SR1 (StemRegenin 1)

StemRegenin 1 increases the number of CD34+ cells after 5 to 7 days with an EC50 of 120 nM. Culture of mPB CD34+ cells with cytokines plus SR1 (1 μM) for 7 days increases the number of CD34+, CD133+, and CD90+ hematopoietic stem and progenitor cell populations 2.6-, 2.3-, and 10-fold, respectively compared to control cells. Continued culture with SR1 (1 μM) for 3 weeks leads to an 11-fold increase in total nucleated cells (TNC), a 73-fold increase in CD34+ cells as compared to control cultures, and a 1118-fold increase in CD34+ cells relative to input cells. Culture of 1×10³ cord blood CD34+ cells for 5 weeks with SR1 (1 μM) results in the production of 1.69×10⁶ colony forming cells. SR1-induces CD34+ cell expansion acts by binding and antagonizing AhR as evident by decreased CYP1B1 and AHRR mRNA levels. ^[1]

SR1 (1 μM) treatment accelerates the proliferation of CD34+ cells and decreased the expression levels of VentX in human CD34+ cells. Ectopic expression of VentX prevents SR1-induced expansion of CD34+ cells. ^[2]

Sequential coculture with bone morphogenetic protein 4 (20 ng/mL), PGE2 (2 μM), and SR1 (0.75 μM) lead to robust Macaca nemestrina iPSC hematopoietic progenitor cell formation. CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cells isolated on the basis of CD34 expression and cultured in SR1 (0.75 μM) expands 3-fold and maintained this long-term repopulating HSC phenotype. ^[3]

SR1 promotes expansion of CB CD34+ cells that contribute to both early and sustained engraftment NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. ^[1]

• AhR ligand binding assay

  Mouse livers are homogenized in buffer (25 mM MOPS, 2 mM EDTA, 0.02% NaN3, and 10% glycerol, pH 7.4) containing 20 mM sodium molybdate and protease inhibitors and centrifuged at 100,000g for 1 h. ligand-treated lysates are incubated at room temperature for 20 min and then photolyzes at 8 cm with 402 nm UV light. Dextran-coated charcoal (1%) is added to the photolyzed samples, which are then centrifuged at 3000g for 10 min to remove free ligand. Labeled samples are resolved using 8% acrylamide-tricine-SDS-PAGE, transferred to PVDF membrane, and visualized using autoradiography. Labeled AHR bands are excised and counted using a γ counter.