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.
Highlights
Identification of effective culture conditions to maintain and possibly expand human HSPCs ex vivo is an important goal for hematological researches
We found that human CD34-positive cells can be maintained in vitro by a combination of CHIR-99021, Forskolin and Oct4-activating compound 1 (OAC1) (CFO) without haematopoietic growth factors
We found that human CD34positive cells cultured with CHIR-99021 (C), a GSK-3 inhibitor, promoted an up to 3.84-fold increase in expression of human HSPC marker gene CD34 (95% confidence interval [CI] 2.06, 5.61; P < 0.001) compared with controls
Summary
Identification of effective culture conditions to maintain and possibly expand human HSPCs ex vivo is an important goal for hematological researches. Previous studies tried to optimize culture conditions with haematopoietic growth factors (HGFs) and exogenous gene expressions to maintain and expand human HSPCs in vitro. These attempts are mostly unsuccessful[1,2,3]. Cardiomyocyte-like cells can be generated by treating human fibroblasts with several small molecular weight chemicals[8]. These chemicals can expand adult stem cells including inducing proliferation of mature primary human hepatocytes and converting rat and mouse mature hepatocytes to proliferative, bi-potent cells in vitro[9,10]
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