A small-molecule/cytokine combination enhances hematopoietic stem cell proliferation via inhibition of cell differentiation

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.

Keywords: Differentiation inhibition; Expansion; Hematopoietic stem cells; Small molecules.

Fig. 1

Effects of small molecules on cell morphology and expansion. a Cell morphology for cultures with various small molecules at three different concentrations. Photos were captured on day 7 (20× objective, scale bar = 50 μm). The vehicle control (VC) was composed of cytokines and DMSO; the negative control (NC) consisted of cytokines only. b CD34-positive rate and fold expansion of CD34⁺ cells for the different concentrations of each single small molecule on day 7. Data are shown as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. C433 CAY10433, SR1 stemregenin1, VPA valproic acid

Fig. 2

Effects of small-molecule combinations on marker purity and cellular fold expansion. a CD34 expression and CD34⁺ cellular fold expansion for different combinations of small molecules. b,c VPA and C433 concentration adjustment. PB CD34⁺ cells were cultured with the three core cytokines, 1 μM stemregenin1 (SR1), and various concentrations of valproic acid (VPA) (b) or CAY10433 (C433) (c). d Final optimization of the compound combination. Data are shown as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. NC negative control, VC vehicle control

Fig. 3

Effects of optimal cytokine combination supplemented with selected small molecules on cell expansion. a Confirmation of PB and cord blood CD34⁺ cells cultured in a 24-well plate. b Optimal cytokine combination screening. Cord blood CD34⁺ cells were primed in the presence or absence of the small-molecule combination supplemented with different groups of cytokines for 7 days. c Representative flow cytometry results of CD34 and CD38 percentage, and phenotypic changes and cellular fold expansion caused by the optimal formula for day 7. All data are shown as means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. CBHSC cord blood hematopoietic stem cell, IgG immunoglobulin G, MNC mononuclear cells, PBHSC peripheral blood hematopoietic stem cell, PC uncultured CBHSCs, TSF TPO + SCF + Flt-3 L, 3TSF IL-3 + TPO + SCF + Flt-3 L, 6TSF IL-6 + TPO + SCF + Flt-3 L, 36TSF IL-6 + IL-3 + TPO + SCF + Flt-3 L, VC vehicle control

Fig. 4

In vitro functional assessments of the SC cocktail-expanded CD34⁺ cells. a Cell cycle analysis. One of three representative experiments is shown, and the percentage of different phases is summarized in the histogram. b HSC-specific gene expression. Results of qualitative RNA-PCR are shown on the left for one representative of three independent RNA samples analyzed per group, and the results of quantitative PCR are shown in the right. c The morphology and numbers of colony-forming units (CFUs). Morphology (20× objective, scale bar = 50 μm) and colony numbers were recorded on day 14 after cell seeding. All data are shown as means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. BFU-E burst-forming unit-erythroid, CFU-GEMM CFU-granulocyte, erythrocyte, macrophage, megakaryocyte, CFU-GM CFU-granulocyte, macrophage, CFU-Mk CFU-megakaryocyte, PC uncultured cord blood HSCs, VC vehicle control

Fig. 5

Functional assessment of the SC cocktail-expanded CD34+ cells in NOD/SCID mice. a The presence of human cells in bone marrow 8 weeks after primary transplantation. b The presence of human cells in peripheral blood 8 weeks after primary transplantation. c The presence of human cells in bone marrow 10 weeks after secondary transplantation. Ten mice were used for each group and five additional mice receiving saline were used to subtract the background signal. Data are shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. PC uncultured cord blood HSCs, VC vehicle control

Fig. 6

Effects of the small-molecule combination on the expression of Notch and Wnt signaling genes in human cord blood CD34⁺ cells. a The relative expression of Notch pathway key genes and target genes. b The relative expression of Wnt pathway key genes and target genes. Data are shown as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. c A schematic summary of the effects of the SC cocktail on CD34⁺ cell proliferation and differentiation. VC vehicle control