A small-molecule/cytokine combination enhances hematopoietic stem cell proliferation via inhibition of cell differentiation
Authors: Yu Zhang
Huihui Wang
Yongping Jiang
Xinxin Ding
Lan Wang
Yupo Ma
Bin Shen
Zhihua Ren
Xin Guan
Journal: Stem Cell Research & Therapy (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Differentiation inhibition; Expansion; Hematopoietic stem cells; Small molecules.
Affiliations: Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China., Biopharmagen Corp, Suzhou, China., Department of Pathology, The State University of New York at Stony Brook, Stony Brook, NY, USA., College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA., Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China. ., Biopharmagen Corp, Suzhou, China. .

Abstract:  Background: Accumulated evidence supports the potent stimulating effects of multiple small molecules on the expansion of hematopoietic stem cells (HSCs) which are important for the therapy of various hematological disorders. Here, we report a novel, optimized formula, named the SC cocktail, which contains a combination of three such small molecules and four cytokines. Methods: Small-molecule candidates were individually screened and then combined at their optimal concentration with the presence of cytokines to achieve maximum capacity for stimulating the human CD34+ cell expansion ex vivo. The extent of cell expansion and the immunophenotype of expanded cells were assessed through flow cytometry. The functional preservation of HSC stemness was confirmed by additional cell and molecular assays in vitro. Subsequently, the expanded cells were transplanted into sublethally irradiated NOD/SCID mice for the assessment of human cell viability and engraftment potential in vivo. Furthermore, the expression of several genes in the cell proliferation and differentiation pathways was analyzed through quantitative polymerase chain reaction (qPCR) during the process of CD34+ cell expansion. Results: The SC cocktail supported the retention of the immunophenotype of hematopoietic stem/progenitor cells remarkably well, by yielding purities of 86.6 ± 11.2% for CD34+ cells and 76.2 ± 10.5% for CD34+CD38- cells, respectively, for a 7-day culture. On day 7, the enhancement of expansion of CD34+ cells and CD34+CD38- cells reached a maxima of 28.0 ± 5.5-fold and 27.9 ± 4.3-fold, respectively. The SC cocktail-expanded CD34+ cells preserved the characteristics of HSCs by effectively inhibiting their differentiation in vitro and retained the multilineage differentiation potential in primary and secondary in vivo murine xenotransplantation trials. Further gene expression analysis suggested that the small-molecule combination strengthened the ability of the cytokines to enhance the Notch pathway for the preservation of HSC stemness, and inhibited the ability of the cytokines to activate the Wnt pathway for HSC differentiation. Conclusions: We developed an optimal small-molecule/cytokine combination for the enhancement of HSC expansion via inhibition of differentiation. This approach indicates promising application for preparation of both the HSCs and the mature, functional hematopoietic cells for clinical transplantation. 

Retracted: No

PubPeer comments:
 Figure 4b: a slight vertical expansion of the Runx1 blot shows higher similarity to the Bmi1 blot than expected. ![file] , 
 #1 We would love to express our appreciation for your interest in our work. After carefully went through the original data, the bands of Runx-1 were mistakenly used as the ones of Bmi1 during the arrangement. We apologize for the careless and thank you so much for pointing it out. We will contact the editor to make a correction asap. The scientific facticity is always our priority and we are glad to have readers like you. , 
 ![file] Please find the corrected figure of the bands, thanks again for your precious comment. ,