Integration of Transcriptome and MicroRNA Profile Analysis of iMSCs Defines Their Rejuvenated State and Conveys Them into a Novel Resource for Cell Therapy in Osteoarthritis.

Although MSCs grant pronounced potential for cell therapies, several factors, such as their heterogeneity restrict their use. To overcome these limitations, iMSCs (MSCs derived from induced pluripotent stem cells (iPSCs) have attracted attention. Here, we analyzed the transcriptome of MSCs, iPSCs and iMSCs derived from healthy individuals and osteoarthritis (OA) patients and explored miRNA-mRNA interactions during these transitions. We performed RNA-seq and gene expression comparisons and Protein-Protein-Interaction analysis followed by GO enrichment and KEGG pathway analyses. MicroRNAs' (miRNA) expression profile using miRarrays and differentially expressed miRNA's impact on regulating iMSCs gene expression was also explored. Our analyses revealed that iMSCs derivation from iPSCs favors the expression of genes conferring high proliferation, differentiation, and migration properties, all of which contribute to a rejuvenated state of iMSCs compared to primary MSCs. Additionally, our exploration of the involvement of miRNAs in this rejuvenated iMSCs transcriptome concluded in twenty-six miRNAs that, as our analysis showed, are implicated in pluripotency. Notably, the identified here interactions between hsa-let7b/i, hsa-miR-221/222-3p, hsa-miR-302c, hsa-miR-181a, hsa-miR-331 with target genes HMGA2, IGF2BP3, STARD4, and APOL6 could prove to be the necessary tools that will convey iMSCs into the ideal mean for cell therapy in osteoarthritis.

Development of a Population Pharmacokinetic Model of Busulfan in Children and Evaluation of Different Sampling Schedules for Precision Dosing.

We develop a population pharmacokinetic model to describe Busulfan pharmacokinetics in paediatric patients and investigate by simulations the impact of various sampling schedules on the calculation of AUC. Seventy-six children had 2 h infusions every 6 h. A two-compartment linear model was found to adequately describe the data. A lag-time was introduced to account for the delay of the administration of the drug through the infusion pump. The mean values of clearance, central volume of distribution, intercompartmental clearance, and peripheral volume of distribution were 10.7 L/h, 39.5 L, 4.68 L/h and 17.5 L, respectively, normalized for a Body Weight (BW) of 70 kg. BW was found to explain a portion of variability with an allometric relationship and fixed exponents of 0.75 on clearance parameters and 1 on volumes. Interindividual variability for clearance and volume of distribution was found to be 28% and 41%, respectively, and interoccasion variability for clearance was found to be 11%. Three sampling schedules were assessed by simulations for bias and imprecision to calculate AUC by a non-compartmental and a model-based method. The latter was found to be superior in all cases, while the non-compartmental was unbiased only in sampling up to 12 h corresponding to a once-daily dosing regimen.

Allogeneic hematopoietic stem cell transplantation in infants is associated with significant morbidity and mortality.

BACKGROUND: Infants are subjected to hematopoietic stem cell transplantation (HSCT) due to malignant and non-malignant diseases. However, specific data concerning the outcome and transplantation-related complications in infants, as a separate age group, are limited. Our aim was to evaluate the impact of infancy on the outcome, toxicity, and complications after HSCT. METHODS: We retrospectively analyzed data of 55 infants that underwent HSCT in our unit from May 1997 until February 2020, emphasizing on the probability of overall survival (OS) and the cumulative incidence (CI) of transplantation-related mortality (TRM) and complications. RESULTS: We report a probability of OS of 61%, a CI of TRM at day 100 and 365 post transplantation of 22% and 30%, respectively, and additionally a CI of graft failure, acute graft-versus-host disease (GvHD), and infectious complications, 18%, 44%, and 39%, respectively. No statistically significant association was detected between the above mentioned parameters and diagnosis, the use of myeloablative or non-myeloablative/reduced toxicity conditioning regimens or the type of donor. CONCLUSIONS: We conclude that HSCT in infancy is associated with significant mortality and morbidity. This is possibly attributed to endogenous, age-related factors. More specifically, infants may be at a higher risk of toxicities due to the immaturity of developing vital organs and the deficiency of the newly adopted immune system that predisposes them to infectious complications. The development of GvHD further augments the danger of infections, in a potential vice-versa relationship. Moreover, there are few data on pharmacokinetics of chemotherapy agents, making safe and efficacious drug administration hard.

Is Carbapenem-resistant Klebsiella pneumoniae Infection in Pediatric Bone Marrow Transplantation Recipients Inevitably Fatal?

Carbapenem resistance, most notably in Klebsiella pneumonia (KPC), results in infections associated with significant morbidity and mortality. Here we report 2 cases of adolescent patients with KPC infection after high-risk bone marrow transplantation, who eventually succumbed from other causes and review the epidemiology and treatment options for KPC infections in this vulnerable population.

HSCT remains the only cure for patients with transfusion-dependent thalassemia until gene therapy strategies are proven to be safe.

Patients with ß-thalassemia suffer from severe anemia, iron overload and multiple complications, that affect their quality of life and well-being. Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-matched sibling donor, performed in childhood, has been the gold standard for thalassemic patients for decades. Unfortunately, siblings are available only for the minority of patients. Fully matched unrelated donors have been the second choice for cure, with equal results as far as overall survival is concerned, having though the cost of frequent and serious complications. On the other hand, haploidentical transplantation is performed more frequently during the last decade, with promising results. Gene therapy represents a novel therapeutic approach, with impressive results from clinical trials, both from gene addition strategies, as well as from the emerging gene editing tools. After reviewing current critical points of HSCT using alternative donors and assessing recently reported safety issues of gene therapy methods, we conclude that, although a breakthrough, the safety of gene therapy remains to be established.

Proliferative and chondrogenic potential of mesenchymal stromal cells from pluripotent and bone marrow cells.

INTRODUCTION: Mesenchymal stromal cells (MSCs) can be derived from a wide range of fetal and adult sources including pluripotent stem cells (PSCs). The properties of PSC-derived MSCs need to be fully characterized, in order to evaluate the feasibility of their use in clinical applications. PSC-MSC proliferation and differentiation potential in comparison with bone marrow (BM)-MSCs is still under investigation. The objective of this study was to determine the proliferative and chondrogenic capabilities of both human induced pluripotent stem cell (hiPSC-) and embryonic stem cell (hESC-) derived MSCs, by comparing them with BM-MSCs. METHODS: MSCs were derived from two hiPSC lines (hiPSC-MSCs), the well characterized Hues9 hESC line (hESC-MSCs) and BM from two healthy donors (BM-MSCs). Proliferation potential was investigated using appropriate culture conditions, with serial passaging, until cells entered into senescence. Differentiation potential to cartilage was examined after in vitro chondrogenic culture conditions. RESULTS: BM-MSCs revealed a fold expansion of 1.18x105 and 2.3x105 while the two hiPSC-MSC lines and hESC-MSC showed 5.88x10¹°, 3.49x108 and 2.88x108, respectively. Under chondrogenic conditions, all MSC lines showed a degree of chondrogenesis. However, when we examined the formed chondrocyte micromasses by histological analysis of the cartilage morphology and immunohistochemistry for the chondrocyte specific markers Sox9 and Collagen II, we observed that PSC-derived MSC lines had formed pink rather than hyaline cartilage, in contrast to BM-MSCs. CONCLUSION: In conclusion, MSCs derived from both hESCs and hiPSCs had superior proliferative capacity compared to BM-MSCs, but they were inefficient in their ability to form hyaline cartilage.

Autosomal dominant hyper-IgE syndrome: When hematopoietic stem cell transplantation should be considered?

AD-HIES or Job's syndrome is a primary immunodeficiency, caused by dominant negative mutations in signal transducer and activator of transcription (STAT) 3. The syndrome is characterized by infectious, immunologic, and non-immunologic manifestations and is associated with significant morbidity, mortality, and development of lymphomas. What has not yet been elucidated is the role of HSCT in the disease treatment spectrum. We review published cases of patients with AD-HIES that underwent HSCT and attempt to clarify at what stage HSCT should be considered and what are the complications.

Toxoplasma gondii: How fatal is it in pediatric allogeneic bone marrow transplantation setting?

Toxoplasmosis is a disease of the immunocompetent population. However, cases of toxoplasma infection associated with immunosuppression have been reported, especially the first months after transplantation. Limited data are available about toxoplasma infection, occurring even many months post-transplant in pediatric patients with nonmalignant and malignant diseases. We report the cases of three patients with early and late disseminated toxoplasmosis and review the literature.

Therapeutic Effects of Mesenchymal Stem Cells Derived From Bone Marrow, Umbilical Cord Blood, and Pluripotent Stem Cells in a Mouse Model of Chemically Induced Inflammatory Bowel Disease.

Acute inflammatory bowel disease (AIBD) is a wide clinical entity including severe gastrointestinal pathologies with common histopathological basis. Epidemiologically increasing diseases, such as necrotizing enterocolitis (NEC), gastrointestinal graft versus host disease (GVHD), and the primary acute phase of chronic inflammatory bowel disease (CIBD), exhibit a high necessity for new therapeutic strategies. Mesenchymal stem cell (MSC) cellular therapy represents a promising option for the treatment of these diseases. In our study, we comparatively assess the efficacy of human MSCs derived from bone marrow (BM), umbilical cord blood (UCB), human embryonic stem cells (ESCs), or human-induced pluripotent stem cells (iPSCs) in a mouse model of chemically induced acute enterocolitis. The laboratory animals were provided ad libitum potable dextrane sulfate sodium solution (DSS) in order to reproduce an AIBD model and then individually exposed intraperitoneally to MSCs derived from BM (BM-MSCs), UCB (UCB-MSCs), ESCs (ESC-MSCs), or iPSCs (iPSC-MSCs). The parameters used to evaluate the cellular treatment efficacy were the animal survival prolongation and the histopathological-macroscopic picture of bowel sections. Although all categories of mesenchymal stem cells led to statistically significant survival prolongation compared to the control group, significant clinical and histopathological improvement was observed only in mice receiving BM-MSCs and UCB-MSCs. Our results demonstrated that the in vivo anti-inflammatory effect of ESC-MSCs and iPSC-MSCs was inferior to that of UCB-MSCs and BM-MSCs. Further investigation will clarify the potential of ESCs and iPSC-derived MSCs in AIBD treatment.

A prospective study on the epidemiology and clinical significance of viral respiratory infections among pediatric oncology patients.

Respiratory infections in oncology are both common and potentially severe. However, there is still a gap in the literature, regarding the epidemiology of viral respiratory infections in children with cancer. We prospectively enrolled 224 patients, from September 2012 to August 2015. The cohort included children with hematologic or solid malignancies receiving chemotherapy, or undergoing hemopoietic stem cell transplantation, outpatients/inpatients exhibiting signs/symptoms of febrile/afebrile upper/lower respiratory infection. Viral infection was diagnosed by detection of ≥1 viruses from a sample at time of enrollment, using the CLART® PneumoVir kit (GENOMICA, Spain). Α detailed questionnaire including demographics and medical history was also completed. Samples were processed in batches, results were communicated as soon as they became available. Children recruited in whom no virus was detected composed the no virus detected group. Viral prevalence was 38.4% in children presenting with respiratory illness. A single virus was found in 30.4%, with RSV being the most frequent. Viral coinfections were detected in 8%. Children with viral infection were more likely to be febrile upon enrollment and to present with lower respiratory signs/symptoms. They had longer duration of illness and they were more likely to receive antibiotics/antifungals. Only 22% of children with influenza received oseltamivir. Mortality was low (2.7%), however, pediatric intensive care unit (PICU) admission and death were correlated with virus detection. In our study mortality was low and PICU admission was related to virus identification. Further research is needed to clarify whether antibiotics in virus-proven infection are of value and underline the importance of oseltamivir's timely administration in influenza.

Epidemiology of viral infections among children undergoing hematopoietic stem cell transplant: Α prospective single-center study.

BACKGROUND: Viral infections are a significant cause of morbidity and mortality in pediatric transplant populations. We analyzed the epidemiology of viral infections in pediatric hematopoietic stem cell transplant (HSCT) patients, including their incidence, associated risk factors, and outcome. METHODS: In a prospective study from September 2011 to September 2015, blood, urine, and stool specimens were monitored weekly from transplantation to day 100 or after if clinically suspected, by use of real-time polymerase chain reaction. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), Herpes simplex virus-1,2, Varicella zoster virus, Human herpes virus-6,7, and Adenovirus infections were monitored. All children and adolescents who underwent HSCT received long-term follow up in the regular outpatient clinics (range 2-48 months). RESULTS: A total of 192 HSCTs (autologous/allogeneic: 53/139) were performed in 165 subjects (median age: 5.6 years). Viruses most commonly isolated were CMV (46.1%), BKV (25.9%) and EBV (22.6%) and were more frequent in allogeneic versus autologous transplants (P < 0.05). Almost all high-risk allogeneic recipients developed EBV infections post-HSCT. EBV-PTLD was the only cause of death among those who developed viral disease. The factors significantly associated with the development of viral infections were recipient's advanced age, unrelated donor, mismatched graft and use of peripheral blood stem cells grafts. CONCLUSIONS: Viral infections were common among our pediatric recipients. Data suggest that monitoring of viral load may be significant to the prevention of viral disease. Particular demographic and transplantation characteristics were associated with the development of viral infections post-HSCT.

Reprogramming of bone marrow derived mesenchymal stromal cells to human induced pluripotent stem cells from pediatric patients with hematological diseases using a commercial mRNA kit.

The potential use of patient-specific induced pluripotent stem cells (hiPSCs) in the study and treatment of hematological diseases requires the setup of efficient and safe protocols for hiPSC generation. We aimed to adopt a reprogramming method for large-scale production of integration-free patient-specific hiPSC-lines in our stem cell processing laboratory, which supports a pediatric hematopoietic stem cell transplant unit located at a tertiary care children's hospital. We describe our 5-year experience in generation of hiPSC-lines from human bone marrow-derived mesenchymal stromal cells (BM-MSCs) using synthetic mRNAs encoding reprogramming factors. We generated hiPSC-lines from pediatric patients with ß-Thalassemia, Sickle Cell Anemia, Blackfan-Diamond Anemia, Severe Aplastic Anemia, DOCK8 Immunodeficiency and 1 healthy control. After optimization of the reprogramming procedure, average reprogramming efficiency of BM-MSCs was 0.29% (range 0.25-0.4). The complete reprogramming process lasted 14-16 days. Three to five hiPSC-colonies per sample were selected, expanded to 5 culture passages and then frozen. The whole procedure took an average time of 1.8 months (range 1.6-2.2). The hiPSC-lines expressed embryonic stem cell markers and exhibited pluripotency. This mRNA reprogramming method can be applicable in a hematopoietic stem cell culture lab setting and would be useful for the clinical translation of patient-specific hiPSCs.

Successful long-term hematological and immunological reconstitution by autologous cord blood transplantation combined with post-transplant immunosuppression in two children with severe aplastic anemia.

aUCBT is a valuable curative option in pediatric patients with refractory idiopathic SAA and no available matched sibling or unrelated donors. Experience in the use of autologous cord blood units in patients with SAA is limited and private for-profit cord blood-banking programs are controversial. We report the successful treatment of two patients with SAA, aged 15 and 24 months, with autologous cord blood combined with immunosuppression. After conditioning with 200 mg/kg cyclophosphamide and ATG, 7.5 mg/kg, 32.2 × 107 /kg, and 3.8 × 107 /kg autologous cord blood nucleated cells were infused, respectively. One of our patients underwent transplantation after failure of IST. Both patients received post-transplant immunosuppression with cyclosporine for 12 months. They remain disease-free 6 years post-transplantation.

Bioassay for Determining Voriconazole Serum Levels in Patients Receiving Combination Therapy with Echinocandins.

Voriconazole levels were determined with high-performance liquid chromatography (HPLC) and a microbiological agar diffusion assay using a Candida parapsilosis isolate in 103 serum samples from an HPLC-tested external quality control program (n = 39), 21 patients receiving voriconazole monotherapy (n = 39), and 7 patients receiving combination therapy (n = 25). The results of the bioassay were correlated with the results obtained from the external quality control program samples and with the HPLC results in sera from patients on voriconazole monotherapy and on combination therapy with an echinocandin (Spearman's rank correlation coefficient [rs], > 0.93; mean ± standard error of the mean [SEM] % difference, <12% ± 3.8%).

Generation of human ß-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with ß-thalassemia (ß-thal) with the aim to generate trangene-free ß-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in ß-thal-iPSC lines. After 10 serial passages in vitro, ß-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free ß-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

Mesenchymal derivatives of genetically unstable human embryonic stem cells are maintained unstable but undergo senescence in culture as do bone marrow-derived mesenchymal stem cells.

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here, we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages, respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations, suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs, which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells.

Transient effect of anti-CD20 therapy in a child with 22q11.2 deletion syndrome and severe steroid refractory cytopenias: a case report.

We report on the development of steroid-refractory recurrent cytopenias in a child with 22q11.2 deletion syndrome. His first hematological complication was autoimmune hemolytic anemia at 3 months of age. Thereafter, he developed severe autoimmune cytopenias of all 3 hematological lineages with poor response to steroids and intravenous immunoglobulin. At the age of 2½ years, a course of anti-CD20 therapy (Rituximab) was given with transient hematological recovery. Because of persistent symptoms, bone marrow transplantation from a matched unrelated donor was performed. Although the data in the use of anti-CD20 therapy in children with 22q11.2 deletion syndrome and autoimmune cytopenias are limited, our experience suggests its potential benefit.

Acute disseminated encephalomyelitis after allogeneic bone marrow transplantation for pure red cell aplasia - a case report and review of the literature.

ADEM is a rare inflammatory demyelinating disease of the CNS, which usually presents after a viral infection or a vaccination. We report a 15-yr-old boy who was diagnosed with ADEM after an HLA-identical sibling allogeneic BMT for transfusion-dependent PRCA. His course was complicated with GVHD affecting the skin and lungs. Five months post-BMT, he developed neurological symptoms including sudden mental status alteration, dysarthria, facial nerve palsy, and acute paraplegia. The MRI revealed multifocal hyperintense lesions mainly in the subcortical white matter of the cerebrum, the brainstem, the basal ganglia, and the thalami. CSF examination was normal. There was no laboratory evidence of infection. The typical MRI findings and an acute monophasic clinical course were consistent with the diagnosis of ADEM. Clinical and radiological improvement was observed after treatment with high-dose steroids and IVIG. Complete neurologic recovery was achieved six months after the onset of symptoms. We present a rare case of ADEM post-BMT and review of the literature.