bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector.
Authors: Kurita Ryo , Sasaki Erika , Hiroyama Takashi , Yokoo Tomoko , Nakazaki Yukoh , Izawa Kiyoko , Ishii Hajime , Tanioka Yoshikuni , Hanazawa Kisaburo , Hashiguchi Takao , Bai Yuan-Son , Soda Yasushi , Watanabe Sumiko , Asano Shigetaka , Tani Kenzaburo 
Published: October 2019

Abstract
Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.
Large Scale Analysis of Foamy Virus Vector Integration Sites in Human CD34+ Cells.
Authors: Trobridge Grant D. , Miller Daniel G. , Jacobs Michael A. , Allen James M. , Olson Erik , Kiem Hans-Peter , Kaul Rajinder , Russell David W. 
Published: October 2019

Abstract
The ability of retroviruses to efficiently integrate into the host cell’s genome has led to their use as gene delivery vehicles for gene therapy. However, integration in the genome can have adverse effects as observed in a gene therapy trial for X-linked SCID using an oncoretroviral vector. Recent studies have shown that an oncoretroviral vector integrated preferentially near transcription start sites and that a lentiviral vector integrated preferentially within genes. Foamy viruses are integrating retroviruses with many properties that distinguish them from onco- or lentiviruses, perhaps the most important characteristic for gene therapy being that they are non-pathogenic. We previously showed that foamy vectors efficiently transduce CD34+ SCID mouse-repopulating cells (SRCs) from human mobilized peripheral blood, demonstrating their potential for hematopoietic stem cell gene therapy. We present here the first large-scale analysis of foamy vector integration sites. Integration sites were determined by infecting human CD34+ cells or normal fibroblasts with a foamy vector carrying a bacterial origin of replication, then rescuing plasmids containing vector provirus-genomic junction sites in bacteria, and sequencing the foamy vector LTR-human genome junctions. Over 1900 unique integration sites in human CD34+ cells and 1000 unique sites in normal human fibroblasts were mapped using the human genome database. The foamy vector did not integrate preferentially into genes. The percentage of integration sites within Refseq genes in human CD34+ cells, human fibroblasts and randomly generated sites was 29, 23, and 32% respectively. Foamy vectors showed only a slight preference for integration within 1 kb 5′ or 3′ of Refseq transcription start sites. In summary, our data show that foamy vectors have a distinct integration site profile relative to oncoretroviral and lentiviral vectors. Future studies will be required to determine if the unique integration site preference of foamy vectors translates into a reduced risk for oncogenesis in gene therapy applications.
Adenovirus-Mediated PD-L1 Over-Expression Has Differential Effects on Allograft Survival in Murine Islet and Heart Transplant Models.
Authors: Gao Wenda , Yamashita Kenichiro , Sullivan Jennifer , Scaria Abraham , Strom Terry B. , Li Xian C. 
Published: October 2019

Abstract
Program death-1 (PD-1) is a negative regulator of the immune system. Blocking PD-1-mediated negative signaling accelerates autoimmune diseases, while engaging PD-1 with recombinant PD-L1Ig fusion protein potentiates the efficacy of co-stimulation blockade in prolonging allograft survival. However, soluble PD-L1Ig itself showed no graft-protecting effect, in contrast to its strong inhibition of T and B cell activation in vitro when applied in a plate-bound form. In this study, we tested the hypothesis that membrane-bound PD-L1 should prolong allograft survival due to its increased ability to crosslink PD-1 receptor. An adenovirus (Ad.PD-L1) was constructed to encode the full-length mPD-L1, followed by green fluorescent protein (GFP) gene linked by an IRES sequence. A control adenovirus (Ad.Ctrl) was similarly constructed that carries only the GFP gene. In islet transplant model, B6AF1 (H-2b/a) islets were infected with the adenoviruses, and then transplanted into C57BL/6 (H-2b) mice induced diabetic by streptozotocin. In heart transplant model, DBA/2 (H-2d) hearts were perfused with adenoviruses, and then transplanted into C57BL/6 mice. PD-L1 over-expression in islet did not prolong graft survival, but accelerated islet rejection (Ad.PD-L1: 9.0+/−3.5 days; Ad.Ctrl: 13.3+/−2.2 days). In contrast, infection with Ad.PD-L1 prolonged heart allograft survival (15.4+/−5.7 days) in C57BL/6 mice, which promptly rejected DBA/2 hearts (7.0+/−2.3 days, p<0.02). Thus, over-expression of membrane-bound PD-L1 has beneficial effect in an organ/tissue specific manner. Strategies other than direct expression of PD-L1 in the islet b cells need to be devised in order to utilize this negative pathway to prevent rejection.
Correlation between Hematopoietic Stem Cell Engraftment Assay Results and Bone Marrow Biopsy, Flow Cytometry, and Cytogenetics Findings.
Authors: Weir Edward G. , Murphy Kathleen , Batista Denise , Griffin Constance A. , Borowitz Michael J. , Ansari-Lari M. Ali 
Published: October 2019

Abstract
Hematopoietic stem cell transplantation following induction chemotherapy is an increasingly successful therapeutic option for patients with leukemia or lymphoma. The use of molecular assays in post-transplant patients has become the standard in evaluating these patients for evidence of engraftment or early recurrence of disease. The detection of residual host cells in the bone marrow (BM) or peripheral blood (PB) following stem cell transplantation often influences subsequent clinical management. The aim of our study is to determine the extent of correlation between the results of PCR-based stem cell engraftment (SCE) assays and BM biopsy (BMBx), multiparameter flow cytometry (FC) and cytogenetics findings in patients who have undergone stem cell transplantation as therapy for hematolymphoid malignancies. We retrospectively reviewed the results of 1103 serial SCE assays performed at The Johns Hopkins Hospital, and 596 of these had temporally corresponding BMBx, FC and/or cytogenetic analysis. Concordance between the results of SCE analysis and those of the latter assays was defined as the detection of similar host/donor compositions. While some cases demonstrated clear discordance between the results, a subset showed an equivocal correlation due to the unclear significance of <5% host DNA by SCE analysis. Of 318 SCE assays with concurrent BMBx, 167(52%) showed concordant results, 104(33%) showed discordant results, and 47(15%) demonstrated an equivocal correlation. Of 221 SCE assays with concurrent FC, 111(50%) showed concordant results, 73(33%) showed discordant results, and 37(17%) demonstrated an equivocal correlation. Additionally, SCE assays were performed on concurrent, paired BM and PB specimens in 168 patients. Concordant results were identified in 141(84%) pairs. Of the remaining 27 pairs, host DNA was detected in the PB of 16 cases in which the BM showed either donor only DNA (6 cases) or <5% host DNA (10 cases). Four cases showed <5% host DNA in the PB and chimeric DNA in the BM. However, donor only DNA was detected in the PB in 7 cases that demonstrated a chimeric BM. Lastly, concurrent SCE analysis and XY FISH analysis was identified in 28 cases. Concordance between these two assays was observed in 24 (86%) cases, whereas one (3%) case was discordant and 3 (11%) cases showed an equivocal correlation. In conclusion, both BMBx and FC show similar but weak correlations to SCE analysis. In contrast, XY FISH analysis demonstrates a strong correlation to SCE analysis. Furthermore, SCE analyses performed on paired PB and BM specimens show an overall good correlation. However, our data suggest that in a subset of cases, SCE analysis performed on PB may detect residual host DNA that is not detectable by SCE analysis of BM, possibly due to the heterogeneity of the marrow composition.
Selection of Cord Blood Units Based on CD34+ Cells for Storage in Cord Blood Bank.
Authors: Vandekerckhove Bart , Van haute Inge , Lootens Nele , De Buck Carine , Verdegem Linda , Van De Steene Viviane , De Smet Saskia , Craeye David 
Published: October 2019

Abstract
Over 170 000 cord blood units are stored worldwide. For the majority of patients in need of an allogeneic donor, a cord blood unit can be found which is identical or has less than 2 HLA mismatches. However, when partially mismatches cord blood units are used for transplantation, the number of CD34+ cells per kg body weight becomes important and should be higher than 1.7 105/kg (Blood2002;100:1611–1618). For this reason, it is important that only cord blood units with high CD34+ cell content are stored. Our standard selection criteria are volume above 40 mL and TNC of 10x108 or higher. Using these criteria, we routinely store 18% of collected units. The mean CD34+ cell content of stored units is 5.2 106 and TNC is 1.38x109. 94%, 52%, 12%, 4% of cord blood units can be used for patients weighing 10, 25, 50 and 75 kg respectively, based on the criterium of 1.7. 105 CD34+ cells/kg patient weight mentioned above.
To optimize CD34+ cell content in the stored units, we compared our present selection method based on TNC and volume (criterium 40/10/?) with a selection method based on volume, TNC and CD34 content. 131 samples with volumes above 40mL and TNC above 8x108 were analysed for CD34+ cell content before processing and subsequently processed in routine. Using this data set, we compared criterium 40/10/? with criteria 40/8/2 (volume:40mL, TNC:8x108, CD34 2x106). We found that both criteria resulted in an equal number of units acceptable for storage. CD34+ cell content increased from 4.5x106 for criterium 40/10/? to 4.8x106 for criterium 40/8/2 whereas TNC dropped from 14.6x109 to 14.1x109. 100%, 47%, 9% and 4 % (criterium 40/8/2) vs 88%, 43%, 9% and 4%(criteria 40/10/?) of cord blood is acceptable for patients weighing 10, 25, 50 and 75 kg resp., demonstrating that the 12% essentially unusable cord blood units are replaced by units which can be used for small children. In conclusion: by including CD34+ cell content in the selection criteria, CD34+ cell content increases so that 100% of the cord blood units can be used for children up to 10 kg. This measure however requires that CD34 measurement is performed on all units with TNC above 8x108 cells.
Comparison of Controlled vs Non-Controlled Rate Freezing of Umbilical Cord Blood Units.
Authors: Mrowiec Zbigniew R. , Fernandez-DeLeon Maryely , Marchioni Misty , Cieszynska Barbara 
Published: October 2019

Abstract
Controlled rate freezing is recognized as a method of choice for the cryopreservation of blood cells. DMSO, the cryoprotectant most commonly used for cryopreservation of cells, is well known to affect cell viability. It is therefore imperative to insure that cells are frozen without any appreciable delay after the addition of DMSO. Addition of cryoprotectant may take between 5–10 minutes per processed cord blood unit. In situations involving large scale processing, such as 5–10 cord blood units simultaneously, delay will occur, which could cause adverse affects on cell viability when controlled rate freezing is utilized
This study was designed to compare the effect of controlled with two non-controlled rate freezing methods on cord blood stem cell viability.
After adding DMSO to final concentration of 10%, each cord blood unit was split into two 25 ml volumes and enclosed in metal freezing canisters. The UCB units were covered by styrofoam sleeves and then placed directly into either a −80°C or a −140°C mechanical freezer. A corresponding portion of each unit was frozen utilizing a Forma Scientific, model 1010 controlled rate freezer.
Approximately 30 days after, matching units were thawed in a 37°C water bath and tested without washing.
Total Nucleated Cell count, Total CD34 count, 7AAD viability and colony forming units assay were performed on fresh and cryopreserved/thawed samples. Statistical pair analysis using student-t test was performed to indicate any statistically significant differences between each procedures.
(table1)
(table2)
Results indicate that all three methods produces similar cell viability as measured by total nucleated cell recovery, total CD34 counts, 7AAD viability and colony forming assays.
Analysis of fresh and frozen samples did not indicate that controlled rate freezing produces a better quality product after thawing compared to two uncontrolled freezing methods.
Controlled rate freezing vs −80oC dump method°
N=5 Fresh Sample Controlled Rate Freezing Non controlled Freezing at −80oC TNC (x10^6) 412.4 ±37.06 397.6±15.65 406.7±24.78 Total CD34+ (10^6) 0.23±0.08 0.19±0.08 p<0.05 0.19±0.07 p<0.05 Viability (%) 97.5±1.67 94.8±2.28 p<0.05 93.4±3.51 p<0.05 Total # of Colonies (x10^6) 0.27±0.09 0.21±0.10 p<0.05 0.20±0.08 p<0.05
Controlled rate freezing vs −140oC dump method
N=5 Fresh Sample Controlled Rate Freezing Non Controlled −140oC TNC (10^6) 365.4±100.87 372.1±102.18 365.9±104.9 Total CD34+ (x10^6) 0.28±0.08 0.023±0.05 p<0.05 0.23±0.05 p<0.05 Viability (5) 96.0±3.94 96.2±2.28 p<0.05 96.2±1.79 Total # of Colonies (10^6) 0.27±0.13 0.27±0.11 0.24±0.15
Long-Term Pre-Transplant Imatinib Can Improve the Outcome of Non-Myeloablative Stem Cell Transplantation without Increasing the Morbidity in Ph+ Chronic Myeloid Leukemia.
Authors: Kim Dong-Wook , Eon Ki-Seong , Kim Yoo-Jin , Kim Hee-Je , Min Chang-Ki , Lee Jong-Wook , Min Woo-Sung , Kim Chun-Choo 
Published: October 2019

Abstract
Background and Objectives: Although non-myeloablative preparative regimens are being increasingly used due to its reduced toxicity associated with allogeneic transplantation, post-transplant relapses are still problematic. The use of imatinib prior to transplant may potentially allow disease control before SCT and accordingly induce the better transplant outcome in CML patients.
Patients and Methods: The role of imatinib before a non-myeloablative stem cell transplantation (NST) was examined in 25 CML patients. Imatinib was pretreated for median 10.8 months. There were 13 men and 12 women with a median age of 35 (19 – 53) years. Of these, 15 patients were in accelerated phase (AP), and rest of them in chronic phase (CP) at the time of diagnosis.
Results: At the time of transplant, all but 1 patient were in complete cytogenetic response and 4 of them were in molecular remission. Normalized BCR-ABL transcript level decreased from 0.65 to 0.00163 during imatinib therapy. All the patients received fludarabine-based regimens. With a median follow-up of 6.9 months (range, 1.3 – 43 months), all the patients are alive and transplant-related mortality was not observed. After transplantation, 4 CP and 7 AP patients additionally achieved molecular remission. Acute graft versus host disease (GVHD) was observed in 4 (16%) of 25 patients, meanwhile chronic GVHD was observed in 10 (59%) of 17 evaluable patients.
Conclusions: This study suggests that the use of imatinib followed by NST can offer an advantage of reduced relapse without increased toxicity. Prospective randomized trial with a larger number of patients is remained to clarify these clinical observations.
Engraftment Syndrome after Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation: Impact of Graft-Versus-Host Disease Prophylaxis and T Cell Dose.
Authors: Dean Robert M. , Donley Kyle M. , Gea-Banacloche Juan , Steinberg Seth M. , Odom Jeanne , Krumlauf Michael , Castro Kathleen , Chinn Tracey , Fowler Daniel H. , Gress Ronald E. , Bishop Michael R. 
Published: October 2019

Abstract
Engraftment syndrome (ES), defined as noninfectious fever, rash, and noncardiogenic pulmonary edema occurring at the time of hematopoietic recovery after stem cell transplantation, may cause significant morbidity in the peri-transplant period. However, its association with GVHD is unclear, and it remains controversial whether T-cell alloreactivity, nonspecific neutrophil activation, or other factors contribute to the pathogenesis of this syndrome. To study this question, we retrospectively analyzed patients enrolled in 3 separate protocols of reduced-intensity allogeneic hematopoietic stem cell transplantation (RIST). Group A included patients with relapsed/refractory hematologic malignancies undergoing RIST with T-cell replete allografts and cyclosporine for GVHD prophylaxis (n=49). Group B consisted of patients with hematologic malignancies receiving T-cell replete allografts and cyclosporine/methotrexate for GVHD prophylaxis (n=20). Group C included patients with metastatic breast cancer undergoing RIST with T-cell depleted allografts (1 x 105 CD3+ cells/kg) and cyclosporine for GVHD prophylaxis (n=17). All patients received an identical reduced-intensity conditioning regimen with fludarabine and cyclophosphamide, followed by infusion of filgrastim-mobilized peripheral blood stem cells and post-transplantation filgrastim until neutrophil recovery. The incidences of ES and acute GVHD varied according to treatment group (ES: chi-squared test, p=0.066; acute GVHD: chi-squared test, p=NS):
Incidence of Engraftment Syndrome and Acute GVHD by Treatment Group
Group Graft Composition GVHD Prophylaxis Engraftment Syndrome Acute GVHD, Grades 2–4 A T-cell replete Cyclosporine 24/49 (49%) 32/49 (65%) B T-cell replete Cyclosporine/Methotrexate 5/20 (25%) 9/20 (45%) C T-cell depleted Cyclosporine 4/17 (24%) 8/17 (47%)
Logistic regression analysis identified hypoxemia requiring oxygen (OR 38.46, 95% CI 6.51 to 227.1; p=0.002), fever greater than 37 degrees C (OR 6.97, 95% CI 2.47 to 19.7; p<0.0001), and the maximum neutrophil count after engraftment (OR 1.07, 95% CI 1.02 to 1.125; p=0.0093) as factors strongly associated with steroid administration for ES. Subjects with acute GVHD displayed a modest trend toward increased frequency of ES (exact Cochran-Armitage trend test, p=0.103). The association of the intensity of GVHD prophylaxis and the maximum neutrophil count with the incidence of ES implies that both alloreactive T cells and donor granulocytes may play a role in the development of this complication after RIST. These results suggest that additional studies are warranted to explore the contribution of alloreactivity to the pathogenesis, prevention, and treatment of ES.
Prior Autologous Hematopoietic Cell Transplantation Confers an Adverse Prognosis for Recipients of Allogeneic Transplantation Following Nonmyeloablative Preparative Conditioning.
Authors: Schiller Gary J. , Emmanouilides Christos , Paquette Ron , Rosen Peter , Gansert Jennifer , Territo Mary C. 
Published: October 2019

Abstract
Allogeneic hematopoietic transplantation following nonmyeloablative preparative conditioning (also know as “mini-dose transplantation”) is frequently applied to patients who, on the basis of prior therapy, organ dysfunction, or co-morbid conditions may be considered ineligible for full, dose-intenstive treatment. This approach assumes that early regimen-related toxicity can be averted by the nonmyeloablative approach. We present the results obtained in a group of consecutive, unselected older adults with advanced hematologic neoplasia who underwent allogeneic transplantation from fully histocompatible siblings or unrelated donors after dose-reduced preparative conditioning in a single center. Thirty-four patients, median age 57 years (range 26–66) underwent allogeneic transplantation from siblings (14 patients) or unrelated donors (20 patients) following preparative conditioning consisting of chemotherapy only (typically fludarabine, cytarabine, and cyclophosphamide) or those drugs plus antithymocyte globulin, respectively. One recipient of a related allograft and one recipient of an unrelated allograft received preparative fludarabine/melphalan and busulfan/fludarabine/ATG, respectively. Diagnoses prior to transplantation included advanced acute leukemia and myelodysplasia (17 patients), nonHodgkin’s or Hodgkin’s lymphoma (11 patients), and CLL or Multiple Myeloma (6 patients). Allogeneic transplantation followed previous autologous transplantation in 17 patients. Time from previous autologous transplantation to nonmyeloablative allogeneic transplantation varied. Prior autograft conferred a significant adverse risk on recipients of allogeneic transplants following nonmyeloablative conditioning. Only two of 17 patients with a history of prior autograft enjoyed long-term survival as opposed to seven of 17 who had not undergone prior autologous transplant. Mortality before day + 100, typically attributed to treatment-related toxicities, occurred in 11 patients; nine of these patients had had prior autologous transplantation. Prior autologous transplantation typically was done for acute myelogenous leukemia in first complete remission and referral for allotransplant may have been an indicator of more aggressive disease biology. In conclusion, allogeneic transplantation following a nonmyeloablative preparative regimen may be associated with significant risk of early mortality in patients who undergo this treatment after prior autologous transplantation, especially for acute leukemia, suggesting that criteria for allogeneic transplantation after nonmyeloablative condition should take this risk into account. Further studies from multiple centers will be required to confirm this observation.