Cleveland, Ohio, United States
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Aljitawi O.S.,University of Kansas Medical Center | Aljitawi O.S.,University of Rochester | Paul S.,University of Kansas Medical Center | Ganguly A.,University of Kansas Medical Center | And 19 more authors.
Blood | Year: 2016

Umbilical cord blood (UCB) engraftment is in part limited by graft cell dose, generally one log less than that of bone marrow (BM)/peripheral blood (PB) cell grafts. Strategies toward increasing hematopoietic stem/progenitor cell (HSPC) homing to BM have been assessed to improve UCB engraftment. Despite recent progress, a complete understanding of how HSPC homing and engraftment are regulated is still elusive. We provide evidence that blocking erythropoietin (EPO)-EPO receptor (R) signaling promotes homing to BM and early engraftment of UCB CD34+ cells. A significant population of UCB CD34+ HSPC expresses cell surface EPOR. Exposure of UCB CD34+ HSPC to EPO inhibits their migration and enhances erythroid differentiation. This migratory inhibitory effect was reversed by depleting EPOR expression on HSPC. Moreover, systemic reduction in EPO levels by hyperbaric oxygen (HBO) used in a preclinical mouse model and in a pilot clinical trial promoted homing of transplanted UCB CD34+ HSPC to BM. Such a systemic reduction of EPO in the host enhanced myeloid differentiation and improved BM homing of UCB CD34+ cells, an effect that was overcome with exogenous EPO administration. Of clinical relevance, HBO therapy before human UCB transplantation was well-tolerated and resulted in transient reduction in EPO with encouraging engraftment rates and kinetics. Our studies indicate that systemic reduction of EPO levels in the host or blocking EPO-EPOR signaling may be an effective strategy to improve BM homing and engraftment after allogeneic UCB transplantation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT02099266). © 2016 by The American Society of Hematology.


PubMed | Indiana University, Cardiovascular Research Institute, Cleveland Cord Blood Center and University of Kansas Medical Center
Type: Journal Article | Journal: Blood | Year: 2016

Umbilical cord blood (UCB) engraftment is in part limited by graft cell dose, generally one log less than that of bone marrow (BM)/peripheral blood (PB) cell grafts. Strategies toward increasing hematopoietic stem/progenitor cell (HSPC) homing to BM have been assessed to improve UCB engraftment. Despite recent progress, a complete understanding of how HSPC homing and engraftment are regulated is still elusive. We provide evidence that blocking erythropoietin (EPO)-EPO receptor (R) signaling promotes homing to BM and early engraftment of UCB CD34


PubMed | Dana-Farber Cancer Institute, City of Hope National Medical Center, Stanford University, Nationwide Childrens Hospital and 26 more.
Type: Journal Article | Journal: Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation | Year: 2016

Alternative graft sources (umbilical cord blood [UCB], matched unrelated donors [MUD], or mismatched unrelated donors [MMUD]) enable patients without a matched sibling donor to receive potentially curative hematopoietic cell transplantation (HCT). Retrospective studies demonstrate comparable outcomes among different graft sources. However, the risk and types of infections have not been compared among graft sources. Such information may influence the choice of a particular graft source. We compared the incidence of bacterial, viral, and fungal infections in 1781 adults with acute leukemia who received alternative donor HCT (UCB, n=568; MUD, n=930; MMUD, n=283) between 2008 and 2011. The incidences of bacterial infection at 1 year were 72%, 59%, and 65% (P<.0001) for UCB, MUD, and MMUD, respectively. Incidences of viral infection at 1 year were 68%, 45%, and 53% (P<.0001) for UCB, MUD, and MMUD, respectively. In multivariable analysis, bacterial, fungal, and viral infections were more common after either UCB or MMUD than after MUD (P<.0001). Bacterial and viral but not fungal infections were more common after UCB than MMUD (P=.0009 and <.0001, respectively). The presence of viral infection was not associated with an increased mortality. Overall survival (OS) was comparable among UCB and MMUD patients with Karnofsky performance status (KPS)90% but was inferior for UCB for patients with KPS<90%. Bacterial and fungal infections were associated with poorer OS. Future strategies focusing on infection prevention and treatment are indicated to improve HCT outcomes.


Ratajczak J.,University of Louisville | Ratajczak J.,Pomeranian Medical University | Zuba-Surma E.,University of Louisville | Klich I.,University of Louisville | And 7 more authors.
Leukemia | Year: 2011

A population of CD133+ Lin- CD45- very small embryonic/epiblast-like stem cells (VSELs) has been purified by multiparameter sorting from umbilical cord blood (UCB). To speed up isolation of these cells, we employed anti-CD133+-conjugated paramagnetic beads followed by staining with Aldefluor to detect aldehyde dehydrogenase (ALDH) activity; we subsequently sorted CD45-/GlyA-/CD133 +/ALDHhigh and CD45-/GlyA-/ CD133+/ALDHlow cells, which are enriched for VSELs, and CD45- /GlyA- /CD133+/ALDHhigh and CD45-/GlyA- /CD133+ /ALDHlow cells, which are enriched for hematopoietic stem/progenitor cells (HSPCs). Although freshly isolated CD45- VSELs did not grow hematopoietic colonies, the same cells, when activated/expanded over OP9 stromal support, acquired hematopoietic potential and grew colonies composed of CD45- hematopoietic cells in methylcellulose cultures. We also observed that CD45 -/GlyA-/CD133+/ALDHhigh VSELs grew colonies earlier than CD45-/GlyA-/CD133+ /ALDHlow VSELs, which suggests that the latter cells need more time to acquire hematopoietic commitment. In support of this possibility, real-time polymerase chain reaction analysis confirmed that, whereas freshly isolated CD45- /GlyA-/CD133+/ALDHhigh VSELs express more hematopoietic transcripts (for example, c-myb), CD45 -/GlyA-/CD133+ /ALDHlow VSELs exhibit higher levels of pluripotent stem cell markers (for example, Oct-4). More importantly, hematopoietic cells derived from VSELs that were co-cultured over OP9 support were able to establish human lympho-hematopoietic chimerism in lethally irradiated non-obese diabetic/severe combined immunodeficiency mice 4-6 weeks after transplantation. Overall, our data suggest that UCB-VSELs correspond to the most primitive population of HSPCs in UCB. © 2011 Macmillan Publishers Limited. All rights reserved.


Wu W.,University of Louisville | Kim C.H.,University of Louisville | Liu R.,University of Louisville | Kucia M.,University of Louisville | And 8 more authors.
Leukemia | Year: 2012

We report that the bone marrow (BM) stroma-released LL-37, a member of the cathelicidin family of antimicrobial peptides, primes/increases the responsiveness of murine and human hematopoietic stem/progenitor cells (HSPCs) to an α-chemokine stromal-derived factor-1 (SDF-1) gradient. Accordingly, LL-37 is upregulated in irradiated BM cells and enhances the chemotactic responsiveness of hematopoietic progenitors from all lineages to a low physiological SDF-1 gradient as well as increasing their (i) adhesiveness, (ii) SDF-1-mediated actin polymerization and (iii) MAPK p42/44 phosphorylation. Mice transplanted with BM cells ex vivo primed by LL-37 showed accelerated recovery of platelet and neutrophil counts by ∼3-5 days compared with mice transplanted with unprimed control cells. These priming effects were not mediated by LL-37 binding to its receptor and depended instead on the incorporation of the CXCR4 receptor into membrane lipid rafts. We propose that LL-37, which has primarily antimicrobial functions and is harmless to mammalian cells, could be clinically applied to accelerate engraftment as an ex vivo priming agent for transplanted human HSPCs. This novel approach would be particularly important in cord blood transplantations, where the number of HSCs available is usually limited. © 2012 Macmillan Publishers Limited All rights reserved.


Brunstein C.G.,University of Minnesota | McKenna D.H.,University of Minnesota | DeFor T.E.,University of Minnesota | Sumstad D.,Molecular and Cellular Therapy Center | And 6 more authors.
Biology of Blood and Marrow Transplantation | Year: 2013

Preclinical data showed that priming CD34+ hematopoietic progenitor cells with complement fragment 3a (C3a) improved homing and engraftment. Thus, we hypothesized that priming of umbilical cord blood (UCB) hematopoietic progenitors with C3a would facilitate homing and could potentially be used to address the need for improved engraftment after UCB transplantation. We primed 1 of 2 UCB units for double UCB transplantation after nonmyeloablative conditioning. This design provided adequate safety and the potential to observe skewed long-term chimerism in favor of the C3a-primed unit as a surrogate measure of efficacy. C3a priming of 1 UCB unit did not result in infusional toxicity. Increased grades 1 to 3 hypertension were the only infusional adverse events observed in 9 (30%) patients. We observed no activation of inflammatory or coagulation pathways downstream of C3a. As tested, C3a priming did not impair engraftment, but did not skew chimerism toward the treated unit. As compared with historical controls, mortality and survival were not adversely affected. Thus, before any additional clinical studies, C3a priming to promote engraftment will require further preclinical optimization. © 2013 American Society for Blood and Marrow Transplantation.


Wuttisarnwattana P.,Chiang Mai University | Gargesha M.,Bioinvision, Inc. | Van'T Hof W.,Cleveland Cord Blood Center | Cooke K.R.,Johns Hopkins University | And 3 more authors.
IEEE Transactions on Medical Imaging | Year: 2016

With its single cell sensitivity over volumes as large as or larger than a mouse, cryo-imaging enables imaging of stem cell biodistribution, homing, engraftment, and molecular mechanisms. We developed and evaluated a highly automated software tool to detect fluorescently labeled stem cells within very large (∼ 200 GB) cryo-imaging datasets. Cell detection steps are: preprocess, remove immaterial regions, spatially filter to create features, identify candidate pixels, classify pixels using bagging decision trees, segment cell patches, and perform 3D labeling. There are options for analysis and visualization. To train the classifier, we created synthetic images by placing realistic digital cell models onto cryo-images of control mice devoid of cells. Very good cell detection results were (precision = 98.49%, recall = 99.97%) for synthetic cryo-images, (precision = 97.81%, recall = 97.71%) for manually evaluated, actual cryo-images, and < 1% false positives in control mice. An α-multiplier applied to features allows one to correct for experimental variations in cell brightness due to labeling. On dim cells (37% of standard brightness), with correction, we improved recall (49.26% → 99.36%) without a significant drop in precision (99.99% → 99.75%). With tail vein injection, multipotent adult progenitor cells in a graft-versus-host-disease model in the first days post injection were predominantly found in lung, liver, spleen, and bone marrow. Distribution was not simply related to blood flow. The lung contained clusters of cells while other tissues contained single cells. Our methods provided stem cell distribution anywhere in mouse with single cell sensitivity. Methods should provide a rational means of evaluating dosing, delivery methods, cell enhancements, and mechanisms for therapeutic cells. © 2015 IEEE.


PubMed | Cleveland Clinic, Ohio State University, Case Western Reserve University, Cleveland Cord Blood Center and EEH Science
Type: Journal Article | Journal: Cytometry. Part B, Clinical cytometry | Year: 2016

The goal of this study was to quantitatively evaluate the reproducibility of current manual counting methods of colony forming units (CFUs) from umbilical cord blood samples METHODS: Fresh and reconstituted frozen cells from 10 cord blood samples were cultured under standard conditions. The number of BFU-Es, CFU-GMs, and CFU-GEMMs were counted by three expert reviewers using the standard microscope method and manually traced CFUs on digital images of cell cultures.The mean colony count based on the traced digital images was 82 (22% CV) and 52 (15% CV) for the fresh and frozen samples, respectively. This was significantly greater than that observed using the microscope, 61 (13% CV) for fresh and 43 (16% CV) for frozen. The difference was mainly due to the reviewers observing more CFU-GMs in the digital images than through the microscope review. All three reviewers agreed on the presence of a colony 72% of the time based on the digital review in both fresh and frozen samples. Reviewer agreement with respect to colony type in the fresh samples was 38% (22%CV), 25% (51%CV), and 6% (115%CV) for BFU-Es, CFU-GMs, and CFU-GEMMs, respectively. Reviewer agreement increased for BFU-Es and CFU-GMs in the frozen samples where fewer colonies were present.Although this study showed marked variability between reviewers, the analysis of manually traced digital images has the potential to improve inter-observer variation when compared to current methods by identifying features that lead to discrepancies in colony counting and providing cases with consensus results. 2016 International Clinical Cytometry Society.

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