Reducing TGF‐β1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling

Abstract Objective As an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34+ cells but also increases CD34− cell numbers. These CD34− cells influenced the ex vivo expansion of CD34+ cells. In this work, the effects of periodically removing CD34− cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated. Materials and methods CD34− cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF‐β1 contents. Results After 10‐day culture, the expansion of CD34+ cells in the CD34− cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34− cells with SR1 addition improved the biological function of expanded CD34+ cells and significantly increased the percentage of self‐renewal symmetric division of CD34+ cells. In addition, the concentration of total TGF‐β1 and activated TGF‐β1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT‐qPCR results showed that the periodic removal of CD34− cells with cooperation from SR1 further reduced the expression of AhR‐related genes. Conclusions Periodic removal of CD34− cells plus cooperation with SR1 improved the expansion of CD34+ cells, maintained better biological function of expanded CD34+ cells and reduced the TGF‐β1 contents by downregulating AhR signalling.

symmetrical division of HSCs is increased by attenuating Aryl hydrocarbon receptor (AhR) signalling in human HSCs in vivo. 7 Therefore, regulation of AhR signalling may be beneficial to increase selfrenewal symmetrical division of HSCs.
AhR is an aromatic compound receptor and a family member of the basic helix-loop-helix-period-aryl hydrocarbon receptor nuclear translocator. 8 Previous studies showed that activation of AhR accelerated the differentiation process of HSCs. [9][10][11] Singh et al 12 found that the exposure of the AhR activator 2,3,7,8-tetra chlorodibenzo-p-dioxin (TCDD) exhibited diminished capacity to reconstitute and home to the marrow of irradiated recipients, and Laiosa et al 13 reported that the long-term self-renewal ability of HSCs from TCDD-exposed foetuses was decreased after initial reconstitution in mouse pregnancy. StemRegenin 1 (SR1) is an antagonist of AhR signalling and facilitates the expansion of HSCs. [14][15][16] The expansion of hESC-derived Lin − CD34 + haematopoietic progenitors was enhanced by SR1 in a concentration-dependent manner. 14 The number of CD34 + , CD133 + and CD90 + haematopoietic stem and progenitor cells with SR1 was significantly increased in the culture of mouse peripheral blood CD34 + cells for 7 days. 15 In summary, the inhibition of AhR signalling was beneficial to improve the expansion of HSCs.
CD34 is a key marker of HSCs. HSCs could lose the CD34 marker and differentiate into CD34 − cells during ex vivo expansion. These increased CD34 − cells influenced the expansion of CD34 + cells through secreting factors. Among these cytokines, transforming growth factor β1 (TGF-β1) is a major cytokine that plays an inhibitory role in haematopoietic function, and the effect depends on the differentiation status of the cells. 17,18 High concentrations of TGF-β1 strongly inhibited the expansion of HSPCs. [19][20][21] The proliferation and differentiation of CD34 + CD38 − cells and CD34 + CD38 + cells were strongly suppressed with TGF-β1 addition. 22 Moreover, different mechanisms have been proposed to explain AhR/TGFβ crosstalk in different cells. 23

| Cell preparation
Cord blood CD34 + cells were obtained by density gradient centrifugation with Ficoll/Histopaque and enriched by a CD34-positive immunomagnetic bead EasySep™ sorting system (Miltenyi Biotec).
To determine the optimal SR1 addition strategy, 0.5 μmol/L SR1 was added, as shown in Table 1. Samples were taken, and the numbers of total cells were counted. The relative expansion fold ratio was calculated to evaluate the expansion of CD34 + cells.
To investigate the effects of removing CD34 − cells with SR1 addition on the expansion of CD34 + cells, experiments were performed as shown in Table 2.

| Cell subpopulation analysis by flow cytometry
A total of 1 × 10 6 cells were stained with mouse anti-human antibodies   According to the proportions in total cells, the numbers of CD34 + cells and CD34 + CD38 − cells were calculated, and the fold expansion was calculated using the numbers divided by the numbers on day 0. and burst-forming units for pure erythroid precursors (BFU-E).

| Secondary expansion assay of expanded CD34 + cells
Expanded CD34 + cells were separated using a Mini MACS magnetic separation system at the end of culture and were reseeded a density of 5 × 10 4 cells/mL in 48-well plates with 500 μL serum-free medium in each well. The serum-free medium was supplemented with 50 ng/mL SCF, 20 ng/mL TPO, and 50 ng/mL Flt3-L. CD34 + cells were cultured for 14 days at 37°C in a humidified incubator with 5% CO 2 . The secondary expansion ability of CD34 + cells was evaluated based on the expansion of total cells and proportion of CD34 + cells on day 14.

| Supernatant human TGF-β1 concentration detection by ELISA
By centrifugation and removal of particulates, cell culture supernatants were collected to detect the concentration of TGF-β1 by enzyme-linked immunosorbent assay (ELISA) (R&D systems ® ).

| Detection of Numb distribution by immunofluorescence staining
A total of 1 × 10 6 cells were exposed to medium containing

| Detection of numb and musashi2 by qRT-PCR
RNA was isolated using a RNeasy Mini kit (Qiagen). cDNA was obtained from equal amounts of RNA using Superscript II reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad) on a CFX 96 C1000™ Thermal cycler (Bio-Rad). Primer sequences are listed in Table 3.

| Statistical analysis
The results are presented as scatter with the mean or mean ± standard deviation of the mean. A t test was used for statistical analysis between the two groups, and ANOVA was used for statistical analysis between three or more groups. P < .05 was considered statistically significant.

| Periodic removal of CD34 − cells in cooperation with SR1 enhanced the expansion of CD34 + cells
To identify the optimal SR1 addition strategy, SR1 was added as de-  were investigated by colony formation assays and secondary expansion assays. In the semisolid culture, the colony number ratio per 10 4 CD34 + cells increased with increasing SR1 addition frequency, and T1 group-expanded CD34 + cells exhibited the highest number of colony-forming units ( Figure 1D). In secondary expansion culture, T1 group-expanded CD34 + cells presented the highest total cell expansion folds and CD34 + cell expansion folds ( Figure 1E,F).
The division mode of CD34 + cells was further evaluated. By detection of the key genes numb and msi2 by RT-PCR, there was a significant downregulation of numb and upregulation of msi2 (Figure 2A,B). By counting cells stained with Numb on day 7, T1 resulted in the highest relative self-renewal symmetric division percentage ratio of CD34 + cells ( Figure 2C,D). According to these results, 0.5 μmol/L SR1 addition at a frequency of 12 h −1 was the optimal addition strategy.
To further promote the expansion of HSCs, CD34 − cells were periodically removed, and the optimal SR1 addition strategy was It was reported that CD34 + CD38 − CD45RA − CD49f + CD90 + cells can be used to characterize human HSPCs. 28 Thus, the percentages of primitive HSPCs (pHSCs, CD34 + CD38 − CD45RA − CD49f + CD90 + cells) were analysed, and the expansion folds of pHSCs were calculated. Higher expansion folds of pHSCs in the CD34 − cell removal plus SR1 group were found on day 10 than in the other groups ( Figure 3C, P < .05).
The biological function of expanded CD34 + cells was evaluated by colony-forming assay and secondary expansion assay ( Figure 3D-F). In the semisolid cultures, the CFU-GEMM, CFU-GM, BFU-E and total CFUs in the CD34 − cell removal plus SR1 group were significantly higher than those in the control group and SR1 group (P < .05).
In secondary expansion cultures, the total cell expansion fold, proportion and expansion fold of CD34 + cells and CD34 + CD38 − cells in the CD34 − cell removal plus SR1 group were also significantly higher than those in the control group and SR1 group (P < .05).
To conclude, removal of CD34 − cells with SR1 further promoted the expansion of CD34 + cells and maintained the biological function of expanded CD34 + cells ex vivo.
The division mode of CD34 + cells was detected (Figure 4).
Compared to the control group, the relative mRNA expression of numb in the CD34 − cell removal plus SR1 group was significantly downregulated relative to that in the control group and SR1 group.
The mRNA expression of msi2 in the CD34 − cell removal plus SR1 group was significantly upregulated relative to that of the control group and SR1 group ( Figure 4A

| Periodic removal of CD34 − cells reduced the content of TGF-β1 in supernatant and further downregulated the AhR signalling gene by cooperating with SR1
By detecting the content of TGF-β1 in the supernatant (Figure 5A), the results indicated that the content of TGF-β1 increased with time. In the CD34 − cell removal plus SR1 group, the concentration of total TGF-β1 was significantly reduced to 99.37 ± 31.33 pg/mL on day 10, which was significantly lower than the 510.31 ± 30.55 pg/ mL in the control group and 539.14 ± 71.55 pg/mL in the SR1 group (P < .05), which showed that periodically removing CD34 − cells reduced the TGF-β1 content. Consistent with total TGF-β1, the level of spontaneously activated TGF-β1 in the SR1 group and the CD34 − cell removal plus SR1 group were both significantly reduced compared with that of the control group ( Figure 5B). p57 kip2 in the CD34 − cell removal plus SR1 group was downregulated relative to that of the control ( Figure 5D). For self-renewal and functional genes of HSCs, the expression of cebp, meis1, runx1, tie2, gata2 and stat5a was not significantly different. The expression of pu.1, hoxa9, hoxb4, hes1 and bmi1 was upregulated in the SR1 group and the CD34 − cell removal plus SR1 group relative to that of the control ( Figure 5E).

| D ISCUSS I ON
The clinical application of HSCs has been limited by insufficient quantities of these cells. Many studies have focused on the ex vivo expansion of HSCs to solve the limitation of HSC numbers. It has been reported that inhibition of AhR signalling is an effective way to decrease differentiation. StemRegenin 1 (SR1) is a common inhibitor of the AHR signalling pathway and has been proven to be a positive regulator of the expansion of HSCs. 14 15 In this work, we also found that SR1 sup-  TGF-β1 was identified as an inhibitor of the expansion of HSCs.
In our work, the contents of total TGF-β1 and activated TGF-β1 were both reduced by removing CD34 − cells with SR1 addition. Consistently, the expression of tgfβr1, smad4 and p57 kip2 in the CD34 − cell removal plus SR1 group was also downregulated. TGF-β1 inhibited haematopoietic function, and its effect depended on its concentration. 19 TGF-β1 was found to strongly inhibit the proliferation and differentiation of primitive cells, including CD34 + CD38 − cells and CD34 + CD38 + cells. 22 SCF-mediated terminal erythroid differentiation was reduced by neutralization of TGF-β1 in human cord blood CD34 + CD38 − Lin − cells. 40 Hexachlorobenzene reduced AhR mRNA expression by enhancing TGF-β1 mRNA levels in human breast cancer cells, 23 and TGF-β1 reduced TCDD-induced AhR gene expression in nontumorigenic prostate epithelial cells. 25 However, some studies showed that TGF-β1 stimulated the synthesis of AhR mRNA in human HepG2 cells 27 and that TGF-β1 was proven to be an endogenous activator of AHR in a dose-responsive manner in H1 L7.5c3 cells. 24 In our work, the expression of the key genes hsp90 and cyp1b1 indicated that removing CD34 − cells and adding SR1 simultaneously further inhibited the AhR signalling pathway. This demonstrated that TGF-β1 may neutralize the inhibition of AhR signalling by SR1. However, a verification test with the supplementation of TGF-β1 and/or neutralizing antibody of TGF-β1 with SR1 may have been more helpful.
In conclusion, removing CD34 − cells and adding SR1 simultaneously reduced the TGF-β1 content, promoted the expansion of CD34 + cells, increased self-renewal symmetric division and maintained better biological function of expanded CD34 + cells by downregulating AhR signalling. These results provide technical support for the optimization of the haematopoietic stem cell culture process ex vivo.

ACK N OWLED G EM ENT
This work was supported by the Science and Technology Innovation Action Plan of Basic Research, Shanghai, China (15JC1401402).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

AUTH O R CO NTR I B UTI O N S
Haibo Cai and Xuejun Zhu conceived the study and designed the experiments. Xuejun Zhu performed the experiment, finished original draft and was responsible for review and editing of manuscript.
Qihao Sun contributed to the review and editing of manuscript.
Haibo Cai was responsible for supervision, review and editing of manuscript and provided funding for the work. Wen-Song Tan was responsible for providing funding.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.