Extrinsic modulation of HSC fate is more practical for clinical application than in vivo approaches that use viral vectors. Among the multiple extrinsic regulators, the chemically well-defined small molecules can be used to regulate the intracellular procedures in a rapid, competitive, and reversible fashion through fine-tuning of their concentration and because of a lack of immunogenicity [3539], which is the reason why we chose this strategy for the induction of ex vivo expansion of CD34+ cells. As demonstrated by accumulated evidence, two categories of compounds, aryl hydrocarbon receptor (AHR) antagonists and histone deacetylase inhibitors (HDACi), have had some striking success in terms of HSC expansion [14, 16, 40, 41]. AHR antagonists can directly bind and restrain AHR, which is partially responsible for HSC quiescence maintenance [42, 43], to induce the expansion of CD34+ cells [14]; an HDACi, on the other hand, promotes HSC proliferation through the regulation of epigenetic plasticity and chromatin structures which are critical for the maintenance of the primitive status of HSCs [16, 40, 4446]. For the purpose of integrating the advantages of these two categories and to maximize their impact on HSC proliferation, we therefore focused on the screening of combinations of these compounds. During this process, as additive negative effects were observed when the selected compounds were combined at their individually determined optimal concentrations, a component adjustment had to be undertaken for the augmentation of the cell yield. SR1 showed lower cytotoxicity than the others, and our previous work also confirmed its capability at 1 μM for better maintenance of CD34+ cells; hence, only two components were adjusted in the compound combination. After multiple rounds of selection, the synergistic effect of these small molecules, coupled with three cytokines, was ultimately acquired which benefits the blocking of HSC differentiation, with a CD34-positive rate of about 92.0% ± 0.5% and primitive-progenitor proportion (CD34+CD38) of about 74.6% ± 7.6%, instead of promoting the total mononuclear cell proliferation.

Unlike previously reports [16], the role of cytokines is irreplaceable and necessary for HSC culture. We found that there were not enough cells left or cell trending death on day 7 in the absence of cytokines during small-molecule combination screening. In addition, TPO, SCF, and Flt3-L have long been confirmed as the core cytokine mix for the best support of HSCs in in-vitro culture [4752]; therefore, they were consequently required for the small-molecule combination screening in the first place. Unexpectedly, we found that the three essential cytokines only expanded a limited number of HSCs whereas the addition of IL-6 and IL-3 significantly strengthened the output. Cells showed robust proliferation in spite of the IL-3 presence; HSC features and signals of function-relevant specific surface markers were lost and might be accompanied by differentiation, and result in the loss of HSC activity [14, 53]. As a result, IL-6 along with the three basic cytokines was identified as the optimal cytokine combination instead of IL-3. Cord blood CD34+ cells cultured in a medium containing these four cytokines plus the small-molecule combination for 7 days could finally reach ~28.0-fold expansion of CD34+ and CD34+CD38 cells with CD34+ purity of 86.6% ± 11.2% and CD34+CD38 proportion of 76.2% ± 10.5%, respectively. Our SC cocktail produced much higher CD34+ expansion folds with higher purity compared with the previously published work with SR1 alone (coupled with cytokines), which reached a 24.2-fold increase in CD34+ cells with a CD34 percentage of 42% ± 1.8% on day 7 [14], and with VPA alone (plus cytokines), which maintained CD34 purity of about 80.0% on day 7 [16]. In summary, these data demonstrated that the capacity for cell proliferation is dramatically influenced by the distinct combination of cytokines and an ideal yield could be obtained by means of the optimal cytokines. Certainly, when referring to the increase in absolute numbers of stem cells, the involvement of a limiting dilution assay can provide more convincing data [5456] by quantitation of SCID-repopulating cells (SRCs) in a given sample before and after in vivo transplantation. In our case, only the absolute number of in-vitro generated cells was calculated; however, we believed that the apparent advantages of the SC cocktail in increasing cell expansion, blocking cell differentiation, and maintaining HSC stemness may benefit the frequency of SRCs in the immunodeficient mouse as well. This experiment will no doubt be evaluated in our future study.

Moreover, in the course of our study, we observed that HSCs cultured with the SC cocktail showed a superb undifferentiated state, affirmed by our functionality studies including cell cycle analysis, HSC-specific gene expression, the CFU assay in vitro, and mouse engraftment in vivo; but they did not notably increase the number of total mononuclear cells. We assumed that the inhibition of HSC differentiation might directly rely on the small-molecule combination. This assumption was further confirmed by the signaling pathway analysis. We observed an ascendant tendency for the Notch target genes in the presence of the compound combination. The upregulation of Notch pathway target genes in the SC cocktail group suggested that this pathway might be activated by the small molecules, and the downstream effects of Notch pathway activation were related to the preservation of HSC properties [31, 32, 57, 58]. Simultaneously, these data implied a stronger proliferation and differentiation ability of cells not treated with the small-molecule cocktail. Besides, based on our results from analysis of the Wnt pathway, we found that the cell differentiation was blocked due to the presence of small molecules. Other research groups, Luis et al. [34] and Famili et al. [33], also found that the canonical Wnt pathway regulates hematopoiesis in a dosage-dependent fashion. Only mildly activated Wnt signaling pathway could enhance the function of HSCs, and a high activated level of the Wnt pathway could enhance the HSC differentiation and impair HSC self-renewal. As shown in our data (Fig. 6b), the Wnt pathway was more increased in the absence of the small-molecule group (VC). On the other hand, the presence of small molecules (SC cocktail) could reduce the HSC stimulation caused by cytokines and only led to a slightly upregulated expression of these genes. Thus it is implied that the blocking of CD34+ cell differentiation in the SC cocktail group may result from the presence of small molecules. It is our plan to perform further investigation for the small-molecule combination using Wnt inhibitors to reveal its exact role on cell differentiation blocking. To sum up, these data demonstrated that the presence of the small-molecule combination can effectively inhibit the prodifferentiation effects of the cytokines without affecting the ability of the cytokines to stimulate cell proliferation.