scholarly journals Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34+-enriched cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Bucar ◽  
André Dargen de Matos Branco ◽  
Márcia F. Mata ◽  
João Coutinho Milhano ◽  
Íris Caramalho ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Cell Number ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Promising Alternative

Abstract Background Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo. Methods UCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed. Results MSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells. Conclusions AT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.

Umbilical Cord Blood Transplantation: A New Alternative Option

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 377-383 ◽  
Author(s):  
William Tse ◽  
Mary J. Laughlin
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Hematopoietic Stem Cell ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Alternative Source ◽  
Hematopoietic Stem

Abstract Allogeneic hematopoietic stem cell transplantation is a life-saving procedure for hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. However, wide application of this procedure is limited by availability of suitably HLA-matched adult donors. Umbilical cord blood (UCB) has being increasingly used as an alternative hematopoietic stem cell source for these patients. To date, over 6000 UCB transplant procedures in children and adults have been performed worldwide using UCB donors. Broader use of UCB for adult patients is however limited by the available infused cell dose. This has prompted intensive research on ex vivo expansion of UCB stem cells and UCB graft-engineering including accessory cells able to improve UCB engraftment and reconstitution and for tissue regenerative potential. Recently, two large European and North American retrospective studies demonstrated that UCB is an acceptable alternative source of hematopoietic stem cells for adult recipients who lack HLA-matched adult donors. UCB is anticipated to address needs in both transplantation and regenerative medicine fields. It has advantages of easy procurement, no risk to donors, low risk of transmitting infections, immediate availability and immune tolerance allowing successful transplantation despite HLA disparity.

Cord Blood Plasma Enhances Migration of Hematopoietic Stem and Progenitor Cells (HSPC)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2959-2959
Author(s):  
James G Farmar ◽  
Alexander Wendling ◽  
Kevin Lynch ◽  
Jose Tomsig ◽  
Mariusz Z Ratajczak ◽  
...  
Keyword(s):  
Stress Response ◽  
Blood Plasma ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Potential Effect ◽  
Hematopoietic Stem

Abstract Abstract 2959 Background: A greater understanding of the mechanisms behind HSPC trafficking is vital to increase the efficacy of HSPC therapy. The composition of adult blood plasma (ABP) is well documented, in particular proteins and metabolites, but very little is known about umbilical cord blood plasma (CBP) which may contain a host of bioactive proteins and lipids released as a stress response during birth as opposed to ABP which is generally regarded as homeostatic. Physiologic stress response may result in altered concentration gradients of these factors thereby creating differing gradients between the marrow and peripheral blood compartments. We sought to investigate factors in CBP and ABP and their effects on HSPC migration. Methods/Results: Both ABP (Research Blood Components, Boston, MA) and CBP was filtered through 0.22 μm filters to remove debris and any cells remaining in the plasma. ABP contained 14% Citrate Phosphate Dextrose Anticoagulant (CPDA) by volume while CBP varied between 28–40% CPDA by volume. ABP samples used in migration assays were diluted to match the CBP concentration with PBS. We screened for the concentrations of 115 known proteins using a multiplexed ELISA assay and compared a pool of 10 CBPs against a pool of 10 ABPs. Umbilical cord blood (UCB) from research units not meeting clinical cell dose threshold was provided by the New York Blood Center (P. Rubinstein, MD). We found 43 proteins were elevated at least two-fold in CBP versus ABP, 16 of which were elevated 10-fold relative to ABP. Out of these 43 proteins, 6 have potential implications on HSPC mobilization: IL8, GCSF (CSF-2), VCAM, MCP1, MIP3, and CXCL10. The concentrations of the proteins in CBP are in pg/mL: IL8 546.85; GCSF 609.91; MCP1 1142.02; MIP3 80.80; VCAM 4, 016, 017.46; and CXCL10 218.27. The fold increase in CBP for these proteins are: IL8 19.39; GCSF 6.39; MCP1 4.12; MIP3 3.48; VCAM 4.20; and CCL10 2.00. The relative contribution of each protein to migration was measured by preparing aliquots of CBP and treating the aliquots with neutralizing antibodies toward each protein (Abcam, Cambridge, MA). Antibodies were incubated at a concentration of 1μg/mL in accordance with recommended concentrations from Abcam. In addition to these 6 proteins, S1P and C3a concentrations were also investigated due to their potential effect in HSPC mobilization. Migration experiments were conducted using Transwell plates (Corning Life Sciences, Lowell, MA). UCB was obtained 24–48 hrs following delivery, and CBP and mononuclear cells were isolated by centrifugation through a Ficoll-Paque density gradient. UCB CD34+ cells were selected by magnetic labeling and sorting using AutoMACS magnetic cell sorter (Miltenyi Biotec, Auburn, CA). UCB HSPCs were placed in upper transwells (8.0 μm pores; 1.5 × 105 cells/well) and the lower well contained CBP, ABP, or fresh RPMI basal media as a control. The cells were allowed to migrate towards the various solutions for 3 h. Cells that migrated were counted and immunophenotyped via hemocytometer and flow cytometry (BD FACS Calibur). In a comparison of migration towards CBP vs. ABP (n = 11 and 10), CBP exhibited an average increase in migration by 157.8 ± 44.1%. Migrations towards CBP depleted of one of the 6 proteins exhibited the following HSPC migrations compared to untreated CBP (100%) were: 48.9 ± 17% for IL8-neutralized (n=6); 90.2 ± 20.4% for GCSF-neutralized (n=5); 102 ± 18.0% for MCP-neutralized (n=5); 71.7 ± 19.8% for MIP3-neutralized (n=6); 35.4 ± 14.7% for VCAM-neutralized (n=4); and 51.7 ± 9.5% for CXCL10-neutralized CBP (n=4). All samples of CBP and ABP used in the migration studies were analyzed for S1P concentration by LC-MS/MS. S1P concentrations in CBP samples ranged from 0.95 to 2.27 (n = 6) times the concentration of S1P in ABP. Additionally, C3a in these CBPs and ABP was analyzed by an ELISA (BD Biosciences, San Jose CA). In CBP samples, C3a varied from 111–297 ng/mL (n = 13) compared to 661 ng/mL in ABP. Conclusion: An improved knowledge of the factors that influence mobilization may provide us with a better approach towards stem cell priming and graft HSPC engineering prior to transplantation. The proteins examined here and the effects of S1P and C3a on HSPC migration may provide novel insights into the factors that influence HSPC trafficking. Further understanding of HSPC migration to proteins released in stress response may be exploited to direct HSPC trafficking in the autologous and allogeneic setting. Disclosures: Lynch: SphynKx Therapeutics LLC: Equity Ownership.

Cytotoxic T Lymphocytes (CTL) Specific for Adenovirus and CMV Can Be Generated from Umbilical Cord Blood for Adoptive Immunotherapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3505-3505
Author(s):  
Catherine M. Bollard ◽  
Patrick Hanley ◽  
Conrad Russell Cruz ◽  
Ann M. Leen ◽  
Jeffrey J. Molldrem ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adoptive Immunotherapy ◽  
Immune Reconstitution ◽  
Mononuclear Cells ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Promising Alternative

Abstract Umbilical cord blood (UCB) transplantation is a promising alternative source of hematopoietic stem cells for patients lacking HLA-matched donors. Nearly 60% of UCB transplants to date have been performed on minority individuals for whom an unrelated donor was not available; moreover, the naïve phenotype of UCB cells may be responsible for the lower incidence and reduced severity of GvHD in these patients. The relatively low cell numbers and naïvety of T lymphocyte populations in UCB grafts has, however, lead to delayed immune reconstitution and higher mortality due to infection. Reactivations of latent viruses such as cytomegalovirus (CMV) are particularly problematic, as is overt infection from adenovirus (Adv). Previous studies have shown that prophylactic adoptive immunotherapy with peripheral blood-derived CTL directed against CMV and Adv can effectively prevent the clinical manifestations of these viruses after hematopoietic stem cell transplant raising the possibility that a similar approach could be developed after UCB transplant. We hypothesized that virus-specific CTL could be generated from UCB for clinical use to restore anti-viral immunity and reduce viral infection post UCB transplant. Bi-virus specific CTL were generated from frozen UCB mononuclear cells using a clinical-grade recombinant adenovirus type5 vector pseudotyped with a type35 fiber carrying a transgene for CMVpp65 as a source of Adv and CMV antigens. UCB-derived dendritic cells were transduced with this Ad5f35pp65 vector as the initial source of antigen presenting cells to stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 rounds of weekly stimulation with autologous UCB-derived EBV-lymphoblastoid cell lines (LCL) transduced with the same vector in the presence of IL-15 and IL-2. UCB from donors of varied HLA types were selected. 40×106 UCB mononuclear cells (available in the 20% fraction of frozen UCB units) were thawed and used in the manufacturing process. After 3 rounds of stimulation, 9 CTL cultures contained a mean of 83% (range 64–94%) CD8+ve T-cells and 27% (range 12–40%) CD4+ve T-cells. Flow cytometric analysis of memory markers after 3 weeks expansion revealed a predominance of CD45RA− CD62L− T-cells (69±18%; range 25–93%) with a smaller population of CD45RA− CD62L+ T-cells (10±5%; range 1–23%). Evaluable UCB CTL lines showed specific cytolytic activity in 51Cr release assays against targets loaded with CMV and Adv antigens. The observed cytotoxicity was specific because unloaded targets and MHC-mismatched targets were not killed. IFNγ ELISPOT assays on CTL lines demonstrated a mean of 209 (range 45–694) and 74 (range 0–188) spot forming cells/1×105 T-cells following incubation with CMV-pp65 and Adv-hexon/penton peptides respectively. No significant response to CMV-IE1 peptides was demonstrated. The expanded UCB CTL had a broad Vβ repertoire and were specific for multiple viral epitopes. In addition, the virus-specific T cells were shown to be expanded only from T-cells with a naïve phenotype (CD45RA+/CCR7+). These results demonstrate that, despite the generally naïve nature of UCB lymphocytes, bi-virus-specific responses can be expanded in vitro and could potentially be used clinically in UCBT patients who develop infectious complications prior to immune reconstitution.

Ex Vivo expansion Of Umbilical Cord Blood CD34+ Cells Under Hypoxic Conditions Using Novel Compound#999 With Cytokines

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4508-4508
Author(s):  
Saadiya Khan ◽  
Alison L. Stewart ◽  
Siddhartha Mukherjee ◽  
Stuart L. Scheiber ◽  
Benjamin L. Ebert ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Fold Increase ◽  
Additive Effect ◽  
Hematopoietic Stem ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
Cells Cultured

Introduction Umbilical cord blood is an increasingly utilized source for hematopoietic stem transplantation. However the limitation is inadequate hematopoietic stem and progenitor cell (HSPC) dose leading to poor engraftment and prolonged neutropenia. Umbilical cord blood transplants (UCBT) were initially restricted to small sized children and adults. The advent of double umbilical cord blood transplants (DUCBT) led to both children and adults transplants with sufficient numbers of HSPCs. However there continue to be issues with insufficient engraftment, extended duration of cytopenia, risk of infections and prolonged duration of hospital stay. There is ongoing research to investigate optimal ex vivo umbilical cord blood (UCB) HSPC expansion with the intention to ensure sustained engraftment, reduce the prolonged periods of neutropenia and curtail the high risk of infectious complications in the immediate post-transplant period. HSPC expansion with cytokines alone produces about 7-fold increase of HSPC over 12-14days. However most IRB approved protocols require that a significant percentage of these cord blood cells be transplanted without manipulation and then the expanded cells be transplanted later. To detect a significant advantage to this expanded fraction we have calculated that HSPCs need to be expanded 8-10 fold. To achieve this we have combined an optimal cytokine combination with hypoxia and the additive of Aryl hydrocarbon Receptor (AhR) antagonist Stem Reginin1 (SR1); previously reported to facilitate HSPC expansion (Boitano et al 2010 Science). Objectives Here we evaluated if there was any potential synergistic effect of combining AhR antagonist SR1 with hypoxia for ex vivo HSPC expansion. Additionally we looked at the effect of adding #999; a small molecule identified using high-throughput screening that selectively expands murine hematopoietic stem cells. Methods UCB derived phenotypic CD34+ cells were cultured in the presence of stem cell factor (SCF), Flt3 ligand (Flt3L) and thrombopoietin (TPO) on a feeder layer of OP9 cells transduced with lentiviral vector expressing red fluorescent protein in both normoxia and hypoxia (3% oxygen). Total cell numbers (TNC) were counted, CD34+ cells were measured through flowcytometry and the self-renewal and multi-lineage differentiation was measured through week-5 cobblestone area forming (CAFC) and colony forming (CFC) assays respectively. Results CD34+ cells cultured in the presence of SCF, Flt3L and TPO (50ng/ml each) resulted in a 100fold expansion of CD34+ cells compared to input cells at 2 weeks. SR1 when added to the above cytokine cocktail led to a 200-fold expansion while #999 used with cytokines resulted in 118-fold expansion at 2weeks. Using both small molecules together in the presence of cytokines did not show an additive effect (207fold increase). Repeating the above experiments in hypoxia (3% oxygen) showed 196-fold increase with cytokines alone, 289-fold increase with SR1, 211-fold increase with #999 and again no additive effect of SR1 and #999 together. CD34+ cells cultured with SR1 or #999 with cytokines produced approximately 1.9 and 1.2 times more CFC than those with cytokines alone respectively. SR1 treated cells on week-5 CAFC showed 3-fold and #999 treated cells 1.3-fold more cobblestones compared to cytokines alone. In hypoxia CD34+ cells cultured with #999 gave rise to more colonies as compared to both SR1 (2-fold more) and cytokines (3-fold more). CAFC data for these are pending. The degree of HSPC expansion with SR1 in addition to cytokines can be increased in hypoxic conditions. # 999 when used with cytokines in hypoxia can also lead to the same degree of HSPC expansion as SR1 in normoxia. The combination of SR1 and #999 showed no additive effect in either normoxia or hypoxia. Conclusion Compound 999 when used in hypoxia leads to a significant expansion of HSPCs compared to cytokines alone or SR1 plus cytokines in normoxia. In vivo xenograft murine studies are been conducted so as to compare and evaluate the engraftment potential of these ex vivo expanded CD34+ cells in irradiated NSG mice. Disclosures: Mukherjee: Onconova Therapeutics: Research Funding. Ebert:Genoptix: Consultancy; Celgene: Consultancy.

Augmentation of umbilical cord blood (UCB) transplantation with ex vivo–expanded UCB cells: results of a phase 1 trial using the AastromReplicell System

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 5061-5067 ◽  
Author(s):  
Jennifer Jaroscak ◽  
Kristin Goltry ◽  
Alan Smith ◽  
Barbara Waters-Pick ◽  
Paul L. Martin ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Phase 1 ◽  
Horse Serum ◽  
Perfusion Culture ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Phase 1 Trial

AbstractAllogeneic stem cell transplantation with umbilical cord blood (UCB) cells is limited by the cell dose a single unit provides recipients. Ex vivo expansion is one strategy to increase the number of cells available for transplantation. Aastrom Biosciences developed an automated continuous perfusion culture device for expansion of hematopoietic stem cells (HSCs). Cells are expanded in media supplemented with fetal bovine serum, horse serum, PIXY321, flt-3 ligand, and erythropoietin. We performed a phase 1 trial augmenting conventional UCB transplants with ex vivo–expanded cells. The 28 patients were enrolled on the trial between October 8, 1997 and September 30, 1998. UCB cells were expanded in the device, then administered as a boost to the conventional graft on posttransplantation day 12. While expansion of total cells and colony-forming units (CFUs) occurred in all cases, the magnitude of expansion varied considerably. The median fold increase was 2.4 (range, 1.0-8.5) in nucleated cells, 82 (range, 4.6-266.4) in CFU granulocyte-macrophages, and 0.5 (range, 0.09-2.45) in CD34+ lineage negative (lin–) cells. CD3+ cells did not expand under these conditions. Clinical-scale ex vivo expansion of UCB is feasible, and the administration of ex vivo–expanded cells is well tolerated. Augmentation of UCB transplants with ex vivo–expanded cells did not alter the time to myeloid, erythroid, or platelet engraftment in 21 evaluable patients. Recipients of ex vivo–expanded cells continue to have durable engraftment with a median follow-up of 47 months (range, 41-51 months). A randomized phase 2 study will determine whether augmenting UCB transplants with ex vivo–expanded UCB cells is beneficial.

scholarly journals INFECTIOUS COMPLICATIONS AFTER UMBILICAL CORD-BLOOD TRANSPLANTATION FROM UNRELATED DONORS

2016 ◽  
Vol 8 ◽  
pp. 2016051 ◽  
Author(s):  
Juan Montoro ◽  
Jaime Sanz
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Cord Blood Transplantation ◽  
Infectious Complications ◽  
Unrelated Donor ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Blood Transplantation

Umbilical cord-blood (UCB) is a well-recognized alternative source of stem cells for unrelated donor hematopoietic stem cell transplantation (HSCT). As compared with other stem cell sources from adult donors, it has the advantages of immediate availability of cells, absence of risk to the donor and reduced risk of graft-versus-host disease despite donor-recipient HLA disparity. However, the use of UCB is limited by the delayed post-transplant hematologic recovery due, at least in part, to the reduced number of hematopoietic cells in the graft and the delayed or incomplete immune reconstitution. As a result, severe infectious complications continue to be a leading cause of morbidity and mortality following UCB transplantation (UCBT). We will address the complex differences in the immune properties of UCB and review the incidence, characteristics, risk factors, and severity of bacterial, fungal and viral infectious complications in patients undergoing UCBT.

Umbilical Cord Blood Stem Cell Transplantation

1993 ◽  
Vol 16 (5_suppl) ◽  
pp. 113-115 ◽  
Author(s):  
R. Miniero ◽  
U. Ramenghi ◽  
N. Crescenzio ◽  
L. Perugini ◽  
A. Busca ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Cord Blood Transplantation ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Alternative Source ◽  
Hematopoietic Stem

Human umbilical cord blood as an alternative source of hematopoietic stem cells for bone marrow reconstitution, has recently been demonstrated to yield successful HLA-matched placental blood grafts in children. It has been shown that cord blood contains sufficient progenitor cells to effect hematological reconstitution. Since then, more than 25 cord blood stem cells (CBSCs) transplants have been performed worldwide for the treatment of a variety of malignant and nonmalignant diseases. The majority of the grafts performed thus far have utilized CBSCs from HLA-identical siblings. However, much of the interest in this setting is devoted to the potential use of CBSCs for HLA-mismatched and unrelated transplants. Preliminary results suggest that allorecognition and graft-versus-host disease may be less intense in CBSCs transplants than in recipients of similarly compatible bone marrow. This review summarizes the results and potential future applications of cord blood transplantation.

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