Production Of Progenitor Cells Research Articles

Stem Cell Dysfunction Exacerbates Muscular Dystrophy

Duchenne muscular dystrophy (DMD), characterized by the absence of the dystrophin protein, has been considered a disease caused by muscle fiber fragility. However, new research shows that impaired muscle stem cells also have an important role in muscle degeneration (Dumont NA et al. Nat Med. doi:10.1038/nm.3990 [published online November 16, 2015]). Through experiments conducted in mice, investigators from the University of Ottawa and their colleagues discovered that, like muscle fiber cells, muscle stem cells express the dystrophin protein. Without dystrophin, muscle stem cells lost their ability to establish cellular polarity, which impaired cell division and reduced the production of muscle progenitor cells needed to form functional muscle fibers. These stem cell defects prevented the repair of damaged muscle tissue, contributing to muscle wasting in DMD.

Taking the Leap: Runx1 in the Formation of Blood from Endothelium.

Blood cell formation in the embryo occurs from multiple anatomic sites and results in the production of hematopoietic stem and progenitor cells that appear in overlapping waves. The transcription factor Runx1 is involved in a dramatic step of this process, for the transition from an endothelial cell that is integrated in a monolayer to a nonadherent circulating blood cell, a process conceptually similar to the epithelial to mesenchymal cell transition. Here we will review the role of Runx1 in the so-called hemogenic endothelium. We will describe the blood cell progenitors for which Runx1 is required, the proximal upstream transcription factors and signaling events that regulate its expression, and some of its important downstream targets in the hemogenic endothelium.

Abstract A11: Myeloid-derived suppressor cells suppress immune surveillance and stimulate cancer cell proliferation/metastasis through activation of mTOR pathway in lal-/- mice

Abstract Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. MDSCs development and homeostasis is controlled by lysosomal acid lipase (LAL), a critical enzyme in the metabolic signaling pathway that hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in lysosomes. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs. LAL-deficient (lal-/-) MDSCs arise from dysregulated production of progenitor cells in the bone marrow. In humans, increased CD14+CD16+ and CD14+CD33+ MDSC subsets have been reported with heterozygote carriers of LAL mutations. Patients with mutations in the LAL gene has been reported to be associated with carcinogenesis. In the LAL-deficient (lal-/-) mouse model, melanoma metastasized massively in allogeneic lal-/- mice, which was suppressed in allogeneic lal+/+ mice due to immune rejection. Here we report for the first time that MDSCs isolated from lal-/- mice directly stimulated B16 melanoma cell proliferation in vitro, and growth and metastasis in vivo. Cytokines i.e., IL-1β and TNFα from MDSCs are required for B16 melanoma cell proliferative function in vitro. Myeloid-specific expression of human LAL (hLAL) in lal-/- mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal-/- MDSCs is mediated, at least in part, through over-activation of the mammalian target of rapamycin (mTOR) pathway. mTOR is a serine/threonine protein kinase that regulates cell growth, proliferation, migration, survival, protein synthesis, and transcription in response to growth factors and mitogens. mTOR is the catalytic subunit of two distinctive complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Unique accessory proteins, regulatory-associated protein of mTOR (RAPTOR), and rapamycin-insensitive companion of mTOR (RICTOR) define the TORC1 and TORC2 complexes, respectively. Affymetrix GeneChip microarray analysis and Ingenuity Pathway Analysis of gene transcripts revealed up-regulation of multiple genes in the mTOR signaling pathway in lal-/- MDSCs. We have shown that inhibition of mTOR in lal-/- MDSCs increased apoptosis, decreased ATP synthesis, decreased ROS production, and recovered impairment of mitochondrial membrane potential. As a result, lal-/- MDSCs reversed the increased cell proliferation, corrected enhanced lal-/- MDSCs development from lineage negative progenitor cells, and reduced systemic MDSC expansion. More importantly, knockdown of mTOR, Raptor or Rictor in lal-/- MDSCs reversed the immune suppression on T cell proliferation and function, as well as suppressed their stimulation on proliferation of cancer cells (including B16 melanoma, LLC and Tramp-C2 cancer cells). Our results indicate that LAL plays a critical role in regulating MDSCs through modulation of the mTOR pathway. MDSCs possess dual functions to overcome immune rejection and facilitate cancer metastasis: 1) suppress immune surveillance, and 2) stimulate cancer cell proliferation, growth, and metastasis. Grant support: This work was supported by National Institutes of Health Grants CA138759, CA152099 (to C. Y.) and HL087001 (to H. D.). Citation Format: Hong Du, Ting Zhao, Xinchun Ding, Katlin Walls, Cong Yan. Myeloid-derived suppressor cells suppress immune surveillance and stimulate cancer cell proliferation/metastasis through activation of mTOR pathway in lal-/- mice. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A11.

Cryopreserved Ex Vivo-Expanded Allogeneic Myeloid Progenitor Cell Product Protects Neutropenic Mice From a Lethal Fungal Infection.

Severe neutropenia induced by chemotherapy or conditioning for hematopoietic cell transplantation often results in morbidity and mortality due to infection by opportunistic pathogens. A system has been developed to generate ex vivo-expanded mouse myeloid progenitor cells (mMPCs) that produce functional neutrophils in vivo upon transplantation in a pathogen challenge model. It has previously been demonstrated that transplantation of large numbers of freshly isolated myeloid progenitors from a single donor provides survival benefit in radiation-induced neutropenic mice. In the present work, an ex vivo-expanded and cryopreserved mMPC product generated from an allogeneic donor pool retains protective activity in vivo in a lethal fungal infection model. Infusion of the allogeneic pooled mMPC product is effective in preventing death from invasive Aspergillus fumigatus in neutropenic animals, and protection is dose dependent. Cell progeny from the mMPC product is detected in the bone marrow, spleen, blood, and liver by flow cytometry 1 week postinfusion but is no longer evident in most animals 4 weeks posttransplant. In this model, the ex vivo-generated pooled allogeneic mMPC product (i) expands and differentiates in vivo; (ii) is functional and prevents death from invasive fungal infection; and (iii) does not permanently engraft or cause allosensitization. These data suggest that an analogous ex vivo-expanded human myeloid progenitor cell product may be an effective off-the-shelf bridging therapy for the infectious complications that develop during hematopoietic recovery following hematopoietic cell transplantation or intensive chemotherapy.

Aryl Hydrocarbon Receptor Antagonism Promotes Hematoendothelial Development from Human Pluripotent Stem Cells

The aryl hydrocarbon receptor (AHR) is an evolutionarily conserved transcription factor originally characterized for its role in mediating biological responses to carcinogenic environmental agents. Recent studies have elucidated the importance of AHR-dependent signaling for normal physiological function in the absence of environmental ligands, most notably the development of Th17 cells, regulatory T-cells, and natural killer (NK) cells from human peripheral blood and umbilical cord blood. Additionally, AHR is highly expressed in hematopoietic stem/progenitor cells (HSPCs), and antagonism of AHR using small molecules results in a marked expansion of umbilical cord blood-derived HSPCs suitable for clinical transplantation. It remains unclear what role, if any, AHR plays during early human hematoendothelial development. We hypothesized inhibition of AHR-mediated cell signaling could promote early human hematopoietic cell development. To model human hematopoiesis, we employed a xenogeneic-free and chemically defined in vitro method to differentiate human embryonic stem cells (hESCs) into endothelial and hematopoietic cells. qRT-PCR analysis demonstrated a significant increase in AHR (13.36±5.52 fold change, p<0.05, n=3) by Day 11 of differentiation relative to undifferentiated hESCs. CYP1A1 and CYP1B1, two downstream targets of AHR-mediated signaling, were similarly upregulated on Day 11 (27.90±6.17 fold change, p<0.05, n=3; 134.28±10.06 fold change, n=3, respectively). Increase in AHR expression mirrored the onset of early hematopoietic progenitor cell differentiation; CD34+ CD43+ and CD34+ CD41a+ cells were markedly increased by Day 12 of hematopoietic differentiation as assessed by flow cytometry (18.9%±3.22, p<0.01, n=7; 8.23±2.00, p<0.05, n=7, respectively). We next modified the relative activity of AHR signaling by differentiating hESCs in the presence of 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD), a prototypical AHR agonist, or StemReginin-1 (SR-1), an AHR antagonist, and assessed its effects on hematopoietic progenitor cell production. Interestingly, we observed a significant increase in the appearance of both CD34+ CD31+ hematoendothelial cells in SR-1 treated hESCs relative to DMSO treated controls (17.63%±1.25, p<0.05, n=3 vs. 11.21±0.63, p<0.05, n=3) at Day 9. Later by Day 12, we also found approximately a two-fold expansion of CD34+ CD45+ hematopoietic progenitor cells in SR-1 treated hESCs relative to DMSO treated controls (16.35%±4.05, p<0.05, n=3 vs. 7.53±0.19, p<0.05, n=3). Treatment with TCDD reciprocally attenuated the development of CD34+ CD45+ progenitor cells at Day 15 relative to DMSO treated controls (3.99%±0.80 vs. 11.79%±1.41, p<0.05, n=3) and resulted in an expansion of terminally differentiated hematopoietic cells (CD34- CD43+: 84.5%±2.78 vs. 70.9±1.58, p<0.05, n=3; CD34- CD45+: 81.75%±1.75 vs. 71.95±2.35, p<0.05, n=3). We confirmed the functionality of the hematopoietic progenitor cells in each group by harvesting non-adherent cells at Day 12 and performing standard colony-forming assays. SR-1 treated cells yielded a 4-fold increase in the total number of colonies generated relative to DMSO treated control cells along with an increased proportion of CFU-M and CFU-GM. We also evaluated whether AHR antagonism could be used to promote NK cell differentiation from hESCs. Using previously optimized and defined NK cell differentiation conditions, we found SR-1 treatment caused an increase in CD56+ CD45+ NK cells relative to DMSO treated controls (26.4% vs. 19.7%, n=2) whereas TCDD treatment caused a decrease (6.7%, n=2). Work assessing how hematopoiesis from hESCs is affected using AHR gene knockouts developed from CRISPR/Cas9-mediated gene deletion is ongoing. Collectively, our results demonstrate AHR antagonism promotes early human hematoendothelial development and may be used as a potential molecular target to enhance hematopoietic cell production from human pluripotent stem cells for clinical applications. DisclosuresNo relevant conflicts of interest to declare.

Altered Hippocampal Neurogenesis and Amygdalar Neuronal Activity in Adult Mice with Repeated Experience of Aggression.

Repeated experience of winning in a social conflict setting elevates levels of aggression and may lead to violent behavioral patterns. Here, we use a paradigm of repeated aggression and fighting deprivation to examine changes in behavior, neurogenesis, and neuronal activity in mice with positive fighting experience. We show that for males, repeated positive fighting experience induces persistent demonstration of aggression and stereotypic behaviors in daily agonistic interactions, enhances aggressive motivation, and elevates levels of anxiety. When winning males are deprived of opportunities to engage in further fights, they demonstrate increased levels of aggressiveness. Positive fighting experience results in increased levels of progenitor cell proliferation and production of young neurons in the hippocampus. This increase is not diminished after a fighting deprivation period. Furthermore, repeated winning experience decreases the number of activated (c-fos-positive) cells in the basolateral amygdala and increases the number of activated cells in the hippocampus; a subsequent no-fight period restores the number of c-fos-positive cells. Our results indicate that extended positive fighting experience in a social conflict heightens aggression, increases proliferation of neuronal progenitors and production of young neurons in the hippocampus, and decreases neuronal activity in the amygdala; these changes can be modified by depriving the winners of the opportunity for further fights.

Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products

Background aimsRemoving DMSO post-thaw results in: reduced infusion reactions, improved recovery and stability of viable CD34+ cells. Validated methods use 5%–8.3% Dextran 40 with 2.5%–4.2% HSA for this purpose. Recent shortages of clinical grade Dextran require identification of suitable alternatives. MethodsPBPC were used to compare a standard 2X wash medium of 5 parts 10% Dextran 40 in saline (DEX) with 1 part 25% HSA (8.3% DEX/ 4.2% HSA) with Hydroxyethyl Starch (HES)-based solutions. Cells in replicate bags were diluted with an equal volume of wash solution, equilibrated 5 minutes, the bag filled with wash medium, pelleted and the supernatant expressed. Bags were restored to the frozen volume in wash medium and tested by single platform flow cytometry and CFU. Total viability, viable TNC, MNC, and CD34+ cell recovery, and CD34+ cell viability were compared immediately post-thaw and after 90 minutes. Results5.2% HES/4.2% HSA did not differ from our standard in CD34 recovery or viability. Due to concerns that high concentrations of HES could affect renal function we tested 0.6% HES/2.5% HSA resulting in significantly poorer CD34 recovery and viability. Results improved using 2.4% HES/4.2% HSA and when 0.6% HES/4.2%HSA was used no significant differences were seen. CFU assays confirmed no differences between the standard dextran arm and HES at 2.4% or 0.6% so long as HSA was at 4.2%. ConclusionsWe conclude that HES from 0.6% to 5.2% with 4.2% HSA is a suitable substitute for Dextran 40 as a reconstitution/washing medium for PBPC products.

Rictor is pivotal for the emergence and function of embryonic hematopoietic stem cells

Engineering hematopoietic stem cells (HSC) from pluripotent stem cells has tremendous clinical significance. Despite efficient production of hematopoietic progenitor cells (HPC), the generation of authentic HSC remains unsuccessful, reflecting poor understanding of regulatory mechanisms specifically regarding HSC ontogeny. Here, we disrupted Rictor, a core mTORC2 component, at distinct developmental stages with Cre/LoxP strategy. Rictor deficiency from embryonic endothelial stage (using Tie2-Cre) had minimal effect on the generation of definitive HPC in vivo and in vitro. In striking contrast, the endothelial emergence of first cohort of HSC was severely blocked in the AGM region. only 1 out of 15 recipients of Tie2-Cre;Rictorf/f AGM cells was successfully repopulated with markedly lower chimerism (15.3%), whereas 12 out of 19 recipients of the control AGM cells were repopulated with a much higher long-term chimerism (54.5 ± 25.1%). Interestingly, deleting Rictor from developing hematopoietic stage (using Vav-Cre), other than within adult bone marrow (using Mx1-Cre), significantly impaired the HSC function in vivo. When E11.5 control AGM and E12.5 FL cells were transplanted, all recipients were repopulated with an average long-term chimerism of 58.2±2.1% and 77.2±11.1% respectively as compared with the markedly reduced chimerism of Vav-Cre;Rictorf/f AGM group (29.9±14.6%) and FL group (37.0±16.3%) respectively. We thus demonstrate that Rictor is vital for endothelial transition to HSC (other than HPC) and for HSC function at embryonic stage (other than in adult), suggesting its hierarchy-and stage-restricted regulatory trait.

Arsenic and Its Methylated Metabolites Inhibit the Differentiation of Neural Plate Border Specifier Cells.

Exposure to arsenic in food and drinking water has been correlated with adverse developmental outcomes, such as reductions in birth weight and neurological deficits. Additionally, studies have shown that arsenic suppresses sensory neuron formation and skeletal muscle myogenesis, although the reason why arsenic targets both of these cell types in unclear. Thus, P19 mouse embryonic stem cells were used to investigate the mechanisms by which arsenic could inhibit cellular differentiation. P19 cells were exposed to 0, 0.1, or 0.5 μM sodium arsenite and induced to form embryoid bodies over a period of 5 days. The expression of transcription factors necessary to form neural plate border specifier (NPBS) cells, neural crest cells and their progenitors, and myocytes and their progenitors were examined. Early during differentiation, arsenic significantly reduced the transcript and protein expression of Msx1 and Pax3, both needed for NPBS cell formation. Arsenic also significantly reduced the protein expression of Sox 10, needed for neural crest progenitor cell production, by 31-50%, and downregulated the protein and mRNA levels of NeuroD1, needed for neural crest cell differentiation, in a time- and dose-dependent manner. While the overall protein expression of transcription factors in the skeletal muscle lineage was not changed, arsenic did alter their nuclear localization. MyoD nuclear translocation was significantly reduced on days 2-5 between 15 and 70%. At a 10-fold lower concentration, monomethylarsonous acid (MMA III) appeared to be just as potent as inorganic arsenic at reducing the mRNA levels Pax3 (79% vs84%), Sox10 (49% vs 65%), and Msx1 (56% vs 56%). Dimethylarsinous acid (DMA III) also reduced protein and transcript expression, but the changes were less dramatic than those with MMA or arsenite. All three arsenic species reduced the nuclear localization of MyoD and NeuroD1 in a similar manner. The early changes in the differentiation of neural plate border specifier cells may provide a mechanism for arsenic to suppress both neurogenesis and myogenesis.

Expression of a surface antigen (MA6) by peripheral blood CD34+ cells is correlated with improved platelet engraftment and may explain delayed platelet engraftment following cord blood transplantation.

CD34(+) cell dose provides a measure of hematopoietic tissue that predicts the rate of engraftment upon transplant. It is positively correlated with multiple measures of hematopoietic recovery, including platelet engraftment. Here we identify a subpopulation of CD34(+) cells that coexpress a surface antigen--MA6, which is more positively correlated with platelet engraftment in a clinical setting than CD34(+) alone. The specific identity and function of MA6 remain to be determined, however, it is expressed by primitive megakaryocyte (MK) progenitors, but is lost with differentiation and is not expressed by platelets. Commitment of CD34(+)MA6(+) cells to the MK lineage was confirmed by in vitro assays and their significance in hematopoietic transplantation explored by flow cytometric analysis of cryopreserved samples of granulocyte colony stimulating factor-mobilized peripheral blood progenitor cell (PBPC) products along with a retrospective analysis of platelet engraftment data. Platelet engraftment by day 21 was predicted by receipt of ≥ 6 × 10(6) CD34(+) cells/kg or ≥ 0.3 × 10(6) CD34(+)MA6(+) cells/kg. Subsequent analysis of cord blood (CB) CD34(+) cells revealed <0.2% coexpressed MA6(+), compared to 8% of PBPC CD34(+) cells. This low proportion of CD34(+)MA6(+) cells may be responsible, at least in part, for the delayed platelet engraftment associated with CB transplantation. However, platelet engraftment is markedly improved in recipients of ex vivo-expanded CB. This may be a consequence of an increased proportion of CD34(+)MA6(+) cells present in the ex vivo-expanded product and also suggests that optimizing ex vivo culture conditions to generate CD34(+)MA6(+) cells might further improve platelet engraftment in CB recipients.

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

SUMMARYNeural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Scaffold-free three-dimensional culture systems for mass production of periosteum-derived progenitor cells

Mesenchymal stem cells (MSCs) are capable of self-renewal and can differentiate into various types of cells for therapeutic purposes. MSCs are frequently cultured in a two-dimensional (2D) system. However, MSCs can lose their differentiation capacity over time in this culture system. In addition, the available surface area for the propagation of cells is limited. Therefore, various three-dimensional (3D) culture systems have been developed. In this study, we developed the scaffold-free 3D culture systems for the expansion of periosteum-derived progenitor cells (PDPCs) as spheres. The spheroid formation of PDPCs was induced using a rotation platform. The spheres maintained their viability and proliferation ability. Moreover, expression levels of the stemness marker genes and proteins were higher in cells grown on 3D culture system than in 2D culture system. In conclusion, a simple and economical 3D culture system has been developed that can increase the potential of PDPCs for clinical use.

Phenotypic and functional comparison of mobilized peripheral blood versus umbilical cord blood megakaryocyte populations

Background aimsHematopoietic stem cell transplantation of mobilized peripheral blood progenitor cell (PBPC) products results in rapid platelet engraftment, whereas the use of cord blood (CB) shows significant delays. The difference in the quality and number of megakaryocyte (MK) progenitors that may be responsible for the delay in platelet engraftment has not been fully defined. The objective of this study was to quantify the cells of the MK lineage in PBPC and CB products to determine whether potential differences exist. MethodsWe examined PBPC or CB for differences in surface markers and subpopulations as well as polyploidization status within the MK lineage. Colony-forming assays were used to determine whether differences exist in the clonogenic MK progenitor cell. Finally, we transplanted PBPC and CB mononuclear cells into NOD/SCID/IL2Rγ−/− (NSG) mice to study platelet engraftment rates. ResultsEquivalent MK populations and polyploidization was observed in PBPCs and CB. MK progenitors were present only in CD34+ cells and had little difference in colony growth between PBPC and CB. Additionally, MK subpopulations were similar in either product with a slightly more progenitor-enriched phenotype in CB. Finally, when PBPC or CB was transplanted at similar doses, equivalent platelet engraftment rates were observed. ConclusionsPBPC and CB contain similar frequencies of MK populations, and, when transplanted in comparable doses, CB is as effective as PBPCs in producing platelet engraftment in vivo. Understanding the differences in MK populations between PBPC and CB could help generate protocols to improve platelet engraftment after CB transplantation.

Mutations of Calreticulin in Myeloproliferative Neoplasms Contribute to Disease Progression Not through Inhibition of Phagocytosis, but through Enhanced Proliferation Production of Myeloid Myelo-Erythroid Progenitor Cells

Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders characterized by overproduction of mature myeloid cells. Recently, somatic mutation of calreticulin (CALR) was frequently found in MPN patients who do not have JAK2 mutation. The CALR mutation in MPN patients usually resulted in loss-of-function of CALR, which may induce impairment of physiological phagocytotic pathway, because surface CALR plays a critical role for macrophages in recognition of low-density lipoprotein receptor-related protein 1 (LRP1) on the targets, mediating pro-phagocytic signals.We hypothesized that the non-functional CALR mutation renders cells resistant to phagocytosis, and impairs the “programmed cell removal” of progenitors or mature blood cells, resulting in accumulation of hematopoietic cells in MPNs. In 135 Japanese MPNs patients enrolled in this study, including polycythemia vera (PV), essential thrombocytosis (ET) or primary myelofibrosis (PMF), 34 patients (25.2%) had CALR mutations, and 80 (59.3%) patients had JAK2 V617F mutation, respectively. CALR mutations were heterozygous in all 34 patients (27 patients with ET, 7 with PMF). On the other hand, JAK2 V617F mutations were found in 26 patients with PV, 39 with ET, and 15 with PMF.The expression levels of pro-phagocytotic CALR were normal in these MPN patients. We then performed in vitro phagocytosis assay to test whether the heterozygous CALR mutation affects engulfment of blood cells by macrophages. Hematopoietic stem cells (HSCs), progenitor cell populations such as common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs) and granulocyte/monocyte progenitors (GMPs), and mature myeloid cells were isolated and opsonized, and were co-cultured with activated macrophages for 2 hours. After the culture, we enumerate macrophages and engulfed cells to analyze phagocytosis index (number of engulfed cells/number of macrophages) (Kuriyama et al. Blood 2012). However, the phagocytosis index was not changed in any of purified hematopoietic cells, irrespective of the presence of CALR or JAK2 mutation. These results strongly suggest that heterozygous, non-functional CALR mutation, and gain-of-function JAK2 mutations should not affect the engulfment process for hematopoietic cells by macrophages.We then investigated the effect of CALR or JAK2 mutations on differentiation and proliferation of stem or progenitor cells in MPNs. We performed colony-forming cell assay of multipotent cells, such as HSCs and CMPs, and evaluated clonal burden of CALR and JAK2 mutations in colonies derived from these stem and progenitor cells. In vitro culture showed that HSCs and CMPs with CALR and JAK2 mutations gave rise to granulocyte/monocyte (GM) or megakaryocyte/erythroid (MegE)-related colonies, whose frequencies were almost identical to those in wild-type controls, suggesting that these mutations do not affect myelo-erythroid lineage commitment at the multipotent stem or progenitor stages. In contrast, when we cultured GMPs and MEPs, frequencies of colonies with CALR or JAK2 mutations were significantly higher as compared to those in HSCs or CMPs (P<0.05); In patients with CALR mutation, 32.5% of HSC-derived colonies had CALR mutations, whereas in MEPs and GMPs, CALR mutations were found in 51.0% and 70%, respectively. In JAK2 mutated MPNs, 17.2% of HSC-derived colonies had JAK2 mutation, whereas 64.7% of MEP- and 87.9% of GMP-derived colonies had this mutation. These results indicate that clones with CALR or JAK2 mutations could contribute more robustly to maintain MEPs and GMPs, and these committed progenitors with mutations might produce higher amounts of mature myelo-erythroid cells, leading to progression of MPNs.Thus, CALR mutation contributes to progression of MPN, not through inhibition of phagocytic clearance, but presumably through enhanced production of myelo-erythroid lineage cells, as JAK2 mutation does. It is important to investigate the mechanism on which the CALR mutation causes overproduction of myelo-erythroid cells in future study. DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetic and Pharmacodynamic Properties of Romiplostim

Background:Chronic Immune thrombocytopenia (ITP) is characterized by low platelet counts, resulting from increased platelet destruction and inadequate platelet production. Romiplostim is a 59 kDa peptibody which binds to and activates the thrombopoietin (TPO) receptor on platelet precursors in the bone marrow, and increases platelet counts. This analysis integrates the pharmacokinetic (PK) and pharmacodynamic (PD) properties of romiplostim in animals, healthy volunteers and patients with ITP, and describes its intricate PK-PD inter-relationship.Methods and Results:In healthy subjects, over a wide range of doses examined, the PK and PD (platelet response) of romiplostim were dependent on both the dose administered and the baseline platelet counts. Following SC administration, platelet counts increased in a dose-dependent fashion after 4 to 9 days, peaking at 12 to 16 days (Wang Clin Pharmacol Ther. 2004;76:628-38). When romiplostim binds to the TPO receptor on megakaryocytes and platelets, the peptibody-receptor complex is internalized and degraded inside the cells. Therefore, as platelet counts increase, a higher number of free receptors are available to clear romiplostim (Wang AAPS J. 2010;12:729-40). Results from rodent studies suggest that as the dose increases, the TPO receptors become saturated and the contribution of the kidney to clearance increases. Additionally, proteolysis plays a role in the clearance of romiplostim; however, the cytochrome P450 enzymes are not involved in protein catabolism (Wang Pharm Res. 2011;28:1931-8), hence there are no known drug-drug interactions or dietary restrictions (Nplate Prescribing Information 2014).Following SC administration, serum concentrations of romiplostim were markedly lower, however, platelet response was similar after the same dose of intravenous (IV) and SC administration (Wang Clin Pharmacol Ther. 2004;76:628-38). This suggests that the PD response is driven by the length of time that the romiplostim concentrations remained above a threshold rather than by the magnitude of concentrations achieved. This effect was verified in a mechanistic PK-PD modeling study in animals (Krzyzanski Pharm Res. 2013;30:655-69). In patients with ITP receiving SC romiplostim at a dose of 1 mcg/kg, the peak platelet response was achieved at 18 days (range 8 to 43; Bussel N Engl J Med. 2006;355:1672-81). Pharmacodynamic model analysis showed that compared with healthy subjects, patients with ITP had a shorter platelet life span and a decreased rate of production of progenitor cells, but no major difference in the time to maturation of megakaryocytes. The PD response in this modeling analysis was not notably affected by age, body weight, sex, and race (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51). The frequency of once-weekly dosing was selected because once every 2 weeks dosing was determined to be inadequate to achieve and maintain platelet counts in the therapeutic range (Bussel N Engl J Med. 2006;355:1672-81). A mechanistic PK-PD model based on data from the healthy subjects further suggested that weekly dosing resulted in a sustained platelet response while dosing less frequently resulted in high fluctuation of platelet counts (Wang AAPS J. 2010;12:729-40). Large inter- and intra-individual variability in the PD response was observed at a given dose; therefore, dose adjustments should be made based on a patient’s platelet counts, using a titrated dosing scheme to prevent having platelet counts over 400 x 109/L (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51).Conclusion:Romiplostim is a peptibody that binds and activates the TPO receptor, and consequently increases platelet production in individuals with chronic ITP. The peptibody-receptor complex is internalized and degraded inside the cells, without involvement of the liver. Romiplostim’s PD response is driven by the length of time that its concentrations remained above a threshold rather than by the magnitude of concentrations achieved. Moreover, weekly dosing has demonstrated a sustained platelet response while less frequent dosing resulted in fluctuating platelet counts. DisclosuresArkam:Amgen Inc.: Employment, Equity Ownership. Off Label Use: Romiplostim is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to corticosteroids, immunoglobulins, or splenectomy. This abstract also describes PK data from healthy volunteers.. Doshi:Amgen Inc.: Employment, Equity Ownership. Yang:Amgen Inc.: Employment, Equity Ownership.

Infusion of a Non HLA-Matched Off-the-Shelf Ex Vivo Expanded Cord Blood Progenitor Cell Product Following Myeloablative Cord Blood Transplantation Is Safe, Decreases the Time to Hematopoietic Recovery, and Results in Excellent Overall Survival

Background: Myeloablative cord blood transplant (CBT) recipients experience delayed hematopoietic recovery and an increased risk of transplant related mortality (TRM). With the primary goal of generating increased numbers of hematopoietic stem and progenitor cells (HSPC) to supplement a conventional cord blood (CB) graft and thereby enhance engraftment and reduce early TRM, we have developed methods to ex vivo expand CB-derived HSPC in the presence of Notch ligand for 14 days. The product is then harvested and cryopreserved for future clinical use. We have previously reported that infusion of non-HLA matched, off-the-shelf ex vivo expanded CB progenitor cells in patients undergoing myeloablative CBT is safe and associated with faster neutrophil (ANC) recovery. We now present updated outcomes data with a median follow-up of 3.2 years (range 1.9-3.7).Methods: Between September 2010 and August 2012, 15 patients with hematologic malignancies were enrolled in this single center Phase I trial to assess the safety of infusing a non-HLA matched expanded CB HSPC product to augment conventional CBT. Conditioning consisted of Fludarabine (75mg/m2), Cytoxan (120mg/kg) and TBI (13.2 Gy). On the day of transplant, 1 or 2 unmanipulated CB units were infused first followed 4 hours later by infusion of the expanded CB product. Donor(s)/host chimerism studies were performed weekly from day 7 to 28, then at days 80, 180 and 1 year on peripheral blood flow sorted into myeloid and lymphoid fractions. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine and MMF beginning on day -3. ANC and platelet engraftment were calculated using cumulative incidence rates to accommodate competing risks. Disease-free survival (DFS) was analyzed using Kaplan-Meier estimates.Results: The median age was 21 years (range, 5-45) and weight 59 kg (range, 23-89). Other patient characteristics and median infused cell doses are shown in Table 1. Four patients (26%) received only 1 unmanipulated CB unit along with the expanded graft. All patients engrafted at a median of 19 days (range, 9-31 days), significantly faster than the 25 days (range, 14-45) observed in 41 recipients of two unmanipulated units otherwise treated identically (p=0.01) (Figure 1A).Similar to our initial expansion trial using partially HLA-matched expanded CB cells, early (day 7) myelomonocytic (CD33 and CD14) recovery was almost entirely (98–100%) due to cells arising from the expansion product. Cells derived from the expansion product were no longer detected at day 14 in all but 2 patients. The median time to platelet recovery (>20 x 10^9/L) was 35 days (range 21–45).The cumulative incidence of platelet recovery by day 100 was significantly higher than that observed in our concurrent control group 92% (CI 95%: 59-99) vs. 69% (95%: 51-81), respectively (p=0.005) (Figure 1B). The probability of 3-year DFS was 86% (95% CI, 57-97). No TRM was observed throughout the study period (Figure 1C), although 2 patients relapsed at days 53 and 219 posttransplant and subsequently died after further therapy. All patients presented with grade II acute GVHD at a median time of 32 days (14-86), with no grade III-IV aGVHD observed (Figure 1D). The skin was the most commonly affected organ (n=12). Eight (53%) patients were treated for pre-engraftment syndrome at a median time of 6 days (range 4-9) and five (33%) patients had GVHD after day 100. At 2 years only 2 patients are still on immunosuppressive therapy for mild chronic GVHD.Conclusions: The results of this Phase I safety study demonstrated that infusion of an off-the-shelf non-HLA matched expanded CB HSPC product to augment a CB graft in the myeloablative setting was safe and led to more rapid neutrophil and platelet recovery compared to our concurrent control group. Importantly, no TRM was observed among the 15 patients on this study, with only 2 deaths due to relapsed disease, leading to an excellent OS of 86% at a median follow-up of 3.2 years. Quite interesting is the observation of no grade 3-4 acute GVHD, even with infusion of a non-HLA matched expanded cell product that may induce increased alloreactivity of the unmanipulated CB unit(s). However, in this preliminary pilot study, it appeared that infusion of the non HLA-matched product was associated with less severe acute GVHD. Based on these very promising results, a prospective multicenter randomized trial utilizing this product is underway. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Infusion of Ex Vivo Expanded Cord Blood Progenitor Cells Reduces the Risk of Bacteremia after Myeloablative Cord Blood Transplant

▪Background: Delayed hematopoietic recovery after myeloablative cord blood transplantation (CBT) is associated with higher infection-related morbidity and mortality when compared to recipients of conventional donor grafts. In particular, prolonged neutropenia, related to low cell doses provided by the cord blood (CB) graft, results in higher rates of infection in the first 100 days post-transplant. To overcome this issue, our group has explored ex vivo expansion of CB-derived CD34+ cells in the presence of Notch ligand. We have previously demonstrated that infusion of this expanded progenitor cell product leads to a significant reduction in the time to neutrophil engraftment in the myeloablative CBT setting [Delaney C, et al, Nat Med 2010]. We now hypothesize that faster neutrophil engraftment is associated with lower risk of bloodstream infections in the first 100 days post-transplant in patients receiving ex-vivo expanded progenitors (EXP) compared to patients receiving conventional CBT (CON).Methods: Between April 2006 and June 2014, a total of 93 consecutive patients were enrolled on multicenter studies coordinated by our Institution. Of these 93, 55 (59%) received single (n=9) or double (n=46) unmanipulated CB graft, while 38 (41%) patients received 1 or 2 unmanipulated CB units plus infusion of either a fresh partially HLA-matched (n=23) or a cryopreserved non-HLA matched (n=15) ex-vivo expanded CB unit. All patients received the same conditioning regimen of Fludarabine (75mg/m2), Cytoxan (120mg/kg) and TBI (13.2 Gy) over 8 days. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. Cox regression model was used to assess the risk of bacterial infection where "failure" was defined as the first occurrence of bloodstream infection within 100 days following CBT.Results: The median patient age was 22 years (range 0.6-45) and 26 (range 3-45) for CON and EXP group respectively (p=0.51); gender, race and CMV serostatus were not different between the 2 groups. Disease distribution was similar in the two groups, with most patients undergoing CBT for AML (n=47), ALL (n=28) and MDS (n=10). Median pre-thaw TNC/kg x107 was 4.97 (range 3.4-16.6) and 11.2 (range 2.7-30.4) for the CON and EXP groups respectively, and pre-thaw CD34 cells/kg x106 was 0.19 for the CON (range 0.04-0.98) and 6.3 for the EXP group (range 0.12-49.7). As expected, time to engraftment was significantly faster for patients receiving the EXP units at 17 days (range 6-41) vs. 25 days in the CON group (range 14-45, p<0.01). Only 1 case of graft failure was observed in the EXP group. The median time to first positive bloodstream infection in the EXP cohort was 36 days (range 3-98) vs. 8 days (range 1-88) in the CON group. The cumulative incidence of bacteremia was significantly lower in the EXP group (n=14) compared to the CON group (n=36) at 37% vs. 65% respectively (p=0.005, Figure 1). The hazard ratio (HR) of bacteremia episodes was significantly lower among patients receiving the expanded cells (HR 0.43; 95% CI 0.23-0.79; p=0.007). Among first documented bacteremic episodes, 5 of 50 (10%) were gram-negative rod bacteremias, all of which were in the CON group.Conclusions: Infections remain a significant risk for morbidity and mortality after myeloablative CBT and are the leading cause of early treatment related mortality. Our data suggest that there was a significant reduction in risk of bacteremia in the first 100 days post myeloablative CBT and a longer time to first infection when patients received ex vivo expanded progenitor cells, either as a partially matched fresh infusion or as an off-the-shelf, non-HLA matched expanded product. Infusion of the expanded cells resulted in reduced time to myeloid engraftment and offered a protective effect against episodes of bacteremia, but the exact mechanisms are presently unknown. It is possible that the expanded cells may home to regions of mucosal injury post TBI and mitigate breakdown thus reducing bacterial translocation. Further research to further define the expanded cells’ mechanism of action with regards to infection reduction is currently underway in animal models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

­Separation of Lymphocyte Populations from Peripheral Blood Progenitor Cell Products Using Affinity Bead Acoustophoresis

Introduction: Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations. We have previously reported the use of a novel ultrasound-based sorting technology, called acoustophoresis, for sorting of platelets (Dykes et al., PloS one 2011) and CD4+ cells from PBPC products (Lenshof et al., Cytometry Part A 2014). Here, we investigated the performance of microfluidic acoustophoresis for the separation of CD8+ lymphocytes from PBPC, and present a method for affinity-bead-mediated acoustic separation of cells which otherwise cannot be discriminated acoustically. In an acoustic standing wave field radiation forces induce movement of particles depending on particle and medium properties, such as for example particle size, density and compressibility. Targeting of cells by affinity specific beads generates cell-bead complexes that exhibit distinct acoustic properties relative to non-targeted cells and, thus, become possible to isolate.Method: PBPC samples (n=16) were obtained from patients and healthy donors. Following density gradient centrifugation, mononuclear cells were labelled with anti-CD8 microbeads (Dynal) and sorted either on an acoustophoresis-microchip (Figure 1) or standard magnetic cell sorting technique for comparison. PBPC samples, target and waste fractions were analysed for purity, separation efficiency, recovery, T-cell function and progenitor cell content.Results: PBPC products contained a mean of 11.6 ± 7.1% CD8+ cells before sorting. Purities obtained with acoustic sorting of CD8+ lymphocytes were 93.3 ± 6.8% compared to 94.4 ± 8.6% for magnetic sorting (n=16). Viabilities of sorted cells were 97.0 ± 3.9% (acoustic) and 97.5 ± 3.5% (magnetic). Mean separation efficiency recovery of acoustic sorted CD8+ cells was 57 ± 19% of the total CD8+ cells compared to a median recovery of magnetic sorted CD8+ cells of 43 ± 17%. Leukocyte subpopulation analysis performed after CD8 selection showed a relative increase of CD4 cells in the non-target fractions due to the removal of CD8 cells. Functional testing of sorted CD8+ lymphocytes showed unimpaired mitogen mediated proliferation capacity after 2-day, 4-day and 6-day stimulation with CD3/CD28. Furthermore, hematopoietic progenitor cell assays revealed a preserved colony forming ability of the post-sorted non-target cellsConclusion: Acoustophoresis is a promising technology to efficiently sort bead-labelled lymphocyte populations from PBPC samples with high purity and recovery without impairing lymphocyte function. Affinity-bead acoustophoresis is, thus, an interesting technology for stem cell processing in PBPC. [Display omitted] DisclosuresLaurell:Acousort AB: shareholder Other. Scheding:Acousort AB: Co-founder and board member Other.

Phase 1 Clinical Investigation of Human Myeloid Progenitor Cells (CLT-008) As a Supportive Care Measure during Chemotherapy for Acute Myeloid Leukemia (AML)

Background: Current management of chemotherapy and radiation-induced neutropenia is unsatisfactory with frequent infection-related deaths. A novel allogeneic human myeloid progenitor cell product, CLT-008, that is culture-derived from G-CSF mobilized HSCs, can be cryopreserved and infused without HLA matching. Cells are intended to engraft transiently and produce mature myeloid effector cells that migrate to tissues and may mitigate the risk of bacterial or fungal infection for weeks, as has been demonstrated in murine models.Methods: To evaluate the safety of CLT-008, a phase 1 dose escalation study was conducted in subjects with AML, MDS or ALL during consolidation (n=18) or with AML or tAML during first or subsequent induction (n=31). Eligible subjects were age 18 or older and treated with a high-dose or standard-dose cytarabine-based regimen. All 18 consolidation subjects and 23 induction subjects received CLT-008. Subjects received CLT-008 on study day 6 (consolidation 106 or 107cells/kg) or on study day 9 (induction 107 or 3x107 cells/kg). G-CSF was given at 5 µg/kg daily from the day of CLT-008 until the ANC was 500/µL. During induction, 8 non-randomly selected controls received G-CSF without CLT-008. Dose limiting toxicities (DLT) were defined as any grade 3-5 infusion reaction, grade 4-5 organ toxicity (not due to the primary malignancy, infection, pre-existing condition, or initiation of new chemotherapy), any Grade III to IV GVHD, or failure of autologous neutrophil recovery at Day 42 not clearly attributable to another cause. All subjects were followed for 49 days and status was assessed at 6 months.Results: Among 41 currently evaluable subjects, 1 experienced a DLT of grade 3 allergic infusion-related reaction which resolved promptly with steroids and antihistamine. There were no other DLTs and no GVHD of any grade. Treatment-emergent adverse events occurring in 3 or more subjects included febrile neutropenia, infection, mucosal inflammation, fatigue, diarrhea, tachycardia and nausea but were not judged related to the product. Among the 23 induction subjects receiving CLT-008 with G-CSF, there was a reduction in mucositis (WHO grade 2 or greater) and in duration of febrile episodes when compared to the 8 control subjects. CLT-008-derived cells were detectable in the peripheral blood for 6 of 23 induction subjects at 2-10% of nucleated cells using STR-PCR; there was no clear effect on ANC from this chimerism. To date, 8 of 23 induction subjects went on to subsequent HSC transplant. Seven engrafted while one died on post-transplant day 10 from an unrelated cause.Conclusions: CLT-008 was safe and well tolerated in this phase 1 study. Only 1 DLT occurred and the pattern of AEs was as expected for this population. Among the induction subjects treated at 3x107 cells/kg, efficacy signals with respect to mucositis and duration of fever were found in the absence of high-level peripheral blood CLT-008 chimerism. These observations suggest that CLT-008-derived myeloid effector cells preferentially migrate to chemotherapy-damaged mucosal tissues where they could function to mitigate infection risk. Further clinical development is planned in a randomized phase 2 study using an enhanced potency iteration of CLT-008, assessment of tissue-level chimerism, and specific ascertainment of outcomes related to infection and chemotherapy sequellae. [Display omitted] DisclosuresWieduwilt:Cellerant Therapeutics: Honoraria, Research Funding. Cerny:Cellerant Therapeutics: Honoraria, Research Funding. Akard:Cellerant Therapeutics: Research Funding. Ustun:Cellerant Therapeutics: Honoraria, Research Funding. Ravandi:Cellerant Therapeutics: Research Funding. Rossetti:Cellerant Therapeutics: Research Funding. Hill:Cellerant Therapeutics: Honoraria, Research Funding. Brown:Cellerant Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Kellerman:Cellerant Therapeutics: Employment, Equity Ownership. Van Syoc:Cellerant Therapeutics: Employment, Equity Ownership. Reed:Cellerant Therapeutics: Employment, Equity Ownership. Andreadis:Cellerant Therapeutics: Research Funding.

Cellular reprogramming for understanding and treating human disease.

In the last two decades we have witnessed a paradigm shift in our understanding of cells so radical that it has rewritten the rules of biology. The study of cellular reprogramming has gone from little more than a hypothesis, to applied bioengineering, with the creation of a variety of important cell types. By way of metaphor, we can compare the discovery of reprogramming with the archeological discovery of the Rosetta stone. This stone slab made possible the initial decipherment of Egyptian hieroglyphics because it allowed us to see this language in a way that was previously impossible. We propose that cellular reprogramming will have an equally profound impact on understanding and curing human disease, because it allows us to perceive and study molecular biological processes such as differentiation, epigenetics, and chromatin in ways that were likewise previously impossible. Stem cells could be called “cellular Rosetta stones” because they allow also us to perceive the connections between development, disease, cancer, aging, and regeneration in novel ways. Here we present a comprehensive historical review of stem cells and cellular reprogramming, and illustrate the developing synergy between many previously unconnected fields. We show how stem cells can be used to create in vitro models of human disease and provide examples of how reprogramming is being used to study and treat such diverse diseases as cancer, aging, and accelerated aging syndromes, infectious diseases such as AIDS, and epigenetic diseases such as polycystic ovary syndrome. While the technology of reprogramming is being developed and refined there have also been significant ongoing developments in other complementary technologies such as gene editing, progenitor cell production, and tissue engineering. These technologies are the foundations of what is becoming a fully-functional field of regenerative medicine and are converging to a point that will allow us to treat almost any disease.