Optimization of small molecules doses for HSC expansion
The proper consentration of selected small molecules which was not cytotoxic for CD34+ cells was determined using MTS assay (Fig. 1). In consistent with the other studies, CD34+ cells cultivated in SB (10 μM), Bpv (5 μM), NAM (2.5 μM) and Pur (4 μM) were viable. However, predetermined concentrations of PD (1 μM), Chir (3 μM) and Pμ (10 μM) were toxic for UCB-HSCs. Therefore, lower concentrations of PD (0.25 μM), Chir (0.37 μM) and Pμ (2.5 μM) were added to the culture medium.
Figure 1. Dose finding and optimization for small-molecules (SB, Bpv, NAM, Pur, PD, Chir, Pμ). In each graph, the middle column corresponds to the reference concentration of the small molecules based on the literatures. Cell viability was measured by MTS assay post 48 hours incubation with small molecules. The negative control in each group was used for normalization of data. Bars indicated as mean ± SD at least five independent replicates. * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001.
SB, Chir and Bpv are sufficient for ex vivo expansion of UCB-CD34+ cells
We next did some serial experiments (Supplementary Fig. 1). In the first round of experiments, isolated UCB-CD34+ cells were cultured in the presence of cytokines (SCF, TPO and Flt3L) and selected small molecules. In the other groups small molecules were deleted one by one from the pool of 7 SMs. Although, individual removal of SB, Chir, Bpv, Pur, NAM and Pμ did not make significant differences in total nuclear cells (TNCs) number compared to the 7SMs group, removal of PD yielded increased total number of mononuclear cells (Fig. 2A). The precise effect of PD on ex vivo expansion of CD34+ cells has been discussed before (24). Additional round of small molecules removal showed that, deletion of NAM and Pur from the cocktail increased the fold expansion of TNCs and CD34+ cells. Furthermore, the groups lacking NAM and Pur had a higher colony forming potential, especially CFU-GM, compared to other groups containing small molecules (Fig. 2B). In next round, by removing Pμ the number of CD34+ cells, CFU-GM and CFU-GEMM colonies was increased significantly compared to the PC group (Fig. 2C). In the final round removal of SB, Chir, or Bpv reduced the expansion of CD34+CD38− cells and abolished formation of CFU-GM and CFU-GEMM colonies, showing that these are essential for CD34+ cell expansion (Fig. 2D). Although, there was no significant difference between the 3SMCs and the positive control in terms of TNC expansion, removal of Bpv slightly increased the TNC fold expansion compared to 3SMs group (118 to 140). Moreover, exclusion of each of the remaining three SMs (SB, Chir, or Bpv) had a dramatic negative impact on the expansion CD34+CD38− cells. Expansion with these three SMs (SB, Chir and Bpv) produced a 2.7-fold increase in the number of CD34+CD38− cells relative to positive control (17 vs. 47). Finally, a CFU assay was performed to determine if the optimal SM cocktail actually promotes the expansion of hUCB-HPCs. As shown in Fig. 2D, the number of total CFUs increased more than 3-fold when CD34+ cells were expanded in the presence of SB, Chir and Bpv for 10 days compared to the positive control. The expanded cells generated significantly more BFU and CFU-GM than the positive control (p<0.01). However, the number of GEMMs in SM group was slightly greater than that of the PC, but the difference was not statistically significant (p>0.05).
Figure 2. Characterization of expanded UCB-CD34+ cells in the presence/absence of different combinations of small molecules. TNC fold expansion, CD34+ cells percentage, fold expansion of CD34+ cells and colony forming potential of UCB-CD34+ cells was evaluated in each experiment. (A) 7 SMs cocktail (SB, PD, Chir, Bpv, NAM, Pur, Pμ) and its derivative groups (B) 6 SMs cocktail (SB, Chir, Bpv, NAM, Pur, Pμ) and its derivative groups (C) 4 SMs cocktail (SB, Chir, Bpv, Pμ) and its derivative groups (D) 3 SMs cocktail (SB, Chir, Bpv). CD34+ cells cultivated in presence of SCF, FLT3L and TPO was used as positive control. Fold expansion was determined by dividing the total number of viable cells expressing the phenotype at the end of the culture by the input number of viable cells expressing the same phenotype (n=3). Statistically significant difference compared with positive control group, *P≤0.05, ** P≤0.01,***P≤0.001.
The ability of 3SMs cocktail to enhance the short-term engraftment potential of ex vivo expanded CD34+ cells in the in utero transplanted NMRI mice
In order to evaluate the in vivo functional capability of the expanded CD34+ cells, we used in utero transplantation model (23). We transplanted 30-50×103 freshly isolated hUCB-CD34+ cells or the cells harvested from the cultures with the same number of input hUCB-CD34+ cells in the presence or absence of SMs cocktail into NMRI mouse embryos, E11.5-E13.5. Two weeks after birth, born mice were treated with human hematopoietic growth factors SCF (4ng/g), IL-3 (4ng/g) and G-CSF (50ng/g) for one week. As shown in Fig.3, by treatment with human hematopoietic factor, the hCD45+ chimerism was distinctly increased compared with initial values, 4 and 8 weeks post transplantation. 16 weeks after transplantation, the average human cell engraftment in the peripheral blood of the mice transplanted with freshly isolated hUCB CD34+ cells was about 1%. While, the percentage of CD45+ cells in 3SMs and positive control transplanted mice was 9 times and 3.4 times (3.6 ± 1 and 3.2 ± 0.3) respectively, compared to the unexpanded cell recipients (Fig. 3). In the other words, ex vivo expansion of hUCB CD34+ cells
with SM cocktail resulted in 1.5 fold increase in human cell engraftment compared to the positive control.
Figure 3. Mean human engraftment levels in the peripheral blood of NMRI mice fetal transplanted with expanded hUCB-CD34+ cells. (A) The percentage of human CD45 cells in the peripheral blood of newborn mice. Each bar indicated mean ±SD for at least 6 independent samples. ****P≤0.0001. (B) Each shape indicates the percentage of human CD45 expression in the peripheral blood of one newborn mouse. Mice with ≥0·2% human cells were considered chimeric.
Ability of the optimal SMs cocktail to modulate the cell signaling pathways
Subsequently, RT-qPCR was performed in order to determine the expression of typical genes involved in HSC stemness. The result shows that the relative expression of the two major genes involved in the proliferation and self-renewal of HSCs, including HOXB4 and GATA2 as well as the HSC-specific marker, CD34, have significantly increased in the presence of 3SMs cocktail after normalization to the level of the PC group. Furthermore, the expression of the CXCR4 gene involved in the migration and transplantation of HSCs has increased dramatically in the presence of 3SMs cocktail. The expression of other genes associated with self-renewal, such as ABCG2, Notch and Bmi1, does not show a significant difference between the groups (Fig. 4).
Figure 4. Treatment by SB, Chir and Bpv modifies the gene expression of UCB-CD34+ cells. Bars represent the mean fold-changes of gene expression in the 3 SMs-expanded cells relative to the positive control group detected by quantitative real-time PCR (n=3), *P≤0.05, **P≤0.01, ***P≤0.001 vs. positive control.