ALBERT

Description: Abstract 2622 Numerous somatic cells can be reprogrammed by ectopic expression of defined transcription factors including OCT4, SOX2, MYC, KLF4 (Yamanaka's factors), NANOG, and LIN28 to pluripotent cells, referred to as induced pluripotent stem (iPS) cells which can then be differentiated into a variety of somatic cell types. Production efficiency of iPS cells by these transcription factors is extremely low, and therefore a large portion of the cells remain unprogrammed or incompletely reprogrammed. Thus, identification of additional factors required for enhancing iPS cells production efficiency has been an intensive research subject. We generated iPS cell from CD34+ cells of human umbilical cord blood that had been frozen in an unseparated state for twenty plus years using lentiviruses expressing Yamanaka's factors. During the iPS cell production processes, we monitored cell surface expression of TRA1-60, a marker for human embryonic stem (hES) cells, within colonies using a live cell staining method. Three to four weeks after gene transduction, TRA1-60 positive cells emerged in about 5% of the colonies. So, we mechanically separated TRA1-60 negative cells from TRA1-60 positive cells in colonies. Further culture did not allow TRA1-60 negative cells to convert to TRA1-60 positive cells even after 10 passages, indicating that TRA1-60 negative cells were stable at an incompletely reprogrammed state. TRA1-60 negative cells were similar to hES cells in morphology and still demonstrated expression of exogenous Yamanaka's factors. TRA1-60 negative cells were distinct from TRA1-60 positive cells with regards to methylation pattern of OCT4 promoter regions and differentiation potential. In contrast to TRA1-60 positive cells, incompletely reprogrammed TRA1-60 negative cells lacked hESC-specific miRNAs (miR-302 and 371 clusters), which are known to be involved in controlling self-renewal and pluripotency of hES cells. We hypothesized that these miRNAs can promote a transition from incompletely to fully reprogrammed iPS cells. To test this hypothesis, we introduced the miRNA clusters of miR-302 and 371 using lentiviruses to TRA1-60 negative incompletely reprogrammed cell to determine whether these miRNAs could convert TRA-60 negative cells to TRA1-60 positive completely reprogrammed cells. Our results showed that these miRNAs were able to convert more than 10% of TRA1-60 negative incompletely reprogrammed cells to TRA1-60 positive iPS cells with characteristics of completely reprogrammed iPS cells, such as differentiation of three germ layers and acquisition of typical hES cell-specific cell cycling patterns with an unrestricted G1 to S phase transition. These results indicate that hES-specific miRNAs have a strong potential to promote partially reprogrammed cord blood CD34+ cells to iPS cells with extensive self-renewal capacity. Our study suggests that current techniques with low iPS cell production efficiency can be improved by ectopic overexpression of hES-specific miRNAs (miR-302 and371 clusters). Disclosures: Broxmeyer: Corduse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Karyotypic abnormalities in cultured embryonic stem cells (ESCs), especially near-diploid aneuploidy, are potential obstacles to ESC use in regenerative medicine. Events causing chromosomal abnormalities in ESCs may be related to events in tumor cells causing chromosomal instability (CIN) in human disease. However, the underlying mechanisms are unknown. Using multiparametric permeabilized-cell flow cytometric analysis, we found that the mitotic-spindle checkpoint, which helps maintain chromosomal integrity during all cell divisions, functions in human and mouse ESCs, but does not initiate apoptosis as it does in somatic cells. This allows an unusual tolerance to polyploidy resulting from failed mitosis, which is common in rapidly proliferating cell populations and which is reduced to near-diploid aneuploidy, which is also common in human neoplastic disease. Checkpoint activation in ESC-derived early-differentiated cells results in robust apoptosis without polyploidy/aneuploidy similar to that in somatic cells. Thus, the spindle checkpoint is “uncoupled” from apoptosis in ESCs and is a likely source of karyotypic abnormalities. This natural behavior of ESCs to tolerate/survive varying degrees of ploidy change could complicate genome-reprogramming studies and stem-cell plasticity studies, but could also reveal clues about the mechanisms of CIN in human tumors.

Description: Ex-vivo expansion of human HSC prior to bone marrow transplantation is still an unrealized goal that could greatly extend the usefulness of this mainstay strategy for treating numerous human hematologic diseases. The safety of this process for potential use in humans depends in large part on the maintenance of karyotypic stability of HSC during expansion, a lack of which could contribute to serious, even fatal, complications such as cancer, and could also contribute to engraftment failure. The spindle checkpoint and its linkage to apoptosis initiation is one of the most important cellular processes that helps maintain chromosomal stability in rapidly proliferating cell populations by removing aneuploid and karyotypically abnormal cells via activation of cell death programs. Detailed understanding of the molecular regulation of this vital cell cycle checkpoint is important to maximize safety of in-vitro HSC expansion techniques. It is widely accepted that mammalian cells enter the next G1-phase with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint that it is from this state that polyploid/aneuploid cells initiate apoptosis. However, definitive biochemical evidence for G1 is scarce or unconvincing; in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to define important factors determining cell fate after mitotic slippage. Results from human and mouse embryonic stem cells that reenter polyploid cell cycles are compared to human somatic hematopoietic cells that die after MS. We now report for the first time that phosphorylation status of pRb, p53, CDK1, and cyclin B1 levels are important for cell fate/apoptosis decision in mitotic-slippage cells, which occurs in a unique, intervening, non-G1, tetraploid subphase. Hyperphosphorylated Rb was extremely abundant in mitotic-slippage cells, a cell signaling event usually associated with early G1-S phase transition. P53 was phosphorylated at sites known to be associated with apoptosis regulation. Cyclin A and B1 were undetectable in mitotic slippage cells; yet, CDK1 was phosphorylated at sites typically associated with its activation. Evidence is also presented raising the possibility of cyclin B1-independent CDK1 activity in mitotic-slippage cells. These findings challenge the current models of spindle checkpoint-apoptosis linkages. Our new model could have important implications for methods to maintain karyotypic stability during ex-vivo HSC expansion.

Description: Although many studies have been conducted on leukemia, only a few have analyzed the metabolomic profiles of various leukemic cells. In this study, the metabolomes of THP-1, U937, KG-1 (acute myelogenous leukemia, AML), K562 (chronic myelogenous leukemia, CML), and cord blood-derived CD34-positive hematopoietic stem cells (HSC) were analyzed using gas chromatography-mass spectrometry, and specific metabolic alterations were found using multivariate statistical analysis. Compared to HSCs, leukemia cell metabolomes were found to have significant alterations, among which three were related to amino acids, three to sugars, and five to fatty acids. Compared to CML, four metabolomes were observed specifically in AML. Given that overall more metabolites are present in leukemia cells than in HSCs, we observed that the activation of glycolysis and oxidative phosphorylation (OXPHOS) metabolism facilitated the incidence of leukemia and the proliferation of leukemic cells. Analysis of metabolome profiles specifically present in HSCs and leukemia cells greatly increases our basic understanding of cellular metabolic characteristics, which is valuable fundamental knowledge for developing novel anticancer drugs targeting leukemia metabolism.

Description: Hematopoietic stem cells (HSCs) reside in hypoxic niches within the bone marrow (BM). Yet, all HSC studies have been performed to date with cells immediately isolated in non-physiologic ambient air, whether or not they are subsequently processed in low oxygen tension. By collecting/manipulating BM in physiologically native conditions of hypoxia where all procedures are performed inside a hypoxic chamber, we demonstrate that brief exposure of mouse BM or human cord blood (CB) to ambient oxygen decreases recovery of phenotypically-defined and functional self-renewing long-term repopulating HSC and concomitantly increases numbers of progenitor cells, a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS). This new phenomenon is exquisitely sensitive to oxygen and great care must be taken to ensure all reagents, solutions, plastics, and anything that will come into contact with the cell suspension, is extensively pre-equilibrated in hypoxia. Up to 5-fold greater numbers of long-term (LT)-HSCs (CD34-CD150+Lin-Sca1+c-kit+CD41-CD48- or CD34-CD135-Lin-Sca1+c-kit+) could be recovered from mouse BM harvested in 3% O2 compared to BM harvested in air, or even BM harvested in 3% O2 and then exposed to air for as little as 30 min before analysis, even if subsequently returned to hypoxia. There was a concomitant decrease in short term-HSCs and multipotent progenitors when BM was harvested in hypoxia, an effect associated with decreased functional cytokine-stimulated colony formation of hematopoietic progenitor cells (HPC: CFU-GM, BFU-E, and CFU-GEMM). Similarly, if human CB was harvested under similar low oxygen conditions, a 3-fold increase in recovered HSCs (Lin-CD34+CD38-CD45RA+CD90+CD49f+) could be achieved compared to CB harvested in air. Using a custom mouse respirator to conduct competitive repopulating transplant experiments completely in a 3% O2 environment revealed an increase in competitive repopulating units (CRUs) up to more than 42 fold was recovered when BM is harvested in hypoxic conditions compared to air harvested BM in primary recipients, thus demonstrating the beneficial effects of hypoxic harvest on functional/transplantable HSCs with secondary transplant capability. These data strongly support the surprising conclusion that, until now, the true numbers of HSCs and the transplantation potency of BM and CB has been routinely and consistently underestimated because of rapid initiation of differentiation of LT-HSCs in ambient air. We present evidence linking mitochondrial function and cyclophilin D to EPHOSS. Genetically or pharmacologically suppressing cyclophilin D function, or p53 gene deletion link production of reactive oxygen species (ROS) to induction of the mitochondrial permeability transition pore (MPTP) as a molecular mechanism of EPHOSS, where rapid ROS generation in HSCs after exposure to “hyperoxic” room air initiates an irreversible cascade of differentiation signals (see illustration). We present additional evidence from gene knock-out model studies implicating roles for miR210 and Hif-1a in EPHOSS. The MPTP inhibitor, cyclosporine A, protects phenotypically-defined as well as functional and transplantable HSCs from EPHOSS during collection in air resulting in at least a 3-fold increase in HSC recovery as well as increased transplantation potency. Thus, pharmacological mitigation of EPHOSS during HSC collections for use in patient transplantation procedures may be clinically advantageous. Because cyclosporine A is already in use clinically, this EPHOSS-reducing strategy may be readily and easily tested for efficacy in a hospital setting. Because many different adult stem cells exist naturally in hypoxic niches, EPHOSS is likely relevant to other stem cells routinely harvested in air. Evidence suggests that aged HSCs may be more sensitive to the deleterious effects of EPHOSS than young HSCs. We propose that metabolic profiling of stem cells, including cancer stem cells, may not accurately represent the metabolism, behavior, and responses of these cells as they exist in their native hypoxic environments because they are harvested and studied in air. Thus, experimental designs that include a consideration of EPHOSS effects may be required to obtain a more complete understanding of stem cell metabolism and biology especially as it relates to stem cell aging or responses of cancer stem cells to chemotherapy. Figure 1 Figure 1. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

Description: Dipeptidylpeptidase 4 (DPP4) is a serine peptidase with enzymatic activity leading to the N terminal cleavage of select penultimate amino acids of proteins. We previously published that the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, as well as the ability of DPP4 T proteins to induce signaling that FL factors can not, have not been previously investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating the here to fore unknown ability of DPP4 cleavage of proteins to alter not only cellular function, but intracellular signaling of growth factors leading to miRNA expression, phosphorylation, and global induction of proteins that the FL form of the protein does not induce. Additionally, and unexpectedly, a 1:1 mixture of the DPP4 T protein and their FL counterpart leads to both overlapping signaling between the FL and T, as well as unique signaling, that is not induced by the FL or T protein alone. This suggests multifaceted, important, and currently unappreciated roles that the DPP4 truncation of proteins may play in the regulation of normal and malignant hematopoiesis as well as other physiologic and pathophysiologic states. From functional data, utilizing the human factor-dependent TF-1 cell line, as well as primary samples from human cord blood (CB) and patients with Acute Myeloid Leukemia (AML), we observed that DPP4 truncation of GM-CSF (T-GM-CSF) and IL-3 (T-IL-3) results in decreased colony stimulating factor (CSF) activity in normal and malignant hematopoietic progenitor cells (HPC). Receptor binding studies confirmed that both T-GM-CSF and T-IL-3 have enhanced receptor affinity compared to their FL form and each can compete to blunt the receptor binding of either FL-GM-CSF or FL-IL-3. In vivo studies demonstrated that either exogenously added T-GM-CSF or T-IL-3 suppressed the effects of exogenously added FL-GM-CSF or FL-IL-3 on progenitor cell numbers per femur and diminished HPC cycling. Investigation of FL vs T mediated signaling alterations was done with TF-1 cells using proteomic and bioinformatic analysis of miRNA expression, and phosphorylation/global protein induction. Both T-GM-CSF and T-IL-3 induced unique signaling that FL-GM-CSF and FL-IL-3 did not, as well as signaling that overlapped with their FL counterparts (Figure 1, Venn Diagram example of global induction of proteins). Additionally, treatment with a 1:1 ratio of the FL/T proteins resulted in both distinctive, and common, signaling compared to that detected with treatment of only FL or T molecules, thus revealing the complexity of the signaling interactions and heretofore unknown activities of DPP4 truncated proteins (Figure 1). Fisher's exact test or B-H multiple testing correction in IPA software were used to statistically generate comparisons of molecules associated with specific signaling induction, and differential as well as overlapping signaling for all GM-CSF vs IL-3 groups. As examples, for both the GM-CSF and IL-3 groups, molecules associated with PI3K/Akt signaling were increased in T and 1:1 groups vs treatment with FL molecules. In contrast, molecules affiliated with CDC42 signaling were induced by treatment with a 1:1 ratio of FL:T molecules but not in the FL or T groups alone for both GM-CSF and IL-3. Interestingly, phosphorylated proteins associated with Flt3 and IL-9 signaling in hematopoietic progenitors was increased in the T and 1:1 groups for GM-CSF and IL-3 compared to the FL groups. Conversely, phosphorylated molecules involved in nucleic acid metabolism were induced by the FL, T and 1:1 of the GM-CSF groups but only by the FL version of IL-3. Further, phosphorylated molecules associated with antigen presentation were only detected in the FL IL-3 and 1:1 IL-3 groups, and molecules functionally associated with carbohydrate metabolism and cellular therapeutics were only detected in the T and 1:1 groups for both GM-CSF and IL-3, and not in the FL stimulated groups. Collectively, these data substantiate that further investigation into the roles and regulation of DPP4, in normal and malignant hematopoiesis, will allow for a better understanding of the significance, and potential clinical utility, of DPP4 activity altering compounds as well as DPP4 truncated molecules. Figure 1 Figure 1. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 2353 Embryonic stem cells (ESCs) are pluripotent, self-renew and can be differentiated into cells of all three germ layers, and nanog, Oct4 and Sox2, form a core of the self-renewal transcription network. Nanog expression is restricted to pluripotent cells and is down regulated upon differentiation; little is known about its regulation. Expression of the OCT4 gene maintains cell pluripotency via a stringent dose-dependent regulation with OCT4 levels above or below required dosages producing cellular differentiation; thus maintenance of a critical amount of OCT4 is necessary to prevent ESC differentiation. Sox2, a high-mobility group domain containing transcription factor, binds to the consensus motif CATTGTT. We recently reported in Blood that Tip110 is an essential gene expressed in earliest cells of adult bone marrow hematopoietic development. Increased TIP110 expression enhanced hematopoietic progenitor cell (HPC) numbers, survival, and cell cycling; decreased Tip110 expression manifested the opposite effect, demonstrating a role for TIP110 in regulation of hematopoiesis. Herein, we investigated TIP110 expression and actions in human (h)ESCs. Quantitative RT-PCR showed that TIP110, as well as Nanog, Oct4 and Sox2 were expressed in a hESC line. hESCs were removed from feeder layers and b-FGF for 5 days, to allow ESC differentiation. TIP110 expression levels were dramatically reduced (by 77%); this was associated with large decreases in expression of NANOG (82%), OCT4 (80%), and SOX2 (85%). We then assessed whether TIP110 might regulate hESC pluripotency. We exogenously over-expressed TIP110 in hESC cells. Feeder layers and b-FGF were withdrawn upon introducing the TIP110 vector and cells cultured for 5 days to test whether sustained TIP110 expression rendered ESCs less sensitive to differentiation. Compared with controls, TIP110 over-expressing cells stained positive for OCT4, NANOG and were negative for Tuji, SMA and AFP, demonstrating that over-expression of TIP110 rendered ESCs less responsive to differentiation. Next, we reduced TIP110 expression by transfection of the hESCs with TIP110 siRNA. Cells were cultured in mTeSR medium on Matrigel-coated dishes for an additional 5 days in order to maintain cells under undifferentiation conditions. TIP 110 siRNA vector expressing cells were negative for OCT4, NANOG, and positive for Tuji, SMA and AFP expression compared with control cells, demonstrating that enforced reduction of TIP110 expression in hESCs causes hESC differentiation. This demonstrated the importance of TIP110 in maintenance of ESC pluripotency. We speculated that TIP110 maintenance of hESC pluripotency might be through regulation of NANOG, OCT4 and SOX2. We silenced TIP110 expression in hESCs by transfection with a TIP110 siRNA vector, previously shown to reduce TIP110 expression by 70%. Cells were cultured in complete 20% KSR hESC medium for an additional 5 days. Expression of these three transcription factors was dramatically decreased, demonstrating that TIP110 is required for maintaining NANOG, OCT4 and SOX2 levels in this hESC line. Reduction of TIP110 expression caused hESC differentiation directly or indirectly through down-regulation of NANOG, OCT4 and SOX2 expression. Thus, TIP110 is preferentially expressed in the undifferentiated state in hESCs and plays a key role in regulating OCT4, SOX2, and NANOG, factors required to maintain pluripotency. Together, our present and previous studies suggest TIP110 expression as a useful marker to distinguish early from more differentiated cells. Modulating TIP110 expression in a controlled fashion may be relevant for cellular engineering and regenerative medicine. Disclosures: No relevant conflicts of interest to declare.