high percentage of treated patients, and may also stimulate other lineages (2-9). In rodents,

Transcription

1 Supplemental Results Lithium increases hematopoietic stem/progenitor cells Lithium increases circulating CD34 + stem cells (1) in humans, increases neutrophil count in a high percentage of treated patients, and may also stimulate other lineages (2-9). In rodents, lithium increases peripheral blood counts and enhances stem and progenitor cell numbers in ex vivo and in vivo assays. These studies led to the hypothesis that the effects of lithium are mediated at the level of the HSC and/or HPC (6, 1, 11). As sophisticated immunophenotypic markers to detect diverse hematopoietic cell types have become available since those early studies were performed, we have re-examined the effect of lithium on hematopoiesis in C7/B6 mice using FCM. Mice received dietary LiCl (or NaCl as control) at a dose that achieves a serum lithium concentration of 1. meq/l (12), similar to therapeutic concentrations in bipolar disorder patients (there was no change in the overall well being of the animals after two-three weeks on lithium). After 2 weeks, bone marrow was isolated and cells were analyzed by FCM. Lithium caused a significant increase in the number of LSK cells compared with NaCl treated animals (Supplemental Figure 1A), consistent with an increase in HSCs and HPCs, and a doubling of the overall marrow cellularity (Supplemental Figure 1B). To confirm this, we also examined expression of the SLAM family receptors CD1, CD48, and CD244 (13) and observed an approximately 2.3-fold increase in the number of CD1 + CD48 - CD244 - cells, an immunophenotypic population highly enriched for HSCs (data not shown). Histological analysis of bone marrow morphology showed no significant differences in maturity or cellular morphology in lithium-treated versus control marrows, as reported previously (not shown). Consistent with published observations in humans and rodents, the percentage of neutrophils in peripheral blood also increased 3-4% in lithium-treated mice (data not shown). Inclusion of CD34 and 1

2 Flk2 in FCM analysis shows that lithium primarily increases the CD34 + Flk2 - population, consistent with an increase in ST-HSCs (Supplemental Figure 1A). GSK-3 as the target of lithium in HSC/HPCs Inhibition of GSK-3 provides a compelling explanation for many of the known effects of lithium (14), but lithium also inhibits inositol monophosphatase and structurally related phosphomonoesterases, some of which are highly sensitive to lithium (1). To test further whether inhibition of GSK-3 explains the hematopoietic effects of lithium, we used alternative, selective GSK-3 inhibitors that are unlikely to have off-target effects that overlap with lithium (16-18). The selective GSK-3 inhibitor, 6-bromo-indirubin 3 -oxime (6BIO) has an IC for GSK-3 in the nanomolar range (17). 6BIO caused a pronounced increase in the number of LSK cells after a 2-week treatment (Supplemental Figure 1C). 6BIO also increased marrow cellularity, similar to lithium. These observations are consistent with Trowbridge et al, who observed an approximately % increase in LSK cells in mice treated with the GSK-3 inhibitor CHIR-911 (19). Goessling et al also reported that 6BIO increases HSCs in mice as measured by long-term competitive repopulation assay (2), consistent with our observation of increased LSK cells after 6BIO treatment. Taken together, these pharmacological data support the hypothesis that lithium increases HSCs through inhibition of GSK-3. To test whether progenitor cells are increased by lithium treatment, we performed colony formation assays. Previous work in the 198's with lithium-treated rodents demonstrated an increase in colony forming units (CFU) in ex vivo assays and by CFU-S formation in short-term transplants (6, 21). To confirm these studies, bone marrow cells were isolated from lithiumtreated mice and cultured in methylcellulose with hematopoietic cytokines. Marrow from lithiumtreated animals showed a two-fold increase in total colony initiating cells compared to control, similar to earlier reports (data not shown) and consistent with more recent observations with 2

3 small molecule GSK-3 inhibitors, which increase CFUs ex vivo and CFU-S approximately 1. to 2-fold (19, 2). To test in a side-by-side comparison whether structurally diverse GSK-3 inhibitors expand HPCs similar to lithium, c-kit + cells were purified from control mice and treated for three days with GSK-3 inhibitors including lithium, 6BIO (17, 2), AR-A14418 (22), and the organometallic GSK-3 inhibitor DW21 (18). (The number of cells after three days was similar in each group (Supplemental Figure 1E). Treated cells were washed and an equal number from each sample was then added to methylcellulose with hematopoietic cytokines and cultured for 1-14 days. Each of the GSK-3 inhibitors induced a marked increase in hematopoietic colony number (Supplemental Figure 1D), strongly supporting the hypothesis that lithium expands the HPC population by inhibiting GSK-3 within hematopoietic cells. Supplemental References 1. Ballin, A., Lehman, D., Sirota, P., Litvinjuk, U., and Meytes, D Increased number of peripheral blood CD34+ cells in lithium-treated patients. Br J Haematol 1: Shopsin, B., Friedmann, R., and Gershon, S Lithium and leukocytosis. Clin Pharmacol Ther 12: Boggs, D.R., Joyce, R.A The Hematopoietic Effects of Lithium. Seminars in Hematology 2: Barr, R.D., and Galbraith, P.R Lithium and hematopoiesis. Canadian Medical Association Journal 128: Tisman, G., Herbert, V., and Rosenblatt, S Evidence that lithium induces human granulocyte proliferation: elevated serum vitamin B 12 binding capacity in vivo and granulocyte colony proliferation in vitro. British Journal of Haematology 24: Joyce, R.A Sequential effects of lithium on haematopoiesis. British Journal of Haematology 6: Bille, P.E., Jensen, M.K., Kaalund Jensen, J.P., and Poulsen, J.C Studies on the haematologic and cytogenetic effect of lithium. Acta Medica Scandinavica 198: Ricci, P., Bandini, G., Franchi, P., Motta, M.R., Visani, G., and Calamandrei, G Haematological effects of lithium carbonate: a study in 6 psychiatric patients. Haematologica 66: Focosi, D., Azzara, A., Kast, R.E., Carulli, G., and Petrini, M. 29. Lithium and hematology: established and proposed uses. Journal of Leukocyte Biology 8:

4 1. Gallicchio, V.S., Messino, M.J., Hulette, B.C., and Hughes, N.K Lithium and hematopoiesis: effective experimental use of lithium as an agent to improve bone marrow transplantation. Journal of Medicine 23: Gallicchio, V.S., and Chen, M.G Modulation of murine pluripotential stem cell proliferation in vivo by lithium carbonate. Blood 6: O'Brien, W.T., Harper, A.D., Jove, F., Woodgett, J.R., Maretto, S., Piccolo, S., and Klein, P.S. 24. Glycogen synthase kinase-3beta haploinsufficiency mimics the behavioral and molecular effects of lithium. Journal of Neuroscience 24: Kiel, M.J., Yilmaz, O.H., Iwashita, T., Terhorst, C., and Morrison, S.J. 2. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.[see comment]. Cell 121: Klein, P.S., and Melton, D.A A molecular mechanism for the effect of lithium on development. Proc. Nat'l. Acad. Sci. U.S.A. 93: Gurvich, N., and Klein, P.S. 22. Lithium and valproic acid: parallels and contrasts in diverse signaling contexts. Pharmacol Ther 96: Bain, J., McLaughlan, H., Elliott, M., and Cohen, P. 23. The specificities of protein kinase inhibitors - an update. Biochem J 371 (Pt. 1): Meijer, L., Skaltsounis, A.L., Magiatis, P., Polychronopoulos, P., Knockaert, M., Leost, M., Ryan, X.P., Vonica, C.A., Brivanlou, A., Dajani, R., et al. 23. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 1: Williams, D.S., Atilla, G.E., Bregman, H., Arzoumanian, A., Klein, P.S., and Meggers, E. 2. Switching on a signaling pathway with an organoruthenium complex. Angew Chem Int Ed Engl. 44: Trowbridge, J.J., Xenocostas, A., Moon, R.T., and Bhatia, M. 26. Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation. Nat Med 12: Goessling, W., North, T.E., Loewer, S., Lord, A.M., Lee, S., Stoick-Cooper, C.L., Weidinger, G., Puder, M., Daley, G.Q., Moon, R.T., et al. 29. Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration. Cell 136: Levitt, L.J., and Quesenberry, P.J The effect of lithium on murine hematopoiesis in a liquid culture system. New England Journal of Medicine 32: Bhat, R., Xue, Y., Berg, S., Hellberg, S., Ormo, M., Nilsson, Y., Radesater, A.C., Jerning, E., Markgren, P.O., Borgegard, T., et al. 23. Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A Journal of Biological Chemistry 278:

5 Supplemental Figure Legends Supplemental figure 1. Lithium and other GSK-3 inhibitors expand HSC/HPCs: (A) Absolute number of Lineage-Sca-1 + ckit + (LSK) fraction, which is enriched for HSCs, and immunophenotypic LT-HSC (LSK; CD34 - Flk2 - ) and ST-HSC (LSK CD34 + Flk-2 - ) fractions in bone marrow harvested from mice treated with control or lithium diet for 2 weeks. (B) Cellularity of bone marrow, thymus and spleen in control and lithium treated mice, shown as the number of nucleated cells/mouse recovered from both femurs and tibias, thymus, and spleen. (C) Percentage (left) and absolute number (right) of HSC/HPCs (as LSK) in mice treated with 6BIO vs control for two weeks. (D) Colony formation assay: Purified total c-kit + cells were treated with lithium, 6BIO, AR-A14418, or Ru (1-OH) (also known as DW12) for 3 days and plated in methylcellulose with hematopoietic cytokines for 12 days. Colonies of >3 viable cells were counted and the mean colony number/, plated cells for each of three separate experiments is shown. (E) The total numbers of c-kit + cells in each drug treatment after 3 days primary culture were shown. Supplemental figure 2. Annexin V staining and cellularity of bone marrow harvested from transplant recipients: (A) Flow cytometric detection of Annexin V using bone marrow cells harvested from 1º recipients after 4 month bone marrow transplantation from both control and. Annexin V was measured in the GFP + LSK gated population. (B) Total number of bone marrow cells and number of GFP + cells recovered in bone marrow from 1º recipients after 4 month transplant. (C) Total number and number of GFP + cells recovered in bone marrow from 2º recipients after 4 months. (D) Total number and number of GFP + cells recovered in bone marrow from 3º recipients after 4 months.

6 Supplemental Figure 3. Increased colony formation induced by requires ß- catenin: Colony formation assay was performed (as in figure 1) using sorted GFP + cells harvested from 1º recipients (A) or 2º recipients (B) of wild-type (left) and ß-catenin KO (right) marrow transduced with control (open boxes) or (filled boxes) lentivirus. Data represent mean number of colonies per well for five mice per construct repeated in three separate experiments. Supplemental Figure 4. Effect of ß-catenin conditional knockout on colony formation in Gsk3-depleted bone marrow: Colony formation assay was performed (as in figure 1) using sorted GFP + cells from each group in 1º (A) and 2º (B) recipients. Data represent mean number of colonies per well for five mice per construct repeated in three separate experiments. 6

7 A. 12 B. 1 Lithium No. of cells(x14) LSK LSK CD34-Flk2- LSK CD34+Flk2- No. of total cells(x17) BM Thymus Spleen Lithium C. Percentage of LSK in BM(%) control 6BIO No. of BM LSK cells(x14) control 6BIO D. 1 colonies/, cells untreated DMSO Li+ 6BIO AR DW21 E. No. of total cells(x1) untreated DMSO Li+ 6BIO AR DW21 Supplemental figure 1. Lithium and other GSK3 inhibitors expand HSC/HPCs: (A) Absolute number of Lineage- Sca-1+cKit+ (LSK) fraction, which is enriched for HSCs, and immunophenotypic LT-HSC (LSK; CD34-Flk2-) and ST-HSC (LSK CD34+Flk-2-) fractions in bone marrow harvested from mice treated with control or lithium diet for 2 weeks. (B) Cellularity of bone marrow, thymus and spleen in control and lithium treated mice, shown as the number of nucleated cells/mouse recovered from both femurs and tibias, thymus, and spleen. (C) Percentage (left) and absolute number (right) of HSC/HPCs (as LSK) in mice treated with 6BIO vs control for two weeks. (D) Colony formation assay: Purified total c-kit+ cells were treated with lithium, 6BIO, AR-A14418, or Ru(1-OH) (also known as DW12) for 3 days and plated in methylcellulose with hematopoietic cytokines for 12 days. Colonies of >3 viable cells were counted and the mean colony number/, plated cells for each of three separate experiments is shown. (E) The total numbers of c-kit+ cells in each drug treatment after 3 days primary culture were shown. Supplemental Figure 1.

8 A. GFP+LSK gated cells relative cell number IgG Annexin V B º recipients No. of BM cells(x16) Total cells GFP+ cells C. No. of BM cells(x16) º recipients Total cells GFP+ cells D. No. of BM cells(x16) º recipients Total cells GFP+ cells Supplemental figure 2. Annexin-V staining and cellularity of bone marrow harvested from transplant recipients: (A) Flow cytometric detection of Annexin V using bone marrow cells harvested from 1º recipients after 4 month bone marrow transplantation from both control and. Annexin V was measured in the GFP+LSK gated population. (B) Total number of bone marrow cells and number of GFP+ cells recovered in bone marrow from 1º recipients after 4 month transplant. (C) Total number and number of GFP+ cells recovered in bone marrow from 2º recipients after 4 months. (D) Total number and number of GFP+ cells recovered in bone marrow from 3º recipients after 4 months. Supplemental figure 2.

9 A. No. of colonies/3, cells WT β catenin KO B. No. of colonies/3, cells WT β catenin KO Supplemental Figure 3. Increased colony formation induced by requires ß-catenin: Colony formation assay was performed (as in figure 1) using sorted GFP+ cells harvested from 1º recipients (A) or 2º recipients (B) of wild-type (left) and ß-catenin KO (right) marrow transduced with control (open boxes) or (filled boxes) lentivirus. Data represent mean number of colonies per well for five mice per construct repeated in three separate experiments. Supplemental figure 3.

10 A. No. of colonies/3, cells WT β catenin KO B. No. of colonies/3, cells WT β catenin KO Supplemental Figure 4. Effect of ß-catenin conditional knockout on colony formation in Gsk3-depleted bone marrow: Colony formation assay was performed (as in figure 1) using sorted GFP+ cells from each group in 1º (A) and 2º (B) recipients. Data represent mean number of colonies per well for five mice per construct repeated in three separate experiments. Supplemental figure 4.