The Retinoid X Receptor Regulates Human Hematopoietic Stem Cell Fate.

Our laboratory has recently demonstrated that competitive inhibition of the enzyme, aldehyde dehydrogenase-1 (ALDH-1), in human hematopoietic stem cells (HSCs) facilitates the 3.4-fold expansion of primitive cells capable of repopulating non-obese diabetic/severe combined immune deficient (NOD/SCID) mice. Since ALDH-1 activity is required for the intracellular production of retinoic acids, we hypothesized that retinoid signaling could be fundamentally involved in human HSC fate determinations. In order to demonstrate a link between ALDH-1 activity and retinoid signaling in human HSCs, we cultured human CD34+CD38−lin- HSC-enriched cells with retinaldehyde, which is a specific substrate for ALDH-1, and found that this induced a 10-fold increase in the expression of cEBP-epsilon, which is a Retinoic Acid Receptor (RAR)-dependent transcription factor. Moreover, when the competitive inhibitor of ALDH-1, diethylaminobenzaldehyde (DEAB), was added to HSC cultures supplemented with retinaldehyde, the expression of cEBP-epsilon was completely blocked, confirming a causal relationship between ALDH-1 activity and retinoid signaling in HSCs. We then sought to determine whether direct agonism or antagonism of the RAR or Retinoid X Receptor (RXR) could alter human HSC fate in short-term culture. We cultured primary human bone marrow (BM) and cord blood (CB) CD34+CD38−lin- HSC-enriched cells with hematopoietic cytokines, thrombopoietin (T), stem cell factor (S) and flt-3 ligand (F) in the presence and absence of several agonists and antagonists of RAR and RXR. Seven day culture of BM or CB CD34+CD38−lin- cells with TSF alone resulted in a loss of CD34+CD38− cells in culture, a pronounced increase in colony forming cell (CFC) activity and a complete loss of primitive SCID-repopulating cells (SRCs) compared to input, reflecting HSC differentiation during culture. Interestingly, the addition of either all-trans retinoic acid (ATRA), an agonist of RAR and RXR, or TTNPB, a selective RAR agonist, induced an accelerated differentiation of HSCs in culture compared to cytokines alone. Surprisingly, the addition of LGD815 (courtesy of Ligand Pharmaceuticals, San Diego, CA), a selective RAR antagonist, had no effect on the differentiation of HSCs that otherwise occurred in response to cytokines alone. However, the addition of LGD101506, a partial antagonist of RXR, preferentially maintained phenotypic CD34+CD38− cells in culture and inhibited CFC production in response to cytokines. Moreover, RXR antagonism maintained or induced the expansion of SRC content in culture, whereas SRC content was completely lost following cytokine culture alone. Taken together, these data demonstrate for the first time that RXR signaling contributes to the intrinsic regulation of human HSC fate. Pharmacologic inhibition of RXR signaling impedes the normal differentiation of HSCs that otherwise occurs in response to proliferation-inducing cytokines. These data provide further evidence for the role of retinoid signaling in the differentiation program of human HSCs and indicate that RXR and its heterodimeric partners are novel targets for the purpose of facilitating human HSC expansion.