Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem and Progenitor Cells from Non-Enriched Umbilical Cord Blood with Azole Compounds

Abstract

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+CD34+ cells in the PB and BM by day 21 compared to non‐expanded and cytokine expanded grafts. The C7 expanded grafts maintained long‐term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre‐culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376–393

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells

“…These usually required supplementation with FBS in order to enrich the formulation to allow for cell expansion, with such basal media varying between Minimum Essential Medium Eagle − Alpha Modification (α-MEM) ( Amsellem et al, 2003 ; de Lima et al, 2008 ; Horwitz et al, 2014 ), Iscove’s modified Dulbecco’s Medium (IMDM) ( Gilmore et al, 2000 ; Çelebi et al, 2012 ) and others (reviewed in Costa et al, 2018 ). With the development of culture media specifically for human HSPC expansion, aligned with growing concerns with the use of FBS as an undefined supplement, formulations were developed to be serum-free, with protocols implementing specialized culture media such as X-VIVO TM 10 medium ( Kögler et al, 1999 ), QBSF-60 serum-free medium ( Qiu et al, 1999 ; Gonçalves et al, 2006 ), StemLine ® stem cell expansion medium ( Tiwari et al, 2013 ) and StemSpan TM serum-free expansion medium ( Delaney et al, 2010 ; Fares et al, 2014 ; Wagner et al, 2016 ; Bari et al, 2018 ). This has benefited cell expansion results but ultimately compromised the applicability of previous optimizations described in the literature.…”

Antioxidant Small Molecule Compound Chrysin Promotes the Self-Renewal of Hematopoietic Stem Cells

“…Under the condition of hypoxia, the expression of CXCR4 statistically increased, which means the ability of homing increased (Mohammadali et al, 2018). The use of p38-MAPK inhibitor (Bari et al, 2018) or antioxidants can maintain low ROS levels, protect HSCs from oxidative stress, and inhibit cell aging and differentiation (Ludin et al, 2014;Cao et al, 2016;Testa et al, 2016). This suggests that antioxidant small molecules may be of potential development value for expansion of HSCs, which can scavenge excessive ROS and repair oxidative damage, thus protecting the biological function of stem cells such as promoting engraftment of HSCs (Naka et al, 2008;Hao et al, 2014;Hu et al, 2014).…”

Expansion of Haematopoietic Stem and Progenitor Cells: Paving the Way for Next- Generation Haematopoietic Stem Cell Transplantation

“…They include P18IN003, P18IN011, and XIE18-6(cyclin-dependent kinase inhibitors) 70 ; NR101 (non-peptidyl small molecule agonist of c-MPL) 71 ; eltrombopag(a human specific thrombopoietin receptor agonist) 72 ; CHIR99021 and rapamycin(Wnt and β catenin pathway modulators) 73 ; and 5-azacytidine, trichostatin A, garcinol, and valproic acid(epigenetic modulators) 74,75 . Others such as resveratrol(a naturally occurring polyphenol) 76 ; serotonin(a monoamine neurotransmitter) 77 ; GW9662(a PPARγ antagonist) 78 ; SB203580, Vx702, BIRB-796 and Ly2228820(potent inhibitors of p38 mitogen-activated protein kinases) 79 ; and C7(a new structural analogue of SB203580) 80 have also been used in HSPC expansion studies. Amongst these compounds, only C7 has been shown to be able to expand HSPC from both non-enriched and CD34enriched grafts that retain both in vitro primitive HSPC phenotype and long-term in vivo functionality in xenotransplantation model 80 .…”