Duygu Uckan

Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys.

Cells in the umbilical cord stroma have gained attention in recent years; however, differentiation to certain lineages in humans has been demonstrated in few studies. Unlike bone marrow MSCs, human umbilical cord stroma cells (HUCSCs) are far from being well characterized. This study attempts to describe proliferation, structural, and differentiation properties of these cells to account for their exceptional nature in many aspects. Cellular dynamics, cellular structure, and the degree of transformations during expansion and differentiation into mesenchymal and neuronal lineages were examined in vitro over a 10‐month period. Comparisons with human bone marrow MSCs regarding differentiation were performed. HUCSCs in culture revealed two distinct cell populations, type 1 and type 2 cells, that possessed differential vimentin and cytokeratin filaments. Corresponding cells were encountered in cord sections displaying region‐specific localization. α‐Smooth muscle actin and desmin filaments, which were evident in cord sections, diminished through passages. No difference was noted regarding type 1 and type 2 cells in differentiation to chondrogenic, adipogenic, and osteogenic lineages, whereas a preferential differentiation was noted in neuronal lineage. Relative success was achieved by production of chondrocytic spheres and osteogenic monolayers, whereas adipocytes were immature compared with bone marrow MSCs. The presence of neuronal markers suggests that they transform into a certain state of maturity under neurogenic induction. Conclusively, HUCSCs retain their original phenotype in culture without spontaneous differentiation, have a limited lifespan, and bear multipotent stem cell characteristics. Given these characteristics, they may be generally considered progenitor cells if manipulated under appropriate conditions and deserve further study to be potentially used in cell‐based therapies.

Life-threatening neurological complications after bone marrow transplantation in children

Summary:Neurological complications may occur in BMT recipients (11–59%), frequently contributing to morbidity or mortality. They are the main causes of death in 10–15%. Life-threatening neurological complications were seen in 11 out of 113 (9.7%) children who underwent BMT from HLA-matched family (n=7) or mismatched donors (n=4) at our institution. Diagnoses of patients with neurological complications were acute myeloblastic leukemia (AML) (five), thalassemia major (two), Fanconi anemia (two), Omenn syndrome (one) and leukodystrophy (one), and the neurological events were seen between days +13 and +85 after transplantation. Minor symptoms including reversible, nonrepetitive seizures were excluded. Cyclosporine A toxicity was diagnosed in six children. The rest of the complications were brain abscess/meningoencephalitis (two), severe hypomagnesemia (one), busulfan toxicity (one), sustained hypertension (three), and intracranial hemorrhage (three). Six patients with neurological complications suffered from >grade II graft-versus-host disease (GvHD), and all were high risk for transplant-related complications. In this study, risk status of the underlying disease, mismatched transplantation, a diagnosis of AML (advanced stage), older age and >grade II GvHD were important adverse factors for the development of severe life-threatening neurological complications.

Mesenchymal Stem Cell Therapy in Necrotizing Enterocolitis: A Rat Study

We evaluated the potential therapeutic use of exogenous human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in an experimental rat model of necrotizing enterocolitis (NEC). Thirty-six newborn Sprague-Dawley rats were randomly divided into three groups: NEC, NEC + hBM-MSC, and a control (control and control + hBM-MSC). NEC was induced by enteral formula feeding, exposure to hypoxia-hyperoxia, and cold stress. After NEC was induced, iron-labeled hBM-MSCs were administered by intraperitoneal injection. All pups were killed on the fourth day following injection, and the terminal ileum was excised for a histopathological and immunohistochemical evaluation. The pups in the NEC + hBM-MSC group showed significant weight gains and improvements in their clinical sickness scores (p < 0.01). Bowel damage severity observed in the histopathological evaluation was significantly lower in the NEC + hBM-MSC group than that in the NEC group (p = 0.012). The number of MSCs homing to the bowel was significantly higher in the NEC + hBM-MSC group than that in the control + hBM-MSC group. In conclusion, this is the first study that has evaluated the effectiveness of hBM-MSCs in a neonatal rat NEC model. MSCs reduced histopathological damage significantly.

Use of Mesenchymal Stem Cells and Darbepoetin Improve Ischemia-Induced Acute Kidney Injury Outcomes

Background: Interest has recently been focused on the possible role of bone marrow-originating stem cells and the therapeutic role of erythropoietin in the recovery of ischemia-induced acute kidney injury (AKI). The aim of the present study was to compare treatment with mesenchymal stem cells (MSCs) to treatment with darbepoetin-α (DPO) or both concomitantly in a rat model of ischemia/reperfusion (I/R) AKI. Methods: Forty male Sprague-Dawley rats were included, and 28 of them were randomly assigned to controls (treated with serum physiologic) or one of the three treatment groups treated with either DPO, MSCs, or both (MSCs and DPO concomitantly) after the induction of I/R injury. Hematocrit, serum creatinine, and BUN levels were obtained at 0, 24, 48, and 72 h of surgery, and renal tissue was obtained at 72 h after nephrectomy for histological analysis. Tissue injury was quantified by standardized histological scoring systems, using light and electron microscopes. Results: Treatment with MSCs or DPO improved renal function compared with controls. However, the improvement observed in renal function in the MSC/DPO group was better than that in the other groups. Histological analysis demonstrated that tissue injury was significantly decreased in rats in the MSC or DPO groups compared to that of the controls; however the best recovery was observed in rats treated with MSCs and DPO concomitantly. Conclusion: These results suggest that concomitant application of DPO and MSCs may be a potential novel renoprotective therapy for patients after having sustained an ischemic renal insult.

Stem cell suspension injected HEMA-lactate-dextran cryogels for regeneration of critical sized bone defects

Abstract HEMA-Lactate-Dextran cryogel scaffolds were produced by cryogelation. Mesencyhmal stem cells (MSC) were isolated from rat bone marrow. Critical sized cranial bone defects were created in rat cranium. Stem cells were injected inside the macropores of the cryogel scaffolds prepared from HEMA-Lactate-Dextran possessing the same dimensions with the defect and placed in the cranial bone. The cryogels placed in the defect without stem cells served as control. After selected time intervals the experimental sites were removed from the animals and new bone formation and tissue integration were investigated by histological analysis. The in vivo results exhibited osseous tissue integration within the implant and mineralized functionally stable bone restoration of the cranial defects. Tissue formation started in the macrospores of the scaffold starting from periphery to the center. A significant ingrowth of connective tissue cells and new blood vessels allowed new bone formation. Histological data demonstrated that new bone per total defect area ratio, were not significantly different in “scaffold-stem cells” group compared to that of “scaffold only” group on all time points. However, the blood vessel density was significantly higher in “scaffold-stem cells” group comparing to that of the “scaffold only” group on day 30. “Scaffold-stem cells” given group gave better tissue response score when compared to “scaffold only” group on day 180.

Bone Marrow Mesenchymal Stem Cells in Patients with Beta Thalassemia Major: Molecular Analysis with Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy Study as a Novel Method

Bone marrow mesenchymal stem cells (BM-MSCs) are the main cellular components of the bone marrow, providing a supportive cellular microenvironment to maintain healthy hematopoiesis. β-thalassemia major (β-TM) is characterized by anemia that is caused by a genetic defect in hemoglobin synthesis and results in ineffective erythropoiesis (IE). The alterations in the microenvironment in thalassemic bone marrow during IE can cause changes in BM-MSCs. This study aimed to investigate global structural and compositional changes in BM-MSCs in β-TM that may provide a basis in understanding interactions of hematopoietic stem cells (HSCs)-MSCs in such a pathological bone marrow microenvironment. Following characterization of morphological, immunophenotypical, and differentiation properties, the changes in healthy and thalassemic BM-MSCs before and after bone marrow transplantation (BMT) were examined by attenuated total reflection-Fourier transform infrared (ATR-FTIR). The significant increase in lipid, protein, glycogen, and nucleic acid contents in thalassemic BM-MSCs with respect to healthy BM-MSCs was attributed to enhanced cell proliferation and BM activity during IE. The significant decreases in the content of mentioned macromolecules in post-transplant group BM-MSCs versus pre-transplant BM-MSCs was interpreted as restoring effect of BMT therapy on IE and defective BM microenvironment. These alterations were also supported by ELISA results of erythropoietin (EPO) and growth differentiation factor (GDF15) in bone marrow plasma samples as a reflection of IE and by MTT proliferation assay on BM-MSCs. Based on these changes, sampling groups were discriminated by cluster analysis. These results provide information for the studies that concentrate on interactions between HSCs-MSCs in bone marrow.

Adipocyte differentiation defect in mesenchymal stromal cells of patients with malignant infantile osteopetrosis.

BACKGROUND Malignant infantile osteopetrosis (MIOP) is a disorder of osteoclasts characterized by defective bone resorption and death in infancy. The multipotent mesenchymal stromal cells (MSC) and their progeny (osteoblasts) are major components of the bone marrow (BM) microenvironment and are found in close contact with cells of hematopoietic origin, including osteoclasts. We hypothesized that MSC defects may be associated with osteoclast dysfunction and osteopetrosis phenotype. METHODS BM MSC, obtained from six patients with MIOP, were expanded in vitro and characterized by morphology, plastic-adherence, immunophenotype and multilineage differentiation potential. RESULTS Physical and immunophenotypic characteristics of patient MSC were similar to healthy age-matched controls. However, an isolated in vitro differentiation defect toward adipogenic lineage was demonstrated in patient MSC and confirmed by low or absent expression of adipogenic transcripts (peroxisome proliferator-activated receptor-gamma, adipophilin, stearoyl-CoA desaturase, leptin and adiponectin) upon induction of adipogenesis. Following BM transplantation, minimal improvement in adipogenic potency of MSC was demonstrated by Oil Red O staining. DISCUSSION MIOP is associated in vitro with a failure of MSC to differentiate into an adipogenic lineage, suggesting a BM microenvironment defect. The defect may contribute to osteoclast dysfunction, or may be attributed to the effect of the osteopetrotic marrow environment. Further investigations should determine the pathophysiologic importance of this novel defect, and could perhaps contribute to consideration of MSC therapy in MIOP.

Tuberculin skin test positivity in pediatric allogeneic BMT recipients and donors in Turkey

Abstract:  The preliminary study was performed to determine the frequency of tuberculin skin test (TST) positivity among 26 patients and their donors screened by TST to investigate whether tuberculin positivity of a recipient or donor influenced the rate of tuberculosis disease, transplant‐related events, and to evaluate the effectiveness of isoniazide (INAH) prophylaxis administered to those with positive TST. The frequency of TST positivity was 23% (n = 6) among recipients and also 23% (n = 6) among donors. Two recipients and five donors with positive TST received INAH prophylaxis for six months. Our use of INAH prophylaxis in transplant patients was very conservative because of the risk of drug interaction. The transplantation procedure was not postponed for either recipient or donor TST positivity. Despite the high frequency of tuberculosis in our country, we have not detected any case of tuberculosis in our center, either among the purified protein derivative‐screened (n = 26) or non‐screened (n = 128) patients except for disseminated tuberculosis infection because of BCG vaccination in two patients with severe combined immunodeficiency. In conclusion, TST positivity in either recipient or donor may not be a contraindication for bone marrow transplantation and the procedure may not be postponed. Pretransplantation TST screening may be needed in countries where tuberculosis is common in the general population.

Hepatitis B immunoglobulin in combination with lamivudine for prevention of hepatitis B virus reactivation in children undergoing bone marrow transplantation

Abstract:  There is little information in literature about the use of hepatitis B immunoglobulin (HBIg) in recipients of bone marrow transplantation (BMT). Here, we report two children who received IV HBIg (Hepatect‐CP) and lamivudine treatment during BMT course for either patient or donor hepatitis B virus (HBV) viremia. A four‐year‐old girl underwent a fully human leukocyte antigen‐matched allogeneic BMT for thalassemia major from her mother positive for hepatitis B surface antigen (HBsAg). A 12‐yr‐old boy with chronic myeloid leukemia, positive for HBsAg and HBV‐DNA received a fully HLA‐matched allogeneic BMT from his sister in the first chronic phase of the disease. HBIg was successfully used in both cases to prevent HBV reactivation of the recipients. The results of our observations are encouraging and we suggest that HBIg in combination with lamivudine may be used in such cases especially in post‐transplant early period to prevent HBV reactivation.

Successful treatment of severe myasthenia gravis developed after allogeneic hematopoietic stem cell transplantation with plasma exchange and rituximab

Myasthenia gravis is among the rare complications after allogeneic hematopoietic stem cell transplantation and is usually associated with chronic GVHD. Herein, we report a 2‐year and 10 months of age female with Griscelli syndrome, who developed severe myasthenia gravis at post‐transplant +22nd month and required respiratory support with mechanical ventilation. She was unresponsive to cyclosporine A, methylprednisolone, intravenous immunoglobulin, and mycophenolate mofetil and the symptoms could only be controlled after plasma exchange and subsequent use of rituximab, in addition to cyclosporine A and mycophenolate mofetil maintenance. She is currently asymptomatic on the 6th month of follow‐up. Pediatr Blood Cancer 2014;61:928–930.