A Co-culture System for Expansion of Nonenriched Cord Blood Stem/Progenitor Cells

2005 ◽  
Vol 4 (4) ◽  
pp. 310-315 ◽  
Author(s):  
Masoud Soleimani . ◽  
Hossein Mozdarani . ◽  
AliAkbar Pourfatholl . ◽  
Yousef Mortazavi . ◽  
Kamran Alimoghaddam . ◽  
...  
2005 ◽  
Vol 33 (7) ◽  
pp. 828-835 ◽  
Author(s):  
Cláudia Lobato da Silva ◽  
Raquel Gonçalves ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3222-3222
Author(s):  
Ping Mao ◽  
Huaxin Duan ◽  
Caixia Wang ◽  
Tingfen Deng ◽  
Changru Luo ◽  
...  

Abstract Abstract 3222 Poster Board III-159 Previous studies of expansion for hematopoietic stem/progenitor cells showed progenitor cells could reach a great expansion but were mainly performed at small culture volume. The poor cell number of expanded cells even great folds of expansion in a no more than 10 ml culture system limited the application for clinic transplantation. Thereof, we expanded umbilical cord blood (UCB) hematopoietic cells at a large scale in 250 ml bioreactor culture system. The mononuclear cell isolated form fresh UCB samples were ex vivo expanded in 250 ml bioreactor with serum-free medium supplied with SCF, FL3 and TPO at concentration 20 ng/ml. Cell number, surface markers and colony forming potential after 7 days of expansion have been evaluated. The sub-lethally irradiated animal models were used to analyze engraftment capability of HSC by transplant of expanded cells in severe combined immunodeficient and nonobese diabetic (NOD/SCID) mice. The detection of human hematopoietic cells in the bone marrow of the mice was performed at 6 wk after the transplant. We have successfully expanded hematopoietic progenitor cells 5 times using the modified bioreactor. The cell viability showed no obvious variations during culture. Total cells increased from 1.76±0.50 ×108 (range 1.2-2.5) to 4.14±0.83×108 (range 3.7-5.6) after expansion, CD34+ cells from 1.25±0.31 ×106 (range 0.96-1.75) to 3.96±0.78×106 (range 2.88-5.04) and CD133+ cells from 1.55±0.69×106 (range 0.96-2.75) to 4.77±0.88×106 (range 4.00-5.04). The colonies per 105 cells of CFU-E /BFU-E, CFU-GM and CFU-Mix increased from 369.6±71.5 to 1648.5±504.3, 42.8±81.4 to 146.4±54.5, 39.1±10.3 to 144.7±38.8, respectively. The positive expression of CD34+ was 0.7%±0.28% at day 0 and 0.9%±0.34% after culture in bioreactor at day 7 and CD133+ cells was 0.8%±0.24% and 1.1%±0.35%. There was no significant survive rate difference between expanded and nonexpanded cells transplantation group. The analysis of multilineage hematopoiesis showed that transplanted human hematopoietic cells is represented in murine bone marrow cells by detection the percentage of human cells identified with human specific anti CD3/19/33/45/61/71 MoAb by FACS and the human specific gene Alu-1 and Cat-1 by PCR. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 360-360
Author(s):  
Quy Le ◽  
Tiffany A. Hylkema ◽  
Sommer Castro ◽  
Jenny L. Smith ◽  
Amanda R. Leonti ◽  
...  

Abstract The CBFA2T3-GLIS2 (CBF/GLIS) fusion is a product of a cryptic translocation exclusively seen in refractory infant AML. Lack of relevant model systems that accurately recapitulate this infant AML has limited progress. To overcome this barrier, we developed an endothelial cell (EC) co-culture system to support malignant transformation, self-renewal, and propagation of leukemia-initiating cells (LIC) in CBF/GLIS-transduced human cord blood hematopoietic stem/progenitor cells (CB HSPCs) ex vivo. Lack of recurrent cooperating mutations suggests that CBF/GLIS fusion might be sufficient for malignant transformation. To test this, we expressed the CBF/GLIS fusion or GFP control in CB HSPCs (CBF/GLIS-CB or GFP-CB) by lentiviral transduction and placed transduced cells in either EC co-culture or myeloid-promoting culture (MC). CBF/GLIS-CB cells expanded faster with prolonged lifespan in EC co-culture compared to MC (Figure 1A). Proliferation of CBF/GLIS-CB cells declined after transfer to either an EC trans-well culture or in suspension culture (Figure 1B), suggesting that direct contact as well as secreted factors are required for optimal growth of transduced cells. The CBF/GLIS fusion has been shown to confer enhanced megakaryocytic differentiation. At 6 weeks, CBF/GLIS-CB cells in EC co-culture formed significantly more megakaryocytic colonies than CBF/GLIS-CB cells grown in MC or CBF/GLIS-GFP cells grown in either condition (Figure 1C). At 12 weeks, CBF/GLIS-CB cells cultured in EC co-culture continued to produce numerous megakaryocytic colonies, demonstrating long lived self-renewal and enhance megakaryocytic differentiation of CBF/GLIS-CB cells co-cultured with ECs. To determine whether the EC niche promotes generation and propagation of LICs, we evaluated the murine engraftment of CBF/GLIS-CB cells expanded on ECs or in MC following 3, 6, 9 and 12 weeks of culture. CBF/GLIS-CB cells cultured in EC co-culture at each time point exhibited robust engraftment that progressed to frank leukemia in vivo (Figure 1D), demonstrating that EC co-culture promotes long-term maintenance of functional LICs. CBF/GLIS-CB cells grown in MC also induced leukemia from 3- and 6-week cultures but then became senescent at 9 and 12 weeks, suggesting limited preservation of the LICs. Flow cytometric analysis of CBF/GLIS-CB cells identified a malignant population that is of the RAM immunophenotype (CD56 hi, CD45 dim, and CD38 dim/-) previously reported in infants with CBF/GLIS AML in both culturing conditions. However, CBF/GLIS-CB cells in EC co-culture constituted an almost homogeneous population that expressed the RAM immunophenotype, whereas only a subset was detected in MC at week 6 (Figure 1E). To determine the fidelity of transformation to primary leukemia, we performed RNA-sequencing of CBF/GLIS-CB cells cultured with ECs or in MC. Unsurpervised clustering analysis demonstrated that the CBF/GLIS-CB cells from weeks 6 and 12 in EC co-culture clustered with primary CBF/GLIS-positive patient samples, but not CBF/GLIS-CB cells cultured in MC nor GFP controls (Figure 1F). Further transcriptome analysis revealed CBF/GLIS and HSC signature genes, previously identified to be associated with CBF/GLIS AML, were both significantly enriched in CBF/GLIS-CB cells grown in EC culture relative to MC (Figure 1G). These results suggested that the signaling pathways that are aberrantly dysregulated in primary CBF/GLIS leukemia are faithfully recapitulated in CBF/GLIS-CB cells co-cultured with ECs. Despite concerted efforts, previous attempts to model CBF/GLIS AML in murine hematopoietic cells have failed to generate overt leukemia. In this study, we demonstrate that in an EC co-culture system, the CBF/GLIS oncogenic fusion is sufficient to transform human CB HSPCs that faithfully recapitulates the morphology, transcriptome and immunophenotype of CBF/GLIS AML as well as highly aggressive leukemia in xenograft models. Furthermore, the EC co-culture system provides a tractable model system to further interrogate the mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy in CBF/GLIS AML. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4176-4176
Author(s):  
Mitsuho Noguchi ◽  
Haruko Tashiro ◽  
Ryosuke Shirasaki ◽  
Moritaka Gotoh ◽  
Kazuo Kawasugi ◽  
...  

Abstract Objectives: We compared the responsiveness to the high dose stimulation of human EPO between the transplant grafts in an in vitro culture system to clarify the characteristics of cord blood and to make clear the repopulation characteristics on human hematopoietic stem/progenitor cells. Methods: Cells (cord blood, bone marrow and peripheral blood stem/progenitor cells) were cultured for six days with G-CSF (100 ng/ml), EPO (10 U/ml), or without additives, and were sorted with antihuman CD133-1 monoclonal antibody. The obtained fractions were further labeled with antihuman CD34 monoclonal antibody and CD133-2 antibody as well as the lineage markers, and their expressions were analyzed. Cells were also assayed with ordinal colony-formation. For the analysis of long-term culture-initiating cells (LTC-ICs), Dexter’s long-term culture method was carried out. The expression of the specific proteins was also analyzed with RT-PCR method. Results. The ratio of the CD34+ cell count to the total cultured mononuclear cell count was not changed between CB, BM and PBSC. However, the ratio of the CD133+ cell count to the total cultured mononuclear cell count was 1.4-fold that of when CB cells were cultured with a high dose of EPO. No significance was observed for BM and PBSC. The count of CD34+CD133+ cells was 1.9-fold that of when CB was cultured with EPO. Dexter’s long-term culture system demonstrated that CB cultured with EPO contained more LTC-ICs and colony-forming cells with a high proliferation potential than that cultured without additives. Discussion: These results indicate that the CB has unique characteristics to respond to a high dose of EPO.


Cytotherapy ◽  
2015 ◽  
Vol 17 (4) ◽  
pp. 428-442 ◽  
Author(s):  
Javad Hatami ◽  
Pedro Z. Andrade ◽  
António Pedro Alves de Matos ◽  
Dusan Djokovic ◽  
Carla Lilaia ◽  
...  

2014 ◽  
Vol 29 (4) ◽  
pp. 457-469 ◽  
Author(s):  
Federica Riva ◽  
Claudia Omes ◽  
Roberto Bassani ◽  
Rossella E Nappi ◽  
Giuliano Mazzini ◽  
...  

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