Primitive Progenitor Cells Research Articles

Drl.3 governs primitive hematopoiesis in zebrafish.

The molecular program controlling hematopoietic differentiation is not fully understood. Here, we describe a family of zebrafish genes that includes a novel hematopoietic regulator, draculin-like 3 (drl.3). We found that drl.3 is expressed in mesoderm-derived hematopoietic cells and is retained during erythroid maturation. Moreover, drl.3 expression correlated with erythroid development in gata1a- and spi1b-depleted embryos. Loss-of-function analysis indicated that drl.3 plays an essential role in primitive erythropoiesis and, to a lesser extent, myelopoiesis that is independent of effects on vasculature, emergence of primitive and definitive progenitor cells and cell viability. While drl.3 depletion reduced gata1a expression and inhibited erythroid development, enforced expression of gata1a was not sufficient to rescue erythropoiesis, indicating that the regulation of hematopoiesis by drl.3 extends beyond control of gata1a expression. Knockdown of drl.3 increased the proportion of less differentiated, primitive hematopoietic cells without affecting proliferation, establishing drl.3 as an important regulator of primitive hematopoietic cell differentiation.

Stem cell programs are retained in human leukemic lymphoblasts.

Leukemic lymphoblasts within different immunophenotypic populations possess stem cell properties. However, whether or not the self-renewal program is retained from stem cells or conferred on progenitors by leukemogenic molecules remains unknown. We have addressed the issue in the context of TEL-AML1-associated acute lymphoblastic leukemia (ALL) by profiling a refined program edited from genes essential for self-renewal of hematopoietic stem cells and B-cell development. Bioinformatic analysis shows that ALL populations are loosely clustered and close to the normal population that contains stem and primitive progenitor cells. This finding indicates that immunophenotypes do not reflect maturation stages in ALL and that the self-renewal program may be retained from stem cells. Results of assessing 'first hit' function of TEL-AML1 in different populations of normal cells demonstrate the molecular model. Therefore, the current study shows a leukemogenic scenario of human ALL in which programs of stem cells are sustained in distinct fractions by leukemogenic mutations.

Id2 Mediates Oligodendrocyte Precursor Cell Maturation Arrest and Is Tumorigenic in a PDGF-Rich Microenvironment

Maturation defects occurring in adult tissue progenitor cells have the potential to contribute to tumor development; however, there is little experimental evidence implicating this cellular mechanism in the pathogenesis of solid tumors. Inhibitor of DNA-binding 2 (Id2) is a transcription factor known to regulate the proliferation and differentiation of primitive stem and progenitor cells. Id2 is derepressed in adult tissue neural stem cells (NSC) lacking the tumor suppressor Tp53 and modulates their proliferation. Constitutive expression of Id2 in differentiating NSCs resulted in maturation-resistant oligodendroglial precursor cells (OPC), a cell population implicated in the initiation of glioma. Mechanistically, Id2 overexpression was associated with inhibition of the Notch effector Hey1, a bHLH transcription factor that we here characterize as a direct transcriptional repressor of the oligodendroglial lineage determinant Olig2. Orthotopic inoculation of NSCs with enhanced Id2 expression into brains of mice engineered to express platelet-derived growth factor in the central nervous system resulted in glioma. These data implicate a mechanism of altered NSC differentiation in glioma development and characterize a novel mouse model that reflects key characteristics of the recently described proneural subtype of glioblastoma multiforme. Such findings support the emerging concept that the cellular and molecular characteristics of tumor cells are linked to the transformation of distinct subsets of adult tissue progenitors.

Induced pluripotent stem cells as a source of hepatocytes.

During the past decade, a series of discoveries has established the potential of the so called terminally differentiated cells to transition to more primitive progenitor cells. The dramatic demonstration of the ability to reprogram differentiated somatic cells to induced pluripotent stem cells (iPSC) that can then give rise to cells of all three germ layers has opened the possibility of generating virtually any cell type in culture, from any given individual. Taking advantage of these concepts, researchers have generated iPSCs by reprogramming a wide variety of somatic cells. In addition to their practical implications, these studies have provided crucial insights into the mechanism of cell plasticity that underlies the transition from one cell type to another. Using concepts derived from research on embryological development, investigators have differentiated iPSCs to cells resembling hepatocytes in many ways. Such hepatocyte-like cells could be of enormous value in disease modeling, drug discovery and regenerative medicine. However, the currently available methods do not yield cells that fully reproduce the characteristics of adult primary hepatocytes. Thus generating hepatocytes from iPSCs is very much a work in progress. In addition to chronicling these exciting developments, this review will discuss the emergent new approaches to generating iPSCs, improving their differentiation to hepatocyte-like cells and maintaining the hepatocyte-like cells in culture for longer survival and better function.

Low-dose Triptolide in combination with Idarubicin could induce apoptosis in leukemia stem and progenitor cells via affecting the intrinsic and extrinsic factors

BackgroundRelapse has been a major hurdle for the success of acute myeloid leukemia chemotherapy, whereas leukemia stem cells (LSCs) have been considered to be responsible for relapse recently. Thus new drug targeting LSCs is urgently needed. Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii, has been used in Traditional Chinese Medicine (TCM) for centuries. Recently, it has been reported that TPL has the potential of depleting quiescent CD34+ primitive CML progenitor cells. Moreover, normal CD34+ hemopoietic stem cells are less sensitive than AML blasts to TPL, suggesting the seclectivity of TPL in targeting LSCs. AimsIn this study, the ability of triptolide to induce apoptosis in leukemia stem and progenitor cells was investigated with the underlying mechanism being explored. MethodsLeukemia stem and progenitor cells were sorted from KG1a cells using flow cytometry with cell cycle being analyzed and then were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR, intracellular ROS measurement colony forming assay were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes, ROS level and colony forming ability. ResultsTPL is highly cytotoxic to leukemia stem and progenitor cells with IC20 of 5.0±0.81nM and IC50 of 20.48±1.6nM after 72h exposure. Leukemia stem and progenitor cells were also exposed to series concentrations of IDA with or without TPL(IC20F5.0nM). TPL significantly enhanced cytotoxicity of IDA to LSCs (IC50-IDA: 285.20±13.7 nM vs. IC50-IDA+TPL: 27.01±0.73 nM, P<0.001) Combination index was also analyzed. Results of combination index showed that synergism effect was observed over a wide range of IC50-IC90 concentrations of IDA and TPL in combination. Moreover, apoptotic ratio of cells treated by IDA with TPL was significantly increased compared to cells treated by IDA alone (24.85±1.70% vs. 76.87±8.34%, P<0.001). What's more, TPL in combination with IDA significantly inhibited the clonogenicity of leukemia stem and progenitor cells (-VE: 2097.3±106.07/well; TPL: 1387.7±40.05/well; IDA: 1252±46.87/well; TPL+IDA: 211±93.18/well, P<0.001). Next, we explore the underlying mechanism of TPL-induced apoptosis in LSCs. The survival of LSCs are dependent of intrinsic factors (Nrf2 and ROS) and extrinsic factors(HIF-1α, VLA4). Induction of ROS while inhibition of protective factor Nrf2 could lead to the apoptosis of LSCs. ROS activation was concentration-dependent when IDA was combined with a constant concentration of TPL (IC20:5nM). The highest activity of intracellular ROS appeared in IDA 200nM+TPL5nM group. Down-regulation of Nrf2 as well as its target genes such as NQO1, GSR and HO-1 was also observed in our study. Moreover, extrinsic factors such as HIF-1α and its down-stream genes such as BNIP3, VEGF and CAIX were also down-regulated in TPL with IDA group. Important extrinsic factors affecting the adhesion of LSCs such as VLA4 and CXCR4 were also down-regulated in TPL with IDA group. ConclusionOur findings indicate that the cytotoxicity of triptolide and the enhancing effect of triptolide on the cytotoxicity of idarubicin in leukemia stem and progenitor cells may be caused by affecting the intrinsic and extrinsic factors. Disclosures:No relevant conflicts of interest to declare.

Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling

Hematopoietic stem and progenitor cells (HSPC) are mostly retained in a quiescent, non-motile mode in the bone marrow (BM), shifting to a cycling, differentiating and migratory state on demand. How HSC replenish the blood with new mature leukocytes on a daily basis while maintaining a constant pool of primitive cells in the BM throughout life is not clear. Recently, we reported that the bioactive lipid Sphingosine 1-Phosphate (S1P) regulates HSPC mobilization via ROS signaling and CXCL12 secretion (Golan et al, Blood 2012). We hypothesize that S1P influences the daily circadian egress of HSPC and their proliferation. We report that S1P levels in the blood are increased following initiation of light at the peak of HSPC egress and are reduced towards the termination of light when circulating HSPC reach a nadir. Interestingly, mice with constitutively low S1P plasma levels due to lack of one of the enzymes that generates S1P (Sphingosine kinase 1), do not exhibit fluctuations of HSPC levels in the blood between day and night. We report that HSPC numbers in the BM are also regulated in a circadian manner. Unexpectedly, we found two different daily peaks: one in the morning, following initiation of light, which is accompanied by increased HSPC egress and the other at night after darkness, which is associated with reduced HSPC egress. In both peaks HSPC begin to cycle and differentiate via up-regulation of reactive oxygen species (ROS) however, the night peak had lower ROS levels. Concomitant with the peak of primitive stem and progenitor cells, we also observed (to a larger extent in the night peak), expansion of a rare activated macrophage/monocyte αSMA/Mac-1 population. This population maintains HSPC in a primitive state via COX2/PGE2 signaling that reduces ROS levels and increases BM stromal CXCL12 surface expression (Ludin et al, Nat. Imm. 2012). We identified two different BM peaks in HSPC levels that are regulated by the nervous system via circadian changes in ROS levels. Augmented ROS levels induce HSPC proliferation, differentiation and motility, which take place in the morning peak; however, they need to be restored to normal levels in order to prevent BM HSPC exhaustion. In the night peak, HSPC proliferate with less differentiation and egress, and activated macrophage/monocyte αSMA/Mac-1 cells are increased to restore ROS levels and activate CXCL12/CXCR4 interactions to maintain a HSPC primitive phenotype. Additionally, S1P also regulates HSPC proliferation, thus mice with low S1P levels share reduced hematopoietic progenitor cells in the BM. Interestingly S1P is required more for the HSPC night peak since in mice with low S1P levels, HSPC peak normally during day time but not at darkness. We suggest that the first peak is initiated via elevation of ROS by norepinephrine that is augmented in the BM following light-driven cues from the brain (Mendez-Ferrer at al, Nature 2008). The morning elevated ROS signal induces a decrease in BM CXCL12 levels and up-regulated MMP-9 activity, leading to HSC proliferation, as well as their detachment from their BM microenvironment, resulting in enhanced egress. Importantly, ROS inhibition by N-acetyl cysteine (NAC) reduced the morning HSPC peak. Since norepinephrine is an inhibitor of TNFα, upon light termination norepinephrine levels decrease and TNFα levels are up-regulated. TNFα induces activation of S1P in the BM, leading to the darkness peak in HSPC levels. S1P was previously shown also to induce PGE2 signaling, essential for HSPC maintenance by the rare activated αSMA/Mac-1 population. Indeed, in mice with low S1P levels, we could not detect a peak in COX2 levels in these BM cells during darkness. We conclude that S1P not only induces HSPC proliferation via augmentation of ROS levels, but also activates PGE2/COX2 signaling in αSMA/Mac-1 population to restore ROS levels and prevent HSPC differentiation and egress during the night peak. We hypothesize that the morning HSPC peak, involves proliferation, differentiation and egress, to allow HSPC to replenish the blood circulation with new cells. In contrast, the second HSPC night peak induces proliferation with reduced differentiation and egress, allowing the renewal of the BM HSPC pool. In summary, we identified two daily circadian peaks in HSPC BM levels that are regulated via light/dark cues and concomitantly allow HSPC replenishment of the blood and immune system, as well as maintenance of the HSPC constant pool in the BM. Disclosures:No relevant conflicts of interest to declare.

Gliosarcoma with ependymal and PNET-like differentiation

A rare case of gliosarcoma which arose in the temporal lobe of a 39-yearold man was reported. The gliomatous area of the tumor showed ependymal differentiation, and also contained immature neuroectodermal tissue resembling a primitive neuroectodermal tumor (PNET) in addition to an ordinary glioblastomatous component. Tumor cells in the PNET-like component were immunoreactive for synaptophysin, CD99, neurogenin 3, and α-internexin, but not for glial fibrillary acidic protein (GFAP), Class III-β tubulin, or Neu N. The mesenchymal area exhibited a compact fascicular proliferation of atypical spindle cells invested by fine reticulin fibrils. In addition, these cells were immunoreactive for Slug and Twist - transcription factors which are involved in the "epithelial-mesenchymal transition (EMT)" phenomenon. Gliosarcomas containing an ependymal or PNET-like component are rare, and to our knowledge, the present case is the first to be reported whose glial element exhibited differentiation toward these two components. The diverse differentiation in the glial element suggests that the tumor most likely originated from primitive neuroepithelial progenitor cells rather than from the neometaplasia of a glioblastoma. The immunoreactivity for transcription factors in the mesenchymal element indicated that EMT might be involved in the pathogenesis of this very rare type of gliosarcoma.

BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells

AbstractMyelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1+/c-Kit+, and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS–AML transformation and survival of leukemic cells.

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro. MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor. ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34+ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor. ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.

Matched miRNA and mRNA signatures from an hESC-based in vitro model of pancreatic differentiation reveal novel regulatory interactions

The differentiation of human pluripotent stem cells (hPSCs) to insulin-expressing beta islet-like cells is a promising in vitro model system for studying the molecular signaling pathways underlying beta cell differentiation, as well as a potential source of cells for the treatment of type 1 diabetes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate many biological processes, including cellular differentiation. We studied the miRNA and mRNA expression profiles of hPSCs at five stages of in vitro differentiation along the pancreatic beta cell lineage (definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor and hormone-expressing endocrine cells) in the context of samples of primary human fetal pancreas and purified adult islet cells using microarray analysis. Bioinformatic analysis of the resulting data identified a unique miRNA signature in differentiated beta islet cells, and predicted the effects of key miRNAs on mRNA expression. Many of the predicted miRNA-mRNA interactions involved mRNAs known to play key roles in the epithelial-mesenchymal transition process and pancreatic differentiation. We validated a subset of the predictions using qRT-PCR, luciferase reporter assays and western blotting, including the known interaction between miR-200 and ZEB2 (involved in epithelial-mesenchymal transition) and the novel interaction between miR-200 and SOX17 (a key transcription factor in specification of definitive endoderm). In addition, we found that miR-30d and let-7e, two miRNAs induced during differentiation, regulated the expression of RFX6, a transcription factor that directs pancreatic islet formation. These findings suggest that precise control of target mRNA expression by miRNAs ensures proper lineage specification during pancreatic development.

KIT Signaling Governs Differential Sensitivity of Mature and Primitive CML Progenitors to Tyrosine Kinase Inhibitors

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34(+)38(-) cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34(+)38(+) cells, SCF strongly induced pAKT(S473) in a phosphoinositide 3-kinase (PI3K)-dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKT(S473) levels remained low in CD34(+)38(-) cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34(+)38(-) compared with CD34(+)38(+) CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells.

Loss of quiescence and impaired function of CD34+/CD38low cells one year following autologous stem cell transplantation

Patients who have undergone autologous stem cell transplantation are subsequently more susceptible to chemotherapy-induced bone marrow toxicity. In the present study, bone marrow primitive progenitor cells were examined one year after autologous stem cell transplantation and compared with normal bone marrow and mobilized peripheral blood stem cells. Post-transplantation bone marrow contained a significantly lower percentage of quiescent cells in the CD34(+)/CD38(low) fraction compared to normal bone marrow. In addition, we observed a strong decrease in stem cell/primitive progenitor frequency in post-transplantation CD34(+) cells as defined by long-term culture assays. Measurement of the levels of reactive oxygen species by flow cytometry revealed comparable levels in post-transplantation and normal bone marrow CD34(+)/CD38(low) cells, while significantly higher levels of reactive oxygen species were observed in CD34(+)/CD38(high) cells following autologous stem cell transplantation compared to normal bone marrow. Moreover, post-transplantation CD34(+) bone marrow cells demonstrated an increased sensitivity to buthionine sulfoximine, a trigger for endogenous production of reactive oxygen species. Gene expression analysis on CD34(+) cells revealed a set of 195 genes, including HMOX1, EGR1, FOS and SIRPA that are persistently down-regulated in mobilized peripheral blood cells and post-transplantation bone marrow compared to normal bone marrow. In conclusion, our data indicate that the diminished regenerative capacity of bone marrow following autologous stem cell transplantation is possibly related to a loss of quiescence and a reduced tolerability to oxidative stress.

Cord blood unit bankability can be predicted by prenatal sonographic parameters

ObjectiveTo identify possible sonographic prenatal parameters and postnatal parameters in order to obtain more bankable cord blood units (CBUs) containing a high number of primitive progenitor cells, allowing CBUs to be used as a source of haematopoietic progenitors for clinical transplantation. Study designProspective study undertaken in the Department of Gynaecology, Obstetrics and Reproductive Science, Second University of Naples, Italy. In total, 219 unrelated CBU donors were enrolled. Ultrasound parameters (biparietal diameter, head circumference, abdominal circumference, femur length, estimation of fetal weight, umbilical artery pulsatility index), collected at hospital admission, together with birth weight and placental weight, were correlated with bankable CBU parameters (CBU volume, total nucleated cell count, CD34+ cell count). ResultsFemur length and abdominal circumference correlated positively with bankable CBUs. Receiver operating curve analysis showed that these parameters can identify bankable CBUs. ConclusionsThis is the first prospective study to show the relationship between ultrasonographic fetal parameters at term and the possibility of obtaining high-quality CBUs. As such, cord blood banking could be improved worldwide by performing low-cost ultrasonographic scans.

Radiation and senescence: cell cycle of primitive hematopoietic progenitor cells (CFU-S13) accelerated in 2-Gy whole-body irradiated senescent mice, which originally decelerated during aging in the steady state

Radiation and senescence: cell cycle of primitive hematopoietic progenitor cells (CFU-S13) accelerated in 2-Gy whole-body irradiated senescent mice, which originally decelerated during aging in the steady state

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

RationaleStage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.ObjectiveWe generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.Methods and ResultsLethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.ConclusionsThese results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.

Quality control measures in cord blood banking in India - critical appraisal and recommendations.

Umbilical cord blood (UCB) is a potential source of primitive hematopoietic stem and progenitor cells (HSPC) available for clinical application to reconstitute the hematopoietic system in patients lacking conventional stem cell donors. In the last decade because the frequency of UCB stem cell transplantation has increased, the quality of UCB available in banks has become an important criterion for the success of any UCB stem cell transplant. Moreover, with the growing numbers of cord blood banks there is not only a need to improvise quality issues but to also develop a more homogenous approach in setting standards and regulations for assessing them. The present article provides an insight on the importance of cord blood banking in India and the impending requirement to provide clarity on certain issues regarding UCB banking. Furthermore, the article discusses about certain practical approaches which could perhaps be referred to as guidelines (and by no means be solely adopted as the only measures) by cord blood banks in India for establishing or maintaining a standard for quality control (QC) measures.

Lineage Tracing of Pf4-Cre Marks Hematopoietic Stem Cells and Their Progeny

The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15–60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage.

The pharmacological activation of adenosine A1 and A 3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice.

This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A1 and A3 receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N (6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N (6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.

Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells

AbstractAbstract 26During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production.As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level. Disclosures:No relevant conflicts of interest to declare.

A Dual Role of Evi-1 During Developmental Hematopoiesis

AbstractAbstract 765The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi-1 expression with aggressiveness in myeloid leukemia. More recent studies suggest involvement of Evi-1 in developmental hematopoiesis. Here, we analyzed zebrafish embryo and human induced pluripotent stem (iPS) cells to understand how Evi-1 modulates early blood development. We found that the zebrafish homologue evi-1 co-localizes with the hematopoietic markers scl, gata1 and gata2 during embryogenesis, indicating a role in primitive hematopoiesis. To analyse the functional role of evi-1, we performed in vivo loss-of-function studies by injecting control and inhibitory morpholino oligonucleotides (MO) suppressing evi-1 pre-mRNA splicing in zebrafish zygotes. To control for off-target effects, three separate MO were designed and injected. N = 100 zebrafish embryos were analyzed per experiment in each group. In situ hybridization analyses performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells as shown by reduced pu.1, mpo and l-plastin expression and suppressed numbers of DsRed positive lyz-expressing cells in Tg(lyz:DsRed) fish. Interestingly, scl expression was not affected, suggesting that evi-1 acts at later stages, regulating specification and/or survival/proliferation of pu.1 expressing progenitor cells. Consistent with this notion, no changes were observed in primitive erythroid progenitor cells as monitored by gata1 and hbae3 expression and flow cytometry analyses of GFP-positive globin-expressing cells in Tg(globin:eGFP) fish. Furthermore, runx1+/cmyb+ cells were strongly reduced, suggesting a separate role of evi-1 in definitive hematopoiesis. Supporting this hypothesis, evi-1 morphants displayed profound suppression of ikzf1+ lymphoid progenitors and rag1+ T-lymphocytes, which was not due to impaired thymus development, since foxn1 expression remained unaltered in evi-1 morphants. Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects HSC proliferation through regulation of Gata2. Indeed, we could show that co-injection of gata2 mRNA was able to rescue the impaired myeloid as well as the HSC phenotype in evi-1 morphants. However, data generated by TUNEL assays indicate that evi-1 also confers apoptosis resistance to developing AGM-HSC. Whether this effect is also mediated by gata2 is currently being explored. We further investigated the effect of EVI-1 during differentiation of hematopoietic cells from human iPS cells. Confirming our results collected in zebrafish embryo, inhibition of EVI-1 strongly reduced the numbers of CD34+ and CD45+ blood progenitor cells and the generation of myeloid colonies in colony forming assays, while leaving SCL expression unaltered. Taken together, our data suggest that Evi-1 plays conserved roles in zebrafish and human hematopoietic development, regulating embryonic myelopoiesis and HSC formation while leaving primitive erythropoiesis unaltered. The precise molecular basis of evi-1 mediated effects on these separate phases of developmental hematopoiesis is subject of ongoing investigations. Disclosures:No relevant conflicts of interest to declare.