Browsing by Author "Goebel, W. Scott"
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Item Characterization and Function of Cryopreserved Bone Marrow from Deceased Organ Donors: A Potential Viable Alternative Graft Source(Elsevier, 2023) Johnstone, Brian H.; Woods, John R.; Goebel, W. Scott; Gu, Dongsheng; Lin, Chieh-Han; Miller, Hannah M.; Musall, Kelsey M.; Sherry, Aubrey M.; Bailey, Barbara J.; Sims, Emily; Sinn, Anthony L.; Pollok, Karen E.; Spellman, Stephen; Auletta, Jeffrey J.; Woods, Erik J.; Pediatrics, School of MedicineDespite the readily available graft sources for allogeneic hematopoietic cell transplantation (alloHCT), a significant unmet need remains in the timely provision of suitable unrelated donor grafts. This shortage is related to the rarity of certain HLA alleles in the donor pool, nonclearance of donors owing to infectious disease or general health status, and prolonged graft procurement and processing times. An alternative hematopoietic progenitor cell (HPC) graft source obtained from the vertebral bodies (VBs) of deceased organ donors could alleviate many of the obstacles associated with using grafts from healthy living donors or umbilical cord blood (UCB). Deceased organ donor-derived bone marrow (BM) can be preemptively screened, cryogenically banked for on-demand use, and made available in adequate cell doses for HCT. We have developed a good manufacturing practice (GMP)-compliant process to recover and cryogenically bank VB-derived HPCs from deceased organ donor (OD) BM. Here we present results from an analysis of HPCs from BM obtained from 250 deceased donors to identify any substantial difference in composition or quality compared with HPCs from BM aspirated from the iliac crests of healthy living donors. BM from deceased donor VBs was processed in a central GMP facility and packaged for cryopreservation in 5% DMSO/2.5% human serum albumin. BM aspirated from living donor iliac crests was obtained and used for comparison. A portion of each specimen was analyzed before and after cryopreservation by flow cytometry and colony-forming unit potential. Bone marrow chimerism potential was assessed in irradiated immunocompromised NSG mice. Analysis of variance with Bonferroni correction for multiple comparisons was used to determine how cryopreservation affects BM cells and to evaluate indicators of successful engraftment of BM cells into irradiated murine models. The t test (with 95% confidence intervals [CIs]) was used to compare cells from deceased donors and living donors. A final dataset of complete clinical and matched laboratory data from 226 cryopreserved samples was used in linear regressions to predict outcomes of BM HPC processing. When compared before and after cryopreservation, OD-derived BM HPCs were found to be stable, with CD34+ cells maintaining high viability and function after thawing. The yield from a single donor is sufficient for transplantation of an average of 1.6 patients (range, 1.2 to 7.5). CD34+ cells from OD-derived HPCs from BM productively engrafted sublethally irradiated immunocompromised mouse BM (>44% and >67% chimerism at 8 and 16 weeks, respectively). Flow cytometry and secondary transplantation confirmed that OD HPCs from BM is composed of long-term engrafting CD34+CD38-CD45RA-CD90+CD49f+ HSCs. Linear regression identified no meaningful predictive associations between selected donor-related characteristics and OD BM HPC quality or yield. Collectively, these data demonstrate that cryopreserved BM HPCs from deceased organ donors is potent and functionally equivalent to living donor BM HPCs and is a viable on-demand graft source for clinical HCT. Prospective clinical trials will soon commence in collaboration with the Center for International Blood and Marrow Research to assess the feasibility, safety, and efficacy of Ossium HPCs from BM (ClinicalTrials.gov identifier NCT05068401).Item Diluted antibiotics for treating traumatized immature teeth(2014) Sabrah, Ala'a Hussein Aref, 1984-; Platt, Jeffrey A., 1958-; Gregory, Richard L.; Hara, Anderson T.; Spolnik, Kenneth Jacob 1950-; Murray, Peter E.; Goebel, W. ScottEndodontic regeneration (ERP) has been successfully used in the treatment of traumatized immature teeth. The procedure has three essential steps: disinfecting the root canal (i.e. triple antibiotic paste (TAP) or double antibiotic paste (DAP)), provoking bleeding inside the canal to form a scaffold upon which pulp stem cells will be deposited and continue root growth, and creating a good coronal seal. Previous research has reported that antibiotic pastes (TAP and DAP) are cytotoxic to stem cells in the concentrations commonly used in endodontic regeneration (1000 mg/mL). To decrease the adverse effects on stem cells and increase the rate of success of the regeneration, defining appropriate antibiotic concentrations for ERP is critical. In this project, five in-vitro experiments were conducted to determine the breakpoint dilutions of both TAP and DAP medicaments, and to prepare a suitable novel pastes containing diluted TAP or DAP medicaments for ERP. In the first experiment, we compared the antibacterial effect of TAP, and DAP against early biofilm formation of Enterococcus faecalis (E. faecalis) and Porphyromonas gingivalis bacteria. In the second study, we investigated the antibacterial effect of various dilutions of TAP and DAP antibiotic medicaments against established E. faecalis biofilm. In the third experiment, we investigated longitudinally the residual antibacterial activity of human radicular dentin treated with 1000, 1 or 0.5 mg/ml of TAP and DAP. In the fourth study, we investigated the cytotoxic effect of various dilutions of TAP and DAP antibiotic medicaments on the survival of human dental pulp stem cells (DPSC). And in the fifth experiment, we investigated the antibacterial and cytotoxic effect of novel intracanal medicaments consisting of methylcellulose (MC) and/or propylene glycol (PG) mixed with 1mg/ml of TAP or DAP. 1 mg/ml of DAP or TAP medicaments had a significant antibacterial effect against early bacterial biofilm formation, and established bacterial biofilm. Furthermore, 1 mg/ml had a residual antibacterial activity comparable to 1000 mg/ml. The novel intracanal medicaments had comparable antibacterial effect to currently used medicaments (1000 mg/ml). Additionally, the novel intracanal medicaments significantly enhanced DPSC metabolic activity, compared to currently used medicaments in endodontic regeneration procedures.Item The Effect of Diluted Triple and Double Antibiotic Pastes on Dental Pulp Stem Cells and Established Enterococcus faecalis Biofilm(Springer, 2015-11) Sabrah, Alaa H. A.; Yassen, Ghaeth H.; Liu, Wai-Ching; Goebel, W. Scott; Gregory, Richard L.; Platt, Jeffrey A.; Department of Restorative Dentistry, IU School of DentistryObjectives To investigate the effect of various dilutions of antibiotic medicaments used in endodontic regeneration on the survival of human dental pulp stem cells (DPSCs) and to determine their antibacterial effect against established Enterococcus faecalis biofilm. Materials and methods The cytotoxic and antibacterial effects of different triple (TAP) and double antibiotic paste (DAP) dilutions (0.125, 0.25, 0.5, 1, and 10 mg/ml) were tested against Enterococcus faecalis established biofilm and DPSC. Established bacterial biofilm were exposed to antibiotic dilutions for 3 days. Then, biofilms were collected, spiral plated, and the numbers of bacterial colony forming units (CFU/ml) were determined. For the cytotoxic effect, lactate dehydrogenase activity assays (LDH) and cell viability assays (WST-1) were used to measure the percentage of DPSC cytotoxicity after 3-day treatment with the same antibiotic dilutions. A general linear mixed model was used for statistical analyses (α = 0.05). Results All antibiotic dilutions significantly decreased the bacterial CFU/ml. For WST-1 assays, all antibiotic dilutions except 0.125 mg/ml significantly reduced the viability of DPSC. For LDH assays, the three lowest tested concentrations of DAP (0.5, 0.25, 0.125 mg/ml) and the two lowest concentrations of TAP (0.25 and 0.125 mg/ml) were non-toxic to DPSC. Conclusions All tested dilutions had an antibacterial effect against E. faecalis. However, 0.125 mg/ml of DAP and TAP showed a significant antibacterial effect with no cytotoxic effects on DPSCs. Clinical relevance Using appropriate antibiotic concentrations of intracanal medicament during endodontic regeneration procedures is critical to disinfect root canal and decrease the adverse effects on stem cells.Item Fabrication of Poly-l-lactic Acid/Dicalcium Phosphate Dihydrate Composite Scaffolds with High Mechanical Strength-Implications for Bone Tissue Engineering(MDPI, 2015) Tanataweethum, Nida; Liu, Wai Ching; Goebel, W. Scott; Li, Ding; Chu, Tien Min; Department of Biomedical Engineering, School of Engineering and TechnologyScaffolds were fabricated from poly-l-lactic acid (PLLA)/dicalcium phosphate dihydrate (DCPD) composite by indirect casting. Sodium citrate and PLLA were used to improve the mechanical properties of the DCPD scaffolds. The resulting PLLA/DCPD composite scaffold had increased diametral tensile strength and fracture energy when compared to DCPD only scaffolds (1.05 vs. 2.70 MPa and 2.53 vs. 12.67 N-mm, respectively). Sodium citrate alone accelerated the degradation rate by 1.5 times independent of PLLA. Cytocompatibility of all samples were evaluated using proliferation and differentiation parameters of dog-bone marrow stromal cells (dog-BMSCs). The results showed that viable dog-BMSCs attached well on both DCPD and PLLA/DCPD composite surfaces. In both DCPD and PLLA/DCPD conditioned medium, dog-BMSCs proliferated well and expressed alkaline phosphatase (ALP) activity indicating cell differentiation. These findings indicate that incorporating both sodium citrate and PLLA could effectively improve mechanical strength and biocompatibility without increasing the degradation time of calcium phosphate cement scaffolds for bone tissue engineering purposes.Item FANCA safeguards interphase and mitosis during hematopoiesis in vivo(Elsevier, 2015-12) Abdul-Sater, Zahi; Cerabona, Donna; Potchanant, Elizabeth Sierra; Sun, Zejin; Enzor, Rikki; He, Ying; Robertson, Kent; Goebel, W. Scott; Nalepa, Grzegorz; Department of Pediatrics, IU School of MedicineThe Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in nonhematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA crosslinking and anti-mitotic chemotherapeutics in primary FANCA-/- cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers.Item How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years - implications for clinical transplantation and stability programs(Baishideng, 2020-05-16) Underwood, John; Rahim, Mahvish; West, Carijo; Britton, Rebecca; Skipworth, Elaine; Graves, Vicki; Sexton, Steven; Harris, Hillary; Schwering, Dave; Sinn, Anthony; Pollok, Karen E.; Robertson, Kent A.; Goebel, W. Scott; Hege, Kerry M.; Pediatrics, School of MedicineBACKGROUND Peripheral blood stem cells (PBSC) are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant. Long term cryopreservation is commonly defined as five years or longer, and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft. Clinical programs, stem cell banks, and regulatory and accrediting agencies interested in product stability would benefit from such data. Thus, we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2Rγnull (NSG) mice. AIM To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units. METHODS PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health. These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice, and the pre-freeze and post-thaw characteristics of the units were compared. Progenitor function was assessed using standard colony-forming assays. CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function. RESULTS Ten PBSC units with mean of 17 years in cryopreservation (range 13.6-18.3 years) demonstrated a mean total cell recovery of 88% ± 12% (range 68%-110%) and post-thaw viability of 69% ± 17% (range 34%-86%). BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw. Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units. All mice demonstrated long-term engraftment at 12 wk with mean 34% ± 24% human CD45+ cells, and differentiation with presence of human CD19+, CD3+ and CD33+ cells. Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies. CONCLUSION We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice, signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.Item In Vivo Effects of Myeloablative Alkylator Therapy on Survival and Differentiation of MGMTP140K-Transduced Human G-CSF-Mobilized Peripheral Blood Cells(Elsevier, 2006-05-01) Cai, Shanbao; Hartwell, Jennifer R.; Cooper, Ryan J.; Juliar, Beth E.; Kreklau, Emi; Abonour, Rafat; Goebel, W. Scott; Pollok, Karen E.; Pediatrics, School of MedicineHigh-intensity alkylator-based chemotherapy is required to eradicate tumors expressing high levels of O6-methylguanine DNA methyltransferase (MGMT). This treatment, however, can lead to life-threatening myelosuppression. We investigated a gene therapy strategy to protect human granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells (MPB) from a high-intensity alkylator-based regimen. We transduced MPB with an oncoretroviral vector that coexpresses MGMTP140K and the enhanced green fluorescent protein (EGFP) (n = 5 donors). At 4 weeks posttransplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, cohorts were not treated or were treated with low- or high-intensity alkylating chemotherapy. In the high-intensity-treated cohort, it was necessary to infuse NOD/SCID bone marrow (BM) to alleviate hematopoietic toxicity. At 8 weeks posttreatment, human CD45+ cells in the BM of mice treated with either regimen were EGFP+ and contained MGMT-specific DNA repair activity. In cohorts receiving low-intensity therapy, both primitive and mature hematopoietic cells were present in the BM. Although B-lymphoid and myeloid cells were resistant to in vivo drug treatment in cohorts that received high-intensity therapy, no human CD34+ cells or B-cell precursors were detected. These data suggest that improved strategies to optimize repair of DNA damage in primitive human hematopoietic cells are needed when using high-intensity anti-cancer therapy.Item Low CD34+ Cell Doses Are Associated with Increased Cost and Worse Outcome after Tandem Autologous Stem Cell Transplantation in Patients with Relapsed or Refractory Germ Cell Tumors(Elsevier, 2018) Hyder, Mustafa A.; Goebel, W. Scott; Ervin, Kirsten D.; Schwartz, Jennifer E.; Robertson, Michael J.; Thakrar, Teresa C.; Albany, Costantine; Farag, Sherif S.; Medicine, School of MedicineTandem autologous stem cell transplantation (ASCT) improves long-term survival of platinum-refractory germ cell tumor (GCT) patients. Studies, predominantly in lymphoma, showed that CD34+ cell doses > 5.0 × 106/kg/single transplant led to decreased resource utilization. Because most GCT patients have received prior cisplatin-based treatment, collecting >10 × 106 CD34+ cells/kg is challenging. We analyzed the effect of CD34+ cell dose on resource utilization and outcome in 131 GCT patients, median age 29.5 years (range, 16 to 58), undergoing tandem ASCT. Of 262 individual transplants performed, 120 were performed as inpatient and 142 as planned outpatient. Overall, median CD34+ dose per transplant was 3.1 × 106/kg (range, .8 to 16.0), with no significant difference between inpatient and outpatient transplants. Patients were divided into quartiles based on the CD34 cell dose infused: Q1, .8 to 1.9 × 106/kg; Q2, 2.0 to 2.9 × 106/kg; Q3, 3.0 to 4.1 × 106/kg; and Q4, 4.2 to 16.0 × 106/kg. For all patients higher CD34+ cell doses were associated with significantly shorter times to neutrophil (P <.001) and platelet recovery (P <.001). For inpatient transplants higher CD34+ doses were significantly associated with shorter length of hospital stay (P <.001); fewer days of filgrastim (P <.001), i.v. antibiotic (P = .012), and antifungal (P = .03) usage; and fewer RBC (P = .001) and platelet units transfused (P <.001), resulting in overall lower cost of care (P < .001). Of the 142 planned outpatient transplants, 100 admissions were required for a median length of hospital stay of 7.0 days (range, 1 to 18). Although there was no significant difference in the rates of hospitalization between patients in different CD34+ cell dose quartiles, a significant trend was observed for shorter hospitalization (P = .01) and fewer RBC (P = .002) and platelet (P = .005) transfusions with higher CD34+ cell dose quartiles. Patients receiving CD34+ cell doses in the lowest dose quartile (Q1) had significantly worse progression-free survival and overall survival compared with patients receiving higher CD34+ cell doses. Overall, resource utilization, including cost of care, is significantly reduced when patients receive higher CD34+ cell doses, indicating greater efforts to improve peripheral blood stem cell collection in this population are needed.Item Outcome of domino hematopoietic stem cell transplantation in human subjects: An international case series(Elsevier, 2018) Belderbos, Mirjam E.; Gennery, Andrew R.; Dvorak, Christopher C.; Blok, Henric-Jan; Eikema, Dirk-Jan; Silva, Juliana M. F.; Veys, Paul; Neven, Bénédicte; Buckley, Rebecca; Cole, Theresa; Cowan, Morton J.; Goebel, W. Scott; Hoenig, Manfred; Kuo, Caroline Y.; Stiehm, E. Richard; Wynn, Robert; Bierings, Marc; Pediatrics, School of MedicineItem Outcomes of Pediatric Patients with Therapy-Related Myeloid Neoplasms(Springer Nature, 2021) Sharma, Akshay; Huang, Sujuan; Li, Ying; Brooke, Russell J.; Ahmed, Ibrahim; Allewelt, Heather B.; Amrolia, Persis; Bertaina, Alice; Bhatt, Neel S.; Bierings, Marc B.; Bies, Joshua; Brisset, Claire; Brondon, Jennifer E.; Dahlberg, Ann; Dalle, Jean-Hugues; Eissa, Hesham; Fahd, Mony; Gassas, Adam; Gloude, Nicholas J.; Goebel, W. Scott; Goeckerman, Erika S.; Harris, Katherine; Ho, Richard; Hudspeth, Michelle P.; Huo, Jeffrey S.; Jacobsohn, David; Kasow, Kimberly A.; Katsanis, Emmanuel; Kaviany, Saara; Keating, Amy K.; Kernan, Nancy A.; Ktena, Yiouli P.; Lauhan, Colette R.; López-Hernandez, Gerardo; Martin, Paul L.; Myers, Kasiani C.; Naik, Swati; Olaya-Vargas, Alberto; Onishi, Toshihiro; Radhi, Mohamed; Ramachandran, Shanti; Ramos, Kristie; Rangarajan, Hemalatha G.; Roehrs, Philip A.; Sampson, Megan E.; Shaw, Peter J.; Skiles, Jodi L.; Somers, Katherine; Symons, Heather J.; de Tersant, Marie; Uber, Allison N.; Versluys, Birgitta; Cheng, Cheng; Triplett, Brandon M.; Pediatrics, School of MedicineLong-term outcomes after allogeneic hematopoietic cell transplantation (HCT) for therapy-related myeloid neoplasms (tMNs) are dismal. There are few multicenter studies defining prognostic factors in pediatric patients with tMNs. We have accumulated the largest cohort of pediatric patients who have undergone HCT for a tMN to perform a multivariate analysis defining factors predictive of long-term survival. Sixty-eight percent of the 401 patients underwent HCT using a myeloablative conditioning (MAC) regimen, but there were no statistically significant differences in the overall survival (OS), event-free survival (EFS), or cumulative incidence of relapse and non-relapse mortality based on the conditioning intensity. Among the recipients of MAC regimens, 38.4% of deaths were from treatment-related causes, especially acute graft versus host disease (GVHD) and end-organ failure, as compared to only 20.9% of deaths in the reduced-intensity conditioning (RIC) cohort. Exposure to total body irradiation (TBI) during conditioning and experiencing grade III/IV acute GVHD was associated with worse OS. In addition, a diagnosis of therapy-related myelodysplastic syndrome and having a structurally complex karyotype at tMN diagnosis were associated with worse EFS. Reduced-toxicity (but not reduced-intensity) regimens might help to decrease relapse while limiting mortality associated with TBI-based HCT conditioning in pediatric patients with tMNs.