Cell Division Tracking Research Articles

Mathematical modeling reveals differential effects of erythropoietin on proliferation and lineage commitment of human hematopoietic progenitors in early erythroid culture.

Erythropoietin is essential for the production of mature erythroid cells, promoting both proliferation and survival. Whether erythropoietin and other cytokines can influence lineage commitment of hematopoietic stem and progenitor cells is of significant interest. To study lineage restriction of the common myeloid progenitor to the megakaryocyte/erythroid progenitor of peripheral blood CD34(+) cells, we have shown that the cell surface protein CD36 identifies the earliest lineage restricted megakaryocyte/erythroid progenitor. Using this marker and carboxyfluorescein succinimidyl ester to track cell divisions in vitro, we have developed a mathematical model that accurately predicts population dynamics of erythroid culture. Parameters derived from the modeling of cultures without added erythropoietin indicate that the rate of lineage restriction is not affected by erythropoietin. By contrast, megakaryocyte/erythroid progenitor proliferation is sensitive to erythropoietin from the time that CD36 first appears at the cell surface. These results shed new light on the role of erythropoietin in erythropoiesis and provide a powerful tool for further study of hematopoietic progenitor lineage restriction and erythropoiesis.

Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process.

BackgroundThe FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.MethodsWe performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.ResultsComputational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.ConclusionsOur results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.

Combination of Tyrosine Kinase Inhibitor with β-Catenin/CBP Modulator C82 Reverses TKI Resistance, Eradicates Quiescent CML Stem/Progenitors Cells, and Overcomes MSC-Associated Microenvironmental Protection

Bcr-Abl tyrosine kinase inhibitors (TKIs) are effective in inducing remissions and improving survival in patients with CML but do not eliminate CML leukemia stem cells (LSCs). Wnt/β-catenin pathway is established to be active in CML and essential for CML LSC, while adult HSCs do not require fully active β-catenin for maintenance. Furthermore, Wnt/β-catenin signaling pathway plays a critical role in TKI resistance and stromal-mediated microenvironmental protection for CML stem and progenitor cells. We propose that combinations of β-catenin inhibitors and TKIs represent a potentially effective therapy by targeting both CML LSCs and leukemia-mediated microenvironmental protection. C82 is a novel β-catenin/CBP modulator that via binding to CBP inhibits the interaction of β-catenin and CBP and thus disrupts Wnt/β-catenin/CBP mediated cell proliferation and self-renewal signaling.CML cell lines and primary CML-BC patient samples were treated with combinations of C82 with different TKIs, including imatinib (IM), nilotinib (NIL), dasatinib (DAS), and ponatinib (PON). Both TKI-sensitive KBM5 (IC50=0.50±0.06µM, EC50=0.32±0.01µM, 48h) and TKI-resistant KBM5-STIT315I (IC50=1.44±0.06µM, EC50=0.36±0.09µM, 48h) cells were sensitive to C82. C82-TKI combinations synergistically induced apoptosis (C82-NIL, CI=0.30±0.07 and C82-DAS, CI=0.20±0.01 in KBM5; C82-NIL, CI=0.24±0.09 and C82-DAS, CI=0.36±0.05 in KBM5-STIT315I at 48h; respectively) and inhibited cell grwoth in both cell lines. KBM5, KBM5-STIT315I and K562 were co-cultured with normal human bone marrow derived-MSCs. Western blot showed that CML/hMSCs co-cultures increased β-catenin, CD44, and survivin proteins in CML cell lines. C82-TKI combinations induced similar degrees of cell death and proliferation inhibition with or without hMSC co-cultures, indicating the combination strategy can overcome MSC-mediated microenvironmental chemoprotection in CML. Western blot analysis showed that C82 significantly inhibited CD44 and survivin expression which was further reduced by C82-TKI combinations in KBM5 and KBM5-STIT315I cells.C82-TKI combinations were evaluated in CML sample (n=6) from heavily-treated and TKI-resistant CML-BC patients. Four out of 6 sample harbored BCR-ABL kinase mutations, including T315I, E255K/V, and H396R. Mononuclear cells from the patients were stained with cell division tracking dye CFSE and then co-cultured with hMSCs. Flow cytometry was performed to identify CD34+CFSEbright and CD34+CFSEdim cells, as quiescent and proliferating population, respectively. When CML cells were treated without hMSC co-culture, C82-TKI combinations exerted stronger synergistic effects in CFSEbright quiescent cells (CI=0.21±0.06, 0.29±0.07, 0.48±0.15, or 0.26±0.03 for combination of C-82 with IM, NIL, DAS, or PON) compared with CFSEdim proliferating cells (CI=0.43±0.05, 0.43±0.17, 0.50±0.20, or 0.44±0.06 for combination of C-82 with IM, NIL, DAS, or PON; respectively). While under co-culture conditions, similar levels of synergy was observed in proliferating (CI=0.39±0.02, 0.23±0.02, 0.32±0.05, or 0.27±0.01 for combination of C-82 with IM, NIL, DAS, or PON) and quiescent cells (CI=0.23±0.02, 0.20±0.01, 0.39±0.10, or 0.20±0.04 for combination of C-82 with IM, NIL, DAS, or PON; respectively). C82-TKI combinations also synergistically induced cell death in CD34+38- CML cells (n=4) and yielded minimum effect on normal bone marrows CD34+ cells (n=3). Invivo studies are ongoing with immunodeficient NOD/SCID/IL2rγnull mice injected with CML-BC patient samples. An open-label, dose-escalation phase I/II study of PRI-724 (active metabolite of C82) for advanced myeloid malignancies (NCT01606579), including CML patients in combination with dasatinb, is enrolling patients at MD Anderson Cancer Center and other centers.Our data demonstrate that β-catenin/CBP signaling pathway plays a critical role in quiescent CML stem/progenitor cells and disruption of the β-catenin/CBP interaction with C82 could overcome MSC-mediated microenvironmental protection for not only proliferating but also quiescent stem/progenitor cells in CML. Combinations of β-catenin/CBP signaling pathway modulator C82 with TKIs represent a potentially promising strategy to tackle TKI resistance and eradicate CML stem/progenitors cells and should be further investigated in larger studies. DisclosuresKouji:PRISM Pharma Co., Ltd: Employment. Cortes:PRISM Pharma Co., Ltd: Clinicaltrial PI for NCT01606579 Other. Andreeff:PRISM Pharma Co., Ltd: Research Funding. Carter:PRISM Pharma Co., Ltd: Research Funding.

Automated, contour-based tracking and analysis of cell behaviour over long time scales in environments of varying complexity and cell density.

Understanding single and collective cell motility in model environments is foundational to many current research efforts in biology and bioengineering. To elucidate subtle differences in cell behaviour despite cell-to-cell variability, we introduce an algorithm for tracking large numbers of cells for long time periods and present a set of physics-based metrics that quantify differences in cell trajectories. Our algorithm, termed automated contour-based tracking for in vitro environments (ACTIVE), was designed for adherent cell populations subject to nuclear staining or transfection. ACTIVE is distinct from existing tracking software because it accommodates both variability in image intensity and multi-cell interactions, such as divisions and occlusions. When applied to low-contrast images from live-cell experiments, ACTIVE reduced error in analysing cell occlusion events by as much as 43% compared with a benchmark-tracking program while simultaneously tracking cell divisions and resulting daughter-daughter cell relationships. The large dataset generated by ACTIVE allowed us to develop metrics that capture subtle differences between cell trajectories on different substrates. We present cell motility data for thousands of cells studied at varying densities on shape-memory-polymer-based nanotopographies and identify several quantitative differences, including an unanticipated difference between two 'control' substrates. We expect that ACTIVE will be immediately useful to researchers who require accurate, long-time-scale motility data for many cells.

Activation Of p53 By MDM2-Inhibition Sensitizes Quiescent Blast Crisis CML Stem/Progenitor Cells To Bcl-2 Inhibitor- and Tyrosine Kinase Inhibitor-Induced Cell Death

The transcriptional activation of pro-apoptotic Bcl-2 family members is a major mechanism of p53-mediated cell death. p53 can also directly contribute to mitochondrial mediated apoptosis by interacting with Bcl-2 family proteins. A number of anti-apoptotic Bcl-2 proteins such as Mcl-1 and Bcl-xL are regulated by the Bcr-Abl tyrosine kinase and confer apoptosis resistance to CML cells. It is therefore not surprising that p53 gene alterations may play a role in the disease progression of CML. The reported p53 mutation rate of 30% suggests potential therapeutic benefit of p53 activation by MDM2 inhibition alone or as sensitization of CML to therapeutic agents.Tyrosine kinase inhibitors (TKIs), the front-line therapy for patients with chronic phase CML, are less effective in patients with blast crisis (BC) CML and inactive against quiescent CML stem/progenitor cells. Here we examine the effects of nutlin3a, which activates p53 by inhibiting MDM2, and its combinations with the Bcl-2 inhibitor ABT-737 and the TKI nilotinib on the viability of proliferating and quiescent CD34+CML stem/progenitor cells obtained from patients with BC CML.Mononuclear cells from patients with BC CML who were resistant to multiple TKIs were stained with the cell division tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM) derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells are distinguishable by flow cytometry (within 7 to 12 days), the cells were treated with nutlin3a, ABT-737, nilotinib, nutlin3a plus ABT-737, or nutlin3a plus nilotinib for 48 hours with or without MSC co-culture. The cells were then stained with a CD34 antibody, and apoptosis (annexin V staining) was determined by flow cytometry. Apoptosis in proliferating and quiescent progenitor cells was defined as percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbrightcells, respectively.Nutlin3a alone was able to decrease viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50 = 2.50 ± 0.92 μM and 2.54 ± 0.60 μM, respectively) and quiescent (IC50 = 3.70 ± 1.22 μM and 4.27 ± 0.77 μM, respectively) CD34+ CML stem/progenitor cells. Although not very active by itself, ABT-737, when combined with nutlin3a (n = 6), induced apoptosis synergistically in proliferating (CI = 0.24 ± 0.12) and more so in quiescent (CI = 0.09 ± 0.10) CD34+CML stem/progenitor cells, even when cells were co-cultured with MSCs (CI = 0.22 ± 0.09 for proliferating and CI = 0.03 ± 0.05 for quiescent stem/progenitor cells). The combination of nutlin3a with nilotinib (n = 4) was also highly synergistic in proliferating stem/progenitors (CI = 0.02 ± 0.04 without and CI = 0.05 ± 0.04 with co-culture) and in quiescent progenitors (CI = 0.07 ± 0.29 without and CI = 0.03 ± 0.06 with co-culture).Treatment of primary CML blast cells with nultin3a increased p53, and pro-apoptotic Bax and Puma levels in all samples examined, indicating activation of transcriptional activity of p53. In addition, nutlin3a treatment decreased the protein levels of anti-apoptotic Bcl-xL and/or Mcl-1. Nilotinib also decreased the expression, both at the RNA and protein levels, of Mcl-1 and Bcl-xL, even in patients who had not responded to nilotinib treatment clinically. ConclusionsHere we demonstrate that activation of p53 induces apoptosis in proliferating and quiescent BC CML stem/progenitor cells and sensitizes to Bcl-2 inhibitor- and TKI-induced cell death by, at least in part, regulating the expression of Bcl-2 family proteins. Results suggest a potential for utilizing this strategy in the treatment of BC CML, and also for the elimination of quiescent CML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Onset of heterogeneity in culture-expanded bone marrow stromal cells

Inconsistencies among in vitro and in vivo experiments using adult mesenchymal stem cells (MSCs) confound development of therapeutic, regenerative medicine applications, and in vitro expansion is typically required to achieve sufficient cell numbers for basic research or clinical trials. Though heterogeneity in both morphology and differentiation capacity of culture-expanded cells is noted, sources and consequences are not well understood. Here, we endeavored to observe the onset of population heterogeneity by conducting long-term continuous in vitro observation of human adult bone marrow stromal cell (BMSC) populations, a subset of which has been shown to be stem cells (also known as bone marrow-derived MSCs). Semi-automated identification and tracking of cell division and migration enabled construction of cell lineage maps that incorporated cell morphology. We found that all BMSCs steadily grew larger over time; this growth was interrupted only when a cell divided, producing two equally sized, morphologically similar daughter cells. However, a finite probability existed that one or both of these daughters then continued to increase in size without dividing, apparently exiting the cell cycle. Thus, larger BMSCs are those cells that have exited the normal cell cycle. These results hold important implications for MSC in vitro culture expansion and biophysical sorting strategies.

Ex vivo expansion of cord blood progenitors impairs their short‐term and long‐term repopulating activity associated with transcriptional dysregulation of signalling networks

Cord blood (CB) has been established to be an alternative source of haematopoietic stem/progenitor cells (HPC) for transplantation. The number of HPC per CB unit is limited, which results in engraftment delay. Ex vivo expansion of HPC improvement must overcome this. Flow cytometry was used to extensively phenotype HPC pre- and post-expansion and CFDA-SE staining was used to track cell divisions. The NSG mouse model was employed in transplantation studies to determine long and short term repopulation in human cells. Gene array analysis was used to evaluate signalling pathways regulated following ex vivo expansion of HPC. expansion of CD34(+) HPC impaired their regenerative function. In this xenograft transplantation model we showed that repopulating activity of CB cells declined following expansion. Expanded HPC had delayed engraftment at early and late stages post-transplant. High resolution division tracking revealed that the cultured HPC had reduced expansion and self-renewal probability and increased differentiation rate compared to non-expanded cells. Gene expression analysis exposed significant modulation of a complex network of genes and pathways that normally maintain HPC proliferation and limit their differentiation. The decline in short-term engraftment is consistent with the loss of rapid SCID repopulating ability r(SRA) by expanded CD34(+) CD38(+) cells recently reported. Our data raise concerns for future clinical applications of expanded HPC alone in transplantation.

Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.

Software for precise tracking of cell proliferation

We have developed a multi-target cell tracking program TADOR, which we applied to a series of fluorescence images. TADOR is based on an active contour model that is modified in order to be free of the problem of locally optimal solutions, and thus is resistant to signal fluctuation and morphological changes. Due to adoption of backward tracing and addition of user-interactive correction functions, TADOR is used in an off-line and semi-automated mode, but enables precise tracking of cell division. By applying TADOR to the analysis of cultured cells whose nuclei had been fluorescently labeled, we tracked cell division and cell-cycle progression on coverslips over an extended period of time.

Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.

High-Resolution Chromatin Immunoprecipitation (ChIP) Sequencing Identifies Novel Binding Targets and Prognostic Role for SOX11 in Mantle Cell Lymphoma

Abstract 585SOX11 belongs to the high-mobility group of transcription factors that bind to DNA and induce large conformational changes that facilitate the binding of other transcription factors. Genome-wide methylation analysis of Mantle cell lymphoma (MCL) patients using Nimblegen arrays identified SOX11 as hypomethylated and overexpressed in comparison to purified naïve B cells, which are the cells from which MCLs originate. Direct sequencing of SOX11 in MCL cell lines and patient samples failed to identify any activating mutations in SOX11, strengthening the role of aberrant promoter hypomethylation in MCL as the cause of its overexpression. SOX11 expression has been previously compared between Non-Hodgkin’s lymphomas and found to be specific for MCL. The function and direct binding targets of SOX11 are unknown.To understand the functional role of SOX11 in MCL, we used high-resolution whole-genome chromatin immunoprecipitation sequencing (ChIP-Seq) to identify the target genes directly bound by SOX11. As a model, genomic DNA from the Granta-519 MCL cell line was immunoprecipitated by SOX11 antibody and IgG and genomic enrichment sites were analyzed by Illumina sequencing. Using stringent statistical criteria (p-value<0.01, fold change≥1.5, and FDR<0.01) we identified 5682 peak positions with significant enrichment of ChIP sequencing reads corresponding to 888 unique genes. Sequencing runs from independent immunoprecipitates had more than 95% overlap in binding targets. These targets were filtered with published gene sets of differentially expressed genes following SOX11 overexpression in MCL cell lines. Pathway analysis revealed cell cycle, FOXm transcriptional network, and TGF-beta receptor signaling as the top three significantly enriched pathways by SOX11 target genes. All three pathways prominently consist of genes with anti-proliferative effects in MCL and B-NHL in general. To test our hypothesis, we transfected SOX11 by lentiviral plasmids into four MCL cell lines with varying levels of intrinsic SOX11 expression. Overexpression of SOX11 led to decreased proliferation in all four cell lines as measured by long-term tracing of living cells using a fluorescent labeled dye to track cell division. Ingenuity Pathway Analysis also identified the tumor promoting Wnt, PKC and IGFR pathways as being significantly enriched by the binding targets. We validated direct binding of SOX11 to RB1, Wnt4 and PKCA by quantitive PCR of ChIP products from Granta-519 cells. RB1 is a key regulator of entry into cell division tumor suppressor in MCL whereas Wnt signaling activates c-myc, which is oncogenic in MCL.Previously published studies have indicated a significant negative prognostic impact of SOX11 expression on overall survival in MCL. We therefore analyzed SOX11 expression in two large independent tissue microarrays from University of Wisconsin (UW) and British Columbia Cancer Agency (BCCA), respectively. MCL patient samples were scored by experienced hematopathologists. High SOX11 expression was associated with better survival: 41 months for SOX11 positive vs. 11 months for SOX11 negative, (p=0.05) in 58 patients in the UW dataset, however the larger BCCA dataset of 140 patients did not show any statistically significant impact of SOX11 expression on survival (p=0.15). These findings suggest that the prognostic impact of SOX11 is heterogenous, and a subset of patients with high SOX11 expression may have improved survival.In conclusion, high-resolution ChIP-Seq identifies more than 800 novel SOX11 direct binding targets. Differential activation of the diverse biological pathways affected by SOX11 target genes may help explain the differential impact of SOX11 expression on patient outcomes. Disclosures:No relevant conflicts of interest to declare.

Physiologic Hypoxia Promotes Maintenance of CML Stem Cells Despite Effective BCR-ABL Inhibition

Abstract 450Chronic myelogenous leukemia (CML) is a disease of hematologic stem cells caused by the BCR-ABL kinase. The use of the ABL kinase inhibitor, imatinib (IM), has dramatically improved the control of CML, but does not cure it. Current evidence suggests that CML stem cells are able to survive IM treatment, and initiate disease recurrence when IM therapy is stopped. The eradication of residual CML stem cells will depend on the identification of mechanisms that support their persistence. The bone marrow microenvironment supports the growth and renewal of hematopoietic stem cells (HSC), and possibly, CML stem cells. Recent studies reveal that physiologic hypoxia (0.1–10%) is a critical component of the microenvironment. We hypothesize that hypoxia maintains CML stem cell function, and that it can do so despite effective BCR-ABL kinase inhibition. To test this hypothesis, we characterized the effect of hypoxia on immunophenotypically-defined CML progenitor and stem cells in the presence and absence of IM. To examine the effect of hypoxia on CML progenitors, we first performed colony forming assays (CFA) on primary chronic phase (CP) CML cells treated with IM (0, 0.25, 1 and 5 μM) under hypoxic (0.5 O2) or normoxic (21% O2) conditions for 96 hours. We found that hypoxia increased the number of colonies by 1.6- and 3.2- fold compared to 21% O2 at 0 and 5μM IM respectively, suggesting that hypoxia favors the maintenance of CML progenitors. To explore the relationship between cell proliferation and hypoxia, we employed CFSE staining to track cell divisions. We found that while both CD34+CD38+ (committed progenitors) and CD34+CD38− (stem cells) CP CML cells proliferated more slowly under hypoxia alone (∼20% decrease in the proliferation index), hypoxia did not further enhance the anti-proliferative effects of IM in both populations. Together, these observations suggest that hypoxia enhances the clonogenic activity of CML progenitors, and that it is able to do so despite the anti-proliferative effects of IM. To rule out the possibility that hypoxia impaired the ability of IM to inhibit BCR-ABL kinase, we also examined the level of phospho-CrkL by intracellular flow cytometry in the CD34+CD38+ and CD34+CD38− CML populations. We found that IM reduced the level of pCrkL in both CD34+CD38+ and CD34+CD38− CML cells, and that hypoxia had no effect on the degree of pCrkL reduction. In line with this result, we also found that hypoxia had no effect on the degree of apoptosis induced by IM. These results indicated that hypoxia increased the clonogenic activity of CML progenitors, and that this occurred in a BCR-ABL-independent manner. These data encouraged us to determine if hypoxia had similar effects on the CML stem cell, which we assayed by performing limiting dilution LTCIC assays, and by assessing the ability of hypoxia-exposed CML cells to engraft immunodeficient NSG mice. In both assays, CML cells incubated under hypoxia showed increases in stem cell capacity (Figure 1). Taken together, our results indicate that physiologic hypoxia maintains the ability of CML cells to act as leukemia stem cells (LSC), and that this can occur in a BCR-ABL-independent manner. In this respect, the functional effects of hypoxia on CML stem cells mirror those on normal HSCs (Danet et al. JCI, 2003). To provide support for this notion, we performed gene expression analysis using Affymetrix U133 Plus 2.0 chips on CD34+ cells obtained from normal individuals, as well as CML patients in CP and blast crisis (BC). Preliminary analyses of the microarray data suggest that the hypoxic response in normal and CD34+ CP cells was remarkably similar, in contrast to the CD34+ BC response. Together, our data indicate that: 1. Physiologic hypoxia enhances the capacity of CP CML progenitors to function as LSCs; 2. Hypoxia enhances CP LSC function independently of BCR-ABL; 3. CP CD34+ CML cells have a similar gene expression response to hypoxia compared to normal CD34+ cells. Our findings suggest that therapies targeting BCR-ABL will not eliminate CP CML stem cells, particularly in the context of a hypoxic microenvironment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Neurobiological Observations of Bone Mesenchymal Stem Cells in vitro and in vivo of Injured Sciatic Nerve in Rabbit

The PKH26 is a fluorescent lipophilic dyes used for the study of Asymmetric cell Divisions (ASDs) and efficiently purifies the stem cell fraction. The aim of this study was to explore the neurobiological characteristics in vitro and in vivo and tracking fate of the transplanted rabbit Bone Marrow-Mesenchymal Stem Cells (rBM-MSCs). A fluorescent microscope was used to determine the changes in cell size, fluorescence intensity during tissue culture, track cell divisions and the distribution of PKH26 dye between daughter cells. The results showed the identification of ASDs based on fluorescence intensity of the PKH26 dye was distributed equally between daughter cells at each division in vitro. The labeling BMSCs with PKH26 showed within the wall of the neurons in the dorsal root ganglia in vivo. Labeled BMSCs which are fibroblastic-like cells in P4 showed oval shaped and less density than P2. Direct examine of the labeled BMSCs in the cryosections at 16 weeks post operation showed the BMSCs were differentiated and appeared as like Schwann cells in an anastomosed sciatic nerve in the Local Treated Group (LTG). In the Systemic Treated Group (STG) sections, the labeled BMSCs were migrated to the anastomosed sciatic nerve, ipsilateral lumber dorsal root ganglia resembling glial and stellate cells and some of the labeled cells migrated to the anterior horn of spinal cord (motor neuron). In conclusion, the biological behaviors of BMSCs in vitro and in vivo showed highly mitosis at P2, activated fibroblast-like cells, differentiated to functional myelinating Schwann-like cells in LTG. The BMSCs in STG migrated and engrafted at the dorsal root ganglia as a neuron and glial cell, glial cells and satellite in the spinal cord.

Use of SNARF-1 to measure murine T cell proliferation in vitro and its application in a novel regulatory T cell suppression assay

The green fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to track the proliferation of T cells in vitro. Such assays often incorporate more than one population of cells, but the paucity of alternative, spectrally distinct dyes suitable for measuring proliferation has hampered the simultaneous tracking of multiple cell populations; furthermore, CFSE is not compatible with green fluorescent protein (GFP), used to identify T cells in various transgenic mice. We have therefore validated the use of the far red dye seminaphthorhodafluor-1 (SNARF)-1 – originally developed to measure intracellular pH – to track murine T cell proliferation in vitro, demonstrating its ability to distinguish multiple cycles of proliferation over three days in a similar fashion to CFSE. The small changes in fluorescence emission attributed to intracellular alkalinisation of proliferating T cells have minimal impact on the ability of SNARF-1 to track cell division and this dye induces minimal cell death at the concentration used in this application. On the basis of these results, we have developed a novel in vitro murine T cell suppression assay, in which the proliferation of both conventional T cells (Tcons) stained with SNARF-1 and regulatory T cells (Tregs) stained with CFSE can be measured simultaneously. We have also demonstrated that SNARF-1 may be used to stain Tcons in assays of suppression involving ‘designer’ Tregs, generated by the transduction of CD4+ T cells with constructs encoding the Foxp3gfp fusion protein.

Bone Morphogenetic Proteins Inhibit CD40L- and IL-21-Induced Ig Production In Human B Cells: Differential Effects of BMP-6 and BMP-7

AbstractAbstract 1745 Introduction:TGF-β has been shown to play an important role in directing class switch recombination in human B cells. Bone morphogenetic proteins (BMPs) are members of the TGF-β family, and we have previously shown that BMP-6 inhibits proliferation of B cell progenitors as well as mature B cells in humans. However, how various BMP members affect Ig production and B-cell maturation to plasma cells have not been investigated. Here we studied the effects of various BMPs in CD27- naive and CD27+ memory B cells from peripheral blood of healthy human donors. Methods:We used flow immunomagnetic beads and flow cytometry cell sorting to purify CD19+CD27- naive and CD19+CD27+ memory B cells from peripheral blood of healthy donors, and investigated BMP induced effects on in vitro proliferation, cell death and Ig production as measured by thymidine incorporation assay, propidium iodide staining and ELISA, respectively. To separate direct inhibition of plasma cell differentiation from indirect effects via suppression of proliferation and induction of apoptosis, CFSE tracking of cell division was combined with immunophenotyping of cultured cells to identify CD27+CD38+ plasma cells. Expression of transcription factors and AICDA were measured by quantitative real-time PCR. Results:BMP-2, -4, -6 and -7 specifically inhibited CD40L- and IL-21-induced production of IgM, IgG and IgA. BMP-6 was the most potent inhibitor, reducing the Ig production by 70% in memory B cells and more than 50% in naive B cells. By investigating the mechanisms for reduced Ig production, we found a striking difference between the structurally similar BMP-6 and BMP-7. BMP-6 directly inhibited differentiation to CD27+CD38+ plasma cells, whereas BMP-7 only had minor effects on differentiation. Instead, BMP-7 mainly affected Ig production indirectly by inducing apoptosis. Furthermore, we explored BMP-6-induced signaling and gene regulation in more detail in memory B cells. BMP-6 up-regulated Id1, Id2 and Id3 gene expression in CD40L- and IL-21-stimulated cells (6.1-fold, 1.7-fold and 4.0-fold induction after 48 hours culture with BMP-6, respectively). In contrast, BMP-6 potently inhibited CD40L- and IL-21-induced upregulation of the transcription factor XBP-1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected, indicating that BMP-6 only modulates late events of plasma cell development. Conclusion:These results show that BMPs potently suppress Ig production in mature human B cells by inhibiting differentiation or by inducing cell death. Disclosures:No relevant conflicts of interest to declare.

BMS-214662 Eliminates Quiescent and Proliferating Acute Myeloid Leukemia Cells through Activation of Protein Kinase Cβ and Enhances the Efficacy of Cytosine Arabinoside

AbstractAbstract 2167Despite major advances in our understanding of the pathophysiology of acute myeloid leukemia (AML), the prognosis remains poor in most patients. While the majority of patients (∼80%) who tolerate induction therapy enter remission, both early and late relapses are a significant problem. It is postulated that, in some cases, these relapses are due to a population of primitive AML stem cells which are not effectively targeted by conventional chemotherapy agents such as cytosine arabinoside (Ara-C) or standard chemotherapy combinations (e.g. DA, ADE or FLAG-IDA). BMS-214662 (Bristol-Myers Squibb), a cytotoxic farnesyltransferase inhibitor (FTI), preferentially kills non-dividing malignant cells (Lee et al, Proc AACR 2001;42:260S) and in a clinical trial as a single agent had anti-leukemia activity in AML (Cortes et al, J Clin Oncology 2005;23:2805). Previous studies have shown that, in chronic myeloid leukemia (CML), BMS-214662 induces mitochondrial apoptosis of primitive progenitor cells via activation of protein kinase Cbeta (PKCβ; Copland et al, Blood 2008;111:2843; Pellicano et al, Blood 2009;114:4186). We therefore assessed the efficacy of BMS-214662, alone and in combination with Ara-C, in patient-derived CD34+ and CD34- AML cells in vitro using a CFSE-based flow cytometry method to track cell division and caspase-3 activity, PARP cleavage, mitochondrial membrane potential (MMP) and measurement of reactive oxygen species (ROS) to assess apoptosis. PKCβ was assessed by Western blotting ± BMS-214662, ± bryostatin-1, a PKCβ modulator. Primary AML cells were cultured for 72 hours in serum free medium supplemented with 5 growth factors (IL-3, IL-6, Flt-3 ligand, G-CSF and SCF). Conditions studied were: (1) no drug control (NDC), (2) BMS-214662 (250nM; IC50 dose), (3) Ara-C 500nM, and (4) BMS-214662 250nm + Ara-C 500nM (BMS-214662+Ara-C). Total viable cells were reduced in all treatment arms compared to NDC. The combination of BMS-214662+Ara-C produced the greatest cytotoxic effect with total viable cells reduced to <20% of NDC after 72 hours (P=0.05). While Ara-C alone did not reduce the number of undivided CFSEmax AML cells compared to NDC, both BMS-214662 alone and BMS-214662+Ara-C resulted in reductions of 47 and 75%, respectively. Furthermore, BMS-214662+Ara-C significantly reduced undivided CFSEmax AML cells compared to either BMS-214662 or Ara-C alone, (P<0.04 and P<0.03, respectively), indicating at least additive activity of this combination. In primary AML cells, BMS-214662 triggered apoptosis via the intrinsic pathway with loss of MMP, generation of ROS, cleavage of PARP and caspase-3 activation. Further experiments demonstrated that PKCβ was up-regulated in a dose-dependent manner in the presence of BMS-214662. Importantly, BMS-214662 at the concentrations effective against AML CD34+ cells did not initiate apoptosis of normal CD34+ hematopoietic cells. Finally, the addition of the PKCβ modulator, bryostatin-1, abrogated the effects of BMS-214662, resulting in reduced activation of caspase-3. These results are interesting as they identify a common mechanism for induction of apoptosis in quiescent CD34+ cells from different myeloid leukemias (all phases of CML and different AML subtypes) which may be unrelated to the FTI activity of this compound. In conclusion, these results indicate that BMS-214662 induces apoptosis of both quiescent and proliferating AML cells via up-regulation of PKCβ and activation of the intrinsic apoptotic pathway. The combination of BMS-214662 with conventional chemotherapy may represent a novel strategy for remission induction therapy in AML, reducing the risk of relapse. Disclosures:Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Copland: Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Co‐culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34+CD38− fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34+, CD133+ and CD38−) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.

Non-Cycling Cells From Acute Myeloid Leukemia Patients Harbor the FLT3-ITD Mutation and Are Insensitive to TKI258, a Potent FLT3-Directed Inhibitor, in Vitro.

Abstract 479The identification of activating mutations in the FLT3 gene and their impact on prognosis has been crucial to the rationale behind the development of FLT3 inhibitors. While it has been shown that some leukemic cells with high tumorigenic potential exist mostly in a dormant state, it is unclear if this quiescent/non-cycling leukemia-initiating fraction carries the FLT3-ITD mutation and if it is successfully targeted by FLT3 inhibitors. As a paradigm, quiescent Ph+ stem cells in CML have been shown to exhibit resistance to bcr-abl targeted inhibitors. Additionally, results from clinical trials suggest that FLT3 inhibitors reduce the leukemic blast count in peripheral blood but are less successful in the bone marrow where factors regulating hematopoietic stem cell quiescence are active. In order to investigate the non-cycling and cycling human leukemic cell boundary, we devised a biological model that allowed us to distinguish non-cycling AML cells from cycling AML progenitors in human FLT3-ITD positive AML samples. CD34+ cells were isolated from AML samples using magnetic cell sorting, labeled with the cell membrane dye PKH26 to enable tracking of cell division, and cultured on murine stroma for 12 days. Non-cycling AML cells were then separated from cycling cells by FACS sorting and were found to retain a CD34+ primitive phenotype in contrast to expanding leukemic blasts. Fluorescence in situ hybridization analyses revealed that non-cycling cells carried leukemic gene rearrangements (trisomy 8, trisomy 13, t[3;21]and t[16;16] in our cases), and were therefore part of the original leukemic clone. PCR for the FLT3-ITD region showed that in four out of five cases, the FLT3-ITD mutation was present in the non-cycling fraction. To examine the distribution of FLT3-ITD to FLT3 wild type (WT) bearing cells, non-cycling AML cells were FACS sorted, DNA extracted and the PCR products subsequently cloned. Bacterial colonies were sequenced and colony-PCR used to determine the ratio of FLT3-ITD to WT bearing colonies for each patient. These data indicated that at least 25% of non-cycling cells (range 25%-100%) harbored the FLT3-ITD mutation. We then assessed the impact of a potent FLT3-directed inhibitor, TKI258 (Novartis), on leukemic cell expansion and the viability of non-cycling cells. TKI258 has been found to induce apoptosis of FLT3-ITD bearing cells of the human acute monocytic leukemia MV4;11 cell line. In our present study, CD34+ AML blasts from the same FLT3-ITD positive patient samples were grown in vitro in the presence of 0 μM, 0.3 μM (IC50 dose) and 1.25 μM TKI258. In stromal cultures, TKI258 significantly reduced leukemic cell expansion (range 2.13 to 20 fold for untreated cultures and 0.07 to 2.27 fold for 1.25 μM TKI258 treated cultures at day 12, p ≤ 0.05). In methylcellulose colony assays, TKI258 exposure resulted in dose-dependent suppression of colony formation of CD34+ FLT3-ITD positive leukemia cells (60% to 81% reduction in the mean plating efficiency of CD34+ AML cells at 0.3 μM TKI258). Despite this striking anti-proliferative effect, the majority of non-cycling cells from AML patients showed resistance to TKI258 (five out of six cases). In these samples, FLT3-ITD positive non-cycling cells could still be detected after treatment with the equivalent highest clinical dose (1.25 μM) of TKI258. Moreover, at a functional level, limiting-dilution experiments on non-cycling AML cells pre-treated with TKI258 showed no impairment in a modified leukemic cobblestone assay at four weeks compared to untreated non-cycling cells. These results suggest that the majority of non-cycling AML cells that harbor FLT3-ITD are unaffected by a FLT3 inhibitor and may constitute an as yet untargeted disease reservoir. Only one FLT3-ITD AML case showed exquisite sensitivity to TKI258 with elimination of the non-cycling fraction observed from 0.3 μM of TKI258 upwards. Possible explanations for this may include specific mutant receptor sensitivities or generic multi-drug resistance mechanisms operating in dormant cells. Interestingly, TKI258 selectively eradicated an ‘intermediate' dividing progenitor population in two of the insensitive cases, an indication that leukemic progenitors may be rendered sensitive to FLT3 inhibition on transiting the dormancy-cycling boundary. Further studies are needed to determine if these findings are representative of a generation of FLT3 inhibitors or specific for TKI258. Disclosures:No relevant conflicts of interest to declare.

Combination of the Hedgehog Pathway Inhibitor LDE225 and Nilotinib Eliminates Chronic Myeloid Leukemia Stem and Progenitor Cells.

Abstract 1428Poster Board I-451Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder arising in a hematopoietic stem cell (HSC). CML is associated with expression of the Philadelphia chromosome (Ph) and its fusion gene product, BCR-ABL, a constitutively active tyrosine kinase. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, all show impressive rates of complete cytogenetic response in chronic phase (CP) CML. However, the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML stem cells not effectively targeted by TKIs. Recent studies have indicated that the Hedgehog (Hh) pathway, a developmental pathway with roles in primitive and adult hematopoiesis, is activated in CML stem cells via upregulation of Smoothened (SMO), a seven-transmembrane domain receptor protein (Dierks et al, Cancer Cell 2008;14:238). We found that Gli1, a downstream target of Hh signalling, is significantly upregulated (6-fold) at the mRNA level in CD34+ enriched CP CML cells compared to normal CD34+ hematopoietic cells by Taqman quantitative RT-PCR. Thus, inhibition of SMO may be an effective therapeutic strategy to reduce the quiescent CML stem cell pool. LDE225 (Novartis Pharma) is a small molecule SMO antagonist which has recently entered Phase 1 clinical evaluation in patients with solid tumors. We assessed the efficacy of LDE225, alone and in combination with nilotinib, in primary CD34+ CML cells in vitro using short- and long-term culture techniques, including colony forming cell (CFC) assays with replates, long-term culture-initiating cell (LTC-IC) assays, CFSE-based flow cytometry to track cell division, Annexin/Viaprobe to measure apoptosis and Ki-67/7AAD cell cycle analysis. In short-term culture, incremental concentrations of LDE225 (0.5 - 1000nM) had no effect on total viable cell counts up to 12 days and did not inhibit cell proliferation as assessed by BrDU incorporation and CFSE staining. LDE225 did not increase apoptosis or alter the cell cycle profile of CD34+ CML cells compared to untreated controls. The lack of response in short-term culture experiments is explained by the stem cell selective nature of LDE225 which affects self-renewal and not proliferation or apoptosis. For long-term culture experiments, CD34+ CML cells were cultured for either 3 or 7 days in serum-free media supplemented with physiological growth factors (IL-3, IL-6, G-CSF, FLT-3 and SCF) with and without LDE225; cells were then washed and put into CFC assays with no added LDE225. Increasing concentrations of LDE225 did not significantly reduce colony read-out in the CFC assays. However, when the CFCs were serially replated there was a highly significant reduction in secondary colony formation and replating efficiency with increasing concentrations of LDE225 up to a plateau at 50nM during the initial 72 hour culture. In the first re-plate, colony formation was reduced by 38% with 5nM (P=0.07) and 62% with 50nM LDE225 (P=0.011; n=3) compared to a “no drug” control. Further reductions in colony formation were seen in second and subsequent replates. In further CFC replating experiments, after 3 and 7 days initial exposure to the combination of LDE225 10nM + nilotinib 5μM, replating efficiency was reduced by 50% (P<0.03) and 74%,(P<0.005) respectively (n=3). Single agent nilotinib resulted in a non-significant increase in colony formation and replating efficiency. In LTC-IC assays, compared to the “no drug” control, CD34+ CML cells showed increased colony formation in the nilotinib arm, indicating that, by inhibiting proliferation, nilotinib exerts a protective effect on CML stem cells (as shown previously for imatinib and dasatinib; Copland et al, Blood 2008;111:2843). The arm containing LDE225 + nilotinib showed a reduction in colony formation compared to both the nilotinib arm (85% reduction) and the “no drug” control (50% reduction). These results confirm the stem cell selectivity of LDE225. In conclusion, LDE225 targets CML stem cells and, in combination with nilotinib, represents a novel therapeutic strategy for targeting both the primitive quiescent CML stem cell population and the bulk CML progenitor population. In vivo studies in murine models of CML are ongoing to help further evaluate the clinical potential of LDE225 in combination with nilotinib. DisclosuresHolyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dorsch: Novartis: Employment. Manley: Novartis: Employment. Bhatia: Novartis: Consultancy. Copland: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Distinct Self-Renewal and Differentiation Phases in the Niche of Infrequently Dividing Hair Follicle Stem Cells

In homeostasis of adult vertebrate tissues, stem cells are thought to self-renew by infrequent and asymmetric divisions that generate another stem cell daughter and a progenitor daughter cell committed to differentiate. This model is based largely on in vivo invertebrate or in vitro mammal studies. Here, we examine the dynamic behavior of adult hair follicle stem cells in their normal setting by employing mice with repressible H2B-GFP expression to track cell divisions and Cre-inducible mice to perform long-term single-cell lineage tracing. We provide direct evidence for the infrequent stem cell division model in intact tissue. Moreover, we find that differentiation of progenitor cells occurs at different times and tissue locations than self-renewal of stem cells. Distinct fates of differentiation or self-renewal are assigned to individual cells in a temporal-spatial manner. We propose that large clusters of tissue stem cells behave as populations whose maintenance involves unidirectional daughter-cell-fate decisions.