and compare their efficiencies. The secondary objective was to select the most appropriate integration-free production method for our institutional iPSC bank using this rare disease as a prototype. Methods Two different integration-free gene transfer methods (episomal and Sendai viral vectors) were tested and compared on the same set of patient samples exhibiting three different mutations associated with osteopetrosis. Generated iPSCs were characterized by standard assays, including immunophenotyping, immunocytochemistry, RT-PCR, embryoid body, and teratoma assays. Karyotype analyses were performed to evaluate genetic stability. Results iPSC lines exhibiting typical ESC-like colony morphology were shown to express pluripotency markers by immunofluorescence staining. Over 90% of the cells were found positive for SSEA-4 and OCT3/4 and negative/weak positive for CD29 by flow cytometry. Immunohistochemical staining of teratoma and spontaneously differentiated embryoid body sections confirmed their trilineage differentiation potential. All iPSC lines expressed pluripotency-related genes. Karyotype analyses were found normal. Direct sequencing of PCR-amplified DNA showed that disease-related mutations were retained in the patient-specific iPSCs. Conclusion Generation of iPSC using SeV and episomal DNA vectors have several advantages over other methods like the ease of production, reliability, high efficiency, and safety, which is required for translational research. Furthermore, owing to the pluripotency and self-renewal capacity, patient-specific iPSCs seem to be ideal cell source for the modeling of a rare genetic bone disease like osteopetrosis to identify osteoclast defects, leading to clinical heterogeneity in osteopetrosis patients, especially among those with different mutations in the same gene. Electronic supplementary material The online version of this article (10.1186/s13287-019-1316-8) contains supplementary material, which is available to authorized users.PubMedWoSScopu

Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 In Vitro

Ribavirin is a guanosine analog and has a broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection whereas the drug itself did not show any toxic effect over the concentration range tested. In silico analysis suggested that Ribarivin has a broad-spectrum impact on SARS-CoV-2, acting at different viral proteins. According to the detailed molecular techniques, Ribavirin was shown to decrease the expression of TMPRSS2 both at mRNA and protein levels 48 hours after treatment. The suppressive effect of Ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that Ribavirin also showed an inhibitory effect on TMPRSS2 enzyme. Based on these results, we hypothesized that Ribavirin may inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. As a conclusion, Ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effect of TMPRSS2 and ACE2 expression.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author