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Abstract
Identification of effective culture conditions to maintain and possibly expand human HSPCs in vitro is an important goal. Recent advances highlight the efficacy of chemicals in maintaining and converting cell fates. We screened 186 chemicals and found that a combination of CHIR-99021, Forskolin and OAC1 (CFO) maintained human CD34-positive cells in vitro. Efficiency of the culture system was characterized using flow cytometry for CD34-positive cells, a colony-forming assay and xeno-transplants. We found that human CD34-positive cells treated with this combination had enhanced expression of human HSPC markers and increased haematopoietic re-populating ability in immune-deficient mice. Single-cell RNA-seq analyses showed that the in vitro cultured human CD34-positive cells were heterogeneous. We found that CFO supports maintenance of human CD34-positive cells by activating HOXA9, GATA2 and AKT-cAMP signaling pathway. These data have implications in therapies requiring maintenance and/or expansion of human HSPCs.
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Details
1 Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China; Stem Cell Institute, Zhejiang University, Hangzhou, China
2 Institute of Hematology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
3 UT-ORNL Graduate School of Genome Science and Technology, The University of Tennessee, Knoxville, TN, USA
4 Core Facilities, Zhejiang University School of Medicine, Hangzhou, China
5 Institute of Mechatronic Control Engineering, Zhejiang University, Hangzhou, China
6 Department of Chemistry, Institute of Microanalytical Systems, Zhejiang University, Hangzhou, China
7 Department of Medicine, Haematology Research Centre, Division of Experimental Medicine, Imperial College London, London, UK
8 Stem Cell Institute, Zhejiang University, Hangzhou, China; Institute of Hematology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Alliance for Atlas of Blood Cells, Hangzhou, China
9 Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China; Stem Cell Institute, Zhejiang University, Hangzhou, China; Institute of Hematology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Alliance for Atlas of Blood Cells, Hangzhou, China; Zhejiang Provincial Key Lab for Tissue Engineering and Regenerative Medicine, Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Hangzhou, China