Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells

Abstract

Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.

Fig. 1

SR1 maintains an HSC phenotype and increases CFU content. (A) Expression of CD34 and CD133 in cultured mPB CD34⁺ cells. (B) Structure of SR1 and LGC006. (C) Phenotype of CD34⁺ mPB at 7 days or (D) CB at 35 days. (E) Weekly TNC counts from control (white bars) or SR1 (1 μM, black bars) cultures of 1,000 CB CD34⁺ cells. (F) Total CFU content of control (white bars) or SR1 (1 μM, black bars) cultures of 1000 CB CD34⁺ cells at 5 weeks.

Fig. 2

Culture of CB CD34⁺ cells with SR1 enhances early and late engraftment. (A) Mice were injected with 1000 (black squares), 3000 (black triangles), 10,000 (black circles), or 30,000 (black diamonds) uncultured CB CD34⁺ cells; or a fraction of the final culture equivalent to the progeny of CB CD34⁺ cells cultured for 21 days with control conditions [10,000 (blue circles)] or SR1 [0.75 μM, 300 (red crosses) or 10,000 (red circles)]. The average percent engraftment of human CD45⁺ cells in the blood (weeks 1 to 8) and BM (week 13) is shown. (B) Representative flow cytometry plots showing myeloid (CD11b and CD33) and lymphoid (CD19 and CD3) engraftment in the bone marrow.

Fig. 3

Culture of CB-derived CD34⁺ cells with SR1 increases SRC numbers. (A) Mice were injected with a fraction of the final culture equivalent to the indicated number of starting CB CD34⁺ cells after culture for 21 days with control conditions (open blue squares) or SR1 [0.75 μM; (open red circles)]. The percentage of human CD45⁺ cells in the BM at 16 weeks is shown. Horizontial lines indictate the average of each group. (B) Linear regression analysis of data from 2A and 3A. Dotted lines represent the 95% confidence intervals. Shapes represent the percent of negative animals for each dose of cells (C) and secondary (D) SRC before and after culture of 2.5 × 10⁵ CB CD34⁺ cells was calculated using Poisson statistics applied to the data in table S5A.

Fig. 4

SR1-induced CD34⁺ cell expansion acts by binding and antagonizing AhR. (A) Percent decrease in CYP1B1 (black squares) and AHRR (black circles) mRNA from mPB CD34⁺ cells treated with SR1 (solid lines) or LGC006 (dashed lines) for 24 h. (B) Quantification of AhR binding shown in fig. S16. (C) Phenotype of GFP⁺ cells 8 days after transduction with lentivirus expressing shRNAs targeting AhR. (D) Relative AhR mRNA expression in sorted CD34⁺GFP⁺ cells (box in C).