Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal

Abstract

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.

Fig. 1. Identification of previously unknown compounds promoting human CD34⁺ cell expansion

(A) Results of primary screen; asterisks denote the compounds that suppress the AhR pathway. (B) Changes in expression levels of AhR targets (AhRR and CYP1B1) measured by quantitative reverse transcription polymerase chain reaction after a 12-hour incubation with selected compounds compared with DMSO (using glyceraldehyde-3-phosphate dehydrogenase and hypoxanthine-guanine phosphoribosyltransferase as control, mean T SD). (C and D) Chemical structure of UM171, the optimized version of UM729, and their comparative activity on expansion of CD34⁺CD45RA⁻ mPB cells after 7-day cultures. The cytostatic/cytotoxic effects of UM729 and UM171 were observed at values above 1 and 0.125 μM, respectively.

Fig. 2. UM171 attenuates cell differentiation and promotes ex vivo expansion of primitive human hematopoietic cells

(A) Wright-stained cytospin preparation of CD34⁺ CB cells at day 0 and after 12 days in fed-batch cultures supplemented with vehicle (DMSO 0.1%), UM171 (35 nM), SR1 (750 nM), or a combination of SR1 (500 nM) and UM171 (35 nM). Arrowheads show macrophages (green) and megakaryocyte (red). (B) Representative FACS profiles of CD34⁺ and CD34⁺CD45RA⁻ populations in fresh (day 0) or cultured (day 12) CB cells. (C) The fold expansion of phenotypically defined cell subsets after 12 days in fed-batch cultures supplemented with indicated compounds [mean ± SD unless specified (ns, not significant); all values are significant when compared with control (black bars): P < 0.05, Mann-Whitney test]. (D) Fold expansion of CFU-GEMMs after 12 days in cultures.

Fig. 3. UM171 promotes expansion of LT-HSCs

(A) LT-HSC frequencies (red lines) and 95% CIs (gray boxes) presented as 1/number of starting cell (day 0) equivalent for each condition; n = 5 independent experiments performed with a pool of two to three human CB units per experiment. Significance level *P < 0.05 (Mann-Whitney test). (B) Levels of human (Hu) engraftment in NSG mice transplanted with different cell doses (column 1); red, 86%; green, 0%. See table S2 for raw data. (C) Representative FACS profiles showing multilineage repopulation of NSG mice (GPA, glycophorin A or CD235a).

Fig. 4. Summary of UM171 effect on cell expansion and differentiation

(A) Heat map showing expression of AhR targets (A) or lineage-specific (B) genes (green, low; red, high) in indicated conditions. (C) Comparison of UM171 with SR1 and DMSO on number and quality of LT-HSCs and progenitors based on results from expansion of 10,000 fresh CD34⁺ CB cells.