Preferential expansion of umbilical cord blood-derived CD34-positive cells on human leukemia inhibitory factor transgenic feeder cells cultured on regenerated silk fibroin film

2013 ◽  
Vol 101B (6) ◽  
pp. 964-971 ◽  
Author(s):  
Xin Yu ◽  
Jingcheng Miao ◽  
Wei Xia ◽  
Zong-jiang Gu
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Silk Fibroin ◽  
Umbilical Cord Blood ◽  
Leukemia Inhibitory Factor ◽  
Human Leukemia ◽  
Feeder Cells ◽  
Regenerated Silk Fibroin ◽  
Silk Fibroin Film ◽  
Cells Cultured

Some characteristics of mast cells cultured from human umbilical cord blood

2000 ◽  
Vol 49 (S1) ◽  
pp. 11-12 ◽  
Author(s):  
H. Y. A. Lau ◽  
T. Obata ◽  
T. Nagakura ◽  
S. M. Chow
Keyword(s):  
Mast Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Cells Cultured

The Hematopoietic Supportive Potential of Human Mesenchymal Stromal Cells Is Associated with Expression of Cadherins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1402-1402
Author(s):  
Christoph Roderburg ◽  
Anke Diehlmann ◽  
Frederik Wein ◽  
Anne Faber ◽  
Ulf Krause ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adipose Tissue ◽  
Cord Blood ◽  
Cell Line ◽  
Umbilical Cord ◽  
Mesenchymal Stromal Cells ◽  
Umbilical Cord Blood ◽  
Stromal Cells ◽  
Culture Conditions ◽  
Cells Cultured

Abstract Self renewal and differentiation of hematopoietic stem cells (HSC) are governed by interaction with the supportive microenvironment of the bone marrow. Secreted factors as well as specific cell adhesion proteins are involved in this interaction. As an in vitro model system, the hematopoietic microenvironment can be mimicked by supportive mesenchymal stromal cells (MSC). We have compared the supportive potential of human MSC from bone marrow (BM) isolated under two different culture conditions (BM-MSC-M1 and BM-MSC-M2), from adipose tissue (AT) and umbilical cord blood (CB) that were all cultivated as described before (Wagner et al. Exp Hematol.2005;11:1402–1416.). As controls we have used the human fibroblast cell line HS68 and the murine fetal liver cell line AFT024. CD34+ cells were isolated from human cord blood and cultured in direct contact with irradiated stromal cells. After four, seven and twelve days the immunophenotype of the hematopoietic cells was analyzed by flow cytometry. Many progenitor cells cultured on BM-MSC or AFT024 maintained a primitive phenotype of CD34+/CD38- cells whereas the proportion of these cells was reduced upon cultivation with CB-MSC and cells cultured on AT-MSC and HS68 displayed a significantly higher expression of CD38 and lower expression of CD34. Furthermore, long term culture initiating-cell (LTC-IC) assays were performed on the different feeder layer. LTC-IC frequency was significantly higher on BM-MSC that were isolated under the two different culture conditions (BM-MSC-M1 1,15 ±0.11%; BM-MSC-M2 1.14±0.08%) and on CB-MSC (1.10±0.13%) compared to AT-MSC (0.32±0.09%) and HS68 (0.67±0.12%). We have compared gene expression profiles of BM-MSC-M1, BM-MSC-M2, CB-MSC, AT-MSC and HS68 by cDNA microarray analysis (51,144 different cDNA clones of the RZPD3 Unigene Set). Differential expression of various genes correlated with the observed differences in supportive potential. Among these were adhesion proteins like N-cadherin, cadherin11, fibronectin1, various integrins (ITGA1, ITGA5 and ITGB1) and VCAM1 as well as secreted proteins including osteonectin, CTGF and SDF1. Westerblot analysis verified on protein level that cadherin11, N-cadherin, and ITGB1 were highly expressed on BM-MSC as compared to AT-MSC and HS68 fibroblasts. In conclusion MSC from human bone marrow or from umbilical cord blood support to a significantly higher degree the maintenance and proliferation of primitive hematopoietic progenitors than MSC derived from adipose tissue. This affinity correlated with up-regulation of cadherin11, N-cadherin and intergrin-beta1 on BM-MSC and CB-MSC.

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.

A novel myeloid-like NK cell progenitor in human umbilical cord blood

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3444-3450 ◽  
Author(s):  
Sonia A. Perez ◽  
Panagiota A. Sotiropoulou ◽  
Dimitra G. Gkika ◽  
Louisa G. Mahaira ◽  
Dimitrios K. Niarchos ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Adherent Cell ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Functional Program ◽  
Hematopoietic Stem ◽  
Cells Cultured

Natural killer (NK) cell differentiation from pluripotent CD34+ human hematopoietic stem cells or oligopotent lymphoid progenitors has already been reported. In the present study, long-term cultures of the CD56−/CD34−myeloid-like adherent cell fraction (ACF) from umbilical cord blood (UCB), characterized by the expression of CD14+ as well as other myeloid markers, were set up with flt3 ligand (FL) and interleukin-15 (IL-15). The UCB/ACF gradually expressed the CD56 marker, which reached fairly high levels (approximately 90% of the cells were CD56+) by day 15. FL plus IL-15–driven ACF/CD56+ cells progressively expressed a mature NK functional program lysing both NK- and lymphokine-activate killer (LAK)–sensitive tumor targets and producing high levels of interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and IL-10 upon stimulation with IL-12 and IL-18. Similar results were obtained when highly purified CD14+ cells from UCB were cultured with FL and IL-15. In contrast, UCB/CD34+ cells cultured under the same conditions showed a delayed expression of CD56 and behaved functionally differently in that they exhibited NK but not LAK cytotoxicity and produced significantly fewer cytokines. Kinetic studies on the phenotype of UCB/ACF or UCB/CD14+ cells cultured in the presence of FL and IL-15 showed a rapid decrease in CD14 expression after day 5, which reached levels of zero by day 20. Approximately 60% of the CD56+ derived from the UCB/ACF or the UCB/CD14+ cells coexpressed CD14 by day 5. Taken together, our data support the role of CD14+ myeloid-like cells within UCB as a novel progenitor for lymphoid NK cells.

Expansion of CD34+ cells isolated from umbilical cord blood using conditioned medium of mixed feeder cells

2008 ◽  
Vol 136 ◽  
pp. S181-S182
Author(s):  
Hyung-Jin Jeon ◽  
Yong-Soo Choi ◽  
Ji-Hae Song ◽  
Jong-Il Kim ◽  
Bu-Young Park ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conditioned Medium ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Feeder Cells ◽  
Cd34 Cells ◽  
Mixed Feeder
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