Comparison of Different Cytokine Conditions Reveals Resveratrol as a New Molecule for Ex Vivo Cultivation of Cord Blood-Derived Hematopoietic Stem Cells

Abstract

Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an interesting source for HSC transplantation. However, the number of collected CB-HSCs is often too low for one transplantation; therefore, ex vivo expansion of CB-HSCs is desirable. Current expansion protocols are based on the use of cytokine combinations, including insulin-like growth factor-binding protein 2 (IGFBP2) and angiopoietin-like proteins, or combinations with "small molecules" such as stemregenin-1. The aim of our project was to compare the potential of different CB-HSC expansion strategies side-by-side by phenotypical analysis in vitro and serial engraftment properties in NOD/SCID/IL2rg-/- (NSG) immunodeficient mice. We further identified resveratrol, a naturally occurring polyphenol, as a new, alternative small molecule combined with cytokines to facilitate serum-free ex vivo expansion of human CB-HSCs. The cultivation in resveratrol preserved the CB-HSC phenotype in vitro most efficiently and was ∼2 times more potent than commonly used cytokine conditions (including stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-6) and the recently established serum-free culture, including IGFBP2 and angiopoietin-like 5. Serial transplantation studies further confirmed resveratrol to support robust multilineage engraftment in primary and secondary NSG recipients. Therefore, our work proposes resveratrol as a new small molecule for improved ex vivo culture and modification of human HSCs based on an efficient ex vivo propagation of the HSC fate.

Significance: Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an important source for HSC transplantations but restricted in their usage because of their low numbers. In gene therapy, modifications of HSCs relies on their ex vivo modification without losing their stemness properties. Therefore, ex vivo cultivation and expansion of CB-HSCs is important for their effective application in HSC transplantation and gene therapy. Several promising protocols for serum-free cultivation of HSCs using different combinations of cytokines or so-called small molecules are described. A direct comparison was performed of three described serum-free cytokine conditions, demonstrating that the natural occurring polyphenol resveratrol is able to support ex vivo cultivation of CB-HSCs. The results show that resveratrol is an additional candidate for improving ex vivo cultures of HSCs for transplantation and gene therapeutic applications in the future.

Keywords: Cord blood; Expansion; Hematopoietic stem cell; Resveratrol; Serial transplantation.

Figure 1.

Ex vivo expansion of CD34⁺ human cord blood (CB)-derived human stem cells (HSCs). Expansion rates of total and HSC-enriched populations after 9 days of ex vivo expansion of human CB-derived CD34⁺ cells in different cytokine combinations. Percentage of CD34- and CD133-positive cells after cultivation was determined via antibody staining and subsequent flow cytometry analysis. Dead cells were excluded via 4′,6-diamidino-2-phenylindole staining. Six independent pools of CB-derived CD34⁺ cells were used. ∗, p < .05; ∗∗, p < .01. (A): Total cell expansion rates relative to input cell numbers. (B): Percentage of CD34⁺ cells after in vitro expansion. (C): Percentage of CD34⁺/CD133⁺ double-positive cells after in vitro expansion. (D): Representative flow cytometry analysis of in vitro cultivated CB-derived HSCs stained for human CD34 and CD133 surface expression. (E): Expansion rates of CD34⁺/CD133⁺ double-positive cells as relative increase based on the CD34⁺/CD133⁺ double-positive cells at the end of expansion shown in (C) and the fold expansion rates (A). Abbreviations: ctrl, control medium containing SCF, THPO, Flt3-L, IL-6; Flt3-L, Fms-related tyrosine kinase 3 ligand; h, human; IL, interleukin; Rvt, medium containing SCF, THPO, Flt3-L, IL-6 plus resveratrol; SCF, stem cell factor; SR-1, medium containing SCF, THPO, Flt3-L, IL-6 plus stemregenin-1; STAI3, medium containing SCF, THPO, Flt3-L, insulin-like growth factor-binding protein 2, angiopoietin-like protein; THPO, thrombopoietin.

Figure 2.

Engraftment of human cells in NOD/SCID/IL2rg^−/− (NSG) mice 9 months after transplantation. Sublethally (2.5 Gy) irradiated NSG mice were transplanted with 20,000 freshly (d0) thawed CD34⁺ cells or their progenies after in vitro cultivation in the respective cytokine conditions. Secondary transplantation was performed with total bone marrow from the indicated primary groups into sublethally irradiated recipients. Next, 100 µl of peripheral blood was stained for human CD45⁺ cells after red blood cell lysis. Approximately 10⁶ total bone marrow cells were stained for the presence of human CD45⁺ cells. (A): Peripheral blood analysis of human chimerism in primary recipients. (B): Human chimerism in bone marrow in primary recipients. (C): Peripheral blood chimerism in secondary recipients. (D): Repopulation of secondary recipients in bone marrow based on human CD45⁺ cells. (E): Fluorescence-activated cell sorting analysis of human chimerism in pooled bone marrow before secondary transplantation. Abbreviations: BM, bone marrow; ctrl, control medium containing SCF, THPO, Flt3-L, IL-6; Flt3-L, Fms-related tyrosine kinase 3 ligand; d0, day 0; h, human; IL, interleukin; PB, peripheral blood; Rvt, medium containing SCF, THPO, Flt3-L, IL-6 plus resveratrol; SCF, stem cell factor; SSC, side scatter; STAI3, medium containing SCF, THPO, Flt3-L, insulin-like growth factor-binding protein 2, angiopoietin-like protein; THPO, thrombopoietin.

Figure 3.

Engraftment of cord blood in primary NOD/SCID/IL2rg^−/− (NSG) mice after in vitro expansion 9 months after transplantation. Sublethally irradiated NSG mice were transplanted with either fresh (d0) or the respective expansion equivalents after culturing 20,000 (A, B) or 100,000 (C, D) CD34⁺ cells per mouse within the different cytokine media. Next, 100 µl of peripheral blood was stained for human CD45⁺ cells after red blood cell lysis. Approximately 10⁶ total bone marrow cells were stained for the presence of human CD45⁺ cells. (A): Peripheral blood analysis of human chimerism after transplantation of 20,000 freshly thawed cells or the expansion equivalents. (B): Bone marrow chimerism of initially transplanted 20,000 CD34⁺ cells or the expansion equivalents. (C): Peripheral blood analysis of human chimerism after transplantation of 100,000 freshly thawed cells or the expansion equivalents. (D): Bone marrow chimerism of initially transplanted 100,000 CD34⁺ cells or the expansion equivalents. Abbreviations: BM, bone marrow; ctrl, control medium containing SCF, THPO, Flt3-L, IL-6; Flt3-L, Fms-related tyrosine kinase 3 ligand; d0, day 0; h, human; IL, interleukin; PB, peripheral blood; Rvt, medium containing SCF, THPO, Flt3-L, IL-6 plus resveratrol; SCF, stem cell factor; STAI3, medium containing SCF, THPO, Flt3-L, insulin-like growth factor-binding protein 2, angiopoietin-like protein; THPO, thrombopoietin.

Figure 4.

Combined data for primary and secondary recipients after normalization. For a better comparison of the in vivo data from the different experiments, the chimerism values of all experiments were normalized and integrated into a single graph as relative engraftment levels. For normalization within each independent experiment, the value of the mouse with the highest chimerism of d0 group was artificially set to 1. All other values were then normalized relative to that value. (A): Peripheral blood chimerism of primary mice. (B): Bone marrow chimerism of primary mice. (C): Peripheral blood of secondary mice. (D): Bone marrow chimerism of secondary mice. Abbreviations: BM, bone marrow; ctrl, control medium containing SCF, THPO, Flt3-L, IL-6; Flt3-L, Fms-related tyrosine kinase 3 ligand; d0, day 0; h, human; IL, interleukin; neg, negative; PB, peripheral blood; rel., relative; Rvt, medium containing SCF, THPO, Flt3-L, IL-6 plus resveratrol; SCF, stem cell factor; STAI3, medium containing SCF, THPO, Flt3-L, insulin-like growth factor-binding protein 2, angiopoietin-like protein; THPO, thrombopoietin.

Figure 5.

Each cytokine condition displayed multilineage engraftment in primary and secondary recipient NOD/SCID/IL2rg^−/− (NSG) mice. Multilineage engraftment of primary (A) and secondary (B) NSG mice was assessed based on staining of ∼10⁶ total bone marrow cells for the presence of human CD19 (lymphoid; white bars) and human CD33 (myeloid; black bars) surface marking on bone marrow cells within the human CD45-positive cell compartment. Combined analysis of all primary and secondary animals transplanted during the present study. Abbreviations: BM, bone marrow; ctrl, control medium containing SCF, THPO, Flt3-L, IL-6; Flt3-L, Fms-related tyrosine kinase 3 ligand; d0, day 0; h, human; IL, interleukin; Rvt, medium containing SCF, THPO, Flt3-L, IL-6 plus resveratrol; SCF, stem cell factor; STAI3, medium containing SCF, THPO, Flt3-L, insulin-like growth factor-binding protein 2, angiopoietin-like protein; THPO, thrombopoietin.