Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells - PubMed (original) (raw)

Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells

John P Chute et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs, although its contribution to stem cell fate remains unknown. In this study, we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover, short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells, the nonobese diabetic/severe combined immunodeficiency mouse repopulating cells, compared with day 0 CD34(+)CD38(-)lin(-) cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist, all-trans-retinoic acid, suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs, suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

ALDH inhibition impedes the differentiation of human HSCs. FACS-sorted CB CD34+CD38−lin− cells were analyzed for ALDH activity levels and compared with the ALDH activity level in the CD34+CD38− progeny of 7-day cultures with thrombopoietin, SCF, and flt3-ligand (TSF) alone versus TSF + DEAB (a). (b) The surface expression of CD34 and CD38 on day 0 CB CD34+CD38−lin− cells (Top) and their progeny after culture with TSF alone (Middle) versus TSF + DEAB (Bottom) is shown. Culture with TSF alone caused a marked increase in CFC content compared with day 0 CD34+CD38−lin− cells, whereas the progeny of DEAB + TSF cultures contained little CFC activity, indicating an inhibition of HSC differentiation during culture (c). ∗ indicates a statistically significant difference between the DEAB + TSF treated group versus TSF alone.

Fig. 2.

Fig. 2.

DEAB treatment promotes the amplification of human SRCs. (a) Representative flow cytometric analysis of human CD45 versus murine CD45 surface staining in NOD/SCID mice is shown at week 8 in mice transplanted with 1 × 103 day 0 CB CD34+CD38−lin− cells (Top), their TSF-cultured progeny (Middle), or their progeny after DEAB + TSF culture (Bottom). (b) A scatter plot of human CD45+ cell engraftment in NOD/SCID mice at 8 weeks posttransplantation is shown with each individual point representing a single transplanted mouse. Mice transplanted with the progeny of CB CD34+CD38−lin− cells cultured with DEAB + TSF demonstrated significantly increased frequency of human engraftment (≥1.0%) and percent huCD45+ cell repopulation compared with day 0 CB CD34+CD38−lin− cells or their progeny after culture with TSF alone. The mean levels of huCD45+ cells per culture condition are indicated by horizontal lines. (c) Multilineage engraftment of CD45+ cells, CD34+ progenitor cells, CD19+ B cells, and CD33/13+ myeloid cells is shown in the BM of a representative NOD/SCID mouse transplanted with the progeny of 2.5 × 103 CB CD34+CD38−lin− cells after DEAB + TSF culture, demonstrating multilineage in vivo differentiation of DEAB treated cells. Shown are IgGPerCP versus IgGFITC control staining (i), huCD45 versus muCD45 staining (ii), IgGFITC versus IgG-phycoerythrin control staining (iii), huCD34 versus huCD38 staining (iv), huCD19 versus huCD3 staining (v), and huCD13 versus huCD33 staining (vi). (d) PCR analysis for a 1,171-bp segment of the human chromosome 17-specific α-satellite region demonstrates engraftment of human cells in secondary mice transplanted with BM cells from primary mice that had been transplanted with limiting dilutions of DEAB + TSF-cultured CB CD34+CD38−lin− cells. Human engraftment is evident at the 0.001–0.1% levels in three of the five mice analyzed. The label “M” represents a 1-kB DNA marker; “Hu” identifies 100% human CB cell DNA; “Mo” identifies 100% mouse BM cell DNA; and “N” identifies the no template control (dH20).

Fig. 3.

Fig. 3.

DEAB inhibits ALDH1 activity and decreases retinoid signaling in HSCs. FACS-sorted CB CD34+CD38−lin− cells (a) were cultured with TSF alone (b), and their phenotype was compared with the progeny of cells cultured with TSF + DEAB (c). For further comparison, CD34+CD38−lin− cells were cultured with ATRA + TSF (d), which induced a marked loss of CD34+ cells and CD34+CD38− cells in culture as compared with input or TSF culture, consistent with accelerated differentiation during culture. The addition of ATRA to TSF also overcame the inhibitory effects of DEAB on HSC differentiation (e), suggesting that exogenous retinoids can overcome the effect of ALDH inhibition on HSCs. (f) Real-time PCR analysis demonstrated a 50% reduction in cEBPε expression in DEAB + TSF-treated cells at day 7 compared with TSF alone. Whereas addition of the substrate for ALDH1, retinaldehyde, markedly increased cEBPε expression in HSCs, the addition of DEAB completely blocked this effect, suggesting that DEAB inhibited ALDH1 activity, thereby impeding retinoid signaling in HSCs.

Fig. 4.

Fig. 4.

Treatment with DEAB sustains HOXB4 expression in HSCs. RNA was isolated from multiple replicates of FACS-sorted CB CD34+CD38−lin− cells at day 0 and their progeny after culture with TSF alone or DEAB + TSF. The RNA was reverse-transcribed, and the expression of HOXB4 and Notch 1 was analyzed by quantitative real-time PCR. (Left) The expression of HOXB4 in CD34+CD38− cells was significantly reduced after short-term culture with TSF, whereas treatment with DEAB prevented the down-regulation of HOXB4 expression over time. (Right) The expression of Notch was also significantly reduced after TSF culture and was not altered by treatment with DEAB.

References

    1. Osawa M., Hanada K., Hamada H., Nakauchi H. Science. 1996;273:242–245. - PubMed
    1. Sorrentino B. Nat. Rev. Immunol. 2004;4:878–888. - PubMed
    1. Varnum-Finney B., Xu L., Brashem-Stein C., Nourigat C., Flowers D., Bakkour S., Pear W., Bernstein I. Nat. Med. 2000;6:1278–1281. - PubMed
    1. Krosl J., Austin P., Beslu N., Kroon E., Humphries R., Savageau G. Nat. Med. 2003;9:1428–1432. - PubMed
    1. Reya T., Duncan A., Ailles L., Domen J., Scherer D., Willert K., Hintz L., Nusse R., Weissman I. Nature. 2003;423:409–414. - PubMed

MeSH terms

Substances

LinkOut - more resources