Primitive Human Cells Research Articles

3105 – ENHANCED OUTPUT OF PRIMITIVE HEMATOPOIETIC CELL PHENOTYPES IN TERATOMAS GENERATED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) IN MORE PERMISSIVE IMMUNODEFICIENT MICE

Investigation of human hematopoietic stem cells (HSCs) has been revolutionized by advances in cell separation and clonal tracking of cells produced in transplanted patients and immunodeficient mice. However, methods able to expand them significantly remain elusive. iPSCs are of continuing interest as an alternative source, although robust conditions that support iPSC generation of human HSC either in vitro or in vivo have also not yet been identified. To determine whether the use of a more compatible in vivo host environment might offer an improvement, we compared the outputs of teratomas generated from human iPSC transplanted (± irradiated mouse fibroblasts engineered to produce human FLT3-L, SCF and IL3) into immunodeficient NOD-Rag1-null- IL2Rgc-null mice with a c-kit deficiency with or without a transgenic source of human IL3, GM-CSF and SCF (NRG-W41± 3GS mice). 6-8 week teratomas generated in the NRG-W41-3GS mice contained 40-fold more human CD34+CD45+ cells than those produced in NRG-W41 mice with yields up to 7 × 106 CD45+ cells. Co-injection of human growth factor-producing mouse fibroblasts further increased the production of human CD34+CD45+ ∼6-fold in the NRG-W41-3GS mice that extended to the phenotypically early GPI80+ and CD49f+ cells within the CD45+CD34+CD38-CD45RA-CD90+ subset. However, despite their display of short-term granulopoietic and erythroid differentiation potential in vitro, no evidence of in vivo engrafting potential was detected. These experiments suggest a persisting failure of conditions in the teratomas to support the acquisition of certain functional properties that primitive human cells require to engraft and set the stage for their future identification.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

Variable aldehyde dehydrogenase activity and effects on chemosensitivity of primitive human leukemic cells.

Aldehyde dehydrogenase (ALDH) activity is an established feature of primitive normal human hematopoietic cells, in which it has been associated with a high expression of the 1A1 isoform of ALDH. High ALDH 1A1 activity has been reported to also characterize cells that propagate malignant populations arising in other tissues, but the regulation and basis of ALDH activity in primary human leukemic cells has not been well studied. We obtained samples from patients with newly diagnosed acute myeloid leukemia (AML; n=21) and chronic myeloid leukemia (CML; n=8) and analyzed different phenotypically and functionally defined subsets for their ALDH activity using the ALDEFLUOR® kit and expression of the ALDH1A1 gene. We detected cells with high ALDH activity (ALDHpos) in all samples from AML and CML patients. These were consistently enriched in the CD34+ population of these samples, but typically not in the CD34+CD38- subset. Leukemic cells with direct clonogenic activity invitro or those able to repopulate the bone marrow of sublethally irradiated non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice were both ALDHpos and ALDHneg. Interestingly, ALDH1A1 transcripts were highest in the ALDHneg leukemic cells and, in studies with leukemic cell lines, exposure to an inhibitor of ALDH activity variably affected sensitivity to daunorubicin. Cells with high ALDH activity are commonly found within the CD34+ population of primary human leukemic cells but, unlike in normal hematopoietic tissues, do not selectively or consistently comprise those with proliferative potential or other distinct functional properties.

RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells

Human bone marrow mesenchymal stem cells (hBMSCs) commitment and differentiation are dictated by bioactive molecules sequestered within their Extra Cellular Matrix (ECM). One common approach to mimic the physiological environment is to functionalize biomaterial surfaces with ECM-derived peptides able to recruit stem cells and trigger their linage-specific differentiation. The objective of this work was to investigate the effect of RGD and BMP-2 ligands crosstalk and density on the extent of hBMSCs osteogenic commitment, without recourse to differentiation medium. RGD peptide promotes cell adhesion via cell transmembrane integrin receptors, while BMP-2 peptide, corresponding to residues 73–92 of Bone Morphogenetic Protein-2, was shown to induce hBMSCs osteoblast differentiation. The immobilization of peptides on aminated glass was ascertained by X-ray Photoelectron Spectroscopy (XPS), the density of grafted peptides was quantified by fluorescence microscopy and the surface roughness was evaluated using Atomic Force Microscopy (AFM). The osteogenic commitment of hBMSCs cultured on RGD and/or BMP-2 surfaces was characterized by immunohistochemistry using STRO-1 as specific stem cells marker and Runx-2 as an earlier osteogenic marker. Biological results showed that the osteogenic commitment of hBMSCs was enhanced on bifunctionalized surfaces as compared to surfaces containing BMP-2, while on RGD surfaces cells mainly preserved their stemness character. These results demonstrated that RGD and BMP-2 mimetic peptides act synergistically to enhance hBMSCs osteogenesis without supplementing the media with osteogenic factors. These findings contribute to the development of biomimetic materials, allowing a deeper understanding of signaling pathways that govern the transition of stem cells towards the osteoblastic lineage. Statement of SignificanceFor a long time, scientists thought that the differentiation of Mesenchymal Stem Cells (MSCs) into bone cells was dictated by growth factors. This manuscript shed light on other ligands that play a crucial role in regulating MSCs fate. In concrete terms, it was demonstrated that the osteoinductive effect of BMP-2 peptide is 2 folds improved in the presence of adhesive RGD peptide. Compared to previous works highlighting this synergistic cooperation between RGD and BMP-2 peptides, the main strength of this work lies to the use of primitive human cells (hMSCs) and well-defined biomimetic material surfaces (controlled surface roughness and peptide densities). This work provides valuable insights to develop custom-designed in vitro cell culture models, capable of targeting the desired cell response.

Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo.

A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells

SummaryDisrupted IKAROS activity is a recurrent feature of some human leukemias, but effects on normal human hematopoietic cells are largely unknown. Here, we used lentivirally mediated expression of a dominant-negative isoform of IKAROS (IK6) to block normal IKAROS activity in primitive human cord blood cells and their progeny. This produced a marked (10-fold) increase in serially transplantable multipotent IK6+ cells as well as increased outputs of normally differentiating B cells and granulocytes in transplanted immunodeficient mice, without producing leukemia. Accompanying T/natural killer (NK) cell outputs were unaltered, and erythroid and platelet production was reduced. Mechanistically, IK6 specifically increased human granulopoietic progenitor sensitivity to two growth factors and activated CREB and its targets (c-FOS and Cyclin B1). In more primitive human cells, IK6 prematurely initiated a B cell transcriptional program without affecting the hematopoietic stem cell-associated gene expression profile. Some of these effects were species specific, thus identifying novel roles of IKAROS in regulating normal human hematopoietic cells.

Adult NK lineage development in humans is strengthened upon emergency

Emergency conditions such as infection demand rapid production and activation of innate immune cells to combat noxious extrinsic agents. In mice, pathogen recognition through Toll like receptors (TLR) and the resulting pro-inflammatory cytokine release induce the expansion of bone marrow hematopoietic stem cells and instruct early differentiation fates so immediate innate cell development is guaranteed. Accordingly, recent work suggests that human primitive cell populations are also capable of microbial components discrimination through TLR, a mechanism that facilitates their differentiation to myeloid lineage cells. We have now found that early lymphoid progenitor cells from adult bone marrow display TLR9 and its ligation promotes the quick development of NK lineage cells by using mechanisms that involve IL-15R up-regulation. Strikingly, this phenomenon is not obvious in their neonatal counterparts, suggesting that contribution of neonatal seminal cells to the emergent cell production in response to viral signals may differ from that of adult cells.

Niche displacement of human leukemic stem cells uniquely allows their competitive replacement with healthy HSPCs.

Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is currently the leading strategy to manage acute myeloid leukemia (AML). However, treatment-related morbidity limits the patient generalizability of HSCT use, and the survival of leukemic stem cells (LSCs) within protective areas of the bone marrow (BM) continues to lead to high relapse rates. Despite growing appreciation for the significance of the LSC microenvironment, it has remained unresolved whether LSCs preferentially situate within normal HSC niches or whether their niche requirements are more promiscuous. Here, we provide functional evidence that the spatial localization of phenotypically primitive human AML cells is restricted to niche elements shared with their normal counterparts, and that their intrinsic ability to initiate and retain occupancy of these niches can be rivaled by healthy hematopoietic stem and progenitor cells (HSPCs). When challenged in competitive BM repopulation assays, primary human leukemia-initiating cells (L-ICs) can be consistently outperformed by HSPCs for BM niche occupancy in a cell dose-dependent manner that ultimately compromises long-term L-IC renewal and subsequent leukemia-initiating capacity. The effectiveness of this approach could be demonstrated using cytokine-induced mobilization of established leukemia from the BM that facilitated the replacement of BM niches with transplanted HSPCs. These findings identify a functional vulnerability of primitive leukemia cells, and suggest that clinical development of these novel transplantation techniques should focus on the dissociation of L-IC-niche interactions to improve competitive replacement with healthy HSPCs during HSCT toward increased survival of patients.

Abstract 2919: Different ROS control mechanisms and mutagenic consequences in primitive subsets of normal human mammary cells.

Abstract Reactive oxygen species (ROS) are known mediators of DNA damage and likely contributors to oncogenesis. However, very little is known about the mechanisms controlling ROS in the cells that make up the normal human mammary gland, in spite of its being a major site of cancer development. To investigate how ROS are generated and their potential role in two functionally distinct, primitive normal human mammary epithelial cell compartments (luminal and basal), we isolated these subsets at high purities by FACS and compared the levels within them of ROS, components that positively and negatively regulate ROS, their responses to oxidative stressors and evidence of ROS-associated DNA damage. The results show that purified progenitors of the cells that line the gland lumen (defined by their EpCAM+ CD49f+ phenotype) contain significantly higher levels of superoxide (O2*) anions and H2O2 than the basal cells (defined as EpCAMlow/- CD49fhigh cells and highly enriched in bipotent progenitors and stem cells). We also find that the elevated levels of these ROS elements in the luminal progenitors are associated with a higher content of mitochondria and higher levels of all 3 superoxide dismutases (SOD-1, 2 and 3). The luminal progenitors are also highly resistant to glutathione depletion and express higher levels of both non-canonical non-glutathione anti-oxidant enzymes and multiple enzymes that control ROS-induced nucleotide damage (i.e., OGG-1, MTH-1, MUTYH) providing a likely explanation for their ability to survive following GSH depletion. Interestingly, we found the mitochondrial antioxidant glutathione peroxidase (GPX)-2 enzyme to be expressed almost exclusively and at high levels in basal mammary cells and its depletion, using a shRNA lentivirus, resulted in loss of progenitor viability/activity by basal but not luminal cells. Luminal progenitors also displayed greater resistance to acute oxidative insults (H2O2 and X-radiation) and displayed an increased accrual of oxidative damage-induced (genomic 8-oxo-dGTP) mutations. Our findings reveal a major difference in the molecular machinery that control ROS levels in normal human basal and luminal mammary progenitors. These correlate with an ability of the luminal progenitors to maintain and tolerate elevated levels of ROS and the continuous acquisition of unrepaired ROS-induced DNA damage, thus suggesting a novel and previously unanticipated vulnerability of these cells to undergo oncogenic transformation. (Supported by Canadian Breast Cancer Foundation/BC-Yukon and the Canadian Cancer Society) Citation Format: Nagarajan Kannan, Maisam Makarem, Long Nguyen, Jeff Dong, Peter Eirew, Connie Eaves. Different ROS control mechanisms and mutagenic consequences in primitive subsets of normal human mammary cells. [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 2919. doi:10.1158/1538-7445.AM2013-2919

Ex vivo expansion of normal and chronic myeloid leukemic stem cells without functional alteration using a NUP98HOXA10homeodomain fusion gene

HOX genes have been implicated as regulators of normal and leukemic stem cell functionality, but the extent to which these activities are linked is poorly understood. Previous studies revealed that transduction of primitive mouse hematopoietic cells with a NUP98HOXA10homeodomain (NA10HD) fusion gene enables a subsequent rapid and marked expansion in vitro of hematopoietic stem cell numbers without causing their transformation or deregulated expansion in vivo. To determine whether forced expression of NA10HD in primitive human cells would have a similar effect, we compared the number of long-term culture-initiating cells (LTC-ICs) present in cultures of lenti-NA10HD versus control virus-transduced CD34+ cells originally isolated from human cord blood and chronic phase (CP) chronic myeloid leukemia (CML) patients. We found that NA10HD greatly increases outputs of both normal and Ph+/BCR-ABL+ LTC-ICs, and this effect is particularly pronounced in cultures containing growth factor-producing feeders. Interestingly, NA10HD did not affect the initial cell cycle kinetics of the transduced cells nor their subsequent differentiation. Moreover, immunodeficient mice repopulated with NA10HD-transduced CP-CML cells for more than 8 months showed no evidence of altered behavior. Thus, NA10HD provides a novel tool to enhance both normal and CP-CML stem cell expansion in vitro, without apparently altering other properties.

Abstract 1013: Enriched miR-126 bioactivity marks the primitive compartment in human AML and regulates leukemia stem cell numbers

Abstract Previous work has shown miRNAs are dysregulated in acute myeloid leukemia (AML), however, there is little known regarding miRNA expression and function in human leukemia stem cells (LSC). In order to elucidate the role of miRNA in LSC, we performed miRNA profiling on fractionated subpopulations of primary AML patient samples. Supervised analysis guided by the in vivo SCID leukemia initiating cell (SL-IC) capacity of each sub-population generated a unique miRNA signature associated with LSC enriched fractions. The biological activity of our top candidate, miR-126, was confirmed at single cell resolution by using a novel bidirectional lentivirus miRNA reporter system in vitro and within primary AML patient samples xenografted into immune-deficient NSG mice. These data suggest that primitive AML cells may express high levels of bioactive miR-126 relative to more “differentiated” blast populations. To test this hypothesis, we FACS sorted miR-126 genetic reporter vector transduced primary AML patient samples and transplanted these populations into immune-compromised secondary mouse recipients. The results of these proof-of-concept experiments demonstrates our ability to prospectively isolate LSC enriched fractions in all 4 AML patient samples tested using only a single biomarker, miR-126. Finally, to understand the functional relevance of miR-126 expression within primitive human AML cells, stable enforced expression and knockdown of miR-126 was achieved using lentiviral vectors. Enforced expression in four primary AML xenografts resulted in a several fold increase of CD34+CD117+ lentivirus marked leukemia cells after 12 weeks. In addition, the miR-126/OE cells showed reduced differentiation marker expression (CD14, CD15) with no significant differences in AML graft size. To determine if the expanded population had SL-IC activity or was a downstream leukemic progenitor, limiting dilution assays were performed by transplantation of FACS sorted lentivirus marked cells into secondary recipient mice for 12 weeks. A 3-20 fold increase in LSC activity was observed with miR-126 forced expression compared to control cells. These data suggest that high levels of miR-126 bioactivity support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. We performed microarray analysis of a primitive human AML cells after miR-126/OE and miR-126/KD. The principal signalling pathway(s) under direct control of miR-126 in primitive AML cells were revealed by subjecting our array data to Gene Set Enrichment Analysis (GSEA) in combination with several published miRNA target prediction algorithms. In summary, this work demonstrates that miR-126 is more abundant and biologically active within the leukemia stem/progenitor cell compartment of the AML functional hierarchy and serves to regulate AML stem cell numbers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1013. doi:1538-7445.AM2012-1013

Potential involvement of human circulating CD14+ monocytes in tissue repair and regeneration

Circulating CD14+ monocytes originate from hematopietic stem cells and are believed to be committed precursors for phagocytes such as macrophages. Recently, we have reported a primitive human cell population called monocyte-derived multipotential cells (MOMCs), which has a fibroblast-like morphology in culture and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from circulating CD14+ monocytes, but circulating precursors still remain undetermined. MOMCs contain progenitors with the capacity to differentiate into a variety of mesenchymal cells in vitro, including bone, cartilage, fat, and skeletal and smooth muscles. Moreover, MOMCs are able to differentiate into the cardiomyogenic and neuronal lineage by co-culturing with primary cultures of embryonic heart and brain, respectively. In addition, MOMCs have capacity of differentiating into endothelium of a mature phenotype with typical morphologic, phenotypic, and functional characteristics. In vitro generation of MOMCs from precursors within circulating monocytes requires their binding to fibronectin and exposure to soluble factors derived from activated platelets. In a rat model of cerebral ischemia, transplantation of MOMCs into the ischemic core results in a significant improvement in neurologic function, but this effect is primarily due to neovascularization through production of a large array of angiogenic factors by MOMCs with some contribution of their differentiation into mature endothelial cells. These findings indicate that circulating CD14+ monocytes are involved in a variety of physiologic functions other than innate and acquired immune responses, such as repair and regeneration of the damaged tissue, through a variety of mechanisms.

Pharmacological Inhibition of the Stress-Related Deacetylase SIRT1 Enhances Eradication of CML stem Cells

Abstract 448BCR-ABL tyrosine kinase inhibitors (TKI) are effective in inducing remissions and prolonging survival of CML patients, but fail to eradicate primitive leukemia stem cells (LSC) which remain a potential source of relapse. New strategies to enhance elimination of residual CML LSC in TKI-treated patients are required. We have previously reported that the stress-related deacetylase SIRT1 is expressed at high levels in CML stem/progenitor cells and that inhibition of SIRT1 expression using lentivirus-expressed shRNA induces apoptosis in CML progenitors and increases their sensitivity to imatinib (IM) by activating p53 signaling (Blood 2010, 116: 200A). These results support an important role for SIRT1 in CML LSC maintenance and TKI resistance, and as a potential molecular target for therapy directed against CML LSC. Tenovin-6 (TV) has been identified as a potent small molecule inhibitor of SIRT1 activity (Cancer Cell 2008, 13:454). Here we evaluated whether pharmacological inhibition of SIRT1 activity using TV could selectively inhibit CML stem/progenitor cells. As with shRNA-mediated knock-down of SIRT1, treatment with TV (0.5 μM) significantly increased apoptosis of CML CD34+ cells (TV16±7% vs. Control 3±2%, p=0.04, n=3), but not normal CD34+ cells (TV 6±2% vs. Control 4±2%, p=0.1, n=3). The combination of IM (2.5 μM) and TV induced significantly increased apoptosis in CML progenitors compared to IM alone, and to a significantly greater extent than in normal cells (CML, TV + IM 40±2% vs. IM 19±3%, p=0.009, n=3; CB, TV + IM 15±4% vs. IM 10±2%, p=0.04, n=3). TV (1 μM) increased apoptosis in both CML CD34+CD38− (TV 42±10% vs. Control 4±3%, p=0.04, n=3) and CD34+CD38+ cells (TV 35±7% vs. Control 8±2%, p=0.03, n=3). CFSE labeling indicated that treatment with TV resulted in increased apoptosis of undivided CML CD34+CD38− cells identified on the basis of high CFSE fluorescence (TV 20±7% vs. Control 2±1%, p=0.04, n=3). The combination of TV with IM resulted in a significant increase in apoptosis in CML CD34+CD38− CFSEhigh cells compared to IM alone (TV plus IM 35±5% vs. IM 10±4%, p=0.03, n=3). Treatment with TV (0.5 μM) reduced CML CFC frequency (70±9% inhibition with TV compared to untreated controls, p=0.009, n=3) without affecting normal CFC frequency. Combination of TV (0.5 μM) with IM resulted in enhanced inhibition of CML CFC compared to IM alone, but did not enhance inhibition of normal CFC (CML: TV plus IM 82±6% inhibition vs. IM 57±10%, p=0.02, n=3; CB: TV plus IM 38±7% inhibition vs. IM 36±9%, p=0.1, n=3). TV treatment effectively inhibited the growth of Baf3 cells expressing T315I-mutated BCR-ABL, and significantly enhanced apoptosis of IM-resistant CML blast crisis CD34+ cells [TV (1 μm) 30±1% vs. Control 19±5%, p=0.04, n=3], suggesting SIRT1 inhibition can also target TKI-resistant CML cells. Ex vivo treatment with TV (1 μM) significantly reduced longer-term (12 weeks) engraftment of CML CD34+ cells in NSG mice following TV treatment (TV treated 0.2*105±0.1*105 human CD45+ cells in murine BM vs. Control 1.8*105±0.6*105, p=0.009, n=5). Significant reduction in engraftment of CD33+ (p=0.008) and CD14+ myeloid cells (p=0.009) was seen. Q-PCR and FISH analysis confirmed that engrafted human cells were leukemic in origin. Interestingly, engraftment of CB CD34+ cells was not reduced after treatment with TV (TV 2.7*106±0.7*106 human CD45+ cells in murine BM, vs. Control 2.4*106±0.8*106, p=0.2, n=6). These results show that SIRT1 inhibition by TV effectively targets primitive human CML cells with in vivo multi-lineage engraftment capacity. Treatment with TV significantly enhanced acetylated p53 levels in CML CD34+ cells, indicating effective inhibition of SIRT1 activity. TV treatment also increased total p53 levels, possibly related to reduced p53 degradation. TV treatment did not increase acetylated p53 or total p53 levels in normal CD34+ cells. Importantly shRNA-mediated knock-down of p53 resulted in significant reduction of TV-induced apoptosis in CML CD34+ cells (13±6% apoptosis with p53 shRNA; 33±7% apoptosis with control shRNA, p=0.04, n=3), indicating that the effects of TV on CML CD34+ cells are related to p53 acetylation and activation. In conclusion, our studies indicate that pharmacological inhibition of SIRT1 can activate p53 and enhance eradication of CML LSC in combination with TKI treatment, and support further evaluation of targeted inhibition of SIRT1 as a therapeutic strategy in CML. Disclosures:No relevant conflicts of interest to declare.

Distinct Human Cell Populations Produce Rapidly Detectable Levels of Neutrophils and Platelets in NOD/SCID-IL-2Receptor Gamma Chain Null Mice Transgenically Engineered to Produce Human Myeloid Growth Factors

Abstract 1892Cord blood (CB) is becoming an increasingly utilized source of cells for cancer patients who are eligible for therapies that require a transplant to rescue them from toxic side effects on their own hematopoietic cells but lack a suitable HLA-matched donor. This strategy is now routinely used in children, but delayed neutrophil and platelet recovery remain unsolved problems and these problems are exacerbated in adults. To address this issue, we first surveyed the variability in 8 individual CB harvests of parameters routinely used to predict the utility of CB units as transplants (i.e., CD34+ and in vitro myeloid clonogenic progenitor cell frequencies). In addition, we compared their 3-week outputs of CD33/15/66+ cells (neutrophils and monocytes) in the marrow and CD41a+ platelets in the blood of sublethally irradiated NSG mice after the IV transplantation of ∼104 CD34+ cells. These latter assessments were based on ongoing experiments in our lab demonstrating that, at this transplant dose, the outputs measured are linearly related to the number of CD34+ cells injected and detect transplantable progenitor cell types that are biologically distinct from cells with longer term repopulating activity. The results showed variation between CBs in all parameters, a marked lack of correlation between %CD34+ cells or % total CFCs in initial cells and %CD41a+ cells regenerated at 3 weeks/104 CD34+ cells transplanted (R=-0.28 and 0.35, respectively), and a weak correlation between the %CD33/15/66+ cells regenerated at 3 weeks/104 CD34+ cells transplanted and %CD34+ cells or % total CFCs in the initial CB cells (R values of 0.46–0.64). However, although engraftment of primitive human cells in NSG mice appears highly efficient, terminal differentiation of the myeloid lineages in these mice is poor. One possible explanation for this deficiency in mature cell output is that several of the murine growth factors responsible for regulating the production and release of these cells into the circulation in mice are not cross-reactive on human cells. We therefore hypothesized that engineering NSG mice to produce the human counterparts might significantly improve the detection of short term repopulating human cells whose maximum clone size might be limiting in NSG mice. Three potential relevant factors are IL-3, GM-CSF and Steel factor. We therefore backcrossed a line of transgenic NS mice we had created to express human IL-3, GM-CSF and Steel factor onto the NSG strain to produce homozygous NSG mice expressing all 3 of these human factors (NSG-3GS mice). We then compared these NSG-3GS mice with NSG mice in terms of their ability to stimulate the production within 3 weeks of human neutrophil-monocytes and platelets from intravenously transplanted CD34+ cells isolated from pooled CB harvests. The results showed that the levels of neutrophils and monocytes generated in the marrow of the NSG-3GS mice were elevated to levels of >50% of the marrow in 90% of the mice, even at the lowest number of CD34+ cells transplanted. Human neutrophils and monocytes were also elevated in the blood of the NSG-3GS mice where, despite the observed “saturation” of the marrow, there was a linear dose-response in the number of human neutrophils and monocytes present in the blood with increasing CD34+ cells infused. These findings are consistent with the reported activities of these molecules in vitro and in patients suggesting their physiological relevance in this murine xenograft model. We next utilized this assay to characterize the cells responsible for the neutrophil/monocyte and platelet repopulating activities detected in NSG-3GS mice. Preliminary assessment of the CD34+CD45RA- population on the basis of CD123 (IL-3 receptor alpha chain) expression indicates that the CD123+ fraction is enriched for short term (3-week) neutrophil/monocyte repopulating activity, while the CD123- fraction is enriched for short term (3-week) platelet repopulating activity. In summary, NSG-3GS mice significantly enhance the output of human cells with short term human myeloid repopulating ability thereby enabling neutrophil/monocyte outputs as well as platelet outputs to be assessed by analysis of peripheral blood samples. We have also used this tool to obtain evidence that these two outputs are derived from distinct cell types. Direct quantification of these may add to future predictions of graft quality. Disclosures:No relevant conflicts of interest to declare.

Imaging-based chemical screens using normal and glioma-derived neural stem cells

The development of optimal culture methods for embryonic, tissue and cancer stem cells is a critical foundation for their application in drug screening. We previously described defined adherent culture conditions that enable expansion of human radial glia-like fetal NS (neural stem) cells as stable cell lines. Similar protocols proved effective in the establishment of tumour-initiating stem cell lines from the human brain tumour glioblastoma multiforme, which we termed GNS (glioma NS) cells. Others have also recently derived more primitive human NS cell lines with greater neuronal subtype differentiation potential than NS cells, which have similarities to the early neuroepithelium, named NES (neuroepithelial stem) cells. In the present paper, we discuss the utility of these cells for chemical screening, and describe methods for a simple high-content live-image-based platform. We report the effects of a panel of 160 kinase inhibitors (Inhibitor Select I and II; Calbiochem) on NES cells, identifying three inhibitors of ROCK (Rho-associated kinase) as promoting the expansion of NES cell cultures. For the GNS cells, we screened a panel of 1000 compounds and confirmed our previous finding of a cytotoxic effect of modulators of neurotransmitter signalling pathways. These studies provide a framework for future higher-throughput screens.

SiRNA-Induced Transient Silencing of PTEN Expression Enhances Human Hematopoietic Cell Engraftment in NOD/SCID/γcnull Mice and Increases Gene Transduction Efficiency.

PTEN (phosphatase and tensin homologue) has been implicated as a regulator of murine hematopoietic stem cell (HSC) self-renewal. In mice, PTEN deletion initially results in a transient expansion of HSC numbers, but the HSC pool subsequently becomes depleted. Therefore, constitutive down-modulation of PTEN would not be desirable for stem cell expansion, however transient depletion of PTEN activity could be beneficial for transplantation and gene therapy applications. Little is understood regarding the role of PTEN in human HSC regulation. We studied the impact of transient inhibition of PTEN expression in human CD34+ cells on their cell cycle profile, their susceptibility to retroviral transduction, their proliferative potential in vitro, and their ability to repopulate NOD/ SCID/γcnull mice. Human G-CSF-mobilized CD34+ cells were transfected with PTEN siRNA (PTEN1 or PTEN2) or control siRNA. Forty-eight hours later PTEN expression was inhibited by 73–100% (PTEN1) and 64–97% (PTEN2) compared to CD34+ cells transfected with control siRNA as measured by real-time RT-PCR. Reduced PTEN protein levels were confirmed by Western blot. Compared to control siRNA treated CD34+ cells, PTEN-silenced CD34+ cells showed a significant decrease in the proportion of cells in the G0 phase of the cell cycle (PTEN1: 10.9%; PTEN2: 13.0%; control: 19.5%; p < 0.05) and a concomitant increase in the proportion of cells in the S + G2/M phase of the cell cycle (PTEN1: 42.1%; PTEN2: 42.4%; control: 37.2%; p < 0.05). We hypothesized that the increased proportion of cycling CD34+ cells may enhance their susceptibility to retroviral transduction for gene therapy applications. Human CD34+ cells were nucleofected with PTEN or control siRNA, cultured for 2 days with cytokines and transduced for 48 hours with MND.MFG.YFP c10 retroviral vectors (MOI=0.3) on fibronectin-coated plates. Transduction efficiencies in the bulk CD34+ cells transfected with PTEN1 siRNA and PTEN2 siRNAs were significantly higher compared with CD34+ cells transfected with a control siRNA as determined by detection of YFP by flow cytometry. The average percentage of YFP+ cells from eight independent transductions were 41.7% (PTEN1, p=0.0001), 38.9% (PTEN2, p=0.003), compared with the control siRNA treated group (29.4%). We next tested whether transient inhibition of PTEN expression could result in improved engraftment of primitive human cells capable of repopulating nonobese diabetic/ severe combined immune-deficient-interleukin-2R−/− (NOD/SCID/γcnull) mice. A total of 25 NOD/SCID/γcnull mice were transplanted with PTEN or control siRNA treated CD34+ cells immediately after nucleofection (PTEN1 n=10; PTEN2 n=5; control n=10) and 9 additional mice were transplanted with untreated human CD34+ cells. Mice transplanted with PTEN1 siRNA- or PTEN2 siRNA-treated CD34+ cells had increased percentages of human cell engraftment as determined by flow cytometry for human CD45 compared to mice transplanted with control siRNA-treated CD34+ cells or untreated CD34+ cells (PTEN1: 37.6%; PTEN2: 38.1%; control: 19.7%; untreated: 19.2%, p = 0.03). NOD/ SCID/γcnull mice transplanted with PTEN1 and PTEN2 siRNA-treated CD34+ cells showed no statistically significant differences in human lymphoid (CD3+, CD20+) or myeloid (CD15+) differentiation compared to control mice but a trend favoring an increased representation of myeloid (CD15+) cells was noted in PTEN1-treated mice (PTEN1: 41.7%; control: 18.8%, p = 0.06). None of these mice developed myeloproliferative disorders or leukemias. Overall, these data suggest that PTEN plays an important role in governing transitions between the quiescent and activated states of primitive human hematopoietic cells, and the transient inhibition of PTEN expression in these cells results in improved retroviral transduction efficiencies and increased engraftment. This approach may find clinical applications in gene therapy for inherited disorders and in adult cord blood stem cell transplantation where the number of HSC is limited.

Transcriptome Analysis of the Normal Human Mammary Cell Commitment and Differentiation Process

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.

Comparative Transcriptome Analysis of Different Subsets of CD34+ Normal and Chronic Myeloid Leukemia Cells Identifies Novel Perturbations in the CML Stem Cell Population.

Chronic myeloid leukemia (CML) arises from a hematopoietic stem cell that acquires a BCR-ABL fusion gene. During the chronic phase of the disease, this cell produces an expanding multi-lineage clone that usually comes to dominate all terminal stages of myelopoiesis. However, eventually, further mutations are acquired which cause progression to a rapidly fatal acute leukemia. To obtain new insights into the molecular perturbations that cause CML stem cells to initiate chronic phase disease, we generated Long Serial Analysis of Gene Expression (LongSAGE) libraries (∼200,000 tags/library) from extracts of highly purified lin−CD34+CD45RA−CD36−CD71−CD7−CD38+ and lin−CD34+CD45RA−CD36−CD71−CD7−CD38− normal bone marrow (BM) and G-CSF-mobilized peripheral blood (G-mPB) cells as well as cells from 3 chronic phase CML patients with predominantly Ph+/BCR-ABL+ cells in both of these very primitive cell subsets. Long term culture-initiating cell (LTC-IC) and direct colony-forming cell (CFC) assays performed on an aliquot of each of these cell populations showed the frequency of LTC-IC were 4 to 130-fold higher in the 34+38− cells than in the matching 34+38+ cells with the opposite trend for the CFCs. Comparison of the tags present in the pooled CML and pooled normal BM and G-mPB libraries revealed many differentially expressed genes. Real-time RT-PCR analysis of lin−CD34+ cells from 14 chronic phase CML patients and 3 normal BMs confirmed the differential expression of 14 candidate transcripts identified by SAGE (changes ranging from 3-fold lower to 80-fold higher in the CML cells, p<0.05). The altered levels of expression of 5 of these genes (i.e., beta-catenin, MLLT3, IL1R1, LY6E and GAS2) were highly correlated with the relative levels of BCR-ABL transcripts in the same cells (r ≥0.6). 5 of the 14 genes (IL1R1, vWF, SOX4, SELL and RHOB) were found to be differentially expressed in the 3-day post-transduction progeny of CD34+ cord blood cells exposed to a BCR/ABL-IRES-GFP vs a control GFP- lentivirus preparation (n=2). 3 (GAS2, DUSP1 and TP53BP2) were upregulated (5 to 11-fold) in imatinib-treated K562 cells (as compared to untreated K562 cells) but their expression remained unchanged in similarly treated KG1 cells (a primitive BCR-ABL-negative human AML cell line) providing further evidence that their deregulated expression is secondary to the kinase activity of p210BCR-ABL. In addition, from a comparison of the meta-library for the 34+38− CML cells with the meta-libraries for both the normal 34+38− and 34+38+ cells, the meta-library for the 34+38+ CML cells and most publicly accessible SAGE data, we were able to identify 65 novel tags in the 34+38− CML cells. 32 of these unique tags were located within conserved genomic regions and >2 kb away from known transcripts, and of these 32, 3 were confirmed to represent novel transcripts using a PCR approach. These results illustrate the potential of SAGE to reveal novel as well as known components in the transcriptomes of rare normal and cancer stem cell populations. Investigation of their roles in primitive human cells transduced with BCR-ABL and BCR-ABL+cell lines indicates the utility of these models for further delineation of the complex effects of BCR-ABL expression in chronic phase CML stem cells.

Human circulating monocytes as multipotential progenitors

Circulating monocytes are believed to be committed precursors for phagocytes, such as macrophages and dendritic cells. Recently, we have reported a primitive human cell population called monocyte-derived multipotential cells (MOMC), which has a fibroblast-like morphology and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. This novel cell type exhibits mixed morphologic and phenotypic features of monocytes, endothelial cells, and mesenchymal cells. MOMC are derived from circulating CD14+ monocytes, and their differentiation requires binding to fibronectin and exposure to one or more soluble factors derived from peripheral blood CD14- cells. MOMC contain progenitors with capacity to differentiate into a variety of non-phagocytes, including bone, cartilage, fat, skeletal and cardiac muscle, neuron, and endothelium. Recent studies by others have also described several distinct human cell populations that are originated from circulating monocytes and have capacity to differentiate into non-phagocytes. These observations together indicate that circulating monocytes are more multipotential than previously thought. In addition, cell transplantation therapies using circulating monocytes are a potential approach for tissue regeneration.

Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells

We recently reported the isolation of a unique subpopulation of human stromal cells from bone marrow (BM) termed marrow-isolated adult multilineage inducible (MIAMI) cells, capable of differentiating in vitro into mature-like cells from all three germ layers. The oxygen tension (pO 2) in BM ranges from 1 to 7%, which prompted us to examine the role of pO 2 in regulating the capacity of MIAMI cells both to self-renew and maintain their pluripotentiality (stemness) or to progress toward osteoblastic differentiation. MIAMI cells were grown under low-pO 2 conditions (1, 3, 5, and 10% oxygen) or air (21% oxygen). The proliferation rate of cells exposed to 3% oxygen (3 days) increased, resulting in cell numbers more than threefold higher than those of cells exposed to air (at 7 days). In cells grown under osteoblastic differentiation conditions, the expression of the osteoblastic markers osteocalcin, bone sialoprotein, osterix, and Runx2 and alkaline phosphatase activity was upregulated when incubated in air; however, it was blocked at low (3%) pO 2. Similarly, biomineralization of long-term cell cultures was high under osteoblastic differentiation conditions in air but was undetectable at low (3%) pO 2. In contrast, low pO 2 upregulated mRNAs for OCT-4, REX-1, telomerase reverse transcriptase, and hypoxia-inducible factor-1α, and increased the expression of SSEA-4 compared to air. Moreover, the expression of embryonic stem cell markers was sustained even under osteogenic culture conditions. Similar results were obtained using commercially available marrow stromal cells. We hypothesize a physiological scenario in which primitive MIAMI cells self-renew while localized to areas of low pO 2 in the bone marrow, but tend to differentiate toward osteoblasts when they are located closer to blood vessels and exposed to higher pO 2. Our results strongly suggest that maintaining developmentally primitive human cells in vitro at low pO 2 would be more physiological and favor stemness over differentiation.