Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon Ex vivo expansion

2011 ◽  
Vol 112 (7) ◽  
pp. 1822-1831 ◽  
Author(s):  
Pedro Z. Andrade ◽  
Cláudia Lobato da Silva ◽  
Francisco dos Santos ◽  
Graça Almeida-Porada ◽  
Joaquim M.S. Cabral
Keyword(s):  
Cord Blood ◽  
Progenitor Cell ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cell Yield ◽  
Hematopoietic Stem ◽  
Cell Enrichment ◽  
Progenitor Cell Yield

scholarly journals Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells

2021 ◽  
Author(s):  
Valentina Orticelli ◽  
Andrea Papait ◽  
Elsa Vertua ◽  
Patrizia Bonassi Signoroni ◽  
Pietro Romele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

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.

Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1068-1068
Author(s):  
Naoko Takebe ◽  
Thomas MacVittie ◽  
Xiangfei Cheng ◽  
Ann M. Farese ◽  
Emily Welty ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

Manipulation of Oxygen Tension and Medium Composition To Enhance CD34+ Cell Numbers and Megakaryocytic Potential of Cord Blood Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 772-772
Author(s):  
Shannon M. Kidd ◽  
Nathalie Brouard ◽  
Kevin Cao ◽  
Simon N. Robinson ◽  
Michael Thomas ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Oxygen Tension ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Medium Composition ◽  
Bleeding Complications ◽  
Striking Difference ◽  
Cell Transplant ◽  
Hematopoietic Stem

Abstract A major disadvantage of cord blood (CB) hematopoietic stem cell transplant is delayed engraftment. Cell dose has been identified as the key variable limiting neutrophil and platelet reconstitution. This has lead to the development of culture systems promoting the ex-vivo expansion of CB hematopoietic stem and progenitor cells (HSC/HPC). Typically such systems involve suspension cultures with cytokine supplementation, where the selected media supplies differing essential nutrients. Physiologically, HSCs are regulated by their interactions with the osteoblastic stem cell niche, a region characterized by low oxygen tension. We therefore investigated the effects of oxygen tension as well as differing medium composition on the ex-vivo expansion of CB HSC/HPC. METHODS: Replilcate cultures of fresh CB CD34+ (3,000 cells/mL) cells were established at 5% (hypoxia) or 20% O2 (normoxia) in αMEM medium supplemented with 20% fetal bovine serum (FBS) or in CellGro serum-free medium. 100ng/mL stem cell factor (SCF), Fms-like tyrosine kinase 3 ligand (Flt3-L), thrombopoietin (Tpo) and granulocyte- colony stimulating factor (G-CSF) were added to each medium. The cellular output was evaluated after 7 and 14 days by counting total nucleated cells (TNC) and flow cytometric analysis measuring HSC/HPC (CD34), myeloid (CD11b), and megakaryocytic (CD41) cell progeny. RESULTS: Cultures established under hypoxic conditions demonstrated a consistent increase in TNC (range 1.15 to 2.27-fold; N=8, p= 0.02) compared to those grown in normoxia. In addition, six of eight CB CD34+ samples showed equivalent or greater TNC production in CellGro versus αMEM/FBS. In accord with this, cultures initiated in CellGro at 5% O2 demonstrated a nearly 2-fold higher incidence (10.1% vs. 5.5%) and content (2.5 ± 0.5 x104vs. 1.3 + 0.3 x104, p=0.001) of CD34+ cells at day 7 than in αMEM/FBS. However the most striking difference between the two culture media was their capacity to support megakaryocyte differentiation in 5% O2. At day 14, a mean of 4.3% of cells cultured in CellGro expressed CD41 corresponding to a mean of 1.8± 0.3 x105 CD41+ cells/culture compared to only 0.12 ± 0.08 x105 CD41 cells (0.03% of cells) in αMEM/FBS cultures (p =0.0001). This reveals a 15-fold difference in megakaryocytic cell production. These data demonstrate that significant increases in TNC, CD34+ and CD41+ fractions can be gained by modifying oxygen tension and medium composition for ex-vivo expansion of CB progenitors. The increase in megakaryocytic cells may be of particular importance in ameliorating bleeding complications and the need for extensive platelet transfusions as a consequence of thrombocytopenia following CB transplant. Figure Figure

Study of In Vitro Proliferation Potential of CD133 Positive Cells Derived from Umbilical Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4039-4039
Author(s):  
Ri Zhang ◽  
Wenjin Gao ◽  
Yuanyuan Sun ◽  
Jingcheng Miao ◽  
Xueguang Zhang
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Low Concentration ◽  
Tgf Β1

Abstract Transforming growth factor-beta 1 (TGF-β1) is known to maintain primitive human hematopoietic stem/progenitor cells with polyfunctional role in a quiescent state and CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. To investigate the specific effect of TGF-β1 on proliferation and differentiation of CD133 positive cells derived from umbilical cord blood (UCB) during short-term culture in vitro, CD133 positive cells from 20 fresh UCB samples were selected using Miltenyi Biotec’s CliniMACS separation device and were cultured in IMDM medium with 20% FCS in the presence of a cytokine combination of SCF, IL-6, thrombopoietin, IL-3 and Flt3-ligand for up to 2 weeks and TGF-β1 with low concentration was also added to the mediumon day 4. The proliferative response was assessed at day 7, day 10 and day 14 by evaluating the following parameters: nucleated cells (NC), clonogenic progenitors (CFU-GEMM,CFU-GM and BFU-E), and immunophenotypes (CD133 and CD34). The results showed that efficacious expansion of various hematopoietic stem/progenitor cells was constantly observed during the culture. The fold expansion of NC on day7, day10 and day14 expansion were 33.59,224.26 and 613.48, respectively. The fold expansion of CFU-GEMM, CFU-GM and BFU-E on day 10 were 24.89, 41.62 and 49.28, respectively, obviously higher than that without ex vivo expansion (P<0.05). The expansions of CD133+, CD133+CD34+ and CD34+ subpopulation on day 14 were up to 25.83-fold, 16.16-fold and 60.54-fold, respectively. Furthermore the expansion systems with TGF-β1 showed more CD133+ cells than control at every time points. Our datas suggested that the CD133+ cells from human UCB have great expansion potential for ex-vivo expansion. The low concentration of TGF-β1 may delay over-differentiation of hematopoietic stem/progenitor cells.

Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3722-3722
Author(s):  
Li Ming Ong ◽  
Xiubo Fan ◽  
Pak Yan Chu ◽  
Florence Gay ◽  
Justina Ang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Insulin Growth Factor ◽  
Nsg Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

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