Background Hematopoietic stem cells (HSCs) have been used to treat the patient with leukemia, lymphoma and other cancers, as well as genetic and immune defects involving the hematopoietic system. However, there is currently no standard or widely accepted ex vivo detection method for HSCs.
Methods: We performed correlation of ex vivo surface marker CD34+CD38-CD90+ and in vivo competitive repopulating unit (CRU) function assay to determine HSC content. 4×105 cells/mL of post-thaw clinical umbilical cord blood (UCB) mononuclear cells (MNCs) were inoculated in serum-free Stemspan® medium including a basal cytokine combination of 100ng/ml SCF, 50ng/ml FL and 100ng/ml TPO (S+F+T) on an mesenchymal stromal cell (MSC) stromal layer. To this cocktail, the following cytokine were added: 15ng/ml of IGFBP1 (A), 20ng/ml of IGFBP2 (B), 10ng/ml of IGF2 (C) and 20ng/ml of ANGPTL3 (D). After 11 days culture, the expanded cord blood cells were analyzed for surface marker CD34+CD38-CD90+ by flow cytometry and transplanted to 8-10 weeks NOD/SCID-IL2γ-/- (NSG) mice by tail vein injection.
Results: There were 11.0±0.3-fold, 11.4±0.2-fold, 10.3±0.3-fold and 12.5±0.8-fold expansion of viable total nucleated cells as well as 6.4±0.2-fold, 5.3±0.1-fold, 8.0±0.2-fold and 8.8±0.5-fold expansion of CD34+CD38-CD90+ cells for the cytokine combinations ① S+T+F, ② S+T+F+B, ③ S+T+F+BCD and ④ S+T+F+ABD. After 4 months transplantation, limiting dilution assay from bone marrow showed that CRU was 1/1.58×106, 1/1.99×106, 1/1.33×106 and 1/1.19×106 in the expanded cells cultured with cytokine combination from ① to ④ compared to 1/3.15×105 in the unexpanded cells when P<0.05. Thus, there was 2.0-fold, 1.7-fold, 2.6-fold and 2.9-fold of in vivo expansion of HSCs for conditions ① to ④. Assessment of donor engraftment in the different organs showed that only 24.0±11.2% of human cells could dominate in bone marrow compared to 55.3±12.2% in spleen, 11.0±10.4% in lung, 8.3±4.6% in liver, 0.8±0.5% in kidney and 0.5±0.6% in heart. Taken as a whole, the results show excellent correlation between the HSC ex vivo surface marker of CD34+CD38-CD90+ and the in vivo CRU functional assay in bone marrow.
Conclusion: The CD34+CD38-CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with in vivo CRU functional assay.
Date: 3th August 2011
Venue: Kiwi Lounge, Grand Copthorne Waterfront Hotel
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