Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment
2011; Elsevier BV; Volume: 40; Issue: 4 Linguagem: Inglês
10.1016/j.exphem.2011.12.005
ISSN1873-2399
AutoresTony Peled, Hadas Shoham, Dorit Aschengrau, Dima Yackoubov, Gabi Frei, Noga Rosenheimer G, Batya Lerrer, Haim Cohen, Arnon Nagler, Eitan Fibach, Amnon Peled,
Tópico(s)PARP inhibition in cancer therapy
ResumoStrategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34+ cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood–derived human CD34+ cells cultured with cytokines. In the presence of NAM, the fraction of CD34+CD38− cells increased and the fraction of differentiated cells (CD14+, CD11b+, and CD11c+) decreased. CD34+ cells cultured with NAM displayed increased migration toward stromal cell derived factor–1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD+-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34+ cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34+ cells. Strategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34+ cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood–derived human CD34+ cells cultured with cytokines. In the presence of NAM, the fraction of CD34+CD38− cells increased and the fraction of differentiated cells (CD14+, CD11b+, and CD11c+) decreased. CD34+ cells cultured with NAM displayed increased migration toward stromal cell derived factor–1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD+-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34+ cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34+ cells. Strategies to expand hematopoietic progenitor cells (HPC) in vitro are of clinical importance to improve the outcome of cord blood transplantations. Exposure of HPC to different combinations of cytokines promotes their exit from the G0 phase of the cell cycle and enables extensive proliferation. Nonetheless, in vitro proliferation is tightly coupled with a commitment to differentiation and reduced self-renewal [1Von Drygalski A. Alespeiti G. Ren L. Adamson J.W. Murine bone marrow cells cultured ex vivo in the presence of multiple cytokine combinations lose radioprotective and long-term engraftment potential.Stem Cells Dev. 2004; 13: 101-111Crossref PubMed Scopus (29) Google Scholar]. Although early-acting cytokines induce robust in vitro expansion of CD34+ cells, expansion of engraftable progenitors is modest [2Xu R. Reems J.A. Umbilical cord blood progeny cells that retain a CD34+ phenotype after ex vivo expansion have less engraftment potential than unexpanded CD34+ cells.Transfusion. 2001; 41: 213-218Crossref PubMed Scopus (29) Google Scholar]. This phenomenon could be explained, at least in part, by an acquired defect in the bone marrow (BM) homing capacity of ex vivo–expanded HPC [3Szilvassy S.J. Bass M.J. Van Zant G. Grimes B. Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by ex vivo expansion.Blood. 1999; 93: 1557-1566PubMed Google Scholar, 4Szilvassy S.J. Meyerrose T.E. Grimes B. Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells.Blood. 2000; 95: 2829-2837PubMed Google Scholar], which is primarily attributed to their active cycling [5Takatoku M. Sellers S. Agricola B.A. et al.Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.J Clin Invest. 2001; 108: 447-455Crossref PubMed Scopus (93) Google Scholar], accompanied by alterations in adhesion and chemokine receptor expression or functionality [6Ahmed F. Ings S.J. Pizzey A.R. et al.Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model.Blood. 2004; 103: 2079-2087Crossref PubMed Scopus (52) Google Scholar]. Therefore, strategies to augment BM homing and engraftment efficacy are particularly important to increase clinical applicability of ex vivo–expanded CD34+ cells [7Hofmeister C.C. Zhang J. Knight K.L. Le P. Stiff P.J. Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche.Bone Marrow Transplant. 2007; 39: 11-23Crossref PubMed Scopus (186) Google Scholar]. Nicotinamide (NAM), a form of vitamin B-3, serves as a precursor of nicotinamide adenine dinucleotide (NAD+) [8Berger F. Ramirez-Hernandez M.H. Ziegler M. The new life of a centenarian: signalling functions of NAD(P).Trends Biochem Sci. 2004; 29: 111-118Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar]. NAM is also a potent inhibitor of enzymes that require NAD+ for their activities [9Banasik M. Komura H. Shimoyama M. Ueda K. Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase.J Biol Chem. 1992; 267: 1569-1575Abstract Full Text PDF PubMed Google Scholar, 10Corda D. Di Girolamo M. Functional aspects of protein mono-ADP-ribosylation.EMBO J. 2003; 22: 1953-1958Crossref PubMed Scopus (243) Google Scholar], such as mono-ADP-ribosyltransferases, poly-ADP-ribose polymerases, CD38, and cyclic ADP ribose/NADase [11Krebs C. Adriouch S. Braasch F. et al.CD38 controls ADP-ribosyltransferase-2-catalyzed ADP-ribosylation of T cell surface proteins.J Immunol. 2005; 174: 3298-3305Crossref PubMed Scopus (74) Google Scholar]. In addition, NAM is a well-established potent inhibitor of the sirtuin family of histone/protein deacetylases, the NAD-dependent class III histone deacetylase (HDAC) [12Denu J.M. Vitamin B3 and sirtuin function.Trends Biochem Sci. 2005; 30: 479-483Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar]. SIRT1, one of the mammalian sirtuins, catalyzes the deacetylation of acetyl-lysine residues by a mechanism whereby NAD+ is cleaved. The reaction results in the release of NAM, which acts as an end-product noncompetitive inhibitor of SIRT1 by binding to a conserved pocket adjacent to NAD(+), thereby blocking NAD(+) hydrolysis [13Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1.J Biol Chem. 2002; 277: 45099-45107Crossref PubMed Scopus (788) Google Scholar]. It was reported that hematopoietic cells derived from SIRT1-deficient mice (SIRT1−/−) display increased in vitro proliferation activity, although their self-renewal and in vivo function were not addressed [14Narala S.R. Allsopp R.C. Wells T.B. et al.SIRT1 acts as a nutrient-sensitive growth suppressor and its loss is associated with increased AMPK and telomerase activity.Mol Biol Cell. 2008; 19: 1210-1219Crossref PubMed Scopus (98) Google Scholar]. Having multiple effects on numerous cells, NAM is implicated in the regulation of cell adhesion, polarity, migration, proliferation, and differentiation [15Glowacki G. Braren R. Firner K. et al.The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse.Protein Sci. 2002; 11: 1657-1670Crossref PubMed Scopus (138) Google Scholar]. NAM was shown to modulate the fate of embryonic stem cells [16Vaca P. Berna G. Martin F. Soria B. Nicotinamide induces both proliferation and differentiation of embryonic stem cells into insulin-producing cells.Transplant Proc. 2003; 35: 2021-2023Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar] and primary nonhematopoietic cells [17Miura M. Kameda Y. Nicotinamide promotes long-term survival and extensive neurite outgrowth in ultimobranchial C cells cultured from chick embryos.J Comp Neurol. 2005; 492: 334-348Crossref PubMed Scopus (8) Google Scholar, 18Sato F. Mitaka T. Mizuguchi T. Mochizuki Y. Hirata K. Effects of nicotinamide-related agents on the growth of primary rat hepatocytes and formation of small hepatocyte colonies.Liver. 1999; 19: 481-488Crossref PubMed Google Scholar, 19Papaccio G. Ammendola E. Pisanti F.A. Nicotinamide decreases MHC class II but not MHC class I expression and increases intercellular adhesion molecule-1 structures in non-obese diabetic mouse pancreas.J Endocrinol. 1999; 160: 389-400Crossref PubMed Scopus (15) Google Scholar]. With regard to hematopoietic cell lines, NAM was reported to inhibit HL-60 cell differentiation mediated by retinoic acid [20Munshi C.B. Graeff R. Lee H.C. Evidence for a causal role of CD38 expression in granulocytic differentiation of human HL-60 cells.J Biol Chem. 2002; 277: 49453-49458Crossref PubMed Scopus (69) Google Scholar], and other studies reported that NAM enhances HL-60 cell differentiation [21Iwata K. Ogata S. Okumura K. Taguchi H. Induction of differentiation in human promyelocytic leukemia HL-60 cell line by niacin-related compounds.Biosci Biotechnol Biochem. 2003; 67: 1132-1135Crossref PubMed Scopus (22) Google Scholar]. Here we studied the effect of NAM on primary cultures of umbilical cord blood (UCB) CD34+ cells. Our results demonstrate that NAM delayed differentiation and enhanced migration, homing, and engraftment of CD34+ cells expanded ex vivo with cytokines. The SIRT1-specific inhibitor, EX-527 [22Solomon J.M. Pasupuleti R. Xu L. et al.Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage.Mol Cell Biol. 2006; 26: 28-38Crossref PubMed Scopus (380) Google Scholar, 23Napper A.D. Hixon J. McDonagh T. et al.Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1.J Med Chem. 2005; 48: 8045-8054Crossref PubMed Scopus (402) Google Scholar], exhibited an effect similar to that of NAM on cultured CD34+ cells, while NAM-related [24Colon-Otero G. Sando J.J. Sims J.L. McGrath E. Jensen D.E. Quesenberry P.J. 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Czapski G. Adamczyk A. Strosznajder R.P. GSK-3beta and oxidative stress in aged brain. Role of poly(ADP- ribose) polymerase-1.Folia Neuropathol. 2007; 45: 220-229PubMed Google Scholar, 29Smets L.A. Loesberg C. Janssen M. Van Rooij H. Intracellular inhibition of mono(ADP-ribosylation) by meta-iodobenzylguanidine: specificity, intracellular concentration and effects on glucocorticoid-mediated cell lysis.Biochim Biophys Acta. 1990; 1054: 49-55Crossref PubMed Scopus (51) Google Scholar] NAD-dependent ADP-ribosyltransferase inhibitors were not effective. Based on these findings, we propose SIRT1 deacetylase as a target accountable for NAM modulating CD34+ cell differentiation in ex vivo cultures. Cells were obtained from UCB samples harvested from consenting mothers after normal full-term deliveries (Sheba Medical Center, Tel-Hashomer, Israel). Samples were collected and frozen according to Rubinstein et al. [30Rubinstein P. Dobrila L. Rosenfield R.E. et al.Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.Proc Natl Acad Sci USA. 1995; 92: 10119-10122Crossref PubMed Scopus (702) Google Scholar]. Before use, cells were thawed, the mononuclear cells purified on a Ficoll-Hypaque gradient, and CD34+ cells were isolated using a MidiMACS CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch, Gladbach, Germany) as described previously [31Peled T. Landau E. Mandel J. et al.Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34(+) cells and increases their engraftment potential in NOD/SCID mice.Exp Hematol. 2004; 32: 547-555Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar]. Purified CD34+ cells were cultured in culture bags (American Fluoroseal Co., Gaithersburg, MD, USA) at 1 ×104 cells/mL (at least 8 mL/bag) in minimum essential medium–α, 10% fetal bovine serum (FBS), and cytokines: thrombopoietin, interleukin (IL)-6, fms-like tyrosine kinase–3 ligand, and stem cell factor, each at a final concentration of 50 ng/mL (Pepro Tech, Inc., Rocky Hill, NJ, USA), with or without NAM (Sigma Aldrich, Milwaukee, WI, USA; catalog number N5535 and Vertellus, Indianapolis IN, USA; catalog number 100547) and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Until week 3, the cultures were topped weekly with the same volume of fresh medium. For long-term experiments, the cultures were weekly demi-depopulated. The number of total nucleated cells (TNC) in culture was determined after dilution (1:10) with phosphate-buffered saline (PBS) by CEDEX, an automatic cell counter (Innovatis AG, Bielefeld, Germany). The CEDEX is an automated cell counting system based on the well-established Trypan Blue exclusion method for determining cell viability. The number of cells in culture was determined by multiplying the number of cells/mL by the culture volume. Fold-expansion was calculated by dividing total number of cells in culture with the culture input number of cells. Number of CD34+ cells and CD34+38− cells were calculated by multiplying percentages with total number of cells in culture/100. Fold-expansion was calculated by dividing the calculated number of CD34+ and CD34+CD38− cells obtained following culture with the number of culture seeded CD34+ and CD34+CD38– cells. For the colony-forming unit (CFUc) assay, 1000 CD34+ cells before culture and 1500 cells after culture were added per 3 mL MethoCult (MethoCult GF+H4435 complete methylcellulose medium with recombinant cytokine and erythropoietin for colony assays of human cells; StemCell Technologies, Vancouver, BC, Canada). After stirring, the mixture was divided into two 35-mm dishes and incubated for 14 days at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the incubation period, colonies (both myeloid and erythroid) were counted under an inverted microscope at 40× magnification. The CFUc content/mL culture was calculated as follows: number of scored colonies per two dishes × total cell number/1500 or 1000. Fold-expansion was calculated by dividing total number of colonies in culture (number/mL × culture volume) with the culture input number of colonies. Cultured and noncultured cells were washed with PBS containing 1% bovine serum albumin and double-stained (at 4°C for 30 minutes) with phycoerythrin (PE) or fluorescein isothiocyanate–conjugated antibodies to CD45, CD34, CXCR4, very late antigen 4 and lymphocyte function–associated antigen 1 (Becton Dickinson, Erembodegem, Belgium) or with differentiation antigens CD38, CD33, CD14, CD15, CD11b, and CD11c (DAKO, Glostrup, Denmark). Cells were then washed in the buffer and analyzed using a flow cytometry (Becton Dickinson, San Jose, CA, USA). The emission of 104 cells was measured using logarithmic amplification and analyzed using CellQuest software (Becton Dickinson, Belgium). Freshly purified CD34+ cells (2 × 106) were incubated at room temperature for 5 minutes with 4 μM PKH26 (PKH26–PE-Cell Linker Kit; Sigma Aldrich, Milwaukee, WI, USA) according to manufacturer’s instructions. Then, an equal volume of 1% FBS was added for 1 minute, and the labeled cells were washed three times in PBS supplemented with 5% human serum albumin. Briefly, cells were washed and resuspended at <107 cells/mL in serum-free medium, and BCECF/AM at a final concentration of 5 μg/mL was added for 10 minutes at 37°C. Uptake of the dye was stopped by the addition of FBS (to give a final concentration of 10%). After labeling, cells were washed three times in PBS with 10% FBS and analyzed by flow cytometry for fluorescence intensity [32Lyons A.B. Parish C.R. Determination of lymphocyte division by flow cytometry.J Immunol Methods. 1994; 171: 131-137Crossref PubMed Scopus (1452) Google Scholar]. Purified CD34+ cells were labeled with PKH2. An aliquot was analyzed with flow cytometry for PKH2 intensity (t = 0) and the rest was cultured with cytokines (i.e., stem cell factor, thrombopoietin, fms-like tyrosine kinase–3, and IL-6), with or without 5 mM NAM. On day 7, cells were harvested and CD34+ cells were reisolated using the MiniMACS CD34 progenitor cell isolation kit and double-stained for CD34 and CD38. CD34 cells and the gated CD34+CD38− cells were analyzed with flow cytometry for PKH2 fluorescence intensity. Minimum essential medium–α plus 1% FBS and 100 ng/mL stromal cell derived factor–1 (SDF-1; R&D Systems Inc, Minneapolis, MN, USA) was placed into the lower chamber of a Costar 24-well Transwell (Corning, Corning, NY, USA). Cells (2 × 105) in 100 μL medium were placed into the upper chamber over a porous membrane (pore size, 5 μm). After 4 hours, cells were collected from the lower chamber and counted. Spontaneous migration was evaluated without SDF-1 in the lower chamber. NOD/LtSz- (nonobese diabetic/severe combined immunodeficient [NOD/SCID]) mice (8–10 weeks old) (Harlan Biotech Israel Ltd., Rehovot, Israel) were sublethally irradiated (with 375 cGy at 67 cGy/min) and 24 hours later inoculated via the tail vein with 10 to 20 million cells stained with BCECF/AM (Calbiochem, Darmstadt, Germany). The experiments were approved by the Animal Care Committee of the Hadassah, Hebrew University Medical Center. Mice were sacrificed at 24 hours post injection. BM samples were obtained by flushing their femurs and tibias with PBS at 4°C. Homing of human cells was detected by flow cytometry. The bright fluorescence of BCECF/AM was sufficient to separate labeled human cells from unlabeled murine cells by at least 1 log. To quantify homing of human progenitor cells, BM cells were stained with allophycocyanin-conjugated anti-human CD34 monoclonal antibodies (Becton Dickinson, Belgium) and BCECF/AM+CD34+ cells were enumerated. For each sample, 100,000 events were acquired and analyzed. The Animal Care Committee of Hadassah, Hebrew University Medical Center, Jerusalem, Israel approved these experiments. To evaluate BM homing after cotransplantation, noncultured cells were stained with BCECF/AM (fluorescein isothiocyanate) and cultured cells stained with PKH (PE), as described. NOD/SCID mice were bred and maintained at the Weizmann Institute, Rehovot, Israel in sterile intraventilated cages (Techniplast, Bugugiatte, Italy) or at Harlan Biotech. The experiments were approved by the Animal Care Committee of the Weizmann Institute and of Harlan Biotech. Eight-week-old mice were sublethally irradiated as described and transplanted with human CB-derived cells. Mice were sacrificed on week 6, and the BM cells were immunophenotyped as described here [31Peled T. Landau E. Mandel J. et al.Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34(+) cells and increases their engraftment potential in NOD/SCID mice.Exp Hematol. 2004; 32: 547-555Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 33Peled T. Mandel J. Goudsmid R.N. et al.Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine.Cytotherapy. 2004; 6: 344-355Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar]. To compare engraftment before and after culture, each CB unit was frozen into two portions. CD34 cells purified from one portion were cultured for 3 weeks as described. The second portion was kept frozen. On the day of transplantation, this portion was thawed and TNC or purified CD34+ cells were transplanted. In some experiments, to avoid donor variability, this procedure was carried out with CD34+ cells pooled from several CB units. Single units were used in experiments where cultured and noncultured TNC were cotransplanted in the same mouse. In these experiments, to avoid clamping of noncultured cells, the two cell populations were mixed just before cell injection. The frequencies of SRC were quantified by a limiting dilution analysis and applying Poisson statistics to the single-hit model as described previously [34Wang J.C. Doedens M. Dick J.E. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay.Blood. 1997; 89: 3919-3924PubMed Google Scholar]. Mice were scored as positively engrafted if at least 0.5% of their marrow cells expressed human CD45. Frequencies of SRC and statistical comparison between individual populations were calculated by maximum likelihood estimator using L-Calc software (StemCell Technologies) [34Wang J.C. Doedens M. Dick J.E. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay.Blood. 1997; 89: 3919-3924PubMed Google Scholar, 35Taswell C. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis.J Immunol. 1981; 126: 1614-1619PubMed Google Scholar]. CD34+ cells were cultured with cytokines, with and without 5 mM NAM, 18 hours before the addition of lysis Tris/saline/azide (1 μM). For the immunoprecipitation, cells were lysed in lysis buffer containing (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA , 1% NP-40, and a cocktail of protease inhibitor). HDAC inhibitors were supplemented in the lysis buffer at the following final concentrations: 5 mM NAM and 1 μM Tris/saline/azide. Cellular lysates were incubated with agarose-conjugated anti-acetyllysine antibody (-Ac-K) (ImmuneChem Pharmaceuticals Inc., Burnaby, BC, Canada) overnight at 4°C on a rotation wheel. Immunocomplexes were washed four times with lysis buffer, boiled, and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western blotting analysis of preimmunoprecipitation (input) and immunoprecipitated samples (-Ac-K) were performed with an anti-Ku70 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The nonparametric Wilcoxon rank test was applied for testing differences between the study groups. All the tests applied were two-tailed, and a p value of ≤0.05 was considered statistically significant. Data were analyzed using SAS software (SAS Institute, Cary, NC, USA). The effect of NAM was determined in CD34+ cells derived from UCB during 3 weeks in cultures supplemented with cytokines (i.e., fms-like tyrosine kinase–3, IL-6, thrombopoietin, and stem cell factor). Analysis included the number of TNCs, CFUc, and phenotypic characterization of hematopoietic progenitors, CD34+ and CD34+CD38− cells (Fig. 1A–L). As early as 1 week post seeding, the number of TNCs (Fig. 1A) and CD34+ cells (Fig. 1B) was substantially lower, while percentages (Fig. 1C, D) and absolute number (Fig. 1E) of CD34+CD38− cells were significantly higher in cultures treated with 2.5 and 5 mM NAM as compared with control cultures treated with cytokines only. The divisional history of seeded CD34+ cells stained with PKH indicated that during the first week in culture, the vast majority of CD34+ cells underwent several cycles under both culture conditions (Fig. 1F, G), with consistent lesser divisions (higher fluorescence intensity) of cells cultured with NAM (Fig. 1H). Slower cycling was particularly prominent in the CD34+CD38− subset (Fig. 1G), which, nevertheless, increased within the expanded cell population from week 1 through week –3 (Fig. 1I). After 3 weeks in culture, fold-expansion of TNCs (Fig. 1J), CFUc (Fig. 1K), and CD34+ cells (Fig. 1L) in NAM-treated cultures reached the values in NAM-nontreated cultures, while fold-expansion of CD34+CD38− cells was superior in cultures treated with NAM throughout the 3-week culture duration (Fig. 1M). Higher concentrations of NAM (10 mM) deteriorated TNC, CD34, and CFU proliferation throughout the culture duration, while a lower concentration of NAM (1 mM) had a slight effect on the expansion of CD34+CD38− cells (Supplementary Figure E1; online only, available at www.exphem.org). Phenotype characterization of lineage differentiated cells in 3-week cultures revealed lessening of differentiation in cultures treated with NAM (2.5 and 5 mM) than in cultures treated with cytokines alone, as demonstrated by significantly lower percentages of CD14, CD11b, CD11c, and CD15+ cells (Supplementary Figure E2; online only, available at www.exphem.org). In order to test the long-term potential of short-term NAM-treated cultures, CD34+ cells from 3 UCB units were cultured for 3 weeks with and without NAM, and subsequently monitored throughout an additional 10 weeks of culture in the absence of NAM. Regardless of NAM treatment, the number of CFUc in long-term expansion cultures are exceeding the number of CD34+ cells, suggesting that the decline in CD34+ cells precedes the decline in CFUc. However, after 9 and 13 weeks in culture, both the numbers of CD34+ cells and their clonogenic activity (CFUc) were significantly increased by the initial treatment of the cultures for 3 weeks with NAM over control cultures not treated with NAM. Moreover, in two out of three cultures initially treated with NAM, numbers of CD34+ cells and CFUc considerably increased throughout the culture duration (from week 7 to week 13) (Fig. 2A, B), suggesting that the 3-week treatment with NAM not only increased the number of cells displaying an early progenitor cell phenotype, but also preserved their potential for long-term expansion. To further test the effect of NAM on in vitro differentiation, IL-3, a cytokine that hastens myeloid differentiation of ex vivo–expanded CD34+ cells [36Nteliopoulos G. Marley S.B. Gordon M.Y. Influence of PI-3K/Akt pathway on Wnt signalling in regulating myeloid progenitor cell proliferation. Evidence for a role of autocrine/paracrine Wnt regulation.Br J Haematol. 2009; 146: 637-651Crossref PubMed Scopus (13) Google Scholar], was added to the medium of cultures supplemented with the four early-acting cytokines and increasing concentrations of NAM (2.5–7.5 mM). After 3 and 6 weeks, both clonogenic activity (Fig. 2C, E) and total cellularity (Fig. 2D, F) were increased, in a dose response, in cultures treated with NAM over control, NAM-nontreated cultures. Furthermore, phase-contrast images (Fig. 2G) visualize the differences in cell morphology between short and long-term cultures treated with and without NAM. Taken together, treatment with NAM delayed differentiation and promoted expansion of progenitors with enhanced self-renewal capacity. The short-term SCID reconstituting capacity of NAM-treated cells was evaluated in transplantation experiments using human anti-CD45 at a threshold level of 0.5% of marrow cellularity. Sublethally irradiated NOD/SCID mice were grafted with 3 × 103 (Fig. 3A) or 6 × 103 (Fig. 3B) purified CD34+ cells , or the entire progeny of the same number of cells was cultured for 3 weeks with or without NAM. Evidence of engraftment was found at 6 weeks post transplantation in 8% of mice grafted with 3 × 103 noncultured CD34+ cells (Fig. 3A). During 3 weeks of culture, a total number of 1 × 106 cells was obtained, irrespective of the presence of NAM. However, while none of the mice grafted with cells cultured with cytokines showed engraftment, exposure to NAM resulted in engraftment in 50% of the mice (Fig. 3A). A donor inoculum of 6 × 103 cells and their entire progeny (2 × 106 cells) resulted in engraftment in 33%, 46%, and 93% of recipients of fresh CD34+ cells, cytokine-cultured cells, and NAM-treated cells, respectively (Fig. 3B). These data suggested a significant advantage in engraftment of cells upon exposure to NAM, over both cultured and fresh CD34+ cells. Short-term SRC frequency was 1 in 18,764 cells (95% confidence interval, 1/44,982–1/7828) and 1 in 16,013 cells (95% confidence interval, 1/38,347–1/6687) for noncultured and cytokine cultured cells, respectively (Fig. 3C, D). NAM-supplemented cultures yielded a short-term SRC frequency of 1 in 2203 cells (95% confidence interval, 1/3964–1/1224) (Fig. 3E). Applying
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