Unsorted Cells Research Articles

Abstract 3953: The omental microenvironment promotes ovarian cancer metastasis and enhances CD-133 mediated self-assembly of ovarian cancer spheroids.

Abstract Objective: Recent evidence suggests that the human omentum functions as a niche that promotes ovarian cancer metastasis. We investigated the role of the omentum in sequestration and enhancement of ovarian cancer stem cells. Materials and Methods: To further explore this unique phenomenon, we examined the ability of cells isolated from ovarian cancer implants at distinct anatomic sites to create multicellular aggregates known as spheroids. In ovarian and other cancers, spheroids promote a pluripotent phenotype and play a critical role in metastasis. Results: We found that ovarian cancer cells recovered from omentum express higher levels of the pluripotency-associated antigen CD133 and create larger numbers of more rapidly growing spheroids than cells recovered from the tubo-ovarian complex. Although CD133+ or CD44+ ovarian cancer cells are equally capable of creating large spheroids, spheroids self-assembled from CD133+ ovarian cancer cells are significantly more tumorigenic in vivo than CD44+, CD133−CD44− or unsorted cells. We also found that spheroids self-assembled from CD133+ ovarian cancer cells migrate to both the ovary and omentum of SCID mice and that CD133+ ovarian cancer cells induce intraperitoneal xenografts when inoculated subcutaneously. In contrast, our data indicate that spheroids self-assembled from CD44+ cells preferentially induce tumor adjacent to their inoculation site. Targeting CD133 expression inhibits the in vivo tumorigencity of spheroids assembled from A2780 ovarian cancer cells. Conclusions: In addition to delineating novel pathways by which the omental microenvironment promotes ovarian cancer metastasis, these findings establish a new paradigm for understanding how ovarian cancer stem cells can be potentially maintained or regenerated within the peritoneal cavity. Strategies to target the omental niche have enormous potential to improve ovarian cancer outcomes. Citation Format: Molly A. Brewer, Guigin Sun, Matthew Anderson, Fanliang Meng, Mei Zhong. The omental microenvironment promotes ovarian cancer metastasis and enhances CD-133 mediated self-assembly of ovarian cancer spheroids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2013-3953

Abstract 233: Therapeutic efficacy of cancer stem cell-based vaccine in an adjuvant setting.

Abstract So far, the majority of cancer stem cell (CSC) studies have been confined to human tumors inoculated into severely immunosuppressed hosts (e.g. SCID mice), where adaptive immune responses are absent, and such hosts are therefore not suitable for the immunologic evaluation of CSCs. The lack of immunocompetent syngeneic animal tumor models where stem cells can be isolated has been the primary obstacle to evaluating the immunogenicity of CSCs. We recently reported the identification of CSC-enriched populations using ALDEFLUOR/ALDH as a marker in murine melanoma D5 and squamous cell cancer SCC7, and evaluated their immunogenicity in two genetically distinct syngeneic immunocompetent hosts. Enriched CSCs are immunogenic and significantly more effective as an antigen source in inducing protective anti-tumor immunity than unsorted tumor cells or purified non-CSCs. We further found that selective targeting of CSCs by CSC-primed antibodies and T cells represents the mechanisms involved in CSC vaccine-conferred protective immunity. If a CSC vaccine is to be clinically relevant, it needs to be evaluated in a therapeutic model. In this study, we examined two models for this purpose. The first model involves the treatment of micrometastatic disease. We initiated vaccination 24 hrs after s.c. inoculation of D5 tumor cells in the syngeneic immunocompetent host (B6 mice), and repeated the vaccination one week later. Comparison was made among the group with PBS-injected controls; mice vaccinated with dendritic cells (DC) pulsed with the lysate of ALDHhigh D5 cells (CSC-TPDC) vs. ALDHlow D5 cells (ALDHlow -TPDC). We found that CSC-TPDC inhibited the tumor growth more than ALDHlow-TPDC and PBS controls, and significantly prolonged the survival of the tumor-bearing mice. The second model involves the treatment of established tumors using CSC-TPDC vaccine as an additional strategy to radiation therapy (RT). Day 7 D5 sc tumors were treated with localized RT with repeat treatments on D8. Vaccine therapy commenced on D8. This combination therapy was repeated twice with one week apart. CSC-TPDC plus RT mediated D5 tumor regression more than ALDHlow-TPDC + RT, RT alone and PBS controls, and prolonged the survival of the tumor-bearing mice. Importantly, we observed metastasis of the D5 tumors to the lung in all the groups except for the one treated with CSC-TPDC plus RT. The findings may help develop novel approaches for cancer treatment by utilizing an autologous cancer stem cell-based vaccine to selectively and immunologically target cancer stem cells. Citation Format: Lin Lu, Huimin Tao, Martin Egenti, Max S. Wicha, Alfred E. Chang, Qiao Li. Therapeutic efficacy of cancer stem cell-based vaccine in an adjuvant setting. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 233. doi:10.1158/1538-7445.AM2013-233

Abstract 5330: Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation.

Abstract Multiple myeloma (MM) remains an incurable clonal plasma cell malignancy. The existence of cancer stem cell-like subpopulation in MM may explain for its unfavourable prognosis and high relapse rate. Studies have shown that CD138neg MM cells were clonogenic and could be serially transplanted, while CD138+ cells could not. We characterized CD138neg cells by checking the presence of such subpopulation and their tumorigenic properties. In concordance with literature, CD138neg cells were present in MM cell lines, and possessed higher clonogenic potential than its non-tumorigenic counterpart as shown in colony formation assay. They were more quiescent, with less than 1% in G2/M phase compared to 5-10% in CD138+ cells. They also demonstrated higher resistance to chemotherapeutic agents, including thalidomide and bortezomib. Since studies in solid tumors have demonstrated that microRNA (miRNA) plays a role in self-renewal, tumorigenicity and chemoresistance in cancer stem cells, we hypothesized that specific miRNAs are the key regulators of the cancer stem cell-like functions in MM clonogenic cells. To search for these candidates, we first identified miRNAs that are differentially expressed among the two subpopulations, CD138+ and CD138neg cells, which were isolated from MM cell lines (NCI-H929, U266 and MM17, our in-house MM cell line) using MACS-immunomagnetic separation system and subjected to Exiqon miRCURY LNA miRNA microarray analysis. Ten candidate miRNAs were identified and selected for further validation by Taqman miRNA assay. By further independent investigation using fifteen MM patient samples, concordantly we found frequent up-regulation of miR-483-5p, miR-1246, miR-1275, miR-1290 and miR-3196 in CD138neg compared to CD138+ cells. MiR-1246 (93.4%, p = 0.006) and miR-1290 (93.4%, p = 0.0015) were chosen for further examination of the potential roles in MM pathogenesis and prognosis because they showed significantly higher expression levels in CD138neg cells than the CD138+ counterparts. Preliminary results showed that inhibiting miR-1246 (p = 0.0478) and miR-1290 (p = 0.0386) reduced cell proliferation in CD138neg cells at 24h or 48h of transfection in unsorted U266 cells, while CD138+ cells were not affected. As predicted using computational algorithms (miRANDA and TargetScan), the downstream targets of the two miRNAs include SEMA6A, STK17A, PHLDA1 and KLF9, which bear functions related to MM pathogenesis or known to be tumor suppressors. Moreover, we also confirmed that their mRNA expressions were down-regulated in U266 CD138neg cells compared with CD138+ cells.Taken together, we suggest that miR-1246 and miR-1290 may play a regulatory role in MM clonogenic cell proliferation. Our findings may provide a novel insight for the microRNA-based therapeutics that specifically targets the clonogenic progenitors, which may prove to be more effective for cure of MM. Citation Format: Coty HY Cheung, Suk-Hang Cheng, Libby Li, Natalie Pui Ha Chan, Rosalina KL Ip, Chi-Keung Cheng, Margaret H.L. Ng. Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2013-5330

All-trans retinoic acid effectively inhibits breast cancer stem cells growth in vitro

To detect the inhibitory effect of all-trans retinoic acid(ATRA) on breast cancer stem cells (CSCs). The inhibitory effect of ATRA on MCF-7 and SK-BR-3 cell lines was analyzed using a Cell Counting Kit-8 (CCK-8). The proportion of CD44(+)CD24(-) tumor cells of the two cell lines were measured before and after the ATRA treatment, and the role of ATRA in the regulation of CSC self-renewing ability was evaluated with a tumor sphere assay. The tumor spheres were grown in an adherent culture to evaluate the ATRA-induced differentiation of breast cancer stem cells. ATRA effectively inhibited the unsorted cells and stem cells, but the CSCs were more sensitive to ATRA. At a concentration of 10(-6) mol/L, the inhibitory rate of MCF-7 unsorted cells and stem cells were (8.66 ± 1.06)% and (21.09 ± 3.25)%, respectively (P = 0.004). For SK-BR-3 cells, the rates were (39.19 ± 1.47)% and (51.22 ± 2.80)%, respectively (P = 0.005). The self-renewing ability of the CSCs was impaired by ATRA at a concentration of 10(-6) mol/L. The rate of MCF-7 and SK-BR-3 stem cells to form tumor sphere was 5.2% (5/96) and 13.5% (13/96), respectively. For the control group, it was 86.5% (83/96) and 93.8% (90/96), respectively (P < 0.001). ATRA also promoted the CD44(+)CD24(-) subpopulation to differentiate. SK-BR-3 stem cells were grown in an adherent culture. After using ATRA, the proportion of CD44(+)CD24(-) cells was (48.1 ± 2.5)% and that of the control group was (86.6 ± 2.5)% (P < 0.001). ATRA effectively inhibits breast NCSCs and CSCs, but CSCs are more sensitive to ATRA. ATRA impairs the self-renewing ability of CSCs and promotes CSCs to differentiate.

CD90 (Thy-1)-Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells

Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105(low) cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD(105). Unsorted cells, CD90(+), CD90(-), CD105(high), and CD105(low) cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome. Transcriptional analysis revealed that the CD90(+) subpopulation was enriched for a more osteogenic subtype relative to the CD105(low) subpopulation. Staining at day 7 for ALP was greatest in the CD90(+) cells, followed by the CD105(low) cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90(+) cells, again followed by the CD105(low) cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90(+) ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90(+) cells showed the most robust bony regeneration. Defects treated with CD90(-) cells, CD105(high) cells, and CD105(low) cells demonstrated some bone formation, but to a lesser degree when compared with the CD90(+) group. While CD105(low) cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90(+) cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.

Impact of Dose Intensification of FluBu2 to FluBu4 on Transplant Related Mortality, Relapse, and Survival After Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia in Remission

Methods: A retrospective review of the patients who relapsed following an allogeneic HCT for a myeloid malignancy at Mayo Clinic in Arizona was performed. The BM sample closest in time prior to relapse was identified and the morphologic, flow cytometric, karyotypic, FISH, PCR and chimerism results were reviewed. Results: Out of 187 patients with a myeloid malignancy, 40 (31 AML, 4 MPN blast crisis, 4 MDS, 1 CML) relapsed at a median of 113 days (range 22-820 days) after HCT. Thirty percent of the relapses occurred after a myeloablative HCT. Donor type was equally distributed between matched related and mismatched/ matched unrelated donors. A cytogenetic and molecular marker with the diagnosis was identifiable in 22/40 and 6/40 patients respectively. 18/40 patients had residual disease present at HCT. 13/40 patients showed an early relapse within the first 90 days. In the remaining patients, time between the last BM and relapse ranged from 38-184 days (median 69). Abnormal results were found only in 9/27 patients in the preceding BM and included abnormal cytogenetic clone identical to diagnosis in 2 patients; abnormal RT-PCR for BCR/ABL in 1, cytogenetic abnormality in donor cell line in 3, and decreased chimerisms in unsorted marrow cells in 3 patients. Conclusions: A 3-month monitoring schedule of BM evaluation using the currently available tools had low sensitivity to predict relapse. Findings that may justify intervention were rare (less than 10% of patients), and were restricted to abnormal cytogenetic and abnormal RT-PCR results. The clinical significance of cytogenetic abnormalities observed in donor cells is not clear. Our findings emphasize that until the optimal surveillance technique and schedule can be defined and standardized, more frequent use of pre-emptive therapy in patients at high risk of relapse may be of benefit.

Annulus fibrosus cells can induce mineralization: an in vitro study

Background contextThere is still no consensus as to whether the calcification observed in degenerate intervertebral discs (IVDs) is a cause or a consequence of disc degeneration. PurposeTo investigate the mineralization potential of healthy (independent of other associated changes) annulus fibrosus (AF) cells under controlled in vitro conditions. Study design/settingIn vitro study to investigate the mineralization potential of the AF cells. MethodsAnnulus fibrosus cells, isolated from bovine IVDs, were grown in monolayer. The effect of cell density, culture time, age of cell source, and passage on the percentage of AF cells with alkaline phosphatase activity (ALPa) was evaluated. Gene expression of mineralization-associated markers was determined. Cells were immunostained for Type I, II, and X collagens. To study mineralization potential, AF cells and AF cells that were sorted into two populations, high (top 5%±1%) or low (bottom 5%±1%) ALPa expressors, were grown in the presence of β-glycerophosphate for 2 weeks. ResultsThe percentage of AF cells that express ALPa changes with time in culture and seeding density for primary immature and mature cell sources but not for passaged cells. Gene expression of ALP, matrix metallopeptidase-13 (MMP-13), osteopontin, and runt-related transcription factor 2 was upregulated by Day 7. Under mineralization-inducing conditions, high ALPa expressors and unsorted AF cells formed von Kossa–positive nodules, composed of hydroxyapatite as determined by electron diffraction analysis. Low ALPa expressors had significantly fewer von Kossa–positive nodules (p<.01) compared with high ALPa expressors. Cells showed colocalization of Type I collagen and ALPa. No Type II collagen was detected suggesting that these were AF cells and not chondrocytes. ConclusionsAnnulus fibrosus cells have mineralizing capability and form hydroxyapatite crystalline deposits when cultured under appropriate conditions. This system could be used to investigate mineralization mechanisms in the AF during pathological calcification and at the AF-bone interface in disc degeneration.

CD200-expressing human basal cell carcinoma cells initiate tumor growth

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

Neural Ganglioside GD2+ Cells Define a Subpopulation of Mesenchymal Stem Cells in Adult Murine Bone Marrow

Background/Aims: Due to the lack of specific markers, the isolation of pure mesenchymal stem cells (MSCs) from murine bone marrow remains an unsolved problem. The present study explored whether the neural ganglioside GD2 could serve as a single surface marker to uniquely distinguish murine bone marrow MSCs (mBM-MSCs) from other marrow elements. Methods: Immunocytochemistry and flow cytometry, in combination with quantitative RT-PCR, were used to identify the expression of GD2 on culture-expanded mBM-MSCs. GD2⁺ and GD2^- fractions from mBM-MSCs cultures were sorted by immunosorting. Flow cytometry was performed to further analyze the biomarkers of GD2-sorted and unsorted cells. Employing CFU-F assay and CCK-8 assay, we examined the clonogenic and proliferative capabilities of GD2-sorted and unsorted cells. Using oil red O and von Kossa staining assay, we also assessed the multi-lineage potential of GD2-sortedand unsorted cells. Results: We found that mBM-MSCs expressed a novel surface marker the neural ganglioside GD2. Importantly, mBM-MSCs were the only cells within bone marrow that expressed this marker. Further studies demonstrated that a homogenous population of MSCs could be obtained from bone marrow cultures in early passages by GD2 immunosorting. Compared to parental cells, GD2⁺-sorted cells not only possessed much higher clonogenic and proliferative capabilities but also had significantly stronger differentiation potential to adipocytes and osteoblasts. Furthermore, GD2⁺-sorted cells displayed enhanced expression of ES markers SSEA-1 and Nanog. Conclusion: Our observations provide the first demonstration that GD2 may serve as a maker for identification and purification of mBM-MSCs. Meanwhile, our study indicates that the cells selected by GD2 are a subpopulation of MSCs with features of primitive precursor cells.

Interindividual variability and co-regulation of DNA methylation differ among blood cell populations

DNA methylation regulates gene expression in a cell-type specific way. Although peripheral blood mononuclear cells (PBMCs) comprise a heterogeneous cell population, most studies of DNA methylation in blood are performed on total mononuclear cells. In this study, we investigated high resolution methylation profiles of 58 CpG sites dispersed over eight immune response genes in multiple purified blood cells from healthy adults and newborns. Adjacent CpG sites showed methylation levels that were increasingly correlated in adult blood vs. cord blood. Thus, while interindividual variability increases from newborn to adult blood, the underlying methylation changes may not be merely stochastic, but seem to be orchestrated as clusters of adjacent CpG sites. Multiple linear regression analysis showed that interindividual methylation variability was influenced by distance of average methylation levels to the closest border (0 or 100%), presence of transcription factor binding sites, CpG conservation across species and age. Furthermore, CD4+ and CD14+ cell types were negative predictors of methylation variability. Concerns that PBMC methylation differences may be confounded by variations in blood cell composition were justified for CpG sites with large methylation differences across cell types, such as in the IFN-γ gene promoter. Taken together, our data suggest that unsorted mononuclear cells are reasonable surrogates of CD8+ and, to a lesser extent, CD4+ T cell methylation in adult peripheral, but not in neonatal, cord blood.

Abstract P1-05-20: Fluorescent Hyaluronan Probes Distinguish Heterogeneous Breast Cancer Cell Subsets and Predict their Invasive Behavior

Abstract Background: Tumor heterogeneity is a determining factor in diagnosis and therapy of breast cancer (Bca). We investigated Bca cellular heterogeneity by a functional fluorescent hyaluronan (HA) probe, and monitored its binding characteristics in relation to the degree of Bca aggression. HA is a polysaccharide ligand for CD44 and RHAMM receptors, which are linked to cell plasticity and predict poor clinical outcome in Bca. Methods: Fluorescent HA probes, multiplexed profiling, and cell sorting strategies were developed and used to detect/capture tumor cell subpopulations with differences in HA binding (HA−/low vs. HAhigh) and HA receptors surface display. Whereas we concentrated on the most metastatic cell line, MDA-MB-231, we also monitored the presence of subpopulations in other Bca lines and showed heterogeneity in all. Results: The HA probe binding was highly heterogeneous, and the highest binding level was detected in highly invasive triple-negative basal subtypes such as MDA-MB-231. HA probes bound to the cell surface but also accumulated in cytoplasm and nucleus. Binding levels were dramatically reduced upon ‘reversion’ of highly malignant cells to a non-malignant phenotype in three-dimensional cultures, suggesting that the level of cellular binding to fluorescent HA probe provides a measure of malignant behavior. Comparison between HAhigh and HA−/low subpopulations revealed surprising differences in morphology, proliferation and invasion in culture, which were retained in two in vivo models. HAhigh subpopulations exhibited higher levels of invasion but surprisingly lower levels of proliferation compared to either unsorted parental cells or the HA−/low subpopulation. Conclusions: Querying HA binding in Bca lines reveals an unexpected heterogeneity with respect to tumor phenotype in vivo, morphology in 3D, invasion and proliferation. These results may aid in diagnosis and therapy of invasive Bca subpopulations and early sorting of cancer patients needing intensive chemotherapy. Keywords: Heterogeneity, 3D cultures, Hyaluronan, CD44, RHAMM/HMMR, Tumor phenotype, Breast cancer, Invasion. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-20.

The stem and roots of Wilms' tumours

AbstractSee related article in EMBO Molecular Medicine http://dx.doi.org/10.1002/emmm.201201516

Functional expression of SK channels in murine detrusor PDGFRα+ cells

We sought to characterize molecular expression and ionic conductances in a novel population of interstitial cells (PDGFRα(+) cells) in murine bladder to determine how these cells might participate in regulation of detrusor excitability. PDGFRα(+) cells and smooth muscle cells (SMCs) were isolated from detrusor muscles of PDGFRα(+)/eGFP and smMHC/Cre/eGFP mice and sorted by FACS. PDGFRα(+) cells were highly enriched in Pdgfra (12 fold vs. unsorted cell) and minimally positive for Mhc (SMC marker), Kit (ICC marker) and Pgp9.5 (neuronal marker). SK3 was dominantly expressed in PDGFRα(+) cells in comparison to SMCs. αSlo (BK marker) was more highly expressed in SMCs. SK3 protein was observed in PDGFRα(+) cells by immunohistochemistry but could not be resolved in SMCs. Depolarization evoked voltage-dependent Ca(2+) currents in SMCs, but inward current conductances were not activated in PDGFRα(+) cells under the same conditions. PDGFRα(+) cells displayed spontaneous transient outward currents (STOCs) at potentials positive to -60 mV that were inhibited by apamin. SK channel modulators, CyPPA and SKA-31, induced significant hyperpolarization of PDGFRα(+) cells and activated SK currents under voltage clamp. Similar responses were not resolved in SMCs at physiological potentials. Single channel measurements confirmed the presence of functional SK3 channels (i.e. single channel conductance of 10 pS and sensitivity to intracellular Ca(2+)) in PDGFRα(+) cells. The apamin-sensitive stabilizing factor regulating detrusor excitability is likely to be due to the expression of SK3 channels in PDGFRα(+) cells because SK agonists failed to elicit resolvable currents and hyperpolarization in SMCs at physiological potentials.

RUNX1c Expression Identifies an Enriched Subpopulation of Hematopoietic Progenitor Cells Derived From Human Pluripotent Stem Cells.

Abstract 2294The ability of human embryonic stem cells (hESCs) to differentiate into any cell lineage makes them an important resource for studies of developmental biology. hESC-derived hematopoietic stem cells (HSCs) developed through in vitro culture systems could provide an unlimited supply of material for replacement of defective blood lineages in vivo. However, to date, there has been only limited ability to isolate functional HSCs from hESCs. Runx1 is a key hematopoietic transcription factor vital to the development of the early embryo. Previous studies have demonstrated the onset of Runx1c to correlate directly with the emergence of definitive HSCs in the murine embryo. Similarly, expression of RUNX1c increases during differentiation of hESCs to hematopoietic cells. This particular feature makes RUNX1c an especially attractive tool for tracking the development of potential HSCs from hESCs. Therefore, we have created a novel reporter system in hESCs wherein both 1 kb of the basal promoter as well as the +24 intronic enhancer of RUNX1c drive expression of the tdTomato fluorochrome. Transgenic hESC lines stably expressing tdTomato from RUNX1c regulatory elements were generated using the Sleeping Beauty transposon system. Successfully engineered clones were selected based on constitutive expression of a GFPzeocin fusion protein which was also included in the transgene. To induce differentiation, the spin-embryoid body (EB) method was used with fully defined media. Differentiating cells were analyzed for tdTomato expression. Sorted tdTomato+ cells were evaluated for RUNX1c expression and hematopoietic stem/progenitor cell potential (CFC assay). Undifferentiated RUNX1c reporter hESC clones were uniformly tdTomato-negative. These clones are shown to express the tdTomato transgene concurrent with the expression of endogenous RUNX1c, as analyzed by flow cytometry and qRT-PCR. Expression was first seen at day 12 with a peak between days 14–16 of differentiation. Cells sorted for expression of tdTomato showed a significant increase of endogenous RUNX1c expression compared to tdTomato-negative cells. Using fluorescence microscopy, we were able to detect tdTomato+ cells with hematopoietic morphology budding from the EBs after 12–15 days. Together, these results validate the accuracy of this reporter system. tdTomato+ cells were found to be enriched for hematopoietic progenitor potential, as demonstrated by increased ability to form hematopoietic colonies in methylcelluose (430 +/− 143 per 105 cells) as compared to both tdTomato negative (131 +/− 15 per 105 cells) and unsorted cells (163 +/− 28 per 105 cells). Interestingly and unexpectedly, flow cytometric analysis demonstrated that only 5–10% of CD34+CD45+ cells expressed tdTomato. The first CD45+ population was found to lack tdTomato expression. Likewise, the first emerging tdTomato+ cells did not express CD45. Of the extracellular markers examined in this study, CD31 and CD43 were found to be expressed on all tdTomato+ cells, while CD31+ and CD43+ populations, though initially tdTomato−, were found to gradually increase in tdTomato expression to 80% and 90% by day 20, respectively. Emerging CD34+ cells lack tdTomato expression with an increase to around 10% of cells, plateauing at day 15. While CD45+ cells initially lack tdTomato expression, they gain expression over time, until days 19–22, when 50–90% of CD45+ cells co-express tdTomato (RUNX1c). Therefore, this hESC RUNX1c reporter system appears to accurately define timing of RUNX1c expression. The finding that emerging RUNX1c+ cells have increased hematopoietic progenitor cell potential mimics what has been found in mice. This, combined with our finding that only a fraction of CD34+CD45+ cells express Runx1c, suggests that one reason hESC-derived hematopoietic cells fail to engraft in immunodeficient mice is that only a small percent of differentiated cells that express hematopoietic surface antigens actually have hematopoietic stem/progenitor cell activity. Current studies focus on more in-depth analysis of tdTomato expressing cells using both single-cell qRT-PCR as well as LTC-IC and engraftment assays to test for functionality. Furthermore, we are currently testing an iPSC line stably expressing the same RUNX1c:tdTomato transgene to test how its RUNX1c expression in iPSCs compares to hESCs during hematopoietic differentiation. Disclosures:No relevant conflicts of interest to declare.

α1 Anti-Trypsin (AAT) Mitigates Hematopoietic Injury and Enhances Bone Marrow Recovery After Total Body Irradiation (TBI)

Abstract 4142 Background:Injury to healthy (non-target) tissues still is a major limitation of radiation therapy, at least in part related to release of cytokines, including TNFα and IL-1β, which amplify tissue damage. Cytokine release is particularly prominent in patients who receive total body irradiation (TBI) before hematopoietic cell transplantation (HCT), as interactions of allogeneic donor cells with patient tissue contribute to the resulting “cytokine storm” and the development of graft versus-host-disease (GVHD). In murine models, administration of alpha-1 anti-trypsin (AAT) in the peri-transplant period decreased GVHD incidence and mortality. AAT, a member of the serine protease inhibitor (serpin) family, is a major protective protein in the circulation and has been used successfully in the clinic for other indications. Methods:We were interested in determining the potential benefits of AAT in preventing toxicity related to the transplant conditioning regimen, specifically TBI. AAT inhibits proteinase-3 (PR3) which, among other targets, cleaves IL-32 thereby leading to activation of TNFa and enhancing the cytokine storm. Since others have also suggested that AAT, via enhanced expression of heme oxygenase-1 (HMOX-1), leads to activation of Nrf2, thereby enhancing transcription of anti-inflammatory cytokines such as IL10 and the IL-1 receptor antagonist (IL1Ra), we determined the overall shift in the cytokine milieu and cell death. Thus, we irradiated male C3H/HeN mice (n = 5 mice per group; 6–8 weeks old) with sub-lethal doses (500, 600 and 700 cGy) of TBI from a 137Cs source. AAT (300 μg/animal) was administered intra-peritoneally 1 hour before and every 48 hours after TBI for a total of 6 doses. The effect of TBI/AAT on hematopoiesis was analyzed by growing granulocyte-macrophage colony forming units (GM-CFU) from bone marrow cells, on days 3, 7 and 14 after TBI. Results:Results were compared to those with cells from albumin-treated controls. Marrows (days 3 and 7) from AAT-treated mice generated higher GM-CFU counts than those from controls (mean = 25 vs. 5 colonies per 25 × 103 cells plated after 500cGy, and 15 vs. 3 colonies per 25 × 103 cells after 600 cGy). Peripheral blood and unsorted bone marrow from AAT-treated mice showed up-regulation of HMOX-1 and Nrf2 (30 and 50 log2 increase, respectively), and enhanced transcription of IL-10 and IL-1Ra (50 and 10 log2, respectively) compared to albumin treated donors. PR3 and TNFα, in contrast, were down-regulated (5 log2 and 7 log2 decrease in comparison to albumin treated controls).The cytokine mitigating effects of AAT were accompanied by attenuation of ATM-p53-dependent DNA damage responses in bone marrow cells (determined on days 3, 7, and 14), showing a 3-fold decrease in p53 protein levels, and a 6-fold decrease in phospho-ATM in comparison to albumin treated controls (peak day 7). In addition, protein levels of caspase 3 and caspase 9 were decreased (2-fold and 5-fold, respectively; peak on day 7) in unsorted marrow cells and spleen lysates of AAT treated mice in comparison to albumin treated controls. Bone marrow histopathology revealed normo-cellularity and a decrease in cleaved caspase 3 staining in AAT-treated mice compared to albumin treated animals. Summary and conclusions:AAT treatment significantly mitigated the hematopoietic toxicity induced by sub-lethal TBI. The mechanism involves cytokine suppression, associated with attenuation of ATM-p53 mediated DNA damage response. Taken together, these data suggest that AAT treatment pre-TBI provides protection against radiation injury. Disclosures:No relevant conflicts of interest to declare.

Identification of Gene Expression Based Prognostic Markers in the Hematopoietic Stem Cells of Patients with Myelodysplastic Syndromes

AbstractAbstract 3857The diagnosis of patients with myelodysplastic syndromes (MDS) is largely dependent on morphologic examination of bone marrow aspirates. Several criteria that form the basis of the classifications and scoring systems most commonly used in clinical practice are affected by operator-dependent variation. In order to identify more standardized molecular markers that would allow a more reliable prediction of prognosis, we have used gene expression profiling (GEP) data on CD34+ cells from MDS patients to determine the relationship between gene expression levels and prognosis in this disorder. GEP data on CD34+ cells from 125 MDS patients with a minimum 12-month follow-up since date of bone marrow sample collection were included in this study. Prediction for overall survival was performed using supervised principal components (“SuperPC”) and lasso penalized Cox proportional hazards regression applying the “Coxnet” algorithm. Supervised principal components analysis was performed on patients randomly split in a training set (n=84) and a test set (n=41), and 139 genes were identified the expression of which was significantly associated with MDS patient survival, including LEF1, CDH1, WT1 and MN1. In order to identify a smaller set of genes associated with patient survival, a second approach aiming at building sparse prediction models was used. A model was generated using the Coxnet algorithm and a predictor consisting of 20 genes was identified. Eight genes identified by the supervised principal components method were in common with the genes identified by the Coxnet model: ADHFE1, BTBD6, CPT1B, LEF1, FRMD6, GPR114, C7orf58 and LOC100286844. The Coxnet predictor outperformed other predictors including one which additionally used clinical information. To validate our findings, we evaluated the performance of our prognostic Coxnet gene signature in an independent gene expression profiling dataset on MDS bone marrow mononuclear cells (Mills et al, Gene Expression Omnibus series GSE15061). Our Coxnet gene signature based on CD34+ cells significantly identified a low-risk patient group in this independent GEP dataset based on unsorted bone marrow mononuclear cells, demonstrating that our signature is robust and may be applicable to bone marrow cells without the need to isolate CD34+ cells. These GEP-based signatures correlating with clinical outcome may significantly contribute to a refined risk classification of MDS. Disclosures:No relevant conflicts of interest to declare.

Leukemic and Hematopoietic Stem Cells Compete for the Same Functional Marrow Niche in a MLL-AF9 Murine Leukemia Model

AbstractAbstract 4897Much is known regarding the location, cellular composition, signaling pathways, and functional role of the normal hematopoietic stem cell (HSC) niche in the bone marrow microenvironment. Microenvironmental cells including osteoblasts, other specialized mesenchymal cells, and vascular endothelial cells exert control over HSC self-renewal, differentiation, and engraftment. Niche occupancy appears to be competitive and limiting in terms of controlling the number of HSCs per organism. Leukemia stem cells (LSCs), through their inherent properties of quiescence and resistance to chemotherapeutic agents, are thought to be one of the principal mechanisms underlying disease relapse in patients. Much less is known regarding the interaction of LSCs and the marrow microenvironment. It is not clear whether LSCs localize to the same niches as HSCs, compete with HSCs for niche occupancy, or share dependence on niche signals, and whether those signals affect tumor responses to chemotherapy. Using a human pre-B ALL xenograft mouse model, Colmone et al (Science 2008) recently showed that leukemic cells may alter the normal microenvironment, resulting in initial homing of transplanted normal HSPCs in distinct atypical niches. Shiozawa et al (JCI 2011) showed that metastatic prostate cancer cells, a tumor type known to target bone, impeded HSC engraftment in a murine model, suggesting competition for the same niche.To investigate the relationship between HSC and LSC niche localization and functional occupancy, we used murine progenitor cells transduced with an MLL-AF9 vector expressing GFP in a murine syngeneic competitive transplantation model. MLL-AF9 cells are highly enriched for LSCs, particularly the c-kit+ compartment (Somervaille Cancer Cell 2006). We found that between approximately 21% and 24% of cells were c-kit+ by FACS in 2 separate experiments. In our model, mice transplanted with unsorted MLL-AF9 cells (1×107) died of AML with a latency of 11–14 days.We cotransplanted a fixed number of MLL-AF9-GFP cells (1×106) with increasing numbers of normal mouse whole bone marrow (WBM) cells, derived from dsRed transgenic mice to facilitate distinction from the GFP+ MLL-AF9 cells, into mice irradiated with 1000 rads: 1×105 [group 1], 1×106 [group 2], 1×107 [group 3], 5×107 [group 4]. Control groups received 1×105 and 1×106 normal WBM cells only. Survival was monitored daily. The control group receiving 1×105 cells only all died with median time to death of 16.5 days from lack of count recovery, those receiving 1×106 cells are still alive 35 days after transplant, indicating that 1×106 cells is adequate to rescue from irradiation. Mice were bled weekly until death and samples were analyzed by flow cytometry. Complete blood counts, blood smears, and splenic sections were obtained from these mice. As expected, there were no circulating blasts detected 7 days post transplant and all mice were healthy. However, 14 days after transplant the percentages of GFP+ leukemic cells detected in the blood were inversely proportional to the number of normal dsRed WBM cells transplanted (group 1 vs. group 2 vs. group 3 vs. group 4 mean percentage of GFP+ cells, 83.97 v 66.53 v 18.73 v 9.275 p< 0.0001). At day 15, mice from group 1, but not from groups 2 to 4, became moribund and were sacrificed. Spleens in this group were heavier than in those mice transplanted with 1×105 normal WBM cells alone and 2 out of 3 showed leucocytosis compared to leucopenia in all mice in the group transplanted with normal cells alone. When mice in the other groups had blood samples taken for analysis while moribund, GFP+ cells were greater than 80% suggesting that mice in group 1 died from complications relating to leukemic infiltration. Confocal microscopy confirmed the colocalization of normal HSPCs and MLL-AF9-GFP LSCs in the niche. Most interestingly, survival was proportional to the numbers of normal WBM cells transplanted, with a continuous delay in leukemic death proportional to the number of normal WBM cells cotransplanted with the same dose of MLL-AF9 cells (Figure 1). Hence, this murine model of leukemia suggests that normal and leukemic cells compete for the same functional niche, that manipulation of the niche could impact on response to anti-leukemic therapies, and that cell dose in the context of stem cell transplantation for leukemia may have an impact on outcome via niche competition. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM+ enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM+ enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.

Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

CD9 Is Expressed on Human Male Germ Cells That Have a Long-Term Repopulation Potential after Transplantation into Mouse Testes1

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.