scholarly journals A Murine Model for Human Cord Blood Transplantation: Near-Term Fetal and Neonatal Peripheral Blood Cells Can Achieve Long-Term Bone Marrow Engraftment in Sublethally Irradiated Adult Recipients

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1089-1099 ◽  
Author(s):  
Andromachi Scaradavou ◽  
Luis Isola ◽  
Pablo Rubinstein ◽  
Yelena Galperin ◽  
Vesna Najfeld ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Blood Transplantation ◽  
Near Term ◽  
Ex Vivo Culture

Abstract The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo–cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.

Cord blood transplantation provides better reconstitution of hematopoietic reservoir compared with bone marrow transplantation

Blood ◽  
2003 ◽  
Vol 102 (3) ◽  
pp. 1138-1141 ◽  
Author(s):  
Francesco Frassoni ◽  
Marina Podestà ◽  
Rita Maccario ◽  
Giovanna Giorgiani ◽  
Gabriele Rossi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Cord Blood Transplantation ◽  
Hematopoietic Progenitor Cell ◽  
Transplant Recipients ◽  
Platelet Recovery ◽  
Blood Transplantation

Abstract Delayed hematopoietic recovery is the main factor precluding a wider use of cord blood (CB) transplants. We hypothesized that this delayed engraftment might not be related to an insufficient number of stem cells in the graft, but to an intrinsic difficulty of these cells to undergo differentiation. To test our hypothesis, 2 groups of children were compared; 12 received a CB transplant and 12 an adult bone marrow (BM) transplant. We studied neutrophil and platelet recovery and, at a median time of approximately 1 year after transplantation, the frequency of colony-forming cells (CFCs) and long-term culture initiating cells (LTC-ICs) in the BM of the 2 groups. Recipients of BM transplants received 1-log more cells and had significantly faster neutrophil and platelet recovery. Conversely, the frequency of committed and early progenitors was significantly higher in the BM of children given CB cells compared with BM transplant recipients (median count of CFC/2 × 104 BM mononuclear cells, 20 versus 11, P = .007; median count of LTC-IC/106 BM mononuclear cells, 8.2 versus 0.2 P = .001). CB, but not adult BM stem cells, can better restore the host hematopoietic progenitor cell reservoir; the delayed engraftment after CB transplantation may reflect the difficulty of CB progenitors to reprogram themselves toward differentiation.

Cobblestone Area-Forming Cell (CAFC) Content of Cord Blood Does Not Predict Engraftment in Double Cord Blood Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2243-2243
Author(s):  
Simon N. Robinson ◽  
Marcos de Lima ◽  
Hong Yang ◽  
William K. Decker ◽  
Dongxia Xing ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Randomized Study ◽  
Clinical Samples ◽  
Hematopoietic Stem ◽  
Blood Transplantation ◽  
Qualitative Measure

Abstract INTRODUCTION: Delayed engraftment remains a serious problem following cord blood (CB) transplantation. It may be due, at least in part, to the limited dose of CB hematopoietic stem and progenitor cells (HSPC) transplanted. Limitations associated with HSPC dose may be reduced by the transplantation of 2 CB units. In an accompanying abstract, de Lima et al. report in detail on an ongoing MDACC randomized study in which patients received double CB units as either 2 unmanipulated units (2×UN), or 1 unmanipulated and 1 ex vivo expanded CB unit (UN+EX). This study has revealed that one CB unit ultimately predominates as the source of long-term, sustained hematopoiesis. We hypothesized that the number of primitive HSPC in the CB unit might ultimately predict which CB unit would ultimately prevail. The in vitro cobblestone area-forming cell (CAFC) assay was used to provide a measure of primitive components of the CB HSPC in each unit. A photomicrograph of a typical cobblestone area (CA) derived from a single CB-derived CAFC is shown. (Figure) CA persisting in in vitro culture for ≥6 weeks (derived from CAFCwk6) represent relatively primitive HSPC and their numbers may provide a qualitative profile for CB units. We hypothesized that of the two CB units transplanted, the one with the greater number of CAFCwk6 would ultimately persist long-term in the patient. METHODS: Clinical samples of 2×UN or UN+EX CB cells were plated in the in vitro CAFC assay.1 The frequency of CAFCwk6 was estimated and total CAFCwk6 numbers transplanted for each CB unit calculated. CAFCwk6 data for each CB unit and engraftment data from patients were analyzed to determine whether the number of CAFCwk6 transplanted was predictive of which CB unit would ultimately be responsible for long-term, sustained engraftment. RESULTS: Preliminary data was accrued from 10 patients.(Table) Six patients received 2×UN and 4 patients received UN+EX (EX indicated by #). CAFCwk6 content at transplant was predictive of which CB unit would ultimately be responsible for long-term, sustained engraftment in only 6 of the 10 cases (60%). CONCLUSION: CAFCwk6 represent a relatively primitive component of the HSPC compartment, however, these data suggest that CAFCwk6 numbers, although possibly a qualitative measure, are not a predictor of long-term sustained engraftment. Figure Figure

Notch-Mediated Expansion of Human Cord Blood Progenitor Cells Results in Rapid Myeloid Reconstitution in Vivo Following Myeloablative Cord Blood Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 212-212 ◽  
Author(s):  
Colleen Delaney ◽  
Carolyn Brashem- Stein ◽  
Howard Voorhies ◽  
Jonathan Gutman ◽  
Mari Dallas ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Fold Increase ◽  
Short Term ◽  
Post Transplant ◽  
Blood Transplantation ◽  
Cell Fractions

Abstract Delayed hematopoietic recovery following cord blood transplantation (CBT) is thought to result from inadequate numbers of progenitor cells in the graft and is associated with increased early transplant related morbidity and mortality. Using an engineered form of the Notch ligand, Delta1, we have previously reported on novel ex vivo expansion methods for generating greatly increased numbers of human CD34 progenitor cells that repopulate immunodeficient mice with markedly enhanced rate and magnitude. We now report results of the initial 6 patients enrolled in a phase I study evaluating the safety and potential efficacy of cord blood (CB) progenitors cultured in the presence Delta1 and recombinant cytokines, with the goal of generating increased numbers of short term repopulating cells capable of providing rapid myeloid engraftment. These patients (AML, n=5; bi-phenotypic leukemia, n=1), were treated with a myeloablative preparative regimen consists of cytoxan 120mg/kg, fludarabine 75mg/m2 and 1320 cGy TBI, followed one day later by infusion of one non-cultured CB unit and then a second unit that has been CD34 enriched and cultured for 16 days as previously described. The median age and weight of the patients enrolled is 28 years (range 11 to 43) and 61.5 kilograms (range 26 to 76). CB units were selected on the basis of cell dose and a requirement of matching at least 4 of 6 loci with the patient (intermediate resolution for HLA-A and B, and high resolution for HLA-DRB1). The non-cultured unit in all patients was 4/6 matched to the patient. The unit used for expansion was 5/6 matched to the patient in two cases, and 4/6 matched in the other four. After culture, there was an average CD34 fold increase of 160 (range 41 to 382) with an average total nucleated cell (TNC) fold increase of 660 (range 146 to 1496). Average infused TNC/kg x107 was 2.9 (range 1.9–5.8) and 4.6 (range 0.6–9.1) for the non-cultured and cultured cells respectively, and infused CD34 cells/kg (x105) was 2.2 (range 1.1–3.4) and 53.4 (range 9.3–133) respectively. No T cells were generated during culture and no toxicities directly attributable to the cultured product, including infusional or increased acute GVHD, have been observed. All patients have engrafted. Relatively rapid engraftment was observed in 5 out of 6 patients treated to date, with a median time to engraftment of 14 days (range 7 to 34), as compared to 25 days (range 16 to 48) in patients (n=17) undergoing an identical transplant regimen at this center, but with 2 non-cultured CB units. The relative contribution of the expanded and non-cultured grafts over time was determined by a DNA-based assay for short tandem repeat loci on peripheral blood sorted cell fractions to include CD3+, CD33+, CD56+, CD14+ and CD19+ cells, beginning day +7 post transplant. In the 5 patients with early myeloid engraftment (≤20 days), engrafted myeloid cells present at day 7 were derived almost entirely from the expanded unit. In three of these five, ANC >500 was observed at days 7, 9 and 16 and was mainly derived from the expanded unit, whereas in the other 2 patients who achieved ANC>500 at day 13 and 20, myeloid engraftment at day 14 was derived from the non-cultured cells. Persistent contribution to engraftment from the expanded cells has been noted in two patients, one through 280 days post transplant but no longer present at one year, and in a more recently treated patient who is currently 75 days post transplant, the expanded cells continue to dominate in CD33, CD14 and CD56, but not CD3, sorted cell fractions. Furthermore, time to platelet engraftment (>20k) has averaged 30 days (range 19–53). Average follow-up time is 277 days (range 70–632). One patient died on day 462 from complications of VZV myelitis; all other patients are alive and in remission. Overall, accelerated myeloid engraftment has been observed in 5/6 patients treated to date as a direct result of Notch-mediated ex vivo expansion of one of two CB units prior to transplantation with ultimate engraftment from the T replete non-cultured unit. These results further suggest that improvement in early myeloid reconstitution may result from provision of short term repopulating cells and/or of cells able to facilitate engraftment of the non-cultured unit. These studies continue with the goal of achieving consistent, rapid engraftment in recipients of hematopoietic cell transplants to decrease morbidity and mortality in the early post-transplant setting.

scholarly journals Early CD3 Peripheral Blood Chimerism Predicts the Long-Term Engrafting Unit Following Myeloablative Double-Cord Blood Transplantation

2012 ◽  
Vol 18 (8) ◽  
pp. 1243-1249 ◽  
Author(s):  
Laura F. Newell ◽  
Filippo Milano ◽  
Ian B. Nicoud ◽  
Stacey Pereira ◽  
Ted A. Gooley ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Cord Blood Transplantation ◽  
Blood Transplantation

Ex Vivo Culture and Amifostine Effects in Human Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4153-4153
Author(s):  
Hun-Mo Ryoo ◽  
Sung Hwa Bae ◽  
Kyung Hee Lee ◽  
Myung Soo Hyun
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Hematopoietic Growth Factors ◽  
Human Cord Blood ◽  
Gm Csf ◽  
Blood Transplantation ◽  
Ex Vivo Culture ◽  
Myeloid Progenitors

Abstract Background Human cord blood(CB) is the third most promising source of hematopoietic stem cells to the bone marrow and peripheral blood for replacing depressed bone marrow function in high dose chemotherapy in patient with cancer. The possibility of cord blood transplantation in adults was limited by the amount of cord blood that could be collected. Cord blood transplantation after ex vivo expansion with cytokines have already been tried in adults. Amifostine is a phosphorylated aminothiol that affords broad cytoprotection from the myelosuppressive effects of antineoplastic agents. Objectives The purposes of this study were to investigate expansion of progenitor and myeloid cells after ex vivo culture of mononuclear cells(MNCs) in umbilical cord blood with growth factor and characterize hematopoietic activities of amifostine. Methods MNCs were cultured and ex vivo expanded into myeloid progenitors by using hematopoietic growth factors(IL-1β, IL-3, IL-6, G-CSF, GM-CSF, SCF, EPO) which are known to stimulate differentiation and proliferation of myeloid progenitors. MNCs exposed to the appropriate amount of amifostine for 15 min were cultured in semisolid media and harvested at 24h intervals, and then apoptosis was assessed by flow cytometry. Results Myeloid colonies were successfully produced from MNCs. Maximal expansion was obtained with the combination of IL-3+SCF+G-CSF+GM-CSF. SCF was thought to be the most important growth factor for expansion of myeloid progenitor. Pretreatment with amifostine for 15 min stimulated formation of hematopoietic colonies at clinically relevant concentrations ranging from 1 to 100μM. Increase in colony number compare to control were comparable after pretreatment with amifostine (10μM), and CFU-GEMM and BFU-E were highly responsive. Further enhancement of colony was not observed after prolonging the duration of pre-incubation exposure to 1, 8 and 24 hours. Amifostine enhanced IL-1 and IL-3 induced formation of CFU-GEMM and BFU-E. Incubation of MNCs with amifostine in suspension culture increased recovery of secondary colonies. Treatment with amifostine retarded cell loss and apoptosis, and promoted cell survival at 24, 48 and 72 hours in cytokine-deficient medium. Conclusions In conclusion, cord blood MNCs can be successfully expanded into myeloid progenitors by using hematopoietic growth factors. This investigation extend the previously recognized hematologic effects of amifostine, and indicate that in addition to its cytoprotective properties, amifostine is a potent stimulant of hematopoietic progenitor growth.

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