Characterization Of Mesenchymal Stem Cells In Mucolipidosis Type Ii (I-Cell Disease)

Mucolipidosis type II (ML-II, I-cell disease) is a fatal inherited lysosomal storage disease caused by a deficiency of the enzyme N-acetylglucosamine-1-phosphotransferase. A characteristic skeletal phenotype is one of the many clinical manifestations of ML-II. Since the mechanisms underlying these skeletal defects in ML-II are not completely understood, we hypothesized that a defect in osteogenic differentiation of ML-II bone marrow mesenchymal stem cells (BM-MSCs) might be responsible for this skeletal phenotype. Here, we assessed and characterized the cellular phenotype of BM-MSCs from a ML-II patient before (BBMT) and after BM transplantation (ABMT), and we compared the results with BM-MSCs from a carrier and a healthy donor. Morphologically, we did not observe differences in ML-II BBMT and ABMT or carrier MSCs in terms of size or granularity. Osteogenic differentiation was not markedly affected by disease or carrier status. Adipogenic differentiation was increased in BBMT ML-II MSCs, but chondrogenic differentiation was decreased in both BBMT and ABMT ML-II MSCs. Immunophenotypically no significant differences were observed between the samples. Interestingly, the proliferative capacity of BBMT and ABMT ML-II MSCs was increased in comparison to MSCs from age-matched healthy donors. These data suggest that MSCs are not likely to cause the skeletal phenotype observed in ML-II, but they may contribute to the pathogenesis of ML-II as a result of lysosomal storage-induced pathology.PubMedWoSScopu

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

Hematopoetik Kök Hücre Hibernasyonunda Nöropeptid Y (NPY)´nin Rolünün İncelenmesi

Hematopoetik kök hücreler (HKH), kök hücre nişi olarak da adlandırılan özel bir mikroçevrede bulunur. Bu mikrocevrede mezenkimal kök hucreler (MKH), endotelyal hücreler (EH), retiküler hücreler bulunur. Perivasküler mikroortamda bulunan EH, MKH, makrofajlar ve HKH yaşamını ve kemik iliği rejenerasyonunu düzenler.Nörotransmitter nöropeptit Y (NPY), makrofajlardan ve osteoblastlardan salınır. NPY, immün ve kemik hücre homeostazını veya bu yapıların vasküler yeniden yapılanmasını, kemik iliği hücrelerinde ifade edilen Y reseptörüyle düzenlerler. Hibernasyonda HKH'ler sıklıkla trabeküler endosteuma yakın bulunurlar. İhtiyaç duyulduğunda bu hücreler çoğalır ve farklılaşır, stres ve yaralanmalarda salgılanan büyüme faktörleri ile hücre döngüsüne girerler. NPY eksikliğinde, HKH’lerin sayıları düşmekte ve kemik iliği rejenerasyonu bozulmaktadır. Önerilen projenin amacı, NPY ve NPY reseptör sinyal yolağı etkisiyle HKH'lerin uyku durumuna geri dönmesi ve HKH havuz boyutu ile kemik iliği fonksiyonunun korunmasında sessizliğin rolünün ortaya çıkmasına ilişkin anahtar bir mekanizmanın belirlenmesidir

Diagnosis: Melanoderma After Hematopoietic Stem Cell Transplantation

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Automated washing of long-term cryopreserved peripheral blood stem cells promotes cell viability and preserves CD34+ cell numbers

Peripheral blood stem cell (PBSC) transplantation has become an established treatment option for a range of malignant and inherited diseases. PBSCs are usually cryopreserved in the presence of dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. However, cryopreservation and thawing of PBSCs may affect cell viability, resulting in delayed engraftment and other risks related to low stem cell numbers [1]. DMSO is commonly used at a concentration of 10% during freezing of PBSCs. However, DMSO itself is toxic and lowering doses of DMSO may have a favorable effect on hematopoietic recovery after transplantation [2, 3]. Generally, PBSCs are thawed and infused without removal of DMSO. This has been associated with a wide range of adverse effects (AEs), ranging from minor to severe life-threatening events [4, 5]. Although not all toxic events can be contributed to DMSO, grafts containing lower concentrations of DMSO typically display a reduced incidence of AEs [5, 6]. To decrease AEs and improve graft quality after thawing, it has been suggested to remove DMSO prior to transplantation [4, 7] using different washing systems [8, 9]. Here, we aimed to remove DMSO from long-term cryopreserved PBSCs using an automated, fully closed system and assessed effects on CD34+ stem cells, viability, and colony-forming capacity

Additional file 1: of Bone marrow derived mesenchymal stem cells ameliorate inflammatory response in an in vitro model of familial hemophagocytic lymphohistiocytosis 2

Figure S1. Flow cytometric immunophenotyping data of MSCs. MSCs positive for CD29 (99.5%), CD44 (99.9%), CD73 (99%), CD90 (98%), and CD105 (99%) expression, compatible with mesenchymal origin; negative for CD34 (0%), CD45 (0%), and CD3 (0%), excluding hematopoietic origin. Assay conducted on Becton Dickinson FACS Aria instrument. PE phycoerythrin, FITC fluorescein isothocyanate. Figure S2. Results of differentiation studies. Alizarin Red staining shows osteogenic differentiation of MSCs following 21 days in culture, 10× (a). Oil red O staining confirmed adipogenic differentiation and accumulation of lipid droplets after 21 days in culture, 2× (b). Scale bar = 100 μm. Olympus IX70, Olympus DP71 digital camera. (DOCX 1056 kb

Hematopoietic stem cell transplantation in a CD3 gamma-deficient infant with inflammatory bowel disease

Partial or total CD3 chain expression defects including CD3 gamma, epsilon, delta, and zeta chain are among the autosomally inherited SCID presenting with T-B+NK+ phenotype with lymphopenia. The clinical findings are generally severe in all except for CD3 gamma deficiency. Here we present a 10-month-old CD3 gamma deficient boy with IBD. The patient had suffered from intractable diarrhea, recurrent pulmonary infections and oral moniliasis since two months of age. Following the first allogeneic HSCT from his HLA-identical (6/6) sister after a reduced intensity regimen, a second transplantation was performed five months later. On day +19 after second transplantation, the CD3 TCR alfa/beta chain expression increased to 66% with development of full donor chimerism (98.6%). A significant improvement in diarrhea, perianal lesions, and rectal fistula was observed suggesting an improvement in inflammatory bowel disease. The patient died at home on day +50 with a sudden respiratory failure secondary to an undetermined infection. The case was interesting being the first reported case with SCID and inflammatory bowel disease who responded very well to HSCT by full recovery of intractable diarrhea, failure to thrive, laboratory findings, and improvement of fistula formation

Intestinal mycobiota composition and changes in children with thalassemia who underwent allogeneic hematopoietic stem cell transplantation

[Background]: Allogeneic hematopoietic stem cell transplantation (HSCT) alters the diversity of the intestinal bacterial microbiota. This study aimed to evaluate human mycobiota composition pre-HSCT and post-HSCT in children with thalassemia.[Method]: Ten children with thalassemia undergoing allogeneic HSCT were enrolled. The stool samples were collected before the transplantation regimen, before the transplant day, and +15, +30 days, and three months after transplantation. Stool samples were also collected from the donor and the patient's caregivers. Gut mycobiota composition was evaluated with metagenomic analysis.[Results]: Pretransplant mycobiota of children with thalassemia (the predominant genus was Saccharomyces, 64.1%) has been shown to approximate the diverse mycobiota compositions of healthy adult donors but becomes altered (lower diversity) following transplant procedures. Three months after HSCT, phyla Ascomycota and Basidiomycota were 83.4% and 15.6%, respectively. The predominant species were Saccaharomyces_uc and Saccharomyces cerevisiae (phylum Ascomycota); we also observed Malassezia restricta and Malassezia globosa (phylum Basidiomycota) (∼13%). On day 90 after HSCT, we observed 65.3% M. restricta and 18.4% M. globosa predominance at the species level in a four-year-old boy with acute graft-versus-host disease (GVHD) (skin and gut involvement) 19 days after transplantation included.[Conclusion]: The mycobiota composition of children with thalassemia altered after HSCT. We observed Malassezia predominance in a child with GVHD. Further studies in children with GVHD will identify this situation.This study was financially supported by the Biocodex Microbiota Foundation-2018,Turkey.Peer reviewe